- Email: [email protected]
Oxysterols and nuclear receptors
Accepted Manuscript Oxysterols and nuclear receptors Liqian Ma, Erik R. Nelson PII:
S0303-7207(19)30022-X
DOI:
https://doi.org/10.1016/j.mce.2019.01.016
Reference:
MCE 10378
To appear in:
Molecular and Cellular Endocrinology
Received Date: 5 November 2018 Revised Date:
8 January 2019
Accepted Date: 16 January 2019
Please cite this article as: Ma, L., Nelson, E.R., Oxysterols and nuclear receptors, Molecular and Cellular Endocrinology (2019), doi: https://doi.org/10.1016/j.mce.2019.01.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title: Oxysterols and Nuclear Receptors
1
Authors: Liqian Ma and Erik R. Nelsona,b,c,d,e* a
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign. Urbana, IL. University of Illinois Cancer Center. Chicago, IL. c Division of Nutritional Sciences, University of Illinois at Urbana-Champaign. Urbana, IL. d Carl R. Woese Institute for Genomic Biology, Anticancer Discovery from Pets to People Theme, University of Illinois at Urbana Champaign, Urbana, IL e Cancer Center at Illinois, University of Illinois at Urbana-Champaign, IL
M AN U
SC
RI PT
b
Abbreviated Title: Oxysterols and Nuclear Receptors. Keywords: cholesterol, oxysterol, 27-hydroxycholesterol; estrogen receptor; liver x receptor; breast cancer; selective estrogen receptor modulator, selective nuclear receptor modulator
EP
TE D
* Corresponding Author: Erik R. Nelson. University of Illinois at Urbana-Champaign. 407 S Goodwin Ave (MC114), Urbana, IL, 61801. Phone: 217-244-5477. Fax: 217-333-1133. E-mail: [email protected].
AC C
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
ACCEPTED MANUSCRIPT
RI PT
Abstract: Oxysterols are derivatives of cholesterol and an important regulator of cholesterol metabolism, in part due to their role as ligands for nuclear receptors, such as the liver x receptors. Oxysterols are also known to be ligands for the RAR-related orphan receptors, involved in normal T cell differentiation. However, increasing evidence supports a role for oxysterols in the progression of several diseases. Here, we review recent developments in oxysterol research, highlighting the biological functions that oxysterols exert through their target nuclear receptors: the liver x receptors, estrogen receptors RAR-related orphan receptor and the glucocorticoid receptor. We also bring the regulation of the immune system into the context of interaction between oxysterols and nuclear receptors, discussing the effect of such interaction on the pro-inflammatory function of macrophages and the development of T cells. Finally, we examine the impact that oxysterols have on various disease models, including cancer, Alzheimer’s disease and atherosclerosis, stressing the role of nuclear receptors if previously identified. This review underscores the need to consider the multifaceted roles of oxysterols in terms of multiple receptor engagements and selective modulation of these receptors.
EP
TE D
M AN U
SC
Funding: This work was funded in part by grants from the Department of Defense Breast Cancer Research Program (BC171214) and the American Institute of Cancer Research (Award 31284) to ERN.
AC C
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
ACCEPTED MANUSCRIPT 42
Introduction:
43 Oxysterols are derivatives of cholesterol produced by the oxidation of cholesterol molecules by enzymatic
45
addition of hydroxyl, carbonyl or epoxide functional groups1. Endogenous oxysterols are commonly produced
46
by non-enzymatic mechanisms2. Specifically, reactive oxygen species (ROS), including alkoxy and peroxyl
47
radicals, can oxidize cholesterol molecules1,3. Cholesterol can also be oxidized by the leukocyte-H2O2-HOCl
48
system during inflammation3. Non-enzymatic oxidation is primarily responsible for the generation of certain
49
oxysterols such as 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βHC)3. However, producing
50
oxysterols by the oxidation of cholesterol on the side-chain is typically catalyzed through enzymatic routes,
51
which are mediated by cytochrome p450 family of enzymes4,5. For instance, cholesterol can be metabolized
52
into 27-hydroxycholesterol (27HC) by CYP27A1, 24-hydroxycholesterol (24HC) by CYP46A1, 25-
53
hydroxycholesterol (25HC) by cholesterol-25-hydroxylase and 22-hydroxycholesterol by CYP11A13,4 (Figure 1).
M AN U
SC
RI PT
44
54
Many oxysterols have been described to bind to and modulate the activity of the liver x receptors (LXRs),
56
thereby completing an important homeostatic loop in cholesterol metabolism (described below). However,
57
several recent studies have implicated oxysterols in the modulation of other nuclear receptors such as the
58
retinoic acid receptor-related orphan receptors (RORs), estrogen receptors (ERs) and glucocorticoid receptors
59
(GRs), as well as non-nuclear receptor mechanisms. Furthermore, it is becoming increasingly apparent that
60
oxysterols play important roles in several pathologies, including cancers, cardiovascular diseases, and
61
neurodegenerative diseases6. This review will discuss our current understanding of oxysterol modulation of
62
nuclear receptors. We will also discuss various pathologies where oxysterols have been described to play a
63
role, and when known, highlight whether nuclear receptor involvement has been examined.
65
EP
AC C
64
TE D
55
Oxysterols and Nuclear Receptors
66 67
Nuclear Receptors
68
Nuclear receptors are a large superfamily consisting of 48 ligand-inducible transcription factors, acting as
69
intracellular receptors that bind to lipophilic ligands capable of crossing the plasma membrane (e.g. steroids)7.
ACCEPTED MANUSCRIPT 70
Conformational changes are commonly triggered upon ligand binding, allowing nuclear receptors to bind to
71
their respective DNA response elements, releasing corepressors and recruiting coactivators to promote
72
transcription of target genes involved in a variety of cellular processes, including cell proliferation and
73
metabolism7,8.
RI PT
74
Nuclear receptors share homology in their structures and functional domains. Five domains are generally
76
shared across typical nuclear receptors: a variable N-terminal region, a conserved DNA binding domain, a
77
variable hinge region, a conserved ligand binding domain and a variable C-terminal region9. After steroid
78
hormones were linked to the progression of prostate cancer and breast cancer, nuclear receptors have gained
79
increasing attention in terms of their roles in disease7,10,11.
M AN U
80
SC
75
Oxysterols and Liver X Receptor Signaling
82
Of the nuclear receptors, the liver X receptors (LXRs) are thought to be the major target of oxysterols,
83
especially in terms of the regulation of cholesterol metabolism. LXRα (NR1H3) and LXRβ (NR1H2) are
84
isoforms of LXR, which serve as cholesterol sensors for the regulation of cholesterol excess12. LXRβ is
85
expressed fairly ubiquitously, while LXRα expression is highest in the liver, with lower expression to various
86
degrees in other tissues13,14. The two isoforms share significant sequence identity, and although speculated,
87
clear evidence of specific biological differences have not been well described. The LXRs are known to
88
heterodimerize with the retinoid X receptors (RXRs) and sit on various DNA response elements, with a direct
89
repeat 4 (DR4) being recognized as the consensus sequence (5′-AGGTCA-NNNN-AGGTCA-3′)12,15,16. In an
90
unliganded state, it is thought that the heterodimer interacts with transcriptionally repressive complex, such as
91
with nuclear receptor corepressor (NCoR), thereby transreprepressing the transcriptional activity of LXR17–19.
92
The transrepression elicited by NCoR complex plays an important role in LXR-mediated anti-inflammatory
93
pathways, such as suppression of Toll-like receptor (TLR)-dependent pro-inflammatory signal expression in
94
macrophages20,21. Upon ligand binding, corepressor proteins are exchanged for coactivators, including NAD+-
95
dependent deacetylases Sirtuin 1 (SIRT1), which allows the RNA polymerase II complex to associate and
96
initiate transcription18,19.
97
AC C
EP
TE D
81
ACCEPTED MANUSCRIPT The first endogenous ligands to be described for the LXRs were 22(R)-hydroxycholesterol (22RHC), 24(S)-
99
hydroxycholesterol (24SHC), and 24(S),25-epoxycholesterol (24,25EC)22. Since then, it has been
100
demonstrated that several oxysterols and certain precursors in the cholesterol-biosynthetic pathway can
101
modulate the activity of the LXRs. These include 22RHC, 25HC, 27HC, 24SHC, 24,25EC and desmosterol23–27.
102
These metabolites have a wide range of affinities or potencies, with 24SHC having an EC50 of 4 µM and 3 µM
103
compared to 27HC at 85 nM and 71 nM for LXRα and LXRβ, respectively22,27. For reference, 27HC is the most
104
abundant oxysterol in circulation with concentrations ranging from 67 ng/mL (0.17 µM) to 199 ng/mL (0.5 µM)
105
while 24SHC circulates from 39 ng/mL (0.1 µM) to 91 ng/mL (0.23 µM)28.
SC
RI PT
98
106
Given the hepatic expression of CYP7A1, and more ubiquitous expression of CYP27A1 (with significant
108
expression observed in cells of the myeloid immune lineage), it would be expected when cholesterol levels are
109
high, so too is its metabolism into oxysterols. Indeed, there is a positive association between circulating
110
cholesterol levels and 27HC in both mice and humans, while statin therapy is associated with decreased
111
plasma 27HC29,30. Furthermore addition of cholesterol to macrophages resulted in increased secretion of 27HC,
112
while inhibiting cholesterol synthesis with simvastatin abolished the production of 27HC by macrophages31,32.
113
Although it is difficult to account for esterification, these studies suggest that increased level, of cholesterol
114
could lead to increased production of 27HC, which can then engage LXRs. Upon binding of an endogenous
115
ligand to the LXR-RXR heterodimer, the corepressor complex dissociates with commensurate recruitment of
116
coactivation complexes, ultimately leading to a transcriptional program aimed at restoring cholesterol
117
homeostasis. Specifically, LXR activation leads to the induction of target genes involved in cholesterol efflux
118
(ATP binding cassette (ABC) transporters A1/G1/G5/G8), apolipoproteins (APO A-1/E), cholesterol metabolism
119
into bile acids (CYP7A1) and decreased intestinal cholesterol absorption33. The removal of cholesterol by
120
reverse cholesterol transport is facilitated by ABC transporters. ABCA1, expressed in multiple cell types,
121
promotes the efflux of free cholesterol to apolipoprotein, in particular APOA1, leading to the formation of
122
discoid lipid-poor HDL15,34. ABCG5 and ABCG8 are half transporters expressed on the apical membrane of
123
enterocyte and on the canalicular membrane of hepatocytes35. The formation of heterodimer by ABCG5 and
124
ABCG8 on hepatocytes can promote the secretion of cholesterol into bile, while on enterocytes can facilitate
125
the excretion of cholesterol back into intestinal lumen12,35. LXR activation is also involved in regulating lipid
AC C
EP
TE D
M AN U
107
ACCEPTED MANUSCRIPT homeostasis by promoting triglyceride synthesis. Genes upregulated in this process are sterol regulatory
127
element-binding protein 1, acetyl-CoA carboxylase, fatty acid synthase and stearoyl-CoA desaturase-134,36,37.
128
The relatively longer-term actions of LXR-mediated homeostasis work in concert with the quicker actions of
129
cholesterol and oxysterols in maintaining sterol regulatory element-binding proteins (especially SREBP 2) in an
130
inactive state bound to the endoplasmic reticulum, thereby reducing the transcription of genes associated with
131
cholesterol metabolism and import38.
RI PT
126
132
Oxysterol and Retinoic acid receptor-related Orphan Receptor (ROR) signaling
134
RORs are nuclear receptors with three subtypes, RORα (NR1F1), RORβ (NR1F2) and RORγ (NR1F3)39.
135
RORs regulate gene transcription by binding to their respective response element as monomers upon ligand
136
binding. Each type of ROR has multiple isoforms expressed in humans and mice, with RORγ having two
137
isoforms, RORγ1 and RORγt40. RORα are involved in a variety of cellular processes, such as cerebellar
138
development, circadian rhythm and lipid metabolism, while RORγ is critical for lymphocyte development and
139
differentiation39,40. Oxysterols, including 22RHC, 24SHC, 25HC and 27HC, have been described as
140
endogenous ligands for both RORα and RORγ41. However, the activities of oxysterols differ, as they have the
141
ability to act as both agonists or inverse agonists. For example, it has been generally found that 24SHC and
142
25HC act as inverse agonists for both RORα and RORγ, while 27HC acts as an agonist for RORγ41. However,
143
a study also identified the ability of 25HC and 22HC to restore RORγ transcriptional activity from a synthetic
144
inhibitor in mammalian cells lines, suggesting some agonistic activity42. Cholesterol and cholesterol sulfate are
145
also the endogenous ligands of ROR, specifically RORα, with cholesterol sulfate having higher affinity to the
146
receptor due to its better fitting in the ligand binding pocket43,44.
M AN U
TE D
EP
AC C
147
SC
133
148
Similar to the LXRs, RORs also participate in cholesterol metabolism. One of the transcriptional targets of
149
RORα activation is CYP7B1, which codes for oxysterol 7α-hydroxylase, an enzyme involved in metabolizing
150
27HC and 25HC to bile acids45. However, RORs’ interaction with oxysterols, especially RORγ and RORγt play
151
very important roles in T helper 17 cell (Th17) differentiation46,47. Th17 cells were shown to preferentially
152
produce two types of oxysterols, 7β, 27-dihydroxycholesterol (7β, 27DHC) and 7α, 27DHC, and these
153
oxysterols rescued the inhibitory effect of ursolic acid on RORγt, while promoting coactivator recruitment to the
ACCEPTED MANUSCRIPT ligand binding domain of RORγt46. In addition, RNA sequencing analysis showed that in Th17 cells, interleukin
155
(IL)-17A, IL-17F, IL-23R, C-C ligand (CCL)20 and C-C chemokine receptor (CCR)6 were regulated by both
156
RORγt and RORα48. Although it is clear that the RORs are required for normal Th17 differentiation and function,
157
the physiological relevance and precise impact of oxysterols on this process remain to be elucidated.
158 159
RI PT
154
Oxysterols and Estrogen Receptor (ER) Signaling
161
Oxysterols were definitively found to bind to and modulate the activity of the estrogen receptors just over 10
162
years ago49,50. There are two main subtypes of nuclear estrogen receptors in mammalian cells, ERα (NR3A1)
163
and ERβ (NR3A2). A membrane-associated G protein-coupled estrogen receptor 1 (GPER1, or GPR30) has
164
also been described, but whether oxysterols can interact with this receptor remains unknown51,52. Nuclear ER
165
signaling comprises ligand binding to ER, followed by dissociation of the heat shock protein from ER, revealing
166
nuclear localization signal, and homo- or hetero-dimerization of two ER monomers51. ER dimers then bind to
167
the respective estrogen response element of the target gene, recruiting co-activators and/or co-repressors,
168
leading to target gene transcription, including trefoil factor 1 (TIFF1) and progesterone receptor (PR)51,52. So-
169
called ‘non-genomic’ or ‘rapid’ ER signaling also exists, where cytosolic ERs engage in kinase-mediated
170
signaling cascades including mobilization of intracellular calcium, stimulation of adenylate cyclase activity and
171
epidermal growth factor receptor activation, ultimately resulting in rapid cellular changes6,52.
M AN U
TE D
EP
172
SC
160
As early as in 2004, oxysterols, including β-epoxycholesterol, 20(S)-hydroxycholesterol and 22RHC, were
174
shown to have estrogenic activity in ERα-expressing HeLa cells53. More recently, a Gal4-ER co-transfection
175
screen of several cholesterol metabolites revealed that 22RHC, 24SHC, 25HC and 27HC significantly inhibited
176
estradiol (E2) activation of both ERα and β, with IC50s ranging from 1-5 µM50. 27HC was subsequently shown
177
to be a selective ER modulator (SERM), with the ability to induce distinct ER conformational changes and to
178
manifest either activating or inhibitory activities, depending on the tissue and cellular context49. 27HC was
179
found to activate the classic ERE-Luciferase reporter (3XERE-TATA-Luc) in transfected HeLa cells, with an
180
EC50 of approximately 1 µM, and when co-treated with estradiol had a Ki of 1.32 µM49. Demonstrating its
AC C
173
ACCEPTED MANUSCRIPT 181
selective-modulatory capabilities, it could activate the ER to promote the cellular proliferation of ER+ breast
182
cancer cell lines, tumors or uteri, but inhibited estrogen-dependent expression of vascular nitric oxide synthase,
183
thereby repressing carotid artery reendothelialization49,50,54,55.
184 Other oxysterols or cholesterol metabolites besides 27HC have also been found to modulate ER activities.
186
7KC was shown to decrease the cytotoxicity of doxorubicin in MCF7 cells, and this effect was dependent on
187
ERα, as fulvestrant inhibition or ERα knockdown restored doxorubicin accumulation in MCF7 cells56. 7KC also
188
induced the expression of TIFF1, an ER target gene56. Structurally similar to 27HC, 25HC upregulates ER
189
target genes, including TIFF1, PR, cathepsin D, cyclin A and cyclin D1 in MCF7 and BG1 cells57. Furthermore,
190
25HC promotes ER+ breast cancer cell proliferation and inhibits hypoxia-inducible factor-1α (HIF-1α)
191
expression induced by hypoxia in cardiomyocytes to prevent cell apoptosis57.
M AN U
SC
RI PT
185
192
Subsequent studies have further confirmed the selective modulator capacity of 27HC. In hematopoietic stem
194
cells (HSCs) during pregnancy, 27HC could induce ERα-dependent HSC mobilization and extramedullary
195
hematopoiesis58. In a cancer cell model, 27HC enhanced ER-mediated transcriptional activity and the
196
recruitment of ER to TIFF1 and growth regulating estrogen receptor binding 1 (GREB1) response elements in
197
an ER+ cancer cell line (MCF7) adapted to long-term estrogen deprivation59. However, in cardiovascular cells,
198
27HC reversed the cardioprotective effect of estrogen by inhibiting estrogen-induced nitric oxide production by
199
vascular cells, indicating an antagonistic activity against ER60. 27HC exhibited a partial-agonist effect on
200
osteoblasts, but ultimately exerted a negative impact on bone quality that could be attenuated by exogenous
201
estrogen61,62. The negative effects on bone are likely a combination of signaling through the ERs and LXRs,
202
and 27HC has been recently been reported to be associated with decreased bone mineral density in
203
postmenopausal women when circulating estrogens are taken into account63. Moreover, 27HC could serve as
204
a partial agonist to ER in ER+ breast cancer cell lines other than MCF7 cells, including T47D and BT48349,54. It
205
has also been suggested that 27HC antagonizes the neuroprotective effects of estrogen signaling through ERα,
206
as an increase in 27HC concentration in the brain is associated with increased neurodegeneration of the
207
hippocampus, decreased expression of ER and a decreased level of hippocampal mitochondria64. 27HC was
208
also shown to exert effects through ERβ: not only was ERβ expression upregulated by 27HC in prostate
AC C
EP
TE D
193
ACCEPTED MANUSCRIPT 209
cancer cell lines, LNCaP and PC3, but the 27HC-induced proliferation of prostate cancer cells was attenuated
210
by the ERβ-specific inhibitor, PHTPP65.
211
Oxysterols and Other Nuclear Receptors
213
The glucocorticoid receptor (NR3C1, GR) is another type of nuclear receptor shown to interact with
214
oxysterols66. Normally, upon ligand (glucocorticoid steroid) binding, GR can dimerize and translocate into the
215
nucleus67,68. In the nucleus, GR dimers bind to GR response elements, recruit coactivators and induce gene
216
transcription (transactivation)67,68. Activated GRs can also complex with other transcription factors, such as
217
nuclear factor-κB (NFκB) and activator protein 1 (AP1), thereby sequestering the transcription factor and
218
repressing the respective target gene transcription (transrepression)67,68. One oxysterol recently identified as a
219
GR ligand is 6-oxo-cholestan-3β,5α-diol (OCDO), which is generated from 5,6α-epoxycholesterol and 5,6β-
220
epoxycholesterol (5,6EC) by cholesterol epoxide hydrolase (ChEH) and 11β-hydroxysteroid-dehydrogenase-
221
type-2 (11βHSD2) in the scenario of breast cancer66,69. By binding and translocating GR into the cellular
222
nucleus, OCDO led to the expression of MMP1, but did not stimulate the expression of cortisol induced-classic
223
GR target genes, SGK1 and MKP1. These data suggest that even though both OCDO and glucocorticoids
224
signal through GR, their actions on GR are distinct66,69.
TE D
M AN U
SC
RI PT
212
225
The estrogen-related receptor (ERR) family is composed of ERRα (NR3B1), ERRβ (NR3B2) and ERRγ
227
(NR3B3), with ERRα being the most studied70. ERRα shares 70% homology with ERα in the DNA-binding
228
domain, but classic ERα ligands, such as 17β-estradiol, do not directly regulate the activity of ERRα70,71. ERRα
229
has been categorized as an orphan receptor due to a lack of endogenous ligand, and is thought to be primarily
230
regulated by other proteins, such as PGC1α72. Recently, Wei et al. identified cholesterol to be a molecule that
231
activates and regulates the transcriptional activity of ERRα70. In addition, ERRα, at least in part, mediates the
232
cholesterol-stimulated osteoclastogenesis and bone resorption70. Whether oxysterols can also regulate the
233
activity of ERRα are unknown, but modeling would suggest that they could interact with the ERRα in a similar
234
way to cholesterol.
235
AC C
EP
226
ACCEPTED MANUSCRIPT Non-Nuclear Receptor Actions of Oxysterols
237
Although the focus of this review is on the interaction between oxysterols and nuclear receptors, it is important
238
to note that the mechanisms of action of oxysterols are not limited to nuclear receptors. Furthermore, the
239
precise mechanisms of action are not known for several of the biological phenomena that oxysterols are
240
implicated in (highlighted below). Oxysterols can also act through GPCRs, including Epstein-Barr virus-induced
241
G-protein coupled receptor 2 (EBI2/GPR183), GPR17, C-X-C chemokine receptor (CXCR)2 and Smoothened
242
(SMO)73–75. EBI2, CXCR2 and GPR17, in particular, were shown to be promiscuously modulated by
243
oxysterols73. In addition, Nedelcu et al. reported that Hedgehog signaling requires the binding of oxysterol to
244
vertebrate SMO75.
SC
RI PT
236
M AN U
245
Besides GPCRs, regulatory proteins, such as SREBPs, are also targets of oxysterols76–78. Specifically,
247
oxysterols can bind to Insulin-induced proteins (INSIGs), prompting INSIGs to bind to the SREBP cleavage-
248
activating protein (SCAP), which then leads to a conformational change in the cytoplasmic loop 6 of SCAP76,77.
249
SCAP is an SREBP-escort protein, which, upon synthesis, promotes the clustering of SREBP in coat protein
250
complex II (COPII)-coated vesicles76,77,79. The SCAP:SREBP complex vesicles then travel to the Golgi complex
251
from endoplasmic reticulum (ER)76,77,79. At the Golgi complex, SREBP is cleaved before entering the nucleus to
252
promote cholesterol synthesis76,77,79. Conformational change of SCAP, induced by oxysterols through INSIGs,
253
prevents the COPII proteins from binding to the MELADL sequence on SCAP, ultimately blocking the transport
254
of SREBPs to the Golgi complex and promotion of cholesterol synthesis76,77. Although cholesterol is the
255
prototypical ligand for the INSIGs, oxysterols have also been shown to bind. Thus, oxysterols likely have two
256
roles in terms of cellular cholesterol homeostasis: inhibition of SREBPs and engagement of the LXRs. One of
257
the SREBP isoforms, SREBP-1a, is highly expressed in macrophages and plays important roles in regulating
258
the activity of the inflammasome, such as the production of pro-inflammatory cytokine IL-1β80.
AC C
EP
TE D
246
259 260
Oxysterols in Health and Disease
261
Oxysterols and the Immune System
262
The roles of oxysterols in the immune system have gained increasing appreciation over the last decade. The
263
interaction of oxysterols with the immune system has been recognized as one of the major ways that
ACCEPTED MANUSCRIPT oxysterols contribute to diseases, and such interaction can be complicated and multifaceted. Demonstrating
265
the complexity of oxysterol regulation of the immune system, some studies have described 25-
266
hydroxycholesterol (25HC) as being immunosuppressive by inhibiting cytotoxic T cell production and the
267
inflammasome activities, while others have found that 7βHC and 25HC induced the production of pro-
268
inflammatory chemokines and cytokines in U937 and THP-1 cells4,81,82. Although some effects of oxysterols
269
have been attributed to specific mechanisms (such as via nuclear receptors or SREBP signaling), many are yet
270
to be explored. Where possible, we have highlighted known mechanisms within this review.
RI PT
264
SC
271 272
LXR activation has been shown to play an important anti-inflammatory role in the immune system, due to the
274
inhibition of pro-inflammatory signal production downstream of NFκB83. Ito et al. showed that LXR activation
275
could disrupt the membrane lipid organization by hindering the recruitment of adaptor proteins, MyD88 and
276
TRAF6, through ABCA1, ultimately resulting in the inhibition of the TLR signaling to NFκB and MAPK
277
effectors84. Non-nuclear receptor-mediated mechanisms are also prevalently used by oxysterols. For example,
278
25HC was identified as one of the anti-inflammatory oxysterols that reduced Il1b transcription and IL1-
279
activating inflammasome activity by repressing SREBP in macrophages85. Studies suggesting an antiviral role
280
of 25HC prompted Cagno et al. to treat cells with 25HC and 27HC prior to Herpes simplex virus 1 (HSV1)
281
infection86. This pre-treatment promoted IL-6 secretion in HeLa cells and NFκB activation in Vero cells, linking
282
25HC and 27HC to a pro-inflammatory role86. It is not clear whether these activities required SREBP or LXR. In
283
the context of cerebral inflammation, Jang et al. identified that 25HC promoted robust nucleotide-binding
284
oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome assembly and
285
activation in macrophages in a potassium efflux/mitochondrial ROS/LXR-dependent manner82. These findings
286
suggest that the actions of oxysterols on the immune function may be selective, cell-type specific and cell-
287
location specific.
AC C
EP
TE D
M AN U
273
288 289
Oxysterols, Nuclear Receptors and Innate Immune System
290
The interaction between LXR and the immune system was first reported by Joseph et al. in that the loss of
291
functional LXRs in mice made them highly susceptible to infection by the intracellular bacteria Listeria
ACCEPTED MANUSCRIPT monocytogenes87. Also, expression of LXR promoted macrophage survival during the infection87. The
293
described effects of LXRs on innate immune cells range from regulation of cholesterol efflux to regulation of
294
inflammatory status. LXRα plays an important role in regulating the activities of Kupffer cells and natural killer T
295
cells88. Endo-Umeda et al. showed that knocking out LXRα leads to an increased level of bone marrow-derived
296
Kupffer cell population (F4/80+CD68+CD11b+) in the liver of mice when they are fed with a high cholesterol
297
diet88. Also, the lack of LXRα led to increased production of pro-inflammatory cytokines by those cells88.
298
Contrary to Kupffer cells, deficiency in LXRα reduced the frequency of natural killer cells and impaired those
299
cells functionally, as illustrated by the decreased capacity to produce IL-4 and interferon (IFN)-γ post α-GalCer
300
stimulation88. Whether specific oxysterols can modulate these LXR-associated activities is likely, given that
301
they are ligands for the LXRs.
M AN U
SC
RI PT
292
302
LXR is not the only nuclear receptor that is involved in innate immunity management. Studies have reported
304
innate immune cell functions regulated by oxysterols through RORα and ER. Tuong et al. identified a crosstalk
305
between RORα and 25HC in regulating macrophage function89. They found that the cholesterol 25 hydroxylase
306
expression was attenuated in RORα-deficient staggerer (sg/sg) mice, along with macrophages exhibiting
307
dysregulated lipid phenotype. Also, knocking down cholesterol 25 hydroxylase with siRNA in wildtype
308
macrophages resulted in lipid dysregulation89. However, treating sg/sg macrophages with 25HC significantly
309
decreased lipid accumulation into lipid droplets by approximately 1.7-fold and rescued the lipid dysregulation
310
phenotype89. ER is another receptor involved in the oxysterol-immunity axis. During pregnancy, 27HC
311
promotes ERα-dependent hematopoietic stem cell mobilization58, and estrogens themselves have been shown
312
to recruit myeloid cells to the tumor niche90. As will be discussed below, 27HC also increases myeloid cell
313
recruitment to metastatic lesions, although it is not yet known whether this is mediated by the ERs or LXRs91.
EP
AC C
314
TE D
303
315
Many studies have suggested an anti-inflammatory effect of oxysterols on innate immune cells, although
316
several of these effects are either LXR/nuclear receptor-independent or have mechanisms that remain
317
unknown. For example, in glial cells, while the oxysterols oxidized on the sterol backbone generally had no
318
effect on pro-inflammatory cytokine production after lipopolysaccharide (LPS) induction, oxysterols oxidized on
319
the side chain generally decreased LPS-induced mRNA expression of IL-1β and macrophages inflammatory
ACCEPTED MANUSCRIPT protein (MIP)-1α, suppressing the inflammatory phenotype of macrophages92. In the same study, it was further
321
shown that 24,25EC, 25HC, and 27HC decreased LPS-induced expression of IL-6 and tumor necrosis factor
322
(TNF)-α92. This finding is further supported by Marengo et al., indicating that an oxysterol mixture decreased
323
the level of reactive oxygen species (ROS) production in human macrophages and increased the level of
324
immunoregulatory cytokine IL-10 secretion93. The relative contributions of LXRs or SREBPs in these processes
325
are not currently known. Besides cytokines, LXR ligands, such as 25HC, can downregulate LPS-induced
326
expression of cyclooxygenase (COX)2 and mPGE2 synthase 1 (mPGES1), both of which are involved in
327
producing prostaglandins, a group of physiologically active and pro-inflammatory lipid compounds94.
SC
RI PT
320
328
7-oxygenated cholesterols have been associated with the development of atherosclerosis, suggesting a pro-
330
inflammatory effect in foamy macrophages during the formation of the atherosclerotic lesion. Similar to those
331
effects of oxysterols that are anti-inflammatory, the mechanisms of oxysterols’ pro-inflammatory effects on the
332
innate immunity largely remain to be elucidated. Proteomics revealed that a mixed treatment of 7βHC and
333
7KC reduced the expressions of glyoxalase 1 and adenylyl cyclase-associated protein 1 (CAP1) in human
334
THP1 cells; both proteins are associated with anti-inflammatory activities95–97. Specifically, glyoxalase 1 inhibits
335
the accumulation of oxidative stress in diabetes mellitus97. Overexpressing CAP1 in monocytes exacerbated
336
adipose tissue inflammation in mice, while suppressing CAP1 expression abolished the resistin-mediated
337
inflammatory activity both in vitro and in vivo96. Another type of pro-inflammatory cytokine, IL-8, was also
338
shown to be upregulated in human macrophages via PI3K and MEK pathway upon 7KC and 7α-
339
hydroxycholesterol (7αHC) treatment98. In this study, the GPCR complement receptor, C5a was implicated in
340
mediating the upregulation of IL-8.
342
TE D
EP
AC C
341
M AN U
329
343 344
Oxysterol, Nuclear Receptors and Adaptive Immune System
345
The interaction between oxysterols and the adaptive immune system is mediated by both LXR and RORγt,
346
with RORγt specifically involved in Th17 differentiation and functionality. LXR activation and signaling usually
347
negatively regulates T cell proliferation99,83. It was shown by Bensinger et al. that LXRβ knockout mice
ACCEPTED MANUSCRIPT displayed unregulated T cell activity, including lymphoid hyperplasia and enhanced responses to antigen
349
challenge100. Also, Ma et al. showed that Tc9 cells, a polarized population of CD8+ T cells with strong anti-
350
tumor activity, are regulated by cholesterol through its derivative, oxysterols, such as 22RHC101. 22RHC
351
inhibited IL-9 expression by activating LXRs, which then resulted in LXR sumoylation and decreased p65
352
binding to the promoter region of Il9 gene, while IL-9 is essential for the survival of Tc9 in vivo101. LXR
353
activation by 25HC and 22RHC also takes part in T regulatory cell (Treg) control102,103. Mice lacking cholesterol
354
25 hydroxylase exhibited increased production of IL-10, while 25HC treatment impaired IL-27-induced Treg
355
differentiation by downregulating IL-10 expression in an LXR-dependent manner102.
SC
RI PT
348
356
Soroosh et al. first described the most potent selective activator for RORγt to be 7β, 27DHC46. They showed
358
that in vivo administration of 7β, 27DHC promoted IL-17 production and in vitro treatment of this oxysterol
359
enhanced IL-17-producing Th17 cell differentiation in a RORγt-dependent manner46. On the flip side, knocking
360
out the enzyme CYP27A1, which is responsible for the production of 7β, 27DHC, significantly reduced IL-17-
361
producing CD4+ and γδ+ T cell population46. Santori et al. also identified “cholesterol biosynthetic intermediate”
362
(CBI) downstream of lanosterol and upstream of zymosterol to be natural RORγt ligand42. In the study,
363
knocking out CYP51 led to smaller lymph node anlagen and stunted development of lymph node structure in
364
embryos, developmental processes mediated by RORγt42. In addition, breeding Sc4molf/f mice to CD4-cre and
365
RORγt-cre mice generated the deletion of Sc4mol in TCRαβ T cells and RORγt-expressing cells. Sc4mol is
366
responsible for modifying the C4-methyl groups of CBIs, and its deletion led to a significant decrease in IL-17-
367
producing CD4+ T cells42. The requirement of CYP51 and Sc4mol in RORγt-mediated adaptive immunity
368
developmental processes dictates the indispensable role of CBI in RORγt signaling.
TE D
EP
AC C
369
M AN U
357
370
Oxysterols and Cancer
371
Since 27HC was first described to be a selective estrogen receptor modulator, the effect of this oxysterol on
372
breast cancer has now been extensively studied in pre-clinical models, which is not surprising given that
373
approximately 60-75% of all breast cancers are ERα+. CYP27A1 protein expression was associated with
374
higher breast tumor grade, while CYP7B1 mRNA expression is associated with a better prognosis, indicating a
375
clinically relevant role for 27HC in breast cancer6,54 . As expected, 27HC promoted the in vitro proliferation of
ACCEPTED MANUSCRIPT cell lines in an ER-dependent manner49,54,55. 25HC has also been shown to promote the proliferation of breast
377
cancer cells through activation of the ER57. 27HC increased the growth of ER+ mammary tumors, while
378
inhibiting the production of 27HC attenuated hypercholesterolemia-driven tumor growth in vivo54,55. Intriguingly,
379
27HC could also promote the transition from epithelial-like to mesenchymal-like (EMT) in breast cancer
380
cells54,104. The synthetic LXR agonist, GW3965, could also promote EMT, indicating that this phenotype is likely
381
LXR mediated54. 7KC also plays a role in breast cancer in that 7KC decreased the cytotoxicity of doxorubicin in
382
ER+ breast cancer cell line MCF7 by upregulating P-glycoprotein expression, while this effect was not
383
observed in ER- cell line, MDA-MB-23156. The requirement of the ERα in 7KC’s effect was further illustrated by
384
knocking down ERα with siRNA, which restored the doxorubicin accumulation in MCF756.
SC
RI PT
376
M AN U
385
While the effects of 27HC and 7KC on breast cancer cells required ERα, ERβ may also play an important role
387
in 27HC signaling in cancer types. Raza et al. demonstrated that 27HC promoted prostate cancer cell
388
proliferation in vitro in LNCaP and PC3 cells through the ER. Specifically, 27HC induced proliferation was
389
significantly reduced in the presence of the ER inhibitor, ICI 182,780 (fulvestrant)65. Likewise, the ERβ-
390
selective antagonist PHTPP reversed the proliferative effect of 27HC on prostate cancer cells, indicating that
391
27HC signals through ERβ to promote their proliferation65. However, these results were contradicted by
392
another study, indicating that 27HC actually decreased the proliferation of prostate cancer cells, via an
393
SREBP2 mediated mechanism105. Furthermore, the expression of CYP27A1 was downregulated in prostate
394
cancers compared to normal tissue, and elevated CYP27A1 expression is associated with a better prognosis
395
for prostate cancer patients. Further work will be required to better characterize the impact of oxysterols on
396
prostate cancer.
AC C
EP
TE D
386
397 398
In addition to breast and prostate cancer, 27HC was shown to affect lung cancer development. Using H1395
399
cell line as a lung cancer model, Hiramitsu et al. found that 27HC advanced ERβ+ lung cancer cell proliferation
400
via phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling106. Chen et al. described the pro-metastatic
401
activity of 25HC in lung adenocarcinoma107. In A549 cells and NCL-H1975 cells, 25HC treatment enhanced
402
snai1 expression, a marker of EMT, in an LXR-dependent manner, which promoted invasion of those cancer
403
cells107.
ACCEPTED MANUSCRIPT 404 Although many oxysterols have been reported to contribute to cancer progression, cholestane-3β, 5α, 6β-triol
406
(triol) was found to have anti-cancer activity108. Not only was the triol treatment able to inhibit the growth of
407
LNCaP CDXR3, DU145 and PC3 prostate cancer cell lines, but also oral administration of this oxysterol was
408
able to slow the progression of xenograft PC3 in NUDE mice108. Triol treatment also inhibited the invasive
409
capacity of DU145 and PC3 cells and downregulated the epithelial to mesenchymal transition-related genes,
410
including N-cadherin, vimentin, Slug, focal adhesion kinase (FAK), phospho-FAK Ser722 and phospho-FAK
411
Tyr861108. Another LXR modulator, dendrogenin A, was reported by de Medina et al. to possess anti-tumor
412
property in vivo109. This cholesterol metabolite was produced by 5,6αEC reacting with histamine and was
413
significantly downregulated in breast tumor, compared to normal tissues109,110. Treating cancer cells with
414
dendrogenin A promoted cancer cell re-differentiation, inhibited OCDO production and induced lethal cancer
415
cell autophagy109,110.
M AN U
SC
RI PT
405
416
Besides the direct effects of oxysterols on cancer cells, these molecules impart an indirect effect on cancer by
418
interplaying with the immune system and creating a microenvironment that promotes cancer invasion and
419
metastasis16,91. Following the study that first identified the pro-metastatic effect of 27HC on breast cancer,
420
Baek et al. reported that 27HC required myeloid immune cell population to promote metastasis54,91. 27HC
421
treatment led to increased levels of polymorphonuclear-neutrophils (PMNs) and γδ+ T cells at distal metastatic
422
sites91. PMNs were further shown to be required for the enrichment of γδ+ T cells, and that both cell
423
populations were required for the full metastatic effect of 27HC91. Specifically, immune depletion of either
424
PMNs or γδ+ T cells reversed the 27HC-driven Met1 breast cancer metastasis to the lungs91. Gene expression
425
revealed that 27HC likely polarizes PMNs into an immune suppressive ‘N2’ phenotype, thereby explaining the
426
observed decreases in CD8+ cytotoxic T cells post 27HC treatment91. Moresco et al. also suggested that
427
oxysterols likely promote breast tumor metastasis by recruiting tumor-promoting neutrophils to the lung
428
metastatic niche, as oxysterol-inactivating enzyme sulfotransferase 2B1b (SULT2B1b)-transduced 4T1 tumor
429
had lower levels of metastasis and infiltration by tumor-promoting neutrophils in vivo111. Raccosta et al.
430
reported an oxysterol-dependent, LXR-independent mechanism by which oxysterols recruit tumor-promoting
431
neutrophils. They showed that bone marrow-derived CD11bhighGr1high myeloid cells migrate toward natural and
AC C
EP
TE D
417
ACCEPTED MANUSCRIPT tumor-released oxysterols112. Specifically, 22RHC led to the activation of CXCR2 on CD11bhighGr1high cells,
433
whose activation led to the production of pro-angiogenic factor, MMP9112. Inactivating CXCR2 inhibited AB1
434
and LLC tumor growth by reducing angiogenesis and immunosuppression112. An LXR-dependent mechanism
435
by which oxysterols create immunosuppressive microenvironment was also illustrated by the same group: by
436
activating LXRα on dendritic cells (DCs), oxysterols inhibit CCR7 expression on DCs, thereby preventing them
437
from migrating to lymph nodes to facilitate T cell activation against tumor cells113. Given that the pro-metastatic
438
effects of oxysterols involve the immune system, it is likely these effects are similar across solid tumors. Indeed,
439
27HC was shown to promote metastasis in pre-clinical models of breast, colon, pancreatic and lung cancers91.
440
Therefore, when examining the role of oxysterols in cancer progression, it is critical that one considers both
441
their direct effects on cancer cells as well as their cancer-cell extrinsic effects.
M AN U
SC
RI PT
432
442 Neurodegenerative Diseases
444
The brain contains approximately 25% of total cholesterol in our body114. In the central nervous system,
445
cholesterol metabolism is tightly controlled and separated from the rest of the body by the blood-brain barrier
446
(BBB)114. Oxysterols such as 27HC can cross the BBB, and play an important role in maintaining cholesterol
447
homeostasis in the central nervous system115,116. However, dysregulated cholesterol homeostasis in the brain,
448
especially along the oxysterol-LXR axis, is implicated in many neurodegenerative diseases, such as
449
Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD)6,114. Besides regulating
450
cholesterol and lipid metabolism, oxysterol signaling through the LXRs also participates in other cellular
451
processes. Among them, 24SHC, 25HC and 27HC have been shown to induce adaptive responses in neuronal
452
cells against cytotoxic oxidative stress induced by 7KC117. This protective effect requires the downstream
453
expression of ABCG1, but not ABCA1117. Additionally, 25HC induces the expression of cholesterol 25
454
hydroxylase via the activation of the LXR, independent of cell type118. In the nervous system, 22HC can
455
upregulate the expression of MAP kinase phosphatase (MKP)-1, which then suppresses the JNK-mediated
456
inflammation in brain astrocytes119. The mechanism of this suppression by 22RHC is via the phosphorylation of
457
Hu antigen R through PKCα, which in turn stabilizes MKP-1 mRNA119. Although the metabotropic glutamate
458
receptor 5 is required for the cytosolic increase in Ca2+, an event upstream of the phosphorylation of Hu, it is
AC C
EP
TE D
443
ACCEPTED MANUSCRIPT 459
not clear whether 22RHC directly binds to this receptor, or whether these activities require interaction with the
460
more traditional oxysterol binding proteins (such as the LXRs or INSIG).
461 24HC is the most abundant oxysterol synthesized in the brain, whereas 27HC is the primary oxysterol
463
produced by peripheral cells, which crosses the BBB114,115,120. While 7KC is associated with a neuro-damaging
464
role, 24HC was shown to have a protective effect during the development of neurodegenerative disorders. For
465
instance, Testa et al. demonstrated that, in SK-N-BE neuroblastoma cells, 24HC could modulate the SIRT1,
466
which then led to the prevention of hyperphosphorylated tau protein accumulation induced by amyloid β (Aβ)
467
monomers121. Okabe et al. also found that sub-lethal concentrations of 24SHC induced adaptive responses
468
and protected the human neuroblastoma SH-SY5Y cells against cytotoxic stress induced by 7KC, dependent
469
of activating LXRβ transcriptional pathway117. However, high concentrations of 24SHC could induce
470
“necroptosis-like” cell death in neuronal cells due to the formation of 24SHC esters120,122. Also, 24HC was
471
shown to increase Aβ accumulation, which in turn contributes to neuronal cell death. Reported by Gamba et al.,
472
24HC promoted not only the binding of Aβ to human neuronal cell lines, SK-N-BE and NT2, but also the pro‐
473
apoptotic and pro‐necrogenic effects of Aβ1–42 peptide on SK-N-BE and NT2, by locally increasing ROS levels
474
through NADPH oxidase123. These works together suggest that 24HC at low concentrations might be protective,
475
but as its levels increase due to dysregulated cholesterol metabolism, this oxysterol could then act to induce
476
cell death.
SC
M AN U
TE D
EP AC C
477
RI PT
462
478
27HC, as the most abundant peripheral oxysterol, can cross the BBB and is therefore also implicated in
479
neuronal cell death. An increased level of 27HC was reported in the brain of patients diagnosed with AD124–126.
480
Also, hypercholesterolemia was linked to accelerated AD pathology and the accumulation of Aβ127,128. One
481
mechanism of cholesterol-linked AD development could be attributed to 27HC accumulation in the brain. Ismail
482
et al. showed that 27HC reduced LXR-mediated brain glucose uptake, GLUT4 expression and spatial memory
483
in mouse model129. The reduced glucose metabolic activity in AD patients has been well-identified in the
484
angular gyrus and some parietotemporal regions130. Moreover, 27HC promoted NFκB binding to the promoter
485
of aspartyl protease BACE1, leading to an increased BACE1 activity and BACE1-mediated cleavage of
ACCEPTED MANUSCRIPT amyloid precursor protein, in a gadd153-dependent manner131. Also, 27HC-induced ROS production could
487
promote IL-6/STAT3 signaling, which was implicated in the induction of nerve cell senescence132. However, it
488
was unclear whether this effect was due to the selective modulatory activity of 27HC on ER. Taken together,
489
limiting dietary cholesterol intake would likely reduce the level of peripheral 27HC, and thus may exhibit clinical
490
value in preventing AD and other related neurodegenerative disorder development29,133. In this regard, there is
491
a positive correlation between plasma cholesterol and 27HC levels in humans, including after dietary
492
consumption of an extra 750mg/day cholesterol for 4 weeks29,133. In mice, a high cholesterol diet results in
493
significantly increased 27HC levels, although dietary restriction experiments have yet to be reported50,54,62.
SC
RI PT
486
494
25HC was reported to be involved in the progression of amyotrophic lateral sclerosis (ALS). Clinical analysis
496
showed that 25HC level was elevated in both cerebral spinal fluid and serum of ALS patients134. Also, the level
497
of 25HC was associated with the level of disease severity in ALS patients134. Mechanistically, 25HC-mediated
498
ALS pathogenesis is likely due to the induction of motor neuronal death, a process dependent of glycogen
499
synthase kinase-3β and LXR, which was shown by Kim et al. in the motor neuron-like cell line (NSC34)
500
expressing mutant G93A superoxide dismutase 1 gene134.
501
TE D
M AN U
495
Atherosclerosis
503
Atherosclerosis is the underlying pathology of many acute cardiovascular syndromes. Atherosclerosis
504
development is characterized by the narrowing of the artery, which results from the accumulation of lipids and
505
inflammatory cells within the intima of arteries. Progression of atherosclerosis is thought to be initiated by
506
endothelial cell dysfunction, leading to the accumulation of low-density lipoprotein-cholesterol (LDL-cholesterol)
507
in the extracellular matrix (ECM). LDL-cholesterol buildup at the extracellular matrix becomes a target of
508
oxidation, and LDLs can be oxidized into oxidized LDL (ox-LDL). Ox-LDL promotes the recruitment of
509
macrophages and the subsequent engulfment of ox-LDL by macrophages transforms macrophages into foam
510
cells6,33,135,136. This transformation is the hallmark of atherosclerotic lesion development137. However, recent
511
reports are questioning whether immune dysregulation may actually precede plaque formation138,139.
512
AC C
EP
502
ACCEPTED MANUSCRIPT Being a byproduct of cholesterol, oxysterols are formed in atherosclerotic plaques both non-enzymatically by
514
LDL oxidation and enzymatically during cholesterol catabolism by cytochrome p450 enzymes that are highly
515
expressed in macrophages33. Due to their prevalence in the atherosclerotic plaques, the role of oxysterols in
516
plaque formation has been heavily investigated. However, the pro-atherogenic properties of oxysterols have
517
been in a long debate largely due to the multifold effects of oxysterols. Macrophages are present in a spectrum
518
of so-called polarities. On the extremes of this polarization spectrum are classically activated, inflammatory M1
519
macrophages, and alternatively activated, anti-inflammatory macrophages. Macrophages across this spectrum
520
are localized within different types of atherosclerotic lesions. Macrophages localized in rupture-prone
521
atherosclerotic plaques more closely reflect the biology of M1 macrophages, while the macrophages that
522
situate in stable plaque more closely resemble the phenotype of M2 macrophages6,93. It has been reported by
523
Marengo et al. that 27HC contributes to the M2 polarization of human macrophages by upregulating the
524
expression of CD36 and CD204 and the production of IL-10, leading to the plaque stabilization93. On the
525
contrary, Gargiulo et al. found that 27HC promoted TLR4 and NFκB activation in U937 cells, which led to the
526
release of pro-inflammatory cytokines, IL-8, IL-1β and TNF-α140. The production of pro-inflammatory cytokines
527
induced by 27HC then contributed to the production of matrix metallopeptidase 9. Although these studies were
528
performed in vitro, the presence of matrix metallopeptidase 9 has the potential to promote plaque instability
529
and rupture140. The pro-inflammatory role of 27HC is likely due to the activation of ERα, as 27HC-induced pro-
530
inflammatory cytokine mRNA expression (IL-1β, IL-6 and TNF-α) was attenuated by ER deficiency60. Ishikawa
531
et al. reported a novel mechanism by which the activation of LXR exerts an atheroprotective role in endothelial
532
cells141. Specifically, GW3965-, T0901317- or 22RHC-induced LXR signaling promotes ER-dependent eNOS
533
activation141. By co-localizing with ERα in the lipid raft and thus functionally coupling with ERα, LXR stimulation
534
could
535
reendothelialization141.
lead
AC C
EP
TE D
M AN U
SC
RI PT
513
to
ERα
Ser118
phosphorylation
by
PI3K/Akt,
eventually
promoting
eNOS-induced
536 537
Structurally similar to 27HC, 25HC was also identified with an atheroprotective role. Li et al. found that Krüppel-
538
like factor 4 (KLF4), a master transcription factor that regulates the anti-inflammatory activity of macrophages,
539
could induce the expression of cholesterol 25 hydroxylase, LXRα and LXRβ in macrophages (RAW264.7)142.
540
Further, the activation of the KLF4-cholesterol 25 hydroxylase-LXR axis promoted M1 to M2 transition in
ACCEPTED MANUSCRIPT 541
macrophages, as both treating macrophages with 25HC and overexpressing KLF4 in macrophages led to a
542
decreased inflammatory activity in macrophages142. Knocking down cholesterol 25 hydroxylase in RAW264.7
543
cells also stimulated the expression of the pro-inflammatory genes and M1 phenotype142.
544 Non-nuclear receptor-mediated mechanisms by which oxysterols contribute to atherosclerosis development
546
have also reported. The β1-Subunit of BKCa channel (KCNMB1) is linked to atheroprotective, in that it
547
modulates vascular contractility143. Interestingly, 7KC has previously been reported to be a competitive inhibitor
548
of the aryl hydrocarbon receptor (AhR)144. In a subsequent report, 7KC was found to contribute to vascular
549
rigidity, and decreases the expression of KCNMB1 protein levels143. The decrease in KCNMB1 was attributed
550
to a decrease in the protein expression of the AhR, although the requirement of the AHR was not specifically
551
tested143. While the AhR is not a member of the nuclear receptor superfamily, it has a very similar mechanism
552
of action in that it is a ligand-activated transcription factor145,146. It will be of interest to evaluate whether other
553
oxysterols can modulate the AhR and to determine the physiological relevance of such engagement.
M AN U
SC
RI PT
545
554 Conclusion
556
Oxysterols play important roles in cholesterol metabolism and the regulation of cholesterol homeostasis.
557
Binding to LXRs, oxysterols promote cholesterol efflux and bile acid synthesis. However, an elevated level of
558
oxysterols has been implicated in the pathophysiology of many age-related diseases, such as breast cancer,
559
atherosclerosis and AD. The main target nuclear receptors mediating the actions of oxysterols are the LXRs,
560
ERs and RORs, although recent reports for the GR and potentially ERRα reveal the possibility for other
561
nuclear receptors being involved in oxysterol biology. The action of oxysterols on each type of receptor is
562
multifaceted. Being a SERM, 27HC can activate or inhibit ER in different cell types. For ROR, oxysterols’
563
actions range from agonist to inverse agonist. It is therefore possible that the multilevel effect of oxysterols on
564
the target receptors leads to diverse phenotypes in cells upon oxysterol stimulation. Also, desmosterol has now
565
been formally demonstrated to be a selective modulator of the LXRs in macrophages147. Specifically,
566
desmosterol activated LXR target gene, ABCA1, while suppressing the SREBP target gene, 24-
567
dehydrocholesterol reductase (DHCR24), and had no effect on fatty acid synthase in mouse and human
568
macrophages147. Indeed, phenotypically, there is also some debate regarding the inflammatory role of
AC C
EP
TE D
555
ACCEPTED MANUSCRIPT oxysterols. While they are generally seen as pro-inflammatory in the innate immune system, an increasing
570
number of studies reported the anti-inflammatory behaviors of oxysterols. The direct effect of oxysterols on T
571
cells are oxysterol- and T cell-type- specific. While 7β, 27DHC promotes Th17 differentiation, 25HC negatively
572
regulates Treg. Though 27HC is recognized as a SERM, other oxysterols’ selective modulatory effects are less
573
clear, although highly likely. Given the evidence that many oxysterols exert variable effects on cells, it is
574
important for future studies to investigate whether other oxysterols are involved in selectively modulating
575
nuclear receptors, such as LXR, in disease conditions.
SC
576
RI PT
569
There is now significant evidence that oxysterols play exacerbating roles across several diseases. Not only can
578
they have direct effects on cancer cells to promote cancer growth, but also oxysterols can modulate pro-tumor
579
host cells, such as myeloid cells, to promote disease progression. Specific mechanisms for many of the
580
described effects of oxysterols in physiology or disease are still unknown. Still, several of the roles of
581
oxysterols are mediated through the LXRs, and thus they have been proposed to be a therapeutic target.
582
However, challenges in targeting this receptor have emerged, as LXRs play an essential role in cholesterol and
583
lipid homeostasis148. While it may be beneficial to target this receptor, its activation leads to
584
hypertriglyceridemia148, which could increase the risk of developing cardiovascular-related diseases or
585
steatosis. Given the potential for LXR ligands to have differential effects depending on the tissue and cellular
586
context, targeting these receptors should proceed with caution. Therefore, it will be of importance for future
587
studies to consider the specific downstream targets upon oxysterol signaling in each of the disease models.
588 589 590 591
References
592
1. Griffiths, W. J. et al. Current trends in oxysterol research. Biochem. Soc. Trans. 44, 652–658 (2016).
593
2. Brzeska, M., Szymczyk, K. & Szterk, A. Current Knowledge about Oxysterols: A Review. J. Food Sci. 81,
594 595 596
AC C
EP
TE D
M AN U
577
R2299–R2308 (2016). 3. Poli, G., Biasi, F. & Leonarduzzi, G. Oxysterols in the pathogenesis of major chronic diseases. Redox Biol. 1, 125–130 (2013).
ACCEPTED MANUSCRIPT 597
4. Vejux, A. & Lizard, G. Cytotoxic effects of oxysterols associated with human diseases: Induction of cell
598
death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis. Mol.
599
Aspects Med. 30, 153–170 (2009).
603 604 605
RI PT
602
218 (2007).
6. He, S. & Nelson, E. R. 27-Hydroxycholesterol, an endogenous selective estrogen receptor modulator. Maturitas 104, 29–35 (2017).
SC
601
5. Norlin, M. & Wikvall, K. Enzymes in the conversion of cholesterol into bile acids. Curr. Mol. Med. 7, 199–
7. Dhiman, V. K., Bolt, M. J. & White, K. P. Nuclear receptors in cancer - uncovering new and evolving roles through genomic analysis. Nat. Rev. Genet. 19, 160–174 (2018).
M AN U
600
8. Sever, R. & Glass, C. K. Signaling by Nuclear Receptors. Cold Spring Harb. Perspect. Biol. 5, (2013).
607
9. Mangelsdorf, D. J. et al. The Nuclear Receptor Superfamily: The Second Decade. Cell 83, 835–839 (1995).
608
10. Huggins, C. Endocrine methods of treatment of cancer of the breast. J. Natl. Cancer Inst. 15, 1–25 (1954).
609
11. Huggins, C. & Hodges, C. V. Studies on prostatic cancer. I. The effect of castration, of estrogen and
TE D
606
610
androgen injection on serum phosphatases in metastatic carcinoma of the prostate. CA. Cancer J. Clin. 22,
611
232–240 (1972).
614
EP
613
12. Maqdasy, S. et al. Once and for all, LXRα and LXRβ are gatekeepers of the endocrine system. Mol. Aspects Med. 49, 31–46 (2016).
AC C
612
13. Auboeuf, D. et al. Tissue distribution and quantification of the expression of mRNAs of peroxisome
615
proliferator-activated receptors and liver X receptor-alpha in humans: no alteration in adipose tissue of
616
obese and NIDDM patients. Diabetes 46, 1319–1327 (1997).
617 618 619 620
14. Heine, G. et al. Liver X receptors control IgE expression in B cells. J. Immunol. Baltim. Md 1950 182, 5276– 5282 (2009). 15. Lee, S. D. & Tontonoz, P. Liver X Receptors at the Intersection of Lipid Metabolism and Atherogenesis. Atherosclerosis 242, 29–36 (2015).
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
RI PT
626
18. Wang, C., Tian, L., Popov, V. M. & Pestell, R. G. Acetylation and Nuclear Receptor Action. J. Steroid Biochem. Mol. Biol. 123, 91–100 (2011).
19. Li, X. et al. SIRT1 deacetylates and positively regulates the nuclear receptor LXR. Mol. Cell 28, 91–106
SC
625
OMEGA 3 FATTY ACIDS. Cell 155, 200–214 (2013).
(2007).
20. Glass, C. K. & Saijo, K. Nuclear receptor transrepression pathways that regulate inflammation in
M AN U
624
17. Li, P. et al. NCoR REPRESSION OF LXRs RESTRICTS MACROPHAGE BIOSYNTHESIS OF INSULIN-SENSITIZING
macrophages and T cells. Nat. Rev. Immunol. 10, 365–376 (2010).
21. Treuter, E. New wrestling rules of anti-inflammatory transrepression by oxysterol receptor LXR revealed. Cell Res. 21, 711–714 (2011).
22. Janowski, B. A. et al. Structural requirements of ligands for the oxysterol liver X receptors LXRα and LXRβ.
TE D
623
Metab. TEM 28, 485–496 (2017).
Proc. Natl. Acad. Sci. U. S. A. 96, 266–271 (1999).
23. Morello, F. et al. LXR-activating oxysterols induce the expression of inflammatory markers in endothelial cells through LXR-independent mechanisms. Atherosclerosis 207, 38–44 (2009).
EP
622
16. Kloudova, A., Guengerich, F. P. & Soucek, P. The role of oxysterols in human cancer. Trends Endocrinol.
24. Lehmann, J. M. et al. Activation of the nuclear receptor LXR by oxysterols defines a new hormone
AC C
621
ACCEPTED MANUSCRIPT
response pathway. J. Biol. Chem. 272, 3137–3140 (1997). 25. Yoshikawa, T. et al. Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter. Mol. Cell. Biol. 21, 2991–3000 (2001). 26. Chen, W., Chen, G., Head, D. L., Mangelsdorf, D. J. & Russell, D. W. Enzymatic Reduction of Oxysterols Impairs LXR Signaling in Cultured Cells and the Livers of Mice. Cell Metab. 5, 73–79 (2007). 27. Fu, X. et al. 27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells. J. Biol. Chem. 276, 38378–38387 (2001).
ACCEPTED MANUSCRIPT
647
648
649
650
651
plasma by high-performance liquid chromatography-mass spectrometry. J. Lipid Res. 45, 776–781 (2004). 29. Karuna, R. et al. Plasma levels of 27-hydroxycholesterol in humans and mice with monogenic disturbances of high density lipoprotein metabolism. Atherosclerosis 214, 448–455 (2011).
RI PT
646
28. Burkard, I., Rentsch, K. M. & von Eckardstein, A. Determination of 24S- and 27-hydroxycholesterol in
30. Thelen, K. M. et al. Effect of pravastatin on plasma sterols and oxysterols in men. Eur. J. Clin. Pharmacol. 62, 9–14 (2006).
31. Hansson, M., Ellis, E., Hunt, M. C., Schmitz, G. & Babiker, A. Marked induction of sterol 27-hydroxylase
SC
645
activity and mRNA levels during differentiation of human cultured monocytes into macrophages. Biochim.
653
Biophys. Acta 1593, 283–289 (2003).
654
M AN U
652
32. Babiker, A. et al. Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-
655
hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport.
656
J. Biol. Chem. 272, 26253–26261 (1997).
33. Brown, A. J. & Jessup, W. Oxysterols and atherosclerosis. Atherosclerosis 142, 1–28 (1999).
658
34. Bełtowski, J. Liver X receptors (LXR) as therapeutic targets in dyslipidemia. Cardiovasc. Ther. 26, 297–316
659
TE D
657
(2008).
35. Baldán, Á., Bojanic, D. D. & Edwards, P. A. The ABCs of sterol transport. J. Lipid Res. 50, S80–S85 (2009).
661
36. Rong, S. et al. Expression of SREBP-1c Requires SREBP-2-mediated Generation of a Sterol Ligand for LXR in
663 664 665 666
AC C
662
EP
660
Livers of Mice. eLife 6, (2017).
37. Calkin, A. & Tontonoz, P. LXR signaling pathways and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 30, (2010).
38. Baek, A. E. & Nelson, E. R. The Contribution of Cholesterol and Its Metabolites to the Pathophysiology of Breast Cancer. Horm. Cancer 7, 219–228 (2016).
667
ACCEPTED MANUSCRIPT 39. Wang, Y., Kumar, N., Crumbley, C., Griffin, P. R. & Burris, T. P. A Second Class of Nuclear Receptors for
668
Oxysterols: Regulation of RORα and RORγ activity by 24S-Hydroxycholesterol (Cerebrosterol). Biochim.
669
Biophys. Acta 1801, 917–923 (2010).
673
674
675
676
RI PT
672
lipid/glucose metabolism, inflammation, and insulin sensitivity. Front. Endocrinol. 4, (2013).
41. Mutemberezi, V., Guillemot-Legris, O. & Muccioli, G. G. Oxysterols: From cholesterol metabolites to key mediators. Prog. Lipid Res. 64, 152–169 (2016).
SC
671
40. Jetten, A. M., Kang, H. S. & Takeda, Y. Retinoic acid-related orphan receptors α and γ: key regulators of
42. Santori, F. R. et al. Identification of Natural RORγ Ligands that Regulate the Development of Lymphoid Cells. Cell Metab. 21, 286–297 (2015).
M AN U
670
43. Kallen, J. A. et al. X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that
677
cholesterol or a cholesterol derivative is the natural ligand of RORalpha. Struct. Lond. Engl. 1993 10, 1697–
678
1707 (2002).
44. Kallen, J., Schlaeppi, J.-M., Bitsch, F., Delhon, I. & Fournier, B. Crystal structure of the human RORalpha
TE D
679
680
Ligand binding domain in complex with cholesterol sulfate at 2.2 A. J. Biol. Chem. 279, 14033–14038
681
(2004).
45. Wada, T., Kang, H. S., Jetten, A. M. & Xie, W. The Emerging Role Of Nuclear Receptor RORα And Its
EP
682
Crosstalk With LXR In Xeno- And Endobiotic Gene Regulation. Exp. Biol. Med. Maywood NJ 233, 1191–1201
684
(2008).
685
686
AC C
683
46. Soroosh, P. et al. Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation. Proc. Natl. Acad. Sci. U. S. A. 111, 12163–12168 (2014).
687
47. Bird, L. T cells: Endogenous agonists for orphan RORγ. Nat. Rev. Immunol. 15, 70–71 (2015).
688
48. Castro, G. et al. RORγt and RORα signature genes in human Th17 cells. PLoS ONE 12, (2017).
689
49. DuSell, C. D., Umetani, M., Shaul, P. W., Mangelsdorf, D. J. & McDonnell, D. P. 27-hydroxycholesterol is an
690
endogenous selective estrogen receptor modulator. Mol. Endocrinol. Baltim. Md 22, 65–77 (2008).
ACCEPTED MANUSCRIPT
691
692
50. Umetani, M. et al. 27-Hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen. Nat. Med. 13, 1185–1192 (2007). 51. Imai, Y. et al. Nuclear Receptors in Bone Physiology and Diseases. Physiol. Rev. 93, 481–523 (2013).
694
52. Wardell, S. E., Nelson, E. R. & McDonnell, D. P. From empirical to mechanism-based discovery of clinically
700
701 702 703 704 705 706 707 708 709
SC
699
54. Nelson, E. R. et al. 27-Hydroxycholesterol Links Hypercholesterolemia and Breast Cancer Pathophysiology. Science 342, 1094–1098 (2013).
M AN U
698
transcriptional activation assay system using HeLa cells. Biosci. Biotechnol. Biochem. 68, 1790–1793 (2004).
55. Wu, Q. et al. 27-Hydroxycholesterol promotes cell-autonomous, ER-positive breast cancer growth. Cell Rep. 5, 637–645 (2013).
56. Wang, C.-W. et al. 7-ketocholesterol and 27-hydroxycholesterol decreased doxorubicin sensitivity in breast cancer cells: estrogenic activity and mTOR pathway. Oncotarget 8, 66033–66050 (2017).
TE D
697
53. Sato, H. et al. Oxysterol regulation of estrogen receptor alpha-mediated gene expression in a
57. Lappano, R. et al. The Cholesterol Metabolite 25-Hydroxycholesterol Activates Estrogen Receptor αMediated Signaling in Cancer Cells and in Cardiomyocytes. PLoS ONE 6, (2011). 58. Oguro, H. et al. 27-Hydroxycholesterol induces hematopoietic stem cell mobilization and extramedullary
EP
696
useful Selective Estrogen Receptor Modulators (SERMs). Steroids 90, 30–38 (2014).
hematopoiesis during pregnancy. J. Clin. Invest. 127, 3392–3401
AC C
695
RI PT
693
59. Simigdala, N. et al. Cholesterol biosynthesis pathway as a novel mechanism of resistance to estrogen deprivation in estrogen receptor-positive breast cancer. Breast Cancer Res. BCR 18, (2016).
710
60. Umetani, M. et al. The Cholesterol Metabolite 27-Hydroxycholesterol Promotes Atherosclerosis via
711
Proinflammatory Processes Mediated by Estrogen Receptor Alpha. Cell Metab. 20, 172–182 (2014).
712
61. DuSell, C. D. et al. The Endogenous Selective Estrogen Receptor Modulator 27-Hydroxycholesterol Is a
713
Negative Regulator of Bone Homeostasis. Endocrinology 151, 3675–3685 (2010).
ACCEPTED MANUSCRIPT
714
62. Nelson, E. R. et al. The Oxysterol, 27-Hydroxycholesterol, Links Cholesterol Metabolism to Bone
715
Homeostasis Through Its Actions on the Estrogen and Liver X Receptors. Endocrinology 152, 4691–4705
716
(2011). 63. Chang, P.-Y. et al. 27-hydroxycholesterol, an endogenous SERM, and risk of fracture in postmenopausal
RI PT
717
718
women: A nested case-cohort study in the Women’s Health Initiative. J. Bone Miner. Res. Off. J. Am. Soc.
719
Bone Miner. Res. (2018). doi:10.1002/jbmr.3576
64. Brooks, S. W., Dykes, A. C. & Schreurs, B. G. A High-Cholesterol Diet Increases 27-Hydroxycholesterol and
SC
720
Modifies Estrogen Receptor Expression and Neurodegeneration in Rabbit Hippocampus. J. Alzheimers Dis.
722
JAD 56, 185–196 (2017).
724
725
726
65. Raza, S. et al. The cholesterol metabolite 27-hydroxycholesterol stimulates cell proliferation via ERβ in prostate cancer cells. Cancer Cell Int. 17, (2017).
66. Voisin, M. et al. Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor. Proc. Natl. Acad. Sci. U. S. A. 114, E9346–E9355 (2017).
TE D
723
M AN U
721
727
67. Weikum, E. R., Knuesel, M. T., Ortlund, E. A. & Yamamoto, K. R. Glucocorticoid receptor control of
728
transcription: precision and plasticity via allostery. Nat. Rev. Mol. Cell Biol. 18, 159–174 (2017).
731
732
733
734
735
736
EP
730
68. Ratman, D. et al. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering. Mol. Cell. Endocrinol. 380, 41–54 (2013).
AC C
729
69. Silvente-Poirot, S., Dalenc, F. & Poirot, M. The Effects of Cholesterol-Derived Oncometabolites on Nuclear Receptor Function in Cancer. Cancer Res. 78, 4803–4808 (2018). 70. Wei, W. et al. Ligand Activation of ERRα by Cholesterol Mediates Statin and Bisphosphonate Effects. Cell Metab. 23, 479–491 (2016). 71. Ascenzi, P., Bocedi, A. & Marino, M. Structure-function relationship of estrogen receptor alpha and beta: impact on human health. Mol. Aspects Med. 27, 299–402 (2006).
ACCEPTED MANUSCRIPT
737
72. Chang, C. & McDonnell, D. P. Molecular Pathways: The Metabolic Regulator Estrogen-Related Receptor
738
Alpha as a Therapeutic Target in Cancer. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 18, 6089–6095
739
(2012).
741
73. Sensi, C. et al. Oxysterols act as promiscuous ligands of class-A GPCRs: in silico molecular modeling and in vitro validation. Cell. Signal. 26, 2614–2620 (2014).
RI PT
740
74. Hannedouche, S. et al. Oxysterols direct immune cell migration through EBI2. Nature 475, 524–527 (2011).
743
75. Nedelcu, D., Liu, J., Xu, Y., Jao, C. & Salic, A. Oxysterol binding to the extracellular domain of Smoothened
745
in Hedgehog signaling. Nat. Chem. Biol. 9, 557–564 (2013).
76. Radhakrishnan, A., Ikeda, Y., Kwon, H. J., Brown, M. S. & Goldstein, J. L. Sterol-regulated transport of
M AN U
744
SC
742
746
SREBPs from endoplasmic reticulum to Golgi: Oxysterols block transport by binding to Insig. Proc. Natl.
747
Acad. Sci. U. S. A. 104, 6511–6518 (2007).
748
77. Sun, L.-P., Seemann, J., Goldstein, J. L. & Brown, M. S. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins. Proc.
750
Natl. Acad. Sci. U. S. A. 104, 6519–6526 (2007).
753
754
755
756
757
mechanisms, both involving SCAP and Insigs. J. Biol. Chem. 279, 52772–52780 (2004).
EP
752
78. Adams, C. M. et al. Cholesterol and 25-hydroxycholesterol inhibit activation of SREBPs by different
79. Matsuda, M. et al. SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver
AC C
751
TE D
749
induced by cholesterol deprivation and insulin elevation. Genes Dev. 15, 1206–1216 (2001). 80. Im, S.-S. et al. Linking Lipid Metabolism to the Innate Immune Response in Macrophages through Sterol Regulatory Element Binding Protein -1a. Cell Metab. 13, 540–549 (2011). 81. Erridge, C., Webb, D. J. & Spickett, C. M. 25-Hydroxycholesterol, 7beta-hydroxycholesterol and 7-
758
ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6
759
signalling in human macrophages. Free Radic. Res. 41, 260–266 (2007).
ACCEPTED MANUSCRIPT
764
765
766 767 768 769 770 771 772
(2013).
RI PT
763
83. Spann, N. J. & Glass, C. K. Sterols and oxysterols in immune cell function. Nat. Immunol. 14, 893–900
84. Ito, A. et al. LXRs link metabolism to inflammation through Abca1-dependent regulation of membrane composition and TLR signaling. eLife 4,
85. Reboldi, A. et al. 25-hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type
SC
762
adrenoleukodystrophy through activation of the NLRP3 inflammasome. Nat. Commun. 7, 13129 (2016).
I interferon. Science 345, 679–684 (2014).
86. Cagno, V. et al. Inhibition of herpes simplex-1 virus replication by 25-hydroxycholesterol and 27-
M AN U
761
82. Jang, J. et al. 25-hydroxycholesterol contributes to cerebral inflammation of X-linked
hydroxycholesterol. Redox Biol. 12, 522–527 (2017).
87. Joseph, S. B. et al. LXR-Dependent Gene Expression Is Important for Macrophage Survival and the Innate Immune Response. Cell 119, 299–309 (2004).
88. Endo-Umeda, K., Nakashima, H., Umeda, N., Seki, S. & Makishima, M. Dysregulation of Kupffer Cells/
TE D
760
773
Macrophages and Natural Killer T Cells in Steatohepatitis in LXRα Knockout Male Mice. Endocrinology
774
(2018). doi:10.1210/en.2017-03141
777
778 779
780 781 782
EP
776
89. Tuong, Z. K. et al. RORα and 25-Hydroxycholesterol Crosstalk Regulates Lipid Droplet Homeostasis in Macrophages. PLoS ONE 11, (2016).
AC C
775
90. Svoronos, N. et al. Tumor Cell–Independent Estrogen Signaling Drives Disease Progression through Mobilization of Myeloid-Derived Suppressor Cells. Cancer Discov. 7, 72–85 (2017). 91. Baek, A. E. et al. The cholesterol metabolite 27 hydroxycholesterol facilitates breast cancer metastasis through its actions on immune cells. Nat. Commun. 8, 864 (2017). 92. Mutemberezi, V. et al. Oxysterol levels and metabolism in the course of neuroinflammation: insights from in vitro and in vivo models. J. Neuroinflammation 15, 74 (2018).
ACCEPTED MANUSCRIPT
783 784 785
93. Marengo, B. et al. Oxysterol mixture and, in particular, 27-hydroxycholesterol drive M2 polarization of human macrophages. BioFactors Oxf. Engl. 42, 80–92 (2016). 94. Guillem-Llobat, P. & Íñiguez, M. A. Inhibition of lipopolysaccharide-induced gene expression by liver X receptor ligands in macrophages involves interference with early growth response factor 1. Prostaglandins
787
Leukot. Essent. Fatty Acids 96, 37–49 (2015).
RI PT
786
95. Ward, L. J., Ljunggren, S. A., Karlsson, H., Li, W. & Yuan, X.-M. Exposure to atheroma-relevant 7-oxysterols
789
causes proteomic alterations in cell death, cellular longevity, and lipid metabolism in THP-1 macrophages.
790
PLOS ONE 12, e0174475 (2017).
792 793
96. Lee, S. et al. Adenylyl Cyclase-Associated Protein 1(CAP1) is a Receptor for Human Resistin and Mediates
M AN U
791
SC
788
Inflammatory Actions of Human Monocytes. Cell Metab. 19, 484–497 (2014). 97. Kim, K. M., Kim, Y. S., Jung, D. H., Lee, J. & Kim, J. S. Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose.
795
Exp. Cell Res. 318, 152–159 (2012).
798 799 800 801 802 803 804 805
C5a receptor. PLOS ONE 12, e0173749 (2017).
99. Zhang, R., Liu, Z., Li, Y. & Wu, B. LXR agonist regulates the proliferation and apoptosis of human T-Cell
EP
797
98. Cho, H. et al. 7α-Hydroxycholesterol induces monocyte/macrophage cell expression of interleukin-8 via
acute lymphoblastic leukemia cells via the SOCS3 pathway. Int. J. Biochem. Cell Biol. 78, 180–185 (2016). 100.
AC C
796
TE D
794
Bensinger, S. J. et al. LXR signaling couples sterol metabolism to proliferation in the acquired immune
response. Cell 134, 97–111 (2008). 101.
Ma, X. et al. Cholesterol negatively regulates IL-9–producing CD8+ T cell differentiation and antitumor
activity. J. Exp. Med. 215, 1555–1569 (2018). 102.
Vigne, S. et al. IL-27-Induced Type 1 Regulatory T-Cells Produce Oxysterols that Constrain IL-10
Production. Front. Immunol. 8, 1184 (2017).
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
phenotype. Oncol. Rep. 26, 389–397 (2011).
RI PT
811
Torres, C. G. et al. 27-hydroxycholesterol induces the transition of MCF7 cells into a mesenchymal
105. Alfaqih, M. A. et al. CYP27A1 Loss Dysregulates Cholesterol Homeostasis in Prostate Cancer. Cancer Res. 77, 1662–1673 (2017). 106.
Hiramitsu, S. et al. Estrogen Receptor Beta-Mediated Modulation of Lung Cancer Cell Proliferation by
27-Hydroxycholesterol. Front. Endocrinol. 9, (2018). 107.
SC
810
104.
Chen, L. et al. 25-Hydroxycholesterol promotes migration and invasion of lung adenocarcinoma cells.
M AN U
809
e0184985 (2017).
Biochem. Biophys. Res. Commun. 484, 857–863 (2017). 108.
Lin, C.-Y. et al. Cholestane-3β, 5α, 6β-triol Suppresses Proliferation, Migration, and Invasion of Human
Prostate Cancer Cells. PLOS ONE 8, e65734 (2013). 109.
de Medina, P. et al. Dendrogenin A arises from cholesterol and histamine metabolism and shows cell
TE D
808
Herold, M. et al. Liver X receptor activation promotes differentiation of regulatory T cells. PloS One 12,
differentiation and anti-tumour properties. Nat. Commun. 4, 1840 (2013). 110.
Poirot, M. & Silvente-Poirot, S. The tumor-suppressor cholesterol metabolite, dendrogenin A, is a new
class of LXR modulator activating lethal autophagy in cancers. Biochem. Pharmacol. 153, 75–81 (2018). 111.
EP
807
103.
Moresco, M. A. et al. Enzymatic Inactivation of Oxysterols in Breast Tumor Cells Constraints Metastasis
AC C
806
ACCEPTED MANUSCRIPT
Formation by Reprogramming the Metastatic Lung Microenvironment. Front. Immunol. 9, (2018). 112.
Raccosta, L. et al. The oxysterol–CXCR2 axis plays a key role in the recruitment of tumor-promoting
neutrophils. J. Exp. Med. 210, 1711–1728 (2013). 113.
Villablanca, E. J. et al. Tumor-mediated liver X receptor-alpha activation inhibits CC chemokine
receptor-7 expression on dendritic cells and dampens antitumor responses. Nat. Med. 16, 98–105 (2010). 114.
Courtney, R. & Landreth, G. E. LXR Regulation of Brain Cholesterol: From Development to Disease.
Trends Endocrinol. Metab. 27, 404–414 (2016).
ACCEPTED MANUSCRIPT 830 831 832
115.
Heverin, M. et al. Crossing the barrier: net flux of 27-hydroxycholesterol into the human brain. J. Lipid
Res. 46, 1047–1052 (2005). 116.
Björkhem, I., Leoni, V. & Svenningsson, P. On the fluxes of side-chain oxidized oxysterols across blood-
brain and blood-CSF barriers and origin of these steroids in CSF (Review). J. Steroid Biochem. Mol. Biol.
834
(2018). doi:10.1016/j.jsbmb.2018.12.009
837 838
839
Okabe, A. et al. Adaptive responses induced by 24S-hydroxycholesterol through liver X receptor
pathway reduce 7-ketocholesterol-caused neuronal cell death. Redox Biol. 2, 28–35 (2013). 118.
Liu, Y. et al. 25-Hydroxycholesterol activates the expression of cholesterol 25-hydroxylase in an LXR-
dependent mechanism. J. Lipid Res. 59, 439–451 (2018). 119.
SC
836
117.
M AN U
835
RI PT
833
Kim, H., Woo, J. H., Lee, J. H., Joe, E. & Jou, I. 22(R)-hydroxycholesterol induces HuR-dependent MAP
840
kinase phosphatase-1 expression via mGluR5-mediated Ca2+/PKCα signaling. Biochim. Biophys. Acta BBA -
841
Gene Regul. Mech. 1859, 1056–1070 (2016).
845 846
TE D
844
Noguchi, N., Urano, Y., Takabe, W. & Saito, Y. New aspects of 24(S)-hydroxycholesterol in modulating
neuronal cell death. Free Radic. Biol. Med. 87, 366–372 (2015). 121.
Testa, G. et al. A silver lining for 24-hydroxycholesterol in Alzheimer’s disease: The involvement of the
neuroprotective enzyme sirtuin 1. Redox Biol. 17, 423–431 (2018). 122.
EP
843
120.
Yamanaka, K., Saito, Y., Yamamori, T., Urano, Y. & Noguchi, N. 24(S)-Hydroxycholesterol Induces
AC C
842
847
Neuronal Cell Death through Necroptosis, a Form of Programmed Necrosis. J. Biol. Chem. 286, 24666–
848
24673 (2011).
849 850 851 852
123.
Gamba, P. et al. Interaction between 24-hydroxycholesterol, oxidative stress, and amyloid-β in
amplifying neuronal damage in Alzheimer’s disease: three partners in crime. Aging Cell 10, 403–417 (2011). 124.
Testa, G. et al. Changes in brain oxysterols at different stages of Alzheimer’s disease: Their involvement
in neuroinflammation. Redox Biol. 10, 24–33 (2016).
857
858
859
860
861
862
863
864
865
126.
Shafaati, M. et al. Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer’s patients
with the Swedish APP 670/671 mutation. J. Lipid Res. 52, 1004–1010 (2011). 127.
Refolo, L. M. et al. Hypercholesterolemia accelerates the Alzheimer’s amyloid pathology in a transgenic
mouse model. Neurobiol. Dis. 7, 321–331 (2000). 128.
Park, S. H. et al. Hypercholesterolemia accelerates amyloid β-induced cognitive deficits. Int. J. Mol.
Med. 31, 577–582 (2013). 129.
RI PT
856
Parkinson’s disease overlap? Exp. Gerontol. 68, 13–18 (2015).
SC
855
Marwarha, G. & Ghribi, O. Does the oxysterol 27-hydroxycholesterol underlie Alzheimer’s disease-
Ismail, M.-A.-M. et al. 27-Hydroxycholesterol impairs neuronal glucose uptake through an IRAP/GLUT4
M AN U
854
125.
system dysregulation. J. Exp. Med. 214, 699–717 (2017).
130. Teune, L. K. et al. Typical cerebral metabolic patterns in neurodegenerative brain diseases. Mov. Disord. 25, 2395–2404 (2010). 131.
Marwarha, G., Raza, S., Prasanthi, J. R. P. & Ghribi, O. Gadd153 and NF-κB Crosstalk Regulates 27-
TE D
853
ACCEPTED MANUSCRIPT
866
Hydroxycholesterol-Induced Increase in BACE1 and β-Amyloid Production in Human Neuroblastoma SH-
867
SY5Y Cells. PLOS ONE 8, e70773 (2013). 132.
Liu, J. et al. The ROS-mediated activation of IL-6/STAT3 signaling pathway is involved in the 27-
EP
868
hydroxycholesterol-induced cellular senescence in nerve cells. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 45,
870
10–18 (2017).
871
872
873
874
875
876
133.
AC C
869
Hirayama, T. et al. Serum concentration of 27-hydroxycholesterol predicts the effects of high-
cholesterol diet on plasma LDL cholesterol level. Hepatol. Res. Off. J. Jpn. Soc. Hepatol. 39, 149–156 (2009). 134.
Kim, S.-M. et al. 25-Hydroxycholesterol is involved in the pathogenesis of amyotrophic lateral sclerosis.
Oncotarget 8, 11855–11867 (2017). 135. Torres, N., Guevara-Cruz, M., Velázquez-Villegas, L. A. & Tovar, A. R. Nutrition and Atherosclerosis. Arch. Med. Res. 46, 408–426 (2015).
ACCEPTED MANUSCRIPT
883
884
885
886 887
888
889
890
891
892
893
894
895
896
897
898
atherosclerotic properties of oxysterols. Lipids Health Dis. 16, (2017). 138.
RI PT
882
Zmysłowski, A. & Szterk, A. Current knowledge on the mechanism of atherosclerosis and pro-
Sima, P., Vannucci, L. & Vetvicka, V. Atherosclerosis as autoimmune disease. Ann. Transl. Med. 6,
(2018). 139.
Libby, P. History of Discovery: Inflammation in Atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 32,
SC
881
137.
2045–2051 (2012). 140.
Gargiulo, S. et al. Relation between TLR4/NF-κB signaling pathway activation by 27-hydroxycholesterol
M AN U
880
2 (2017).
and 4-hydroxynonenal, and atherosclerotic plaque instability. Aging Cell 14, 569–581 (2015). 141.
Ishikawa, T. et al. LXRβ/estrogen receptor-α signaling in lipid rafts preserves endothelial integrity. J.
Clin. Invest. 123, 3488–3497 (2013). 142.
Li, Z. et al. Krüppel-Like Factor 4 Regulation of Cholesterol-25-Hydroxylase and Liver X Receptor
TE D
879
Foks, A. C. & Bot, I. Preface: Pathology and Pharmacology of Atherosclerosis. Eur. J. Pharmacol. 816, 1–
Mitigates Atherosclerosis Susceptibility. Circulation 136, 1315–1330 (2017). 143.
Son, Y. et al. 7-Ketocholesterol induces the reduction of KCNMB1 in atherosclerotic blood vessels.
Biochem. Biophys. Res. Commun. 457, 324–327 (2015). 144.
EP
878
136.
Savouret, J. F. et al. 7-ketocholesterol is an endogenous modulator for the arylhydrocarbon receptor. J.
AC C
877
Biol. Chem. 276, 3054–3059 (2001). 145.
Larigot, L., Juricek, L., Dairou, J. & Coumoul, X. AhR signaling pathways and regulatory functions.
Biochim. Open 7, 1–9 (2018). 146.
Beischlag, T. V., Morales, J. L., Hollingshead, B. D. & Perdew, G. H. The Aryl Hydrocarbon Receptor
Complex and the Control of Gene Expression. Crit. Rev. Eukaryot. Gene Expr. 18, 207–250 (2008).
ACCEPTED MANUSCRIPT
899
147.
Muse, E. D. et al. Cell-specific discrimination of desmosterol and desmosterol mimetics confers
900
selective regulation of LXR and SREBP in macrophages. Proc. Natl. Acad. Sci. U. S. A. 115, E4680–E4689
901
(2018).
903
148.
Fessler, M. B. The challenges and promise of targeting the Liver X Receptors for treatment of
inflammatory disease. Pharmacol. Ther. 181, 1–12 (2018).
904
AC C
EP
TE D
M AN U
SC
905
RI PT
902
Figure Legends:
EP
TE D
M AN U
SC
RI PT
Figure 1: Chemical structures and metabolic pathways for several common oxysterols. ROS: reactive oxygen species. DHCR24: 24-dehydrocholesterol reductase. CYP46A1: cytochrome P450 46A1. CYP27A1: cytochrome P450 27A1. CYP11A1: cytochrome P450 11A1. 7βHC: 7β-hydroxycholesterol. 7KC: 7ketocholesterol. 24HC: 24-hydroxycholesterol. 27HC:27-hydroxycholesterol. 25HC: 25-hydroxycholesterol 22HC: 22-hydroxycholesterol.
AC C
906 907 908 909 910 911 912
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
H
RI PT
H
H
H
H
SC M AN U
HO
DHCR24
H
R OS
OH
EP
TE D
7βHC
H H
H
HO
H
AC C
H
H H
H OH
HO
H
H
O H
7KC HO
H
Cholesterol
Cho l
CYP11A1
S O R
H
H
Desmoste
HO
H
H
H
22HC
ACCEPTED MANUSCRIPT Highlights:
EP
TE D
M AN U
SC
RI PT
Oxysterols play important roles in cholesterol regulation and homeostasis Certain oxysterols serve as ligands for nuclear receptors There is increasing interest in the roles of oxysterols in different diseases Review of oxysterols in disease, with emphasis on the roles of nuclear receptors
AC C
• • • •
S0303-7207(19)30022-X
DOI:
https://doi.org/10.1016/j.mce.2019.01.016
Reference:
MCE 10378
To appear in:
Molecular and Cellular Endocrinology
Received Date: 5 November 2018 Revised Date:
8 January 2019
Accepted Date: 16 January 2019
Please cite this article as: Ma, L., Nelson, E.R., Oxysterols and nuclear receptors, Molecular and Cellular Endocrinology (2019), doi: https://doi.org/10.1016/j.mce.2019.01.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title: Oxysterols and Nuclear Receptors
1
Authors: Liqian Ma and Erik R. Nelsona,b,c,d,e* a
Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign. Urbana, IL. University of Illinois Cancer Center. Chicago, IL. c Division of Nutritional Sciences, University of Illinois at Urbana-Champaign. Urbana, IL. d Carl R. Woese Institute for Genomic Biology, Anticancer Discovery from Pets to People Theme, University of Illinois at Urbana Champaign, Urbana, IL e Cancer Center at Illinois, University of Illinois at Urbana-Champaign, IL
M AN U
SC
RI PT
b
Abbreviated Title: Oxysterols and Nuclear Receptors. Keywords: cholesterol, oxysterol, 27-hydroxycholesterol; estrogen receptor; liver x receptor; breast cancer; selective estrogen receptor modulator, selective nuclear receptor modulator
EP
TE D
* Corresponding Author: Erik R. Nelson. University of Illinois at Urbana-Champaign. 407 S Goodwin Ave (MC114), Urbana, IL, 61801. Phone: 217-244-5477. Fax: 217-333-1133. E-mail: [email protected].
AC C
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
ACCEPTED MANUSCRIPT
RI PT
Abstract: Oxysterols are derivatives of cholesterol and an important regulator of cholesterol metabolism, in part due to their role as ligands for nuclear receptors, such as the liver x receptors. Oxysterols are also known to be ligands for the RAR-related orphan receptors, involved in normal T cell differentiation. However, increasing evidence supports a role for oxysterols in the progression of several diseases. Here, we review recent developments in oxysterol research, highlighting the biological functions that oxysterols exert through their target nuclear receptors: the liver x receptors, estrogen receptors RAR-related orphan receptor and the glucocorticoid receptor. We also bring the regulation of the immune system into the context of interaction between oxysterols and nuclear receptors, discussing the effect of such interaction on the pro-inflammatory function of macrophages and the development of T cells. Finally, we examine the impact that oxysterols have on various disease models, including cancer, Alzheimer’s disease and atherosclerosis, stressing the role of nuclear receptors if previously identified. This review underscores the need to consider the multifaceted roles of oxysterols in terms of multiple receptor engagements and selective modulation of these receptors.
EP
TE D
M AN U
SC
Funding: This work was funded in part by grants from the Department of Defense Breast Cancer Research Program (BC171214) and the American Institute of Cancer Research (Award 31284) to ERN.
AC C
24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
ACCEPTED MANUSCRIPT 42
Introduction:
43 Oxysterols are derivatives of cholesterol produced by the oxidation of cholesterol molecules by enzymatic
45
addition of hydroxyl, carbonyl or epoxide functional groups1. Endogenous oxysterols are commonly produced
46
by non-enzymatic mechanisms2. Specifically, reactive oxygen species (ROS), including alkoxy and peroxyl
47
radicals, can oxidize cholesterol molecules1,3. Cholesterol can also be oxidized by the leukocyte-H2O2-HOCl
48
system during inflammation3. Non-enzymatic oxidation is primarily responsible for the generation of certain
49
oxysterols such as 7-ketocholesterol (7KC) and 7β-hydroxycholesterol (7βHC)3. However, producing
50
oxysterols by the oxidation of cholesterol on the side-chain is typically catalyzed through enzymatic routes,
51
which are mediated by cytochrome p450 family of enzymes4,5. For instance, cholesterol can be metabolized
52
into 27-hydroxycholesterol (27HC) by CYP27A1, 24-hydroxycholesterol (24HC) by CYP46A1, 25-
53
hydroxycholesterol (25HC) by cholesterol-25-hydroxylase and 22-hydroxycholesterol by CYP11A13,4 (Figure 1).
M AN U
SC
RI PT
44
54
Many oxysterols have been described to bind to and modulate the activity of the liver x receptors (LXRs),
56
thereby completing an important homeostatic loop in cholesterol metabolism (described below). However,
57
several recent studies have implicated oxysterols in the modulation of other nuclear receptors such as the
58
retinoic acid receptor-related orphan receptors (RORs), estrogen receptors (ERs) and glucocorticoid receptors
59
(GRs), as well as non-nuclear receptor mechanisms. Furthermore, it is becoming increasingly apparent that
60
oxysterols play important roles in several pathologies, including cancers, cardiovascular diseases, and
61
neurodegenerative diseases6. This review will discuss our current understanding of oxysterol modulation of
62
nuclear receptors. We will also discuss various pathologies where oxysterols have been described to play a
63
role, and when known, highlight whether nuclear receptor involvement has been examined.
65
EP
AC C
64
TE D
55
Oxysterols and Nuclear Receptors
66 67
Nuclear Receptors
68
Nuclear receptors are a large superfamily consisting of 48 ligand-inducible transcription factors, acting as
69
intracellular receptors that bind to lipophilic ligands capable of crossing the plasma membrane (e.g. steroids)7.
ACCEPTED MANUSCRIPT 70
Conformational changes are commonly triggered upon ligand binding, allowing nuclear receptors to bind to
71
their respective DNA response elements, releasing corepressors and recruiting coactivators to promote
72
transcription of target genes involved in a variety of cellular processes, including cell proliferation and
73
metabolism7,8.
RI PT
74
Nuclear receptors share homology in their structures and functional domains. Five domains are generally
76
shared across typical nuclear receptors: a variable N-terminal region, a conserved DNA binding domain, a
77
variable hinge region, a conserved ligand binding domain and a variable C-terminal region9. After steroid
78
hormones were linked to the progression of prostate cancer and breast cancer, nuclear receptors have gained
79
increasing attention in terms of their roles in disease7,10,11.
M AN U
80
SC
75
Oxysterols and Liver X Receptor Signaling
82
Of the nuclear receptors, the liver X receptors (LXRs) are thought to be the major target of oxysterols,
83
especially in terms of the regulation of cholesterol metabolism. LXRα (NR1H3) and LXRβ (NR1H2) are
84
isoforms of LXR, which serve as cholesterol sensors for the regulation of cholesterol excess12. LXRβ is
85
expressed fairly ubiquitously, while LXRα expression is highest in the liver, with lower expression to various
86
degrees in other tissues13,14. The two isoforms share significant sequence identity, and although speculated,
87
clear evidence of specific biological differences have not been well described. The LXRs are known to
88
heterodimerize with the retinoid X receptors (RXRs) and sit on various DNA response elements, with a direct
89
repeat 4 (DR4) being recognized as the consensus sequence (5′-AGGTCA-NNNN-AGGTCA-3′)12,15,16. In an
90
unliganded state, it is thought that the heterodimer interacts with transcriptionally repressive complex, such as
91
with nuclear receptor corepressor (NCoR), thereby transreprepressing the transcriptional activity of LXR17–19.
92
The transrepression elicited by NCoR complex plays an important role in LXR-mediated anti-inflammatory
93
pathways, such as suppression of Toll-like receptor (TLR)-dependent pro-inflammatory signal expression in
94
macrophages20,21. Upon ligand binding, corepressor proteins are exchanged for coactivators, including NAD+-
95
dependent deacetylases Sirtuin 1 (SIRT1), which allows the RNA polymerase II complex to associate and
96
initiate transcription18,19.
97
AC C
EP
TE D
81
ACCEPTED MANUSCRIPT The first endogenous ligands to be described for the LXRs were 22(R)-hydroxycholesterol (22RHC), 24(S)-
99
hydroxycholesterol (24SHC), and 24(S),25-epoxycholesterol (24,25EC)22. Since then, it has been
100
demonstrated that several oxysterols and certain precursors in the cholesterol-biosynthetic pathway can
101
modulate the activity of the LXRs. These include 22RHC, 25HC, 27HC, 24SHC, 24,25EC and desmosterol23–27.
102
These metabolites have a wide range of affinities or potencies, with 24SHC having an EC50 of 4 µM and 3 µM
103
compared to 27HC at 85 nM and 71 nM for LXRα and LXRβ, respectively22,27. For reference, 27HC is the most
104
abundant oxysterol in circulation with concentrations ranging from 67 ng/mL (0.17 µM) to 199 ng/mL (0.5 µM)
105
while 24SHC circulates from 39 ng/mL (0.1 µM) to 91 ng/mL (0.23 µM)28.
SC
RI PT
98
106
Given the hepatic expression of CYP7A1, and more ubiquitous expression of CYP27A1 (with significant
108
expression observed in cells of the myeloid immune lineage), it would be expected when cholesterol levels are
109
high, so too is its metabolism into oxysterols. Indeed, there is a positive association between circulating
110
cholesterol levels and 27HC in both mice and humans, while statin therapy is associated with decreased
111
plasma 27HC29,30. Furthermore addition of cholesterol to macrophages resulted in increased secretion of 27HC,
112
while inhibiting cholesterol synthesis with simvastatin abolished the production of 27HC by macrophages31,32.
113
Although it is difficult to account for esterification, these studies suggest that increased level, of cholesterol
114
could lead to increased production of 27HC, which can then engage LXRs. Upon binding of an endogenous
115
ligand to the LXR-RXR heterodimer, the corepressor complex dissociates with commensurate recruitment of
116
coactivation complexes, ultimately leading to a transcriptional program aimed at restoring cholesterol
117
homeostasis. Specifically, LXR activation leads to the induction of target genes involved in cholesterol efflux
118
(ATP binding cassette (ABC) transporters A1/G1/G5/G8), apolipoproteins (APO A-1/E), cholesterol metabolism
119
into bile acids (CYP7A1) and decreased intestinal cholesterol absorption33. The removal of cholesterol by
120
reverse cholesterol transport is facilitated by ABC transporters. ABCA1, expressed in multiple cell types,
121
promotes the efflux of free cholesterol to apolipoprotein, in particular APOA1, leading to the formation of
122
discoid lipid-poor HDL15,34. ABCG5 and ABCG8 are half transporters expressed on the apical membrane of
123
enterocyte and on the canalicular membrane of hepatocytes35. The formation of heterodimer by ABCG5 and
124
ABCG8 on hepatocytes can promote the secretion of cholesterol into bile, while on enterocytes can facilitate
125
the excretion of cholesterol back into intestinal lumen12,35. LXR activation is also involved in regulating lipid
AC C
EP
TE D
M AN U
107
ACCEPTED MANUSCRIPT homeostasis by promoting triglyceride synthesis. Genes upregulated in this process are sterol regulatory
127
element-binding protein 1, acetyl-CoA carboxylase, fatty acid synthase and stearoyl-CoA desaturase-134,36,37.
128
The relatively longer-term actions of LXR-mediated homeostasis work in concert with the quicker actions of
129
cholesterol and oxysterols in maintaining sterol regulatory element-binding proteins (especially SREBP 2) in an
130
inactive state bound to the endoplasmic reticulum, thereby reducing the transcription of genes associated with
131
cholesterol metabolism and import38.
RI PT
126
132
Oxysterol and Retinoic acid receptor-related Orphan Receptor (ROR) signaling
134
RORs are nuclear receptors with three subtypes, RORα (NR1F1), RORβ (NR1F2) and RORγ (NR1F3)39.
135
RORs regulate gene transcription by binding to their respective response element as monomers upon ligand
136
binding. Each type of ROR has multiple isoforms expressed in humans and mice, with RORγ having two
137
isoforms, RORγ1 and RORγt40. RORα are involved in a variety of cellular processes, such as cerebellar
138
development, circadian rhythm and lipid metabolism, while RORγ is critical for lymphocyte development and
139
differentiation39,40. Oxysterols, including 22RHC, 24SHC, 25HC and 27HC, have been described as
140
endogenous ligands for both RORα and RORγ41. However, the activities of oxysterols differ, as they have the
141
ability to act as both agonists or inverse agonists. For example, it has been generally found that 24SHC and
142
25HC act as inverse agonists for both RORα and RORγ, while 27HC acts as an agonist for RORγ41. However,
143
a study also identified the ability of 25HC and 22HC to restore RORγ transcriptional activity from a synthetic
144
inhibitor in mammalian cells lines, suggesting some agonistic activity42. Cholesterol and cholesterol sulfate are
145
also the endogenous ligands of ROR, specifically RORα, with cholesterol sulfate having higher affinity to the
146
receptor due to its better fitting in the ligand binding pocket43,44.
M AN U
TE D
EP
AC C
147
SC
133
148
Similar to the LXRs, RORs also participate in cholesterol metabolism. One of the transcriptional targets of
149
RORα activation is CYP7B1, which codes for oxysterol 7α-hydroxylase, an enzyme involved in metabolizing
150
27HC and 25HC to bile acids45. However, RORs’ interaction with oxysterols, especially RORγ and RORγt play
151
very important roles in T helper 17 cell (Th17) differentiation46,47. Th17 cells were shown to preferentially
152
produce two types of oxysterols, 7β, 27-dihydroxycholesterol (7β, 27DHC) and 7α, 27DHC, and these
153
oxysterols rescued the inhibitory effect of ursolic acid on RORγt, while promoting coactivator recruitment to the
ACCEPTED MANUSCRIPT ligand binding domain of RORγt46. In addition, RNA sequencing analysis showed that in Th17 cells, interleukin
155
(IL)-17A, IL-17F, IL-23R, C-C ligand (CCL)20 and C-C chemokine receptor (CCR)6 were regulated by both
156
RORγt and RORα48. Although it is clear that the RORs are required for normal Th17 differentiation and function,
157
the physiological relevance and precise impact of oxysterols on this process remain to be elucidated.
158 159
RI PT
154
Oxysterols and Estrogen Receptor (ER) Signaling
161
Oxysterols were definitively found to bind to and modulate the activity of the estrogen receptors just over 10
162
years ago49,50. There are two main subtypes of nuclear estrogen receptors in mammalian cells, ERα (NR3A1)
163
and ERβ (NR3A2). A membrane-associated G protein-coupled estrogen receptor 1 (GPER1, or GPR30) has
164
also been described, but whether oxysterols can interact with this receptor remains unknown51,52. Nuclear ER
165
signaling comprises ligand binding to ER, followed by dissociation of the heat shock protein from ER, revealing
166
nuclear localization signal, and homo- or hetero-dimerization of two ER monomers51. ER dimers then bind to
167
the respective estrogen response element of the target gene, recruiting co-activators and/or co-repressors,
168
leading to target gene transcription, including trefoil factor 1 (TIFF1) and progesterone receptor (PR)51,52. So-
169
called ‘non-genomic’ or ‘rapid’ ER signaling also exists, where cytosolic ERs engage in kinase-mediated
170
signaling cascades including mobilization of intracellular calcium, stimulation of adenylate cyclase activity and
171
epidermal growth factor receptor activation, ultimately resulting in rapid cellular changes6,52.
M AN U
TE D
EP
172
SC
160
As early as in 2004, oxysterols, including β-epoxycholesterol, 20(S)-hydroxycholesterol and 22RHC, were
174
shown to have estrogenic activity in ERα-expressing HeLa cells53. More recently, a Gal4-ER co-transfection
175
screen of several cholesterol metabolites revealed that 22RHC, 24SHC, 25HC and 27HC significantly inhibited
176
estradiol (E2) activation of both ERα and β, with IC50s ranging from 1-5 µM50. 27HC was subsequently shown
177
to be a selective ER modulator (SERM), with the ability to induce distinct ER conformational changes and to
178
manifest either activating or inhibitory activities, depending on the tissue and cellular context49. 27HC was
179
found to activate the classic ERE-Luciferase reporter (3XERE-TATA-Luc) in transfected HeLa cells, with an
180
EC50 of approximately 1 µM, and when co-treated with estradiol had a Ki of 1.32 µM49. Demonstrating its
AC C
173
ACCEPTED MANUSCRIPT 181
selective-modulatory capabilities, it could activate the ER to promote the cellular proliferation of ER+ breast
182
cancer cell lines, tumors or uteri, but inhibited estrogen-dependent expression of vascular nitric oxide synthase,
183
thereby repressing carotid artery reendothelialization49,50,54,55.
184 Other oxysterols or cholesterol metabolites besides 27HC have also been found to modulate ER activities.
186
7KC was shown to decrease the cytotoxicity of doxorubicin in MCF7 cells, and this effect was dependent on
187
ERα, as fulvestrant inhibition or ERα knockdown restored doxorubicin accumulation in MCF7 cells56. 7KC also
188
induced the expression of TIFF1, an ER target gene56. Structurally similar to 27HC, 25HC upregulates ER
189
target genes, including TIFF1, PR, cathepsin D, cyclin A and cyclin D1 in MCF7 and BG1 cells57. Furthermore,
190
25HC promotes ER+ breast cancer cell proliferation and inhibits hypoxia-inducible factor-1α (HIF-1α)
191
expression induced by hypoxia in cardiomyocytes to prevent cell apoptosis57.
M AN U
SC
RI PT
185
192
Subsequent studies have further confirmed the selective modulator capacity of 27HC. In hematopoietic stem
194
cells (HSCs) during pregnancy, 27HC could induce ERα-dependent HSC mobilization and extramedullary
195
hematopoiesis58. In a cancer cell model, 27HC enhanced ER-mediated transcriptional activity and the
196
recruitment of ER to TIFF1 and growth regulating estrogen receptor binding 1 (GREB1) response elements in
197
an ER+ cancer cell line (MCF7) adapted to long-term estrogen deprivation59. However, in cardiovascular cells,
198
27HC reversed the cardioprotective effect of estrogen by inhibiting estrogen-induced nitric oxide production by
199
vascular cells, indicating an antagonistic activity against ER60. 27HC exhibited a partial-agonist effect on
200
osteoblasts, but ultimately exerted a negative impact on bone quality that could be attenuated by exogenous
201
estrogen61,62. The negative effects on bone are likely a combination of signaling through the ERs and LXRs,
202
and 27HC has been recently been reported to be associated with decreased bone mineral density in
203
postmenopausal women when circulating estrogens are taken into account63. Moreover, 27HC could serve as
204
a partial agonist to ER in ER+ breast cancer cell lines other than MCF7 cells, including T47D and BT48349,54. It
205
has also been suggested that 27HC antagonizes the neuroprotective effects of estrogen signaling through ERα,
206
as an increase in 27HC concentration in the brain is associated with increased neurodegeneration of the
207
hippocampus, decreased expression of ER and a decreased level of hippocampal mitochondria64. 27HC was
208
also shown to exert effects through ERβ: not only was ERβ expression upregulated by 27HC in prostate
AC C
EP
TE D
193
ACCEPTED MANUSCRIPT 209
cancer cell lines, LNCaP and PC3, but the 27HC-induced proliferation of prostate cancer cells was attenuated
210
by the ERβ-specific inhibitor, PHTPP65.
211
Oxysterols and Other Nuclear Receptors
213
The glucocorticoid receptor (NR3C1, GR) is another type of nuclear receptor shown to interact with
214
oxysterols66. Normally, upon ligand (glucocorticoid steroid) binding, GR can dimerize and translocate into the
215
nucleus67,68. In the nucleus, GR dimers bind to GR response elements, recruit coactivators and induce gene
216
transcription (transactivation)67,68. Activated GRs can also complex with other transcription factors, such as
217
nuclear factor-κB (NFκB) and activator protein 1 (AP1), thereby sequestering the transcription factor and
218
repressing the respective target gene transcription (transrepression)67,68. One oxysterol recently identified as a
219
GR ligand is 6-oxo-cholestan-3β,5α-diol (OCDO), which is generated from 5,6α-epoxycholesterol and 5,6β-
220
epoxycholesterol (5,6EC) by cholesterol epoxide hydrolase (ChEH) and 11β-hydroxysteroid-dehydrogenase-
221
type-2 (11βHSD2) in the scenario of breast cancer66,69. By binding and translocating GR into the cellular
222
nucleus, OCDO led to the expression of MMP1, but did not stimulate the expression of cortisol induced-classic
223
GR target genes, SGK1 and MKP1. These data suggest that even though both OCDO and glucocorticoids
224
signal through GR, their actions on GR are distinct66,69.
TE D
M AN U
SC
RI PT
212
225
The estrogen-related receptor (ERR) family is composed of ERRα (NR3B1), ERRβ (NR3B2) and ERRγ
227
(NR3B3), with ERRα being the most studied70. ERRα shares 70% homology with ERα in the DNA-binding
228
domain, but classic ERα ligands, such as 17β-estradiol, do not directly regulate the activity of ERRα70,71. ERRα
229
has been categorized as an orphan receptor due to a lack of endogenous ligand, and is thought to be primarily
230
regulated by other proteins, such as PGC1α72. Recently, Wei et al. identified cholesterol to be a molecule that
231
activates and regulates the transcriptional activity of ERRα70. In addition, ERRα, at least in part, mediates the
232
cholesterol-stimulated osteoclastogenesis and bone resorption70. Whether oxysterols can also regulate the
233
activity of ERRα are unknown, but modeling would suggest that they could interact with the ERRα in a similar
234
way to cholesterol.
235
AC C
EP
226
ACCEPTED MANUSCRIPT Non-Nuclear Receptor Actions of Oxysterols
237
Although the focus of this review is on the interaction between oxysterols and nuclear receptors, it is important
238
to note that the mechanisms of action of oxysterols are not limited to nuclear receptors. Furthermore, the
239
precise mechanisms of action are not known for several of the biological phenomena that oxysterols are
240
implicated in (highlighted below). Oxysterols can also act through GPCRs, including Epstein-Barr virus-induced
241
G-protein coupled receptor 2 (EBI2/GPR183), GPR17, C-X-C chemokine receptor (CXCR)2 and Smoothened
242
(SMO)73–75. EBI2, CXCR2 and GPR17, in particular, were shown to be promiscuously modulated by
243
oxysterols73. In addition, Nedelcu et al. reported that Hedgehog signaling requires the binding of oxysterol to
244
vertebrate SMO75.
SC
RI PT
236
M AN U
245
Besides GPCRs, regulatory proteins, such as SREBPs, are also targets of oxysterols76–78. Specifically,
247
oxysterols can bind to Insulin-induced proteins (INSIGs), prompting INSIGs to bind to the SREBP cleavage-
248
activating protein (SCAP), which then leads to a conformational change in the cytoplasmic loop 6 of SCAP76,77.
249
SCAP is an SREBP-escort protein, which, upon synthesis, promotes the clustering of SREBP in coat protein
250
complex II (COPII)-coated vesicles76,77,79. The SCAP:SREBP complex vesicles then travel to the Golgi complex
251
from endoplasmic reticulum (ER)76,77,79. At the Golgi complex, SREBP is cleaved before entering the nucleus to
252
promote cholesterol synthesis76,77,79. Conformational change of SCAP, induced by oxysterols through INSIGs,
253
prevents the COPII proteins from binding to the MELADL sequence on SCAP, ultimately blocking the transport
254
of SREBPs to the Golgi complex and promotion of cholesterol synthesis76,77. Although cholesterol is the
255
prototypical ligand for the INSIGs, oxysterols have also been shown to bind. Thus, oxysterols likely have two
256
roles in terms of cellular cholesterol homeostasis: inhibition of SREBPs and engagement of the LXRs. One of
257
the SREBP isoforms, SREBP-1a, is highly expressed in macrophages and plays important roles in regulating
258
the activity of the inflammasome, such as the production of pro-inflammatory cytokine IL-1β80.
AC C
EP
TE D
246
259 260
Oxysterols in Health and Disease
261
Oxysterols and the Immune System
262
The roles of oxysterols in the immune system have gained increasing appreciation over the last decade. The
263
interaction of oxysterols with the immune system has been recognized as one of the major ways that
ACCEPTED MANUSCRIPT oxysterols contribute to diseases, and such interaction can be complicated and multifaceted. Demonstrating
265
the complexity of oxysterol regulation of the immune system, some studies have described 25-
266
hydroxycholesterol (25HC) as being immunosuppressive by inhibiting cytotoxic T cell production and the
267
inflammasome activities, while others have found that 7βHC and 25HC induced the production of pro-
268
inflammatory chemokines and cytokines in U937 and THP-1 cells4,81,82. Although some effects of oxysterols
269
have been attributed to specific mechanisms (such as via nuclear receptors or SREBP signaling), many are yet
270
to be explored. Where possible, we have highlighted known mechanisms within this review.
RI PT
264
SC
271 272
LXR activation has been shown to play an important anti-inflammatory role in the immune system, due to the
274
inhibition of pro-inflammatory signal production downstream of NFκB83. Ito et al. showed that LXR activation
275
could disrupt the membrane lipid organization by hindering the recruitment of adaptor proteins, MyD88 and
276
TRAF6, through ABCA1, ultimately resulting in the inhibition of the TLR signaling to NFκB and MAPK
277
effectors84. Non-nuclear receptor-mediated mechanisms are also prevalently used by oxysterols. For example,
278
25HC was identified as one of the anti-inflammatory oxysterols that reduced Il1b transcription and IL1-
279
activating inflammasome activity by repressing SREBP in macrophages85. Studies suggesting an antiviral role
280
of 25HC prompted Cagno et al. to treat cells with 25HC and 27HC prior to Herpes simplex virus 1 (HSV1)
281
infection86. This pre-treatment promoted IL-6 secretion in HeLa cells and NFκB activation in Vero cells, linking
282
25HC and 27HC to a pro-inflammatory role86. It is not clear whether these activities required SREBP or LXR. In
283
the context of cerebral inflammation, Jang et al. identified that 25HC promoted robust nucleotide-binding
284
oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome assembly and
285
activation in macrophages in a potassium efflux/mitochondrial ROS/LXR-dependent manner82. These findings
286
suggest that the actions of oxysterols on the immune function may be selective, cell-type specific and cell-
287
location specific.
AC C
EP
TE D
M AN U
273
288 289
Oxysterols, Nuclear Receptors and Innate Immune System
290
The interaction between LXR and the immune system was first reported by Joseph et al. in that the loss of
291
functional LXRs in mice made them highly susceptible to infection by the intracellular bacteria Listeria
ACCEPTED MANUSCRIPT monocytogenes87. Also, expression of LXR promoted macrophage survival during the infection87. The
293
described effects of LXRs on innate immune cells range from regulation of cholesterol efflux to regulation of
294
inflammatory status. LXRα plays an important role in regulating the activities of Kupffer cells and natural killer T
295
cells88. Endo-Umeda et al. showed that knocking out LXRα leads to an increased level of bone marrow-derived
296
Kupffer cell population (F4/80+CD68+CD11b+) in the liver of mice when they are fed with a high cholesterol
297
diet88. Also, the lack of LXRα led to increased production of pro-inflammatory cytokines by those cells88.
298
Contrary to Kupffer cells, deficiency in LXRα reduced the frequency of natural killer cells and impaired those
299
cells functionally, as illustrated by the decreased capacity to produce IL-4 and interferon (IFN)-γ post α-GalCer
300
stimulation88. Whether specific oxysterols can modulate these LXR-associated activities is likely, given that
301
they are ligands for the LXRs.
M AN U
SC
RI PT
292
302
LXR is not the only nuclear receptor that is involved in innate immunity management. Studies have reported
304
innate immune cell functions regulated by oxysterols through RORα and ER. Tuong et al. identified a crosstalk
305
between RORα and 25HC in regulating macrophage function89. They found that the cholesterol 25 hydroxylase
306
expression was attenuated in RORα-deficient staggerer (sg/sg) mice, along with macrophages exhibiting
307
dysregulated lipid phenotype. Also, knocking down cholesterol 25 hydroxylase with siRNA in wildtype
308
macrophages resulted in lipid dysregulation89. However, treating sg/sg macrophages with 25HC significantly
309
decreased lipid accumulation into lipid droplets by approximately 1.7-fold and rescued the lipid dysregulation
310
phenotype89. ER is another receptor involved in the oxysterol-immunity axis. During pregnancy, 27HC
311
promotes ERα-dependent hematopoietic stem cell mobilization58, and estrogens themselves have been shown
312
to recruit myeloid cells to the tumor niche90. As will be discussed below, 27HC also increases myeloid cell
313
recruitment to metastatic lesions, although it is not yet known whether this is mediated by the ERs or LXRs91.
EP
AC C
314
TE D
303
315
Many studies have suggested an anti-inflammatory effect of oxysterols on innate immune cells, although
316
several of these effects are either LXR/nuclear receptor-independent or have mechanisms that remain
317
unknown. For example, in glial cells, while the oxysterols oxidized on the sterol backbone generally had no
318
effect on pro-inflammatory cytokine production after lipopolysaccharide (LPS) induction, oxysterols oxidized on
319
the side chain generally decreased LPS-induced mRNA expression of IL-1β and macrophages inflammatory
ACCEPTED MANUSCRIPT protein (MIP)-1α, suppressing the inflammatory phenotype of macrophages92. In the same study, it was further
321
shown that 24,25EC, 25HC, and 27HC decreased LPS-induced expression of IL-6 and tumor necrosis factor
322
(TNF)-α92. This finding is further supported by Marengo et al., indicating that an oxysterol mixture decreased
323
the level of reactive oxygen species (ROS) production in human macrophages and increased the level of
324
immunoregulatory cytokine IL-10 secretion93. The relative contributions of LXRs or SREBPs in these processes
325
are not currently known. Besides cytokines, LXR ligands, such as 25HC, can downregulate LPS-induced
326
expression of cyclooxygenase (COX)2 and mPGE2 synthase 1 (mPGES1), both of which are involved in
327
producing prostaglandins, a group of physiologically active and pro-inflammatory lipid compounds94.
SC
RI PT
320
328
7-oxygenated cholesterols have been associated with the development of atherosclerosis, suggesting a pro-
330
inflammatory effect in foamy macrophages during the formation of the atherosclerotic lesion. Similar to those
331
effects of oxysterols that are anti-inflammatory, the mechanisms of oxysterols’ pro-inflammatory effects on the
332
innate immunity largely remain to be elucidated. Proteomics revealed that a mixed treatment of 7βHC and
333
7KC reduced the expressions of glyoxalase 1 and adenylyl cyclase-associated protein 1 (CAP1) in human
334
THP1 cells; both proteins are associated with anti-inflammatory activities95–97. Specifically, glyoxalase 1 inhibits
335
the accumulation of oxidative stress in diabetes mellitus97. Overexpressing CAP1 in monocytes exacerbated
336
adipose tissue inflammation in mice, while suppressing CAP1 expression abolished the resistin-mediated
337
inflammatory activity both in vitro and in vivo96. Another type of pro-inflammatory cytokine, IL-8, was also
338
shown to be upregulated in human macrophages via PI3K and MEK pathway upon 7KC and 7α-
339
hydroxycholesterol (7αHC) treatment98. In this study, the GPCR complement receptor, C5a was implicated in
340
mediating the upregulation of IL-8.
342
TE D
EP
AC C
341
M AN U
329
343 344
Oxysterol, Nuclear Receptors and Adaptive Immune System
345
The interaction between oxysterols and the adaptive immune system is mediated by both LXR and RORγt,
346
with RORγt specifically involved in Th17 differentiation and functionality. LXR activation and signaling usually
347
negatively regulates T cell proliferation99,83. It was shown by Bensinger et al. that LXRβ knockout mice
ACCEPTED MANUSCRIPT displayed unregulated T cell activity, including lymphoid hyperplasia and enhanced responses to antigen
349
challenge100. Also, Ma et al. showed that Tc9 cells, a polarized population of CD8+ T cells with strong anti-
350
tumor activity, are regulated by cholesterol through its derivative, oxysterols, such as 22RHC101. 22RHC
351
inhibited IL-9 expression by activating LXRs, which then resulted in LXR sumoylation and decreased p65
352
binding to the promoter region of Il9 gene, while IL-9 is essential for the survival of Tc9 in vivo101. LXR
353
activation by 25HC and 22RHC also takes part in T regulatory cell (Treg) control102,103. Mice lacking cholesterol
354
25 hydroxylase exhibited increased production of IL-10, while 25HC treatment impaired IL-27-induced Treg
355
differentiation by downregulating IL-10 expression in an LXR-dependent manner102.
SC
RI PT
348
356
Soroosh et al. first described the most potent selective activator for RORγt to be 7β, 27DHC46. They showed
358
that in vivo administration of 7β, 27DHC promoted IL-17 production and in vitro treatment of this oxysterol
359
enhanced IL-17-producing Th17 cell differentiation in a RORγt-dependent manner46. On the flip side, knocking
360
out the enzyme CYP27A1, which is responsible for the production of 7β, 27DHC, significantly reduced IL-17-
361
producing CD4+ and γδ+ T cell population46. Santori et al. also identified “cholesterol biosynthetic intermediate”
362
(CBI) downstream of lanosterol and upstream of zymosterol to be natural RORγt ligand42. In the study,
363
knocking out CYP51 led to smaller lymph node anlagen and stunted development of lymph node structure in
364
embryos, developmental processes mediated by RORγt42. In addition, breeding Sc4molf/f mice to CD4-cre and
365
RORγt-cre mice generated the deletion of Sc4mol in TCRαβ T cells and RORγt-expressing cells. Sc4mol is
366
responsible for modifying the C4-methyl groups of CBIs, and its deletion led to a significant decrease in IL-17-
367
producing CD4+ T cells42. The requirement of CYP51 and Sc4mol in RORγt-mediated adaptive immunity
368
developmental processes dictates the indispensable role of CBI in RORγt signaling.
TE D
EP
AC C
369
M AN U
357
370
Oxysterols and Cancer
371
Since 27HC was first described to be a selective estrogen receptor modulator, the effect of this oxysterol on
372
breast cancer has now been extensively studied in pre-clinical models, which is not surprising given that
373
approximately 60-75% of all breast cancers are ERα+. CYP27A1 protein expression was associated with
374
higher breast tumor grade, while CYP7B1 mRNA expression is associated with a better prognosis, indicating a
375
clinically relevant role for 27HC in breast cancer6,54 . As expected, 27HC promoted the in vitro proliferation of
ACCEPTED MANUSCRIPT cell lines in an ER-dependent manner49,54,55. 25HC has also been shown to promote the proliferation of breast
377
cancer cells through activation of the ER57. 27HC increased the growth of ER+ mammary tumors, while
378
inhibiting the production of 27HC attenuated hypercholesterolemia-driven tumor growth in vivo54,55. Intriguingly,
379
27HC could also promote the transition from epithelial-like to mesenchymal-like (EMT) in breast cancer
380
cells54,104. The synthetic LXR agonist, GW3965, could also promote EMT, indicating that this phenotype is likely
381
LXR mediated54. 7KC also plays a role in breast cancer in that 7KC decreased the cytotoxicity of doxorubicin in
382
ER+ breast cancer cell line MCF7 by upregulating P-glycoprotein expression, while this effect was not
383
observed in ER- cell line, MDA-MB-23156. The requirement of the ERα in 7KC’s effect was further illustrated by
384
knocking down ERα with siRNA, which restored the doxorubicin accumulation in MCF756.
SC
RI PT
376
M AN U
385
While the effects of 27HC and 7KC on breast cancer cells required ERα, ERβ may also play an important role
387
in 27HC signaling in cancer types. Raza et al. demonstrated that 27HC promoted prostate cancer cell
388
proliferation in vitro in LNCaP and PC3 cells through the ER. Specifically, 27HC induced proliferation was
389
significantly reduced in the presence of the ER inhibitor, ICI 182,780 (fulvestrant)65. Likewise, the ERβ-
390
selective antagonist PHTPP reversed the proliferative effect of 27HC on prostate cancer cells, indicating that
391
27HC signals through ERβ to promote their proliferation65. However, these results were contradicted by
392
another study, indicating that 27HC actually decreased the proliferation of prostate cancer cells, via an
393
SREBP2 mediated mechanism105. Furthermore, the expression of CYP27A1 was downregulated in prostate
394
cancers compared to normal tissue, and elevated CYP27A1 expression is associated with a better prognosis
395
for prostate cancer patients. Further work will be required to better characterize the impact of oxysterols on
396
prostate cancer.
AC C
EP
TE D
386
397 398
In addition to breast and prostate cancer, 27HC was shown to affect lung cancer development. Using H1395
399
cell line as a lung cancer model, Hiramitsu et al. found that 27HC advanced ERβ+ lung cancer cell proliferation
400
via phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling106. Chen et al. described the pro-metastatic
401
activity of 25HC in lung adenocarcinoma107. In A549 cells and NCL-H1975 cells, 25HC treatment enhanced
402
snai1 expression, a marker of EMT, in an LXR-dependent manner, which promoted invasion of those cancer
403
cells107.
ACCEPTED MANUSCRIPT 404 Although many oxysterols have been reported to contribute to cancer progression, cholestane-3β, 5α, 6β-triol
406
(triol) was found to have anti-cancer activity108. Not only was the triol treatment able to inhibit the growth of
407
LNCaP CDXR3, DU145 and PC3 prostate cancer cell lines, but also oral administration of this oxysterol was
408
able to slow the progression of xenograft PC3 in NUDE mice108. Triol treatment also inhibited the invasive
409
capacity of DU145 and PC3 cells and downregulated the epithelial to mesenchymal transition-related genes,
410
including N-cadherin, vimentin, Slug, focal adhesion kinase (FAK), phospho-FAK Ser722 and phospho-FAK
411
Tyr861108. Another LXR modulator, dendrogenin A, was reported by de Medina et al. to possess anti-tumor
412
property in vivo109. This cholesterol metabolite was produced by 5,6αEC reacting with histamine and was
413
significantly downregulated in breast tumor, compared to normal tissues109,110. Treating cancer cells with
414
dendrogenin A promoted cancer cell re-differentiation, inhibited OCDO production and induced lethal cancer
415
cell autophagy109,110.
M AN U
SC
RI PT
405
416
Besides the direct effects of oxysterols on cancer cells, these molecules impart an indirect effect on cancer by
418
interplaying with the immune system and creating a microenvironment that promotes cancer invasion and
419
metastasis16,91. Following the study that first identified the pro-metastatic effect of 27HC on breast cancer,
420
Baek et al. reported that 27HC required myeloid immune cell population to promote metastasis54,91. 27HC
421
treatment led to increased levels of polymorphonuclear-neutrophils (PMNs) and γδ+ T cells at distal metastatic
422
sites91. PMNs were further shown to be required for the enrichment of γδ+ T cells, and that both cell
423
populations were required for the full metastatic effect of 27HC91. Specifically, immune depletion of either
424
PMNs or γδ+ T cells reversed the 27HC-driven Met1 breast cancer metastasis to the lungs91. Gene expression
425
revealed that 27HC likely polarizes PMNs into an immune suppressive ‘N2’ phenotype, thereby explaining the
426
observed decreases in CD8+ cytotoxic T cells post 27HC treatment91. Moresco et al. also suggested that
427
oxysterols likely promote breast tumor metastasis by recruiting tumor-promoting neutrophils to the lung
428
metastatic niche, as oxysterol-inactivating enzyme sulfotransferase 2B1b (SULT2B1b)-transduced 4T1 tumor
429
had lower levels of metastasis and infiltration by tumor-promoting neutrophils in vivo111. Raccosta et al.
430
reported an oxysterol-dependent, LXR-independent mechanism by which oxysterols recruit tumor-promoting
431
neutrophils. They showed that bone marrow-derived CD11bhighGr1high myeloid cells migrate toward natural and
AC C
EP
TE D
417
ACCEPTED MANUSCRIPT tumor-released oxysterols112. Specifically, 22RHC led to the activation of CXCR2 on CD11bhighGr1high cells,
433
whose activation led to the production of pro-angiogenic factor, MMP9112. Inactivating CXCR2 inhibited AB1
434
and LLC tumor growth by reducing angiogenesis and immunosuppression112. An LXR-dependent mechanism
435
by which oxysterols create immunosuppressive microenvironment was also illustrated by the same group: by
436
activating LXRα on dendritic cells (DCs), oxysterols inhibit CCR7 expression on DCs, thereby preventing them
437
from migrating to lymph nodes to facilitate T cell activation against tumor cells113. Given that the pro-metastatic
438
effects of oxysterols involve the immune system, it is likely these effects are similar across solid tumors. Indeed,
439
27HC was shown to promote metastasis in pre-clinical models of breast, colon, pancreatic and lung cancers91.
440
Therefore, when examining the role of oxysterols in cancer progression, it is critical that one considers both
441
their direct effects on cancer cells as well as their cancer-cell extrinsic effects.
M AN U
SC
RI PT
432
442 Neurodegenerative Diseases
444
The brain contains approximately 25% of total cholesterol in our body114. In the central nervous system,
445
cholesterol metabolism is tightly controlled and separated from the rest of the body by the blood-brain barrier
446
(BBB)114. Oxysterols such as 27HC can cross the BBB, and play an important role in maintaining cholesterol
447
homeostasis in the central nervous system115,116. However, dysregulated cholesterol homeostasis in the brain,
448
especially along the oxysterol-LXR axis, is implicated in many neurodegenerative diseases, such as
449
Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD)6,114. Besides regulating
450
cholesterol and lipid metabolism, oxysterol signaling through the LXRs also participates in other cellular
451
processes. Among them, 24SHC, 25HC and 27HC have been shown to induce adaptive responses in neuronal
452
cells against cytotoxic oxidative stress induced by 7KC117. This protective effect requires the downstream
453
expression of ABCG1, but not ABCA1117. Additionally, 25HC induces the expression of cholesterol 25
454
hydroxylase via the activation of the LXR, independent of cell type118. In the nervous system, 22HC can
455
upregulate the expression of MAP kinase phosphatase (MKP)-1, which then suppresses the JNK-mediated
456
inflammation in brain astrocytes119. The mechanism of this suppression by 22RHC is via the phosphorylation of
457
Hu antigen R through PKCα, which in turn stabilizes MKP-1 mRNA119. Although the metabotropic glutamate
458
receptor 5 is required for the cytosolic increase in Ca2+, an event upstream of the phosphorylation of Hu, it is
AC C
EP
TE D
443
ACCEPTED MANUSCRIPT 459
not clear whether 22RHC directly binds to this receptor, or whether these activities require interaction with the
460
more traditional oxysterol binding proteins (such as the LXRs or INSIG).
461 24HC is the most abundant oxysterol synthesized in the brain, whereas 27HC is the primary oxysterol
463
produced by peripheral cells, which crosses the BBB114,115,120. While 7KC is associated with a neuro-damaging
464
role, 24HC was shown to have a protective effect during the development of neurodegenerative disorders. For
465
instance, Testa et al. demonstrated that, in SK-N-BE neuroblastoma cells, 24HC could modulate the SIRT1,
466
which then led to the prevention of hyperphosphorylated tau protein accumulation induced by amyloid β (Aβ)
467
monomers121. Okabe et al. also found that sub-lethal concentrations of 24SHC induced adaptive responses
468
and protected the human neuroblastoma SH-SY5Y cells against cytotoxic stress induced by 7KC, dependent
469
of activating LXRβ transcriptional pathway117. However, high concentrations of 24SHC could induce
470
“necroptosis-like” cell death in neuronal cells due to the formation of 24SHC esters120,122. Also, 24HC was
471
shown to increase Aβ accumulation, which in turn contributes to neuronal cell death. Reported by Gamba et al.,
472
24HC promoted not only the binding of Aβ to human neuronal cell lines, SK-N-BE and NT2, but also the pro‐
473
apoptotic and pro‐necrogenic effects of Aβ1–42 peptide on SK-N-BE and NT2, by locally increasing ROS levels
474
through NADPH oxidase123. These works together suggest that 24HC at low concentrations might be protective,
475
but as its levels increase due to dysregulated cholesterol metabolism, this oxysterol could then act to induce
476
cell death.
SC
M AN U
TE D
EP AC C
477
RI PT
462
478
27HC, as the most abundant peripheral oxysterol, can cross the BBB and is therefore also implicated in
479
neuronal cell death. An increased level of 27HC was reported in the brain of patients diagnosed with AD124–126.
480
Also, hypercholesterolemia was linked to accelerated AD pathology and the accumulation of Aβ127,128. One
481
mechanism of cholesterol-linked AD development could be attributed to 27HC accumulation in the brain. Ismail
482
et al. showed that 27HC reduced LXR-mediated brain glucose uptake, GLUT4 expression and spatial memory
483
in mouse model129. The reduced glucose metabolic activity in AD patients has been well-identified in the
484
angular gyrus and some parietotemporal regions130. Moreover, 27HC promoted NFκB binding to the promoter
485
of aspartyl protease BACE1, leading to an increased BACE1 activity and BACE1-mediated cleavage of
ACCEPTED MANUSCRIPT amyloid precursor protein, in a gadd153-dependent manner131. Also, 27HC-induced ROS production could
487
promote IL-6/STAT3 signaling, which was implicated in the induction of nerve cell senescence132. However, it
488
was unclear whether this effect was due to the selective modulatory activity of 27HC on ER. Taken together,
489
limiting dietary cholesterol intake would likely reduce the level of peripheral 27HC, and thus may exhibit clinical
490
value in preventing AD and other related neurodegenerative disorder development29,133. In this regard, there is
491
a positive correlation between plasma cholesterol and 27HC levels in humans, including after dietary
492
consumption of an extra 750mg/day cholesterol for 4 weeks29,133. In mice, a high cholesterol diet results in
493
significantly increased 27HC levels, although dietary restriction experiments have yet to be reported50,54,62.
SC
RI PT
486
494
25HC was reported to be involved in the progression of amyotrophic lateral sclerosis (ALS). Clinical analysis
496
showed that 25HC level was elevated in both cerebral spinal fluid and serum of ALS patients134. Also, the level
497
of 25HC was associated with the level of disease severity in ALS patients134. Mechanistically, 25HC-mediated
498
ALS pathogenesis is likely due to the induction of motor neuronal death, a process dependent of glycogen
499
synthase kinase-3β and LXR, which was shown by Kim et al. in the motor neuron-like cell line (NSC34)
500
expressing mutant G93A superoxide dismutase 1 gene134.
501
TE D
M AN U
495
Atherosclerosis
503
Atherosclerosis is the underlying pathology of many acute cardiovascular syndromes. Atherosclerosis
504
development is characterized by the narrowing of the artery, which results from the accumulation of lipids and
505
inflammatory cells within the intima of arteries. Progression of atherosclerosis is thought to be initiated by
506
endothelial cell dysfunction, leading to the accumulation of low-density lipoprotein-cholesterol (LDL-cholesterol)
507
in the extracellular matrix (ECM). LDL-cholesterol buildup at the extracellular matrix becomes a target of
508
oxidation, and LDLs can be oxidized into oxidized LDL (ox-LDL). Ox-LDL promotes the recruitment of
509
macrophages and the subsequent engulfment of ox-LDL by macrophages transforms macrophages into foam
510
cells6,33,135,136. This transformation is the hallmark of atherosclerotic lesion development137. However, recent
511
reports are questioning whether immune dysregulation may actually precede plaque formation138,139.
512
AC C
EP
502
ACCEPTED MANUSCRIPT Being a byproduct of cholesterol, oxysterols are formed in atherosclerotic plaques both non-enzymatically by
514
LDL oxidation and enzymatically during cholesterol catabolism by cytochrome p450 enzymes that are highly
515
expressed in macrophages33. Due to their prevalence in the atherosclerotic plaques, the role of oxysterols in
516
plaque formation has been heavily investigated. However, the pro-atherogenic properties of oxysterols have
517
been in a long debate largely due to the multifold effects of oxysterols. Macrophages are present in a spectrum
518
of so-called polarities. On the extremes of this polarization spectrum are classically activated, inflammatory M1
519
macrophages, and alternatively activated, anti-inflammatory macrophages. Macrophages across this spectrum
520
are localized within different types of atherosclerotic lesions. Macrophages localized in rupture-prone
521
atherosclerotic plaques more closely reflect the biology of M1 macrophages, while the macrophages that
522
situate in stable plaque more closely resemble the phenotype of M2 macrophages6,93. It has been reported by
523
Marengo et al. that 27HC contributes to the M2 polarization of human macrophages by upregulating the
524
expression of CD36 and CD204 and the production of IL-10, leading to the plaque stabilization93. On the
525
contrary, Gargiulo et al. found that 27HC promoted TLR4 and NFκB activation in U937 cells, which led to the
526
release of pro-inflammatory cytokines, IL-8, IL-1β and TNF-α140. The production of pro-inflammatory cytokines
527
induced by 27HC then contributed to the production of matrix metallopeptidase 9. Although these studies were
528
performed in vitro, the presence of matrix metallopeptidase 9 has the potential to promote plaque instability
529
and rupture140. The pro-inflammatory role of 27HC is likely due to the activation of ERα, as 27HC-induced pro-
530
inflammatory cytokine mRNA expression (IL-1β, IL-6 and TNF-α) was attenuated by ER deficiency60. Ishikawa
531
et al. reported a novel mechanism by which the activation of LXR exerts an atheroprotective role in endothelial
532
cells141. Specifically, GW3965-, T0901317- or 22RHC-induced LXR signaling promotes ER-dependent eNOS
533
activation141. By co-localizing with ERα in the lipid raft and thus functionally coupling with ERα, LXR stimulation
534
could
535
reendothelialization141.
lead
AC C
EP
TE D
M AN U
SC
RI PT
513
to
ERα
Ser118
phosphorylation
by
PI3K/Akt,
eventually
promoting
eNOS-induced
536 537
Structurally similar to 27HC, 25HC was also identified with an atheroprotective role. Li et al. found that Krüppel-
538
like factor 4 (KLF4), a master transcription factor that regulates the anti-inflammatory activity of macrophages,
539
could induce the expression of cholesterol 25 hydroxylase, LXRα and LXRβ in macrophages (RAW264.7)142.
540
Further, the activation of the KLF4-cholesterol 25 hydroxylase-LXR axis promoted M1 to M2 transition in
ACCEPTED MANUSCRIPT 541
macrophages, as both treating macrophages with 25HC and overexpressing KLF4 in macrophages led to a
542
decreased inflammatory activity in macrophages142. Knocking down cholesterol 25 hydroxylase in RAW264.7
543
cells also stimulated the expression of the pro-inflammatory genes and M1 phenotype142.
544 Non-nuclear receptor-mediated mechanisms by which oxysterols contribute to atherosclerosis development
546
have also reported. The β1-Subunit of BKCa channel (KCNMB1) is linked to atheroprotective, in that it
547
modulates vascular contractility143. Interestingly, 7KC has previously been reported to be a competitive inhibitor
548
of the aryl hydrocarbon receptor (AhR)144. In a subsequent report, 7KC was found to contribute to vascular
549
rigidity, and decreases the expression of KCNMB1 protein levels143. The decrease in KCNMB1 was attributed
550
to a decrease in the protein expression of the AhR, although the requirement of the AHR was not specifically
551
tested143. While the AhR is not a member of the nuclear receptor superfamily, it has a very similar mechanism
552
of action in that it is a ligand-activated transcription factor145,146. It will be of interest to evaluate whether other
553
oxysterols can modulate the AhR and to determine the physiological relevance of such engagement.
M AN U
SC
RI PT
545
554 Conclusion
556
Oxysterols play important roles in cholesterol metabolism and the regulation of cholesterol homeostasis.
557
Binding to LXRs, oxysterols promote cholesterol efflux and bile acid synthesis. However, an elevated level of
558
oxysterols has been implicated in the pathophysiology of many age-related diseases, such as breast cancer,
559
atherosclerosis and AD. The main target nuclear receptors mediating the actions of oxysterols are the LXRs,
560
ERs and RORs, although recent reports for the GR and potentially ERRα reveal the possibility for other
561
nuclear receptors being involved in oxysterol biology. The action of oxysterols on each type of receptor is
562
multifaceted. Being a SERM, 27HC can activate or inhibit ER in different cell types. For ROR, oxysterols’
563
actions range from agonist to inverse agonist. It is therefore possible that the multilevel effect of oxysterols on
564
the target receptors leads to diverse phenotypes in cells upon oxysterol stimulation. Also, desmosterol has now
565
been formally demonstrated to be a selective modulator of the LXRs in macrophages147. Specifically,
566
desmosterol activated LXR target gene, ABCA1, while suppressing the SREBP target gene, 24-
567
dehydrocholesterol reductase (DHCR24), and had no effect on fatty acid synthase in mouse and human
568
macrophages147. Indeed, phenotypically, there is also some debate regarding the inflammatory role of
AC C
EP
TE D
555
ACCEPTED MANUSCRIPT oxysterols. While they are generally seen as pro-inflammatory in the innate immune system, an increasing
570
number of studies reported the anti-inflammatory behaviors of oxysterols. The direct effect of oxysterols on T
571
cells are oxysterol- and T cell-type- specific. While 7β, 27DHC promotes Th17 differentiation, 25HC negatively
572
regulates Treg. Though 27HC is recognized as a SERM, other oxysterols’ selective modulatory effects are less
573
clear, although highly likely. Given the evidence that many oxysterols exert variable effects on cells, it is
574
important for future studies to investigate whether other oxysterols are involved in selectively modulating
575
nuclear receptors, such as LXR, in disease conditions.
SC
576
RI PT
569
There is now significant evidence that oxysterols play exacerbating roles across several diseases. Not only can
578
they have direct effects on cancer cells to promote cancer growth, but also oxysterols can modulate pro-tumor
579
host cells, such as myeloid cells, to promote disease progression. Specific mechanisms for many of the
580
described effects of oxysterols in physiology or disease are still unknown. Still, several of the roles of
581
oxysterols are mediated through the LXRs, and thus they have been proposed to be a therapeutic target.
582
However, challenges in targeting this receptor have emerged, as LXRs play an essential role in cholesterol and
583
lipid homeostasis148. While it may be beneficial to target this receptor, its activation leads to
584
hypertriglyceridemia148, which could increase the risk of developing cardiovascular-related diseases or
585
steatosis. Given the potential for LXR ligands to have differential effects depending on the tissue and cellular
586
context, targeting these receptors should proceed with caution. Therefore, it will be of importance for future
587
studies to consider the specific downstream targets upon oxysterol signaling in each of the disease models.
588 589 590 591
References
592
1. Griffiths, W. J. et al. Current trends in oxysterol research. Biochem. Soc. Trans. 44, 652–658 (2016).
593
2. Brzeska, M., Szymczyk, K. & Szterk, A. Current Knowledge about Oxysterols: A Review. J. Food Sci. 81,
594 595 596
AC C
EP
TE D
M AN U
577
R2299–R2308 (2016). 3. Poli, G., Biasi, F. & Leonarduzzi, G. Oxysterols in the pathogenesis of major chronic diseases. Redox Biol. 1, 125–130 (2013).
ACCEPTED MANUSCRIPT 597
4. Vejux, A. & Lizard, G. Cytotoxic effects of oxysterols associated with human diseases: Induction of cell
598
death (apoptosis and/or oncosis), oxidative and inflammatory activities, and phospholipidosis. Mol.
599
Aspects Med. 30, 153–170 (2009).
603 604 605
RI PT
602
218 (2007).
6. He, S. & Nelson, E. R. 27-Hydroxycholesterol, an endogenous selective estrogen receptor modulator. Maturitas 104, 29–35 (2017).
SC
601
5. Norlin, M. & Wikvall, K. Enzymes in the conversion of cholesterol into bile acids. Curr. Mol. Med. 7, 199–
7. Dhiman, V. K., Bolt, M. J. & White, K. P. Nuclear receptors in cancer - uncovering new and evolving roles through genomic analysis. Nat. Rev. Genet. 19, 160–174 (2018).
M AN U
600
8. Sever, R. & Glass, C. K. Signaling by Nuclear Receptors. Cold Spring Harb. Perspect. Biol. 5, (2013).
607
9. Mangelsdorf, D. J. et al. The Nuclear Receptor Superfamily: The Second Decade. Cell 83, 835–839 (1995).
608
10. Huggins, C. Endocrine methods of treatment of cancer of the breast. J. Natl. Cancer Inst. 15, 1–25 (1954).
609
11. Huggins, C. & Hodges, C. V. Studies on prostatic cancer. I. The effect of castration, of estrogen and
TE D
606
610
androgen injection on serum phosphatases in metastatic carcinoma of the prostate. CA. Cancer J. Clin. 22,
611
232–240 (1972).
614
EP
613
12. Maqdasy, S. et al. Once and for all, LXRα and LXRβ are gatekeepers of the endocrine system. Mol. Aspects Med. 49, 31–46 (2016).
AC C
612
13. Auboeuf, D. et al. Tissue distribution and quantification of the expression of mRNAs of peroxisome
615
proliferator-activated receptors and liver X receptor-alpha in humans: no alteration in adipose tissue of
616
obese and NIDDM patients. Diabetes 46, 1319–1327 (1997).
617 618 619 620
14. Heine, G. et al. Liver X receptors control IgE expression in B cells. J. Immunol. Baltim. Md 1950 182, 5276– 5282 (2009). 15. Lee, S. D. & Tontonoz, P. Liver X Receptors at the Intersection of Lipid Metabolism and Atherogenesis. Atherosclerosis 242, 29–36 (2015).
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
RI PT
626
18. Wang, C., Tian, L., Popov, V. M. & Pestell, R. G. Acetylation and Nuclear Receptor Action. J. Steroid Biochem. Mol. Biol. 123, 91–100 (2011).
19. Li, X. et al. SIRT1 deacetylates and positively regulates the nuclear receptor LXR. Mol. Cell 28, 91–106
SC
625
OMEGA 3 FATTY ACIDS. Cell 155, 200–214 (2013).
(2007).
20. Glass, C. K. & Saijo, K. Nuclear receptor transrepression pathways that regulate inflammation in
M AN U
624
17. Li, P. et al. NCoR REPRESSION OF LXRs RESTRICTS MACROPHAGE BIOSYNTHESIS OF INSULIN-SENSITIZING
macrophages and T cells. Nat. Rev. Immunol. 10, 365–376 (2010).
21. Treuter, E. New wrestling rules of anti-inflammatory transrepression by oxysterol receptor LXR revealed. Cell Res. 21, 711–714 (2011).
22. Janowski, B. A. et al. Structural requirements of ligands for the oxysterol liver X receptors LXRα and LXRβ.
TE D
623
Metab. TEM 28, 485–496 (2017).
Proc. Natl. Acad. Sci. U. S. A. 96, 266–271 (1999).
23. Morello, F. et al. LXR-activating oxysterols induce the expression of inflammatory markers in endothelial cells through LXR-independent mechanisms. Atherosclerosis 207, 38–44 (2009).
EP
622
16. Kloudova, A., Guengerich, F. P. & Soucek, P. The role of oxysterols in human cancer. Trends Endocrinol.
24. Lehmann, J. M. et al. Activation of the nuclear receptor LXR by oxysterols defines a new hormone
AC C
621
ACCEPTED MANUSCRIPT
response pathway. J. Biol. Chem. 272, 3137–3140 (1997). 25. Yoshikawa, T. et al. Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter. Mol. Cell. Biol. 21, 2991–3000 (2001). 26. Chen, W., Chen, G., Head, D. L., Mangelsdorf, D. J. & Russell, D. W. Enzymatic Reduction of Oxysterols Impairs LXR Signaling in Cultured Cells and the Livers of Mice. Cell Metab. 5, 73–79 (2007). 27. Fu, X. et al. 27-hydroxycholesterol is an endogenous ligand for liver X receptor in cholesterol-loaded cells. J. Biol. Chem. 276, 38378–38387 (2001).
ACCEPTED MANUSCRIPT
647
648
649
650
651
plasma by high-performance liquid chromatography-mass spectrometry. J. Lipid Res. 45, 776–781 (2004). 29. Karuna, R. et al. Plasma levels of 27-hydroxycholesterol in humans and mice with monogenic disturbances of high density lipoprotein metabolism. Atherosclerosis 214, 448–455 (2011).
RI PT
646
28. Burkard, I., Rentsch, K. M. & von Eckardstein, A. Determination of 24S- and 27-hydroxycholesterol in
30. Thelen, K. M. et al. Effect of pravastatin on plasma sterols and oxysterols in men. Eur. J. Clin. Pharmacol. 62, 9–14 (2006).
31. Hansson, M., Ellis, E., Hunt, M. C., Schmitz, G. & Babiker, A. Marked induction of sterol 27-hydroxylase
SC
645
activity and mRNA levels during differentiation of human cultured monocytes into macrophages. Biochim.
653
Biophys. Acta 1593, 283–289 (2003).
654
M AN U
652
32. Babiker, A. et al. Elimination of cholesterol in macrophages and endothelial cells by the sterol 27-
655
hydroxylase mechanism. Comparison with high density lipoprotein-mediated reverse cholesterol transport.
656
J. Biol. Chem. 272, 26253–26261 (1997).
33. Brown, A. J. & Jessup, W. Oxysterols and atherosclerosis. Atherosclerosis 142, 1–28 (1999).
658
34. Bełtowski, J. Liver X receptors (LXR) as therapeutic targets in dyslipidemia. Cardiovasc. Ther. 26, 297–316
659
TE D
657
(2008).
35. Baldán, Á., Bojanic, D. D. & Edwards, P. A. The ABCs of sterol transport. J. Lipid Res. 50, S80–S85 (2009).
661
36. Rong, S. et al. Expression of SREBP-1c Requires SREBP-2-mediated Generation of a Sterol Ligand for LXR in
663 664 665 666
AC C
662
EP
660
Livers of Mice. eLife 6, (2017).
37. Calkin, A. & Tontonoz, P. LXR signaling pathways and atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 30, (2010).
38. Baek, A. E. & Nelson, E. R. The Contribution of Cholesterol and Its Metabolites to the Pathophysiology of Breast Cancer. Horm. Cancer 7, 219–228 (2016).
667
ACCEPTED MANUSCRIPT 39. Wang, Y., Kumar, N., Crumbley, C., Griffin, P. R. & Burris, T. P. A Second Class of Nuclear Receptors for
668
Oxysterols: Regulation of RORα and RORγ activity by 24S-Hydroxycholesterol (Cerebrosterol). Biochim.
669
Biophys. Acta 1801, 917–923 (2010).
673
674
675
676
RI PT
672
lipid/glucose metabolism, inflammation, and insulin sensitivity. Front. Endocrinol. 4, (2013).
41. Mutemberezi, V., Guillemot-Legris, O. & Muccioli, G. G. Oxysterols: From cholesterol metabolites to key mediators. Prog. Lipid Res. 64, 152–169 (2016).
SC
671
40. Jetten, A. M., Kang, H. S. & Takeda, Y. Retinoic acid-related orphan receptors α and γ: key regulators of
42. Santori, F. R. et al. Identification of Natural RORγ Ligands that Regulate the Development of Lymphoid Cells. Cell Metab. 21, 286–297 (2015).
M AN U
670
43. Kallen, J. A. et al. X-ray structure of the hRORalpha LBD at 1.63 A: structural and functional data that
677
cholesterol or a cholesterol derivative is the natural ligand of RORalpha. Struct. Lond. Engl. 1993 10, 1697–
678
1707 (2002).
44. Kallen, J., Schlaeppi, J.-M., Bitsch, F., Delhon, I. & Fournier, B. Crystal structure of the human RORalpha
TE D
679
680
Ligand binding domain in complex with cholesterol sulfate at 2.2 A. J. Biol. Chem. 279, 14033–14038
681
(2004).
45. Wada, T., Kang, H. S., Jetten, A. M. & Xie, W. The Emerging Role Of Nuclear Receptor RORα And Its
EP
682
Crosstalk With LXR In Xeno- And Endobiotic Gene Regulation. Exp. Biol. Med. Maywood NJ 233, 1191–1201
684
(2008).
685
686
AC C
683
46. Soroosh, P. et al. Oxysterols are agonist ligands of RORγt and drive Th17 cell differentiation. Proc. Natl. Acad. Sci. U. S. A. 111, 12163–12168 (2014).
687
47. Bird, L. T cells: Endogenous agonists for orphan RORγ. Nat. Rev. Immunol. 15, 70–71 (2015).
688
48. Castro, G. et al. RORγt and RORα signature genes in human Th17 cells. PLoS ONE 12, (2017).
689
49. DuSell, C. D., Umetani, M., Shaul, P. W., Mangelsdorf, D. J. & McDonnell, D. P. 27-hydroxycholesterol is an
690
endogenous selective estrogen receptor modulator. Mol. Endocrinol. Baltim. Md 22, 65–77 (2008).
ACCEPTED MANUSCRIPT
691
692
50. Umetani, M. et al. 27-Hydroxycholesterol is an endogenous SERM that inhibits the cardiovascular effects of estrogen. Nat. Med. 13, 1185–1192 (2007). 51. Imai, Y. et al. Nuclear Receptors in Bone Physiology and Diseases. Physiol. Rev. 93, 481–523 (2013).
694
52. Wardell, S. E., Nelson, E. R. & McDonnell, D. P. From empirical to mechanism-based discovery of clinically
700
701 702 703 704 705 706 707 708 709
SC
699
54. Nelson, E. R. et al. 27-Hydroxycholesterol Links Hypercholesterolemia and Breast Cancer Pathophysiology. Science 342, 1094–1098 (2013).
M AN U
698
transcriptional activation assay system using HeLa cells. Biosci. Biotechnol. Biochem. 68, 1790–1793 (2004).
55. Wu, Q. et al. 27-Hydroxycholesterol promotes cell-autonomous, ER-positive breast cancer growth. Cell Rep. 5, 637–645 (2013).
56. Wang, C.-W. et al. 7-ketocholesterol and 27-hydroxycholesterol decreased doxorubicin sensitivity in breast cancer cells: estrogenic activity and mTOR pathway. Oncotarget 8, 66033–66050 (2017).
TE D
697
53. Sato, H. et al. Oxysterol regulation of estrogen receptor alpha-mediated gene expression in a
57. Lappano, R. et al. The Cholesterol Metabolite 25-Hydroxycholesterol Activates Estrogen Receptor αMediated Signaling in Cancer Cells and in Cardiomyocytes. PLoS ONE 6, (2011). 58. Oguro, H. et al. 27-Hydroxycholesterol induces hematopoietic stem cell mobilization and extramedullary
EP
696
useful Selective Estrogen Receptor Modulators (SERMs). Steroids 90, 30–38 (2014).
hematopoiesis during pregnancy. J. Clin. Invest. 127, 3392–3401
AC C
695
RI PT
693
59. Simigdala, N. et al. Cholesterol biosynthesis pathway as a novel mechanism of resistance to estrogen deprivation in estrogen receptor-positive breast cancer. Breast Cancer Res. BCR 18, (2016).
710
60. Umetani, M. et al. The Cholesterol Metabolite 27-Hydroxycholesterol Promotes Atherosclerosis via
711
Proinflammatory Processes Mediated by Estrogen Receptor Alpha. Cell Metab. 20, 172–182 (2014).
712
61. DuSell, C. D. et al. The Endogenous Selective Estrogen Receptor Modulator 27-Hydroxycholesterol Is a
713
Negative Regulator of Bone Homeostasis. Endocrinology 151, 3675–3685 (2010).
ACCEPTED MANUSCRIPT
714
62. Nelson, E. R. et al. The Oxysterol, 27-Hydroxycholesterol, Links Cholesterol Metabolism to Bone
715
Homeostasis Through Its Actions on the Estrogen and Liver X Receptors. Endocrinology 152, 4691–4705
716
(2011). 63. Chang, P.-Y. et al. 27-hydroxycholesterol, an endogenous SERM, and risk of fracture in postmenopausal
RI PT
717
718
women: A nested case-cohort study in the Women’s Health Initiative. J. Bone Miner. Res. Off. J. Am. Soc.
719
Bone Miner. Res. (2018). doi:10.1002/jbmr.3576
64. Brooks, S. W., Dykes, A. C. & Schreurs, B. G. A High-Cholesterol Diet Increases 27-Hydroxycholesterol and
SC
720
Modifies Estrogen Receptor Expression and Neurodegeneration in Rabbit Hippocampus. J. Alzheimers Dis.
722
JAD 56, 185–196 (2017).
724
725
726
65. Raza, S. et al. The cholesterol metabolite 27-hydroxycholesterol stimulates cell proliferation via ERβ in prostate cancer cells. Cancer Cell Int. 17, (2017).
66. Voisin, M. et al. Identification of a tumor-promoter cholesterol metabolite in human breast cancers acting through the glucocorticoid receptor. Proc. Natl. Acad. Sci. U. S. A. 114, E9346–E9355 (2017).
TE D
723
M AN U
721
727
67. Weikum, E. R., Knuesel, M. T., Ortlund, E. A. & Yamamoto, K. R. Glucocorticoid receptor control of
728
transcription: precision and plasticity via allostery. Nat. Rev. Mol. Cell Biol. 18, 159–174 (2017).
731
732
733
734
735
736
EP
730
68. Ratman, D. et al. How glucocorticoid receptors modulate the activity of other transcription factors: a scope beyond tethering. Mol. Cell. Endocrinol. 380, 41–54 (2013).
AC C
729
69. Silvente-Poirot, S., Dalenc, F. & Poirot, M. The Effects of Cholesterol-Derived Oncometabolites on Nuclear Receptor Function in Cancer. Cancer Res. 78, 4803–4808 (2018). 70. Wei, W. et al. Ligand Activation of ERRα by Cholesterol Mediates Statin and Bisphosphonate Effects. Cell Metab. 23, 479–491 (2016). 71. Ascenzi, P., Bocedi, A. & Marino, M. Structure-function relationship of estrogen receptor alpha and beta: impact on human health. Mol. Aspects Med. 27, 299–402 (2006).
ACCEPTED MANUSCRIPT
737
72. Chang, C. & McDonnell, D. P. Molecular Pathways: The Metabolic Regulator Estrogen-Related Receptor
738
Alpha as a Therapeutic Target in Cancer. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 18, 6089–6095
739
(2012).
741
73. Sensi, C. et al. Oxysterols act as promiscuous ligands of class-A GPCRs: in silico molecular modeling and in vitro validation. Cell. Signal. 26, 2614–2620 (2014).
RI PT
740
74. Hannedouche, S. et al. Oxysterols direct immune cell migration through EBI2. Nature 475, 524–527 (2011).
743
75. Nedelcu, D., Liu, J., Xu, Y., Jao, C. & Salic, A. Oxysterol binding to the extracellular domain of Smoothened
745
in Hedgehog signaling. Nat. Chem. Biol. 9, 557–564 (2013).
76. Radhakrishnan, A., Ikeda, Y., Kwon, H. J., Brown, M. S. & Goldstein, J. L. Sterol-regulated transport of
M AN U
744
SC
742
746
SREBPs from endoplasmic reticulum to Golgi: Oxysterols block transport by binding to Insig. Proc. Natl.
747
Acad. Sci. U. S. A. 104, 6511–6518 (2007).
748
77. Sun, L.-P., Seemann, J., Goldstein, J. L. & Brown, M. S. Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins. Proc.
750
Natl. Acad. Sci. U. S. A. 104, 6519–6526 (2007).
753
754
755
756
757
mechanisms, both involving SCAP and Insigs. J. Biol. Chem. 279, 52772–52780 (2004).
EP
752
78. Adams, C. M. et al. Cholesterol and 25-hydroxycholesterol inhibit activation of SREBPs by different
79. Matsuda, M. et al. SREBP cleavage-activating protein (SCAP) is required for increased lipid synthesis in liver
AC C
751
TE D
749
induced by cholesterol deprivation and insulin elevation. Genes Dev. 15, 1206–1216 (2001). 80. Im, S.-S. et al. Linking Lipid Metabolism to the Innate Immune Response in Macrophages through Sterol Regulatory Element Binding Protein -1a. Cell Metab. 13, 540–549 (2011). 81. Erridge, C., Webb, D. J. & Spickett, C. M. 25-Hydroxycholesterol, 7beta-hydroxycholesterol and 7-
758
ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6
759
signalling in human macrophages. Free Radic. Res. 41, 260–266 (2007).
ACCEPTED MANUSCRIPT
764
765
766 767 768 769 770 771 772
(2013).
RI PT
763
83. Spann, N. J. & Glass, C. K. Sterols and oxysterols in immune cell function. Nat. Immunol. 14, 893–900
84. Ito, A. et al. LXRs link metabolism to inflammation through Abca1-dependent regulation of membrane composition and TLR signaling. eLife 4,
85. Reboldi, A. et al. 25-hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type
SC
762
adrenoleukodystrophy through activation of the NLRP3 inflammasome. Nat. Commun. 7, 13129 (2016).
I interferon. Science 345, 679–684 (2014).
86. Cagno, V. et al. Inhibition of herpes simplex-1 virus replication by 25-hydroxycholesterol and 27-
M AN U
761
82. Jang, J. et al. 25-hydroxycholesterol contributes to cerebral inflammation of X-linked
hydroxycholesterol. Redox Biol. 12, 522–527 (2017).
87. Joseph, S. B. et al. LXR-Dependent Gene Expression Is Important for Macrophage Survival and the Innate Immune Response. Cell 119, 299–309 (2004).
88. Endo-Umeda, K., Nakashima, H., Umeda, N., Seki, S. & Makishima, M. Dysregulation of Kupffer Cells/
TE D
760
773
Macrophages and Natural Killer T Cells in Steatohepatitis in LXRα Knockout Male Mice. Endocrinology
774
(2018). doi:10.1210/en.2017-03141
777
778 779
780 781 782
EP
776
89. Tuong, Z. K. et al. RORα and 25-Hydroxycholesterol Crosstalk Regulates Lipid Droplet Homeostasis in Macrophages. PLoS ONE 11, (2016).
AC C
775
90. Svoronos, N. et al. Tumor Cell–Independent Estrogen Signaling Drives Disease Progression through Mobilization of Myeloid-Derived Suppressor Cells. Cancer Discov. 7, 72–85 (2017). 91. Baek, A. E. et al. The cholesterol metabolite 27 hydroxycholesterol facilitates breast cancer metastasis through its actions on immune cells. Nat. Commun. 8, 864 (2017). 92. Mutemberezi, V. et al. Oxysterol levels and metabolism in the course of neuroinflammation: insights from in vitro and in vivo models. J. Neuroinflammation 15, 74 (2018).
ACCEPTED MANUSCRIPT
783 784 785
93. Marengo, B. et al. Oxysterol mixture and, in particular, 27-hydroxycholesterol drive M2 polarization of human macrophages. BioFactors Oxf. Engl. 42, 80–92 (2016). 94. Guillem-Llobat, P. & Íñiguez, M. A. Inhibition of lipopolysaccharide-induced gene expression by liver X receptor ligands in macrophages involves interference with early growth response factor 1. Prostaglandins
787
Leukot. Essent. Fatty Acids 96, 37–49 (2015).
RI PT
786
95. Ward, L. J., Ljunggren, S. A., Karlsson, H., Li, W. & Yuan, X.-M. Exposure to atheroma-relevant 7-oxysterols
789
causes proteomic alterations in cell death, cellular longevity, and lipid metabolism in THP-1 macrophages.
790
PLOS ONE 12, e0174475 (2017).
792 793
96. Lee, S. et al. Adenylyl Cyclase-Associated Protein 1(CAP1) is a Receptor for Human Resistin and Mediates
M AN U
791
SC
788
Inflammatory Actions of Human Monocytes. Cell Metab. 19, 484–497 (2014). 97. Kim, K. M., Kim, Y. S., Jung, D. H., Lee, J. & Kim, J. S. Increased glyoxalase I levels inhibit accumulation of oxidative stress and an advanced glycation end product in mouse mesangial cells cultured in high glucose.
795
Exp. Cell Res. 318, 152–159 (2012).
798 799 800 801 802 803 804 805
C5a receptor. PLOS ONE 12, e0173749 (2017).
99. Zhang, R., Liu, Z., Li, Y. & Wu, B. LXR agonist regulates the proliferation and apoptosis of human T-Cell
EP
797
98. Cho, H. et al. 7α-Hydroxycholesterol induces monocyte/macrophage cell expression of interleukin-8 via
acute lymphoblastic leukemia cells via the SOCS3 pathway. Int. J. Biochem. Cell Biol. 78, 180–185 (2016). 100.
AC C
796
TE D
794
Bensinger, S. J. et al. LXR signaling couples sterol metabolism to proliferation in the acquired immune
response. Cell 134, 97–111 (2008). 101.
Ma, X. et al. Cholesterol negatively regulates IL-9–producing CD8+ T cell differentiation and antitumor
activity. J. Exp. Med. 215, 1555–1569 (2018). 102.
Vigne, S. et al. IL-27-Induced Type 1 Regulatory T-Cells Produce Oxysterols that Constrain IL-10
Production. Front. Immunol. 8, 1184 (2017).
812
813
814
815
816
817
818
819
820
821
822
823
824
825
826
827
828
829
phenotype. Oncol. Rep. 26, 389–397 (2011).
RI PT
811
Torres, C. G. et al. 27-hydroxycholesterol induces the transition of MCF7 cells into a mesenchymal
105. Alfaqih, M. A. et al. CYP27A1 Loss Dysregulates Cholesterol Homeostasis in Prostate Cancer. Cancer Res. 77, 1662–1673 (2017). 106.
Hiramitsu, S. et al. Estrogen Receptor Beta-Mediated Modulation of Lung Cancer Cell Proliferation by
27-Hydroxycholesterol. Front. Endocrinol. 9, (2018). 107.
SC
810
104.
Chen, L. et al. 25-Hydroxycholesterol promotes migration and invasion of lung adenocarcinoma cells.
M AN U
809
e0184985 (2017).
Biochem. Biophys. Res. Commun. 484, 857–863 (2017). 108.
Lin, C.-Y. et al. Cholestane-3β, 5α, 6β-triol Suppresses Proliferation, Migration, and Invasion of Human
Prostate Cancer Cells. PLOS ONE 8, e65734 (2013). 109.
de Medina, P. et al. Dendrogenin A arises from cholesterol and histamine metabolism and shows cell
TE D
808
Herold, M. et al. Liver X receptor activation promotes differentiation of regulatory T cells. PloS One 12,
differentiation and anti-tumour properties. Nat. Commun. 4, 1840 (2013). 110.
Poirot, M. & Silvente-Poirot, S. The tumor-suppressor cholesterol metabolite, dendrogenin A, is a new
class of LXR modulator activating lethal autophagy in cancers. Biochem. Pharmacol. 153, 75–81 (2018). 111.
EP
807
103.
Moresco, M. A. et al. Enzymatic Inactivation of Oxysterols in Breast Tumor Cells Constraints Metastasis
AC C
806
ACCEPTED MANUSCRIPT
Formation by Reprogramming the Metastatic Lung Microenvironment. Front. Immunol. 9, (2018). 112.
Raccosta, L. et al. The oxysterol–CXCR2 axis plays a key role in the recruitment of tumor-promoting
neutrophils. J. Exp. Med. 210, 1711–1728 (2013). 113.
Villablanca, E. J. et al. Tumor-mediated liver X receptor-alpha activation inhibits CC chemokine
receptor-7 expression on dendritic cells and dampens antitumor responses. Nat. Med. 16, 98–105 (2010). 114.
Courtney, R. & Landreth, G. E. LXR Regulation of Brain Cholesterol: From Development to Disease.
Trends Endocrinol. Metab. 27, 404–414 (2016).
ACCEPTED MANUSCRIPT 830 831 832
115.
Heverin, M. et al. Crossing the barrier: net flux of 27-hydroxycholesterol into the human brain. J. Lipid
Res. 46, 1047–1052 (2005). 116.
Björkhem, I., Leoni, V. & Svenningsson, P. On the fluxes of side-chain oxidized oxysterols across blood-
brain and blood-CSF barriers and origin of these steroids in CSF (Review). J. Steroid Biochem. Mol. Biol.
834
(2018). doi:10.1016/j.jsbmb.2018.12.009
837 838
839
Okabe, A. et al. Adaptive responses induced by 24S-hydroxycholesterol through liver X receptor
pathway reduce 7-ketocholesterol-caused neuronal cell death. Redox Biol. 2, 28–35 (2013). 118.
Liu, Y. et al. 25-Hydroxycholesterol activates the expression of cholesterol 25-hydroxylase in an LXR-
dependent mechanism. J. Lipid Res. 59, 439–451 (2018). 119.
SC
836
117.
M AN U
835
RI PT
833
Kim, H., Woo, J. H., Lee, J. H., Joe, E. & Jou, I. 22(R)-hydroxycholesterol induces HuR-dependent MAP
840
kinase phosphatase-1 expression via mGluR5-mediated Ca2+/PKCα signaling. Biochim. Biophys. Acta BBA -
841
Gene Regul. Mech. 1859, 1056–1070 (2016).
845 846
TE D
844
Noguchi, N., Urano, Y., Takabe, W. & Saito, Y. New aspects of 24(S)-hydroxycholesterol in modulating
neuronal cell death. Free Radic. Biol. Med. 87, 366–372 (2015). 121.
Testa, G. et al. A silver lining for 24-hydroxycholesterol in Alzheimer’s disease: The involvement of the
neuroprotective enzyme sirtuin 1. Redox Biol. 17, 423–431 (2018). 122.
EP
843
120.
Yamanaka, K., Saito, Y., Yamamori, T., Urano, Y. & Noguchi, N. 24(S)-Hydroxycholesterol Induces
AC C
842
847
Neuronal Cell Death through Necroptosis, a Form of Programmed Necrosis. J. Biol. Chem. 286, 24666–
848
24673 (2011).
849 850 851 852
123.
Gamba, P. et al. Interaction between 24-hydroxycholesterol, oxidative stress, and amyloid-β in
amplifying neuronal damage in Alzheimer’s disease: three partners in crime. Aging Cell 10, 403–417 (2011). 124.
Testa, G. et al. Changes in brain oxysterols at different stages of Alzheimer’s disease: Their involvement
in neuroinflammation. Redox Biol. 10, 24–33 (2016).
857
858
859
860
861
862
863
864
865
126.
Shafaati, M. et al. Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer’s patients
with the Swedish APP 670/671 mutation. J. Lipid Res. 52, 1004–1010 (2011). 127.
Refolo, L. M. et al. Hypercholesterolemia accelerates the Alzheimer’s amyloid pathology in a transgenic
mouse model. Neurobiol. Dis. 7, 321–331 (2000). 128.
Park, S. H. et al. Hypercholesterolemia accelerates amyloid β-induced cognitive deficits. Int. J. Mol.
Med. 31, 577–582 (2013). 129.
RI PT
856
Parkinson’s disease overlap? Exp. Gerontol. 68, 13–18 (2015).
SC
855
Marwarha, G. & Ghribi, O. Does the oxysterol 27-hydroxycholesterol underlie Alzheimer’s disease-
Ismail, M.-A.-M. et al. 27-Hydroxycholesterol impairs neuronal glucose uptake through an IRAP/GLUT4
M AN U
854
125.
system dysregulation. J. Exp. Med. 214, 699–717 (2017).
130. Teune, L. K. et al. Typical cerebral metabolic patterns in neurodegenerative brain diseases. Mov. Disord. 25, 2395–2404 (2010). 131.
Marwarha, G., Raza, S., Prasanthi, J. R. P. & Ghribi, O. Gadd153 and NF-κB Crosstalk Regulates 27-
TE D
853
ACCEPTED MANUSCRIPT
866
Hydroxycholesterol-Induced Increase in BACE1 and β-Amyloid Production in Human Neuroblastoma SH-
867
SY5Y Cells. PLOS ONE 8, e70773 (2013). 132.
Liu, J. et al. The ROS-mediated activation of IL-6/STAT3 signaling pathway is involved in the 27-
EP
868
hydroxycholesterol-induced cellular senescence in nerve cells. Toxicol. Vitro Int. J. Publ. Assoc. BIBRA 45,
870
10–18 (2017).
871
872
873
874
875
876
133.
AC C
869
Hirayama, T. et al. Serum concentration of 27-hydroxycholesterol predicts the effects of high-
cholesterol diet on plasma LDL cholesterol level. Hepatol. Res. Off. J. Jpn. Soc. Hepatol. 39, 149–156 (2009). 134.
Kim, S.-M. et al. 25-Hydroxycholesterol is involved in the pathogenesis of amyotrophic lateral sclerosis.
Oncotarget 8, 11855–11867 (2017). 135. Torres, N., Guevara-Cruz, M., Velázquez-Villegas, L. A. & Tovar, A. R. Nutrition and Atherosclerosis. Arch. Med. Res. 46, 408–426 (2015).
ACCEPTED MANUSCRIPT
883
884
885
886 887
888
889
890
891
892
893
894
895
896
897
898
atherosclerotic properties of oxysterols. Lipids Health Dis. 16, (2017). 138.
RI PT
882
Zmysłowski, A. & Szterk, A. Current knowledge on the mechanism of atherosclerosis and pro-
Sima, P., Vannucci, L. & Vetvicka, V. Atherosclerosis as autoimmune disease. Ann. Transl. Med. 6,
(2018). 139.
Libby, P. History of Discovery: Inflammation in Atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 32,
SC
881
137.
2045–2051 (2012). 140.
Gargiulo, S. et al. Relation between TLR4/NF-κB signaling pathway activation by 27-hydroxycholesterol
M AN U
880
2 (2017).
and 4-hydroxynonenal, and atherosclerotic plaque instability. Aging Cell 14, 569–581 (2015). 141.
Ishikawa, T. et al. LXRβ/estrogen receptor-α signaling in lipid rafts preserves endothelial integrity. J.
Clin. Invest. 123, 3488–3497 (2013). 142.
Li, Z. et al. Krüppel-Like Factor 4 Regulation of Cholesterol-25-Hydroxylase and Liver X Receptor
TE D
879
Foks, A. C. & Bot, I. Preface: Pathology and Pharmacology of Atherosclerosis. Eur. J. Pharmacol. 816, 1–
Mitigates Atherosclerosis Susceptibility. Circulation 136, 1315–1330 (2017). 143.
Son, Y. et al. 7-Ketocholesterol induces the reduction of KCNMB1 in atherosclerotic blood vessels.
Biochem. Biophys. Res. Commun. 457, 324–327 (2015). 144.
EP
878
136.
Savouret, J. F. et al. 7-ketocholesterol is an endogenous modulator for the arylhydrocarbon receptor. J.
AC C
877
Biol. Chem. 276, 3054–3059 (2001). 145.
Larigot, L., Juricek, L., Dairou, J. & Coumoul, X. AhR signaling pathways and regulatory functions.
Biochim. Open 7, 1–9 (2018). 146.
Beischlag, T. V., Morales, J. L., Hollingshead, B. D. & Perdew, G. H. The Aryl Hydrocarbon Receptor
Complex and the Control of Gene Expression. Crit. Rev. Eukaryot. Gene Expr. 18, 207–250 (2008).
ACCEPTED MANUSCRIPT
899
147.
Muse, E. D. et al. Cell-specific discrimination of desmosterol and desmosterol mimetics confers
900
selective regulation of LXR and SREBP in macrophages. Proc. Natl. Acad. Sci. U. S. A. 115, E4680–E4689
901
(2018).
903
148.
Fessler, M. B. The challenges and promise of targeting the Liver X Receptors for treatment of
inflammatory disease. Pharmacol. Ther. 181, 1–12 (2018).
904
AC C
EP
TE D
M AN U
SC
905
RI PT
902
Figure Legends:
EP
TE D
M AN U
SC
RI PT
Figure 1: Chemical structures and metabolic pathways for several common oxysterols. ROS: reactive oxygen species. DHCR24: 24-dehydrocholesterol reductase. CYP46A1: cytochrome P450 46A1. CYP27A1: cytochrome P450 27A1. CYP11A1: cytochrome P450 11A1. 7βHC: 7β-hydroxycholesterol. 7KC: 7ketocholesterol. 24HC: 24-hydroxycholesterol. 27HC:27-hydroxycholesterol. 25HC: 25-hydroxycholesterol 22HC: 22-hydroxycholesterol.
AC C
906 907 908 909 910 911 912
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT
H
RI PT
H
H
H
H
SC M AN U
HO
DHCR24
H
R OS
OH
EP
TE D
7βHC
H H
H
HO
H
AC C
H
H H
H OH
HO
H
H
O H
7KC HO
H
Cholesterol
Cho l
CYP11A1
S O R
H
H
Desmoste
HO
H
H
H
22HC
ACCEPTED MANUSCRIPT Highlights:
EP
TE D
M AN U
SC
RI PT
Oxysterols play important roles in cholesterol regulation and homeostasis Certain oxysterols serve as ligands for nuclear receptors There is increasing interest in the roles of oxysterols in different diseases Review of oxysterols in disease, with emphasis on the roles of nuclear receptors
AC C
• • • •