StAR, a bridge from ApoE, LDL, and HDL cholesterol trafficking to mitochondrial metabolism

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StAR, a bridge from ApoE, LDL, and HDL cholesterol trafficking to mitochondrial metabolism Colin R. Jefcoate1,2 and Michele Campaigne Larsen1 Abstract

Cholesterol trafficking from serum lipoproteins converges on steroidogenic acute regulator (StAR/STARD1) at the outer mitochondrial membrane (OMM). 195S-Phospho-StAR directs cholesterol released by lipid droplets or endosomes to the inner mitochondrial membrane (IMM) for metabolism at CYP11A1. A helical N-terminal domain slows StAR import, providing time to channel OMM cholesterol to the IMM. Import requires OMM StAR renewal through translation. StAR transcription is activated by CRTC2 binding to cAMP response element-binding protein (CREB), initiated by protein kinase A phosphorylation of salt-inducible kinase forms. mRNA formation is restrained by slow splicing, as established by singlemolecule fluorescence in situ hybridization (smFISH) imaging of primary and spliced RNA at StAR gene loci. Resolved 3.5kb mRNA molecules slowly exit loci, associating singly with individual perinuclear mitochondria. 30 UTR sequestration by AKAP1 and TIS11b effects the OMM location and enhanced mRNA turnover. Mitochondrial fusion, stimulated by cAMP and MFN2, enhances StAR activity, while initiating slower import at mitochondrial-associated endoplasmic reticulum membrane (MAM) contacts, directed by VDAC2 and translocator protein (18 kDa) (TSPO). IMM StAR additionally prevents cholesterolinduced respiratory stress. Addresses 1 Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI, 53705, USA 2 Endocrinology and Reproductive Physiology Program, University of Wisconsin, Madison, WI, 53705, USA Corresponding author: Jefcoate, Colin R. ([email protected])

Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205 This review comes from a themed issue on Adrenal Cortex Edited by André Lacroix and Enzo Lalli For a complete overview see the Issue and the Editorial Available online 9 August 2019 https://doi.org/10.1016/j.coemr.2019.07.011 2451-9650/© 2019 Published by Elsevier Ltd.

Keywords STARD proteins, Cholesterol trafficking, CYP11A, SIK/CRTC2, Steroidogenesis, Mitochondria.

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Abbreviations StAR (STARD1), steroidogenic acute regulator; ER, endoplasmic reticulum; CE, cholesterol ester; CYP11A1 (P450-scc), cytochrome P450 11A1; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane; NTD, N-terminal helical domain; CTD, C-terminal domain; PKA, protein kinase A; SIK, salt-inducible kinase.

Introduction: steroidogenic acute regulator as a participant in cholesterol trafficking The steroidogenesis acute regulator (StAR [STARD1]) plays a unique role in steroid-producing cells by both directing cholesterol entry into the mitochondria for metabolism and playing a key role in cholesterol trafficking. The latter includes cholesterol uptake from lowdensity lipoprotein (LDL) and high-density lipoprotein (HDL), synthesis in the endoplasmic reticulum (ER), and storage in lipid droplets (LDs) as cholesterol esters (CEs) (Figure 1). StAR-mediated metabolism is balanced against cholesterol export back to HDL, mediated by the ATP-dependent pumps, ABCA1 and ABCG1 [1]. The mitochondrial fusionefission cycle overlaps and impacts steps that we will describe for StAR [2]. We will focus on StAR in steroidogenic cells and the complementary activities of STARD3 and STARD4 [3,4]. Questions are raised about how StAR activity extends beyond cholesterol transport and functions in nonsteroidogenic cell types.

Translation-coupled StAR mediates cholesterol transfer to mitochondrial CYP11A1 In the 1960s, Ferguson and Garren [75,76] discovered a novel coupling between cholesterol metabolism and protein translation. Simpson and Boyd [77] recognized that a form of cytochrome P450 located in the inner mitochondrial membrane (IMM) of adrenal mitochondria metabolized cholesterol (now CYP11A1). Protein translation targeted a pool of ‘reactive cholesterol’ in IMMs, which equilibrated with spectrally identified CYP11A1echolesterol complexes [5]. In primary bovine adrenal cells, cholesterol rapidly accumulated in mitochondria after the addition of inhibitors of translation but remained inactive [6]. A CYP11A1 inhibitor (aminoglutethimide) produced large adrenocorticotropic hormone (ACTH)-induced mitochondrial cholesterol accumulations that peaked in 30 min. Critically, restraints on cholesterol were bypassed Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205

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Figure 1

Role of StAR in cholesterol cell trafficking and steroid synthesis. In mice, cells receive cholesterol (CH) from both LDL and HDL but export primarily to HDL. Three processes deliver CH to StAR; a fourth process removes CH. Process 1, ApoE/LDL receptor (LDLR): (a) CH is taken up from the ApoE component of LDL at LDLRs and is delivered to the endosome network with recycling of ApoE. (b) CH and fatty acid esters (CE) are transferred to late endosomes, where CE is converted to CH by acid CE esterases. CH is transferred within endosomes by the NPC2 transporter to STARD3 and subsequently to the ER through intermembrane contacts. CH enters mitochondria from MAM complexes. StAR enhances this transfer but is not essential. Inhibition of steroidogenesis by NPC inhibitors (U18666) implicates direct transfer to the OMM via StAR. NPC1 does not transfer CH to mitochondria in absence of StAR. Process 2, HDL/SRB1: CH enters cells from HDL via SRB1, with transfers through microfilaments to lipid droplet structures, where ACAT sustains CE. cAMP activation of hormone-sensitive lipase (HSL) directly transfers CH to StAR. Process 3, synthesis: CH in the ER is additionally sustained by synthesis from acetyl CoA, which, however, is blocked when SCAP is bound by CH through retention of the mediator, SREBP2. CH also transfers at MAM contact sites. Process 4, export to HDL: CH released from lipid droplets is transferred from cells by STARD4 to transmembrane transporters ABCA1 and ABCG1 to ApoA1 and HDL. StAR transfers CH from the OMM to IMM where metabolism to pregnenolone occurs primarily at CYP11A1 but in small part to 27-hydroxy-CH at CYP27A1. HO–CH derivatives activate LXR receptors that drive expression of ABCA1, ABCG1, SREBP, ApoE and, to lesser extent, StAR. CE, cholesterol ester; ER, endoplasmic reticulum; StAR, steroidogenesis acute regulator; HSL, hormone-sensitive lipase; OMM, outer mitochondrial membrane; NPC1, Niemann-Pick C1; LDL, low-density lipoprotein; HDL, high-density lipoprotein; MAM, mitochondrialassociated endoplasmic reticulum membrane; SCAP, SREBP-cleavage activating protein.

by hydroxyl cholesterol metabolites [5]. Cycloheximide caused cholesterol to accumulate in the outer mitochondrial membrane (OMM), without access to CYP11A1 in the IMM. Actin microfilaments also controlled access of cholesterol to CYP11A1, thus establishing the importance of cholesterol trafficking to the mitochondria [7]. cAMPstimulated proteins that matched these characteristics, with sizes from 37 kDa to 28 kDa, were identified by Pon et al [8] in rat adrenal cells. Primary bovine adrenal cells produced equivalent protein sets when stimulated by ACTH in fasciculata cells and by angiotensin or Kþ ions [9], mediated by Ca2þ-dependent kinases in glomerulosa cells. The origin of these protein sets was identified by cloning of the StAR gene in the study by Clark et al [10]. Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205

StAR identification has allowed retrospective mechanistic characterization of the earlier work with stressed rats and cultured primary adrenal cells. These acute StAR physiological processes include the finding that IMM cholesterol accumulates when adrenal blood flow, which is substantial, is restricted [11]. This cholesterol pool is normally removed completely by CYP11A1 metabolism. Such physiological findings raise questions about whether the large accumulation of IMM StAR has a protective function and, therefore, whether N-terminal domain (NTD) cleavage is more than a termination process. Recent work has made advances using the convenient mouse MA10 Leydig and human adrenal H295 tumor cell models. However, both lack the acute www.sciencedirect.com

StAR directs cholesterol trafficking to mitochondrial metabolism Jefcoate and Larsen

translation-sensitive hormonal response found in early passage Y-1 adrenal tumor cells, which parallel the early studies with primary cells.

Structural features of StAR participation in cholesterol movement The single crystal structure reveals StAR as a two-part protein: a 68-amino-acid helical NTD and a 210amino-acid C-terminal domain that binds cholesterol [12]. The StAR cholesterol transfer activity depends on phosphorylation by protein kinase A (PKA) at 195S [13], a step that takes place as the protein emerges from the ribosome [8]. The N-terminal sequence contains three conserved sites for cleavage by mitochondrial metalloproteases [14], which generate the shorter phosphorylated and unphosphorylated forms, previously detected as gel spots [8,9]. The Miller lab used a novel COS1 cell reconstitution system to establish that StAR can function without the NTD on the OMM and is inactive when the N-terminal sequence is replaced to accelerate import and also when reaching the mitochondrial matrix [15]. Significantly, most loss-of-function human mutations concentrated in the C-terminal domain [16]. StAR cholesterol intermembrane transfer activity is terminated after import and N-terminal cleavage. However, the three conserved cleavage sites suggest the NTD involvement in some way [17]. Protein synthesis is necessary to renew the activity at the OMM. This recovery only takes 3e5 min in adrenal cells [18]. Thus, only StAR synthesized in a 30-min pulse is inhibited by cycloheximide and o-phenanthroline, whereas metabolism of 25-HO-cholesterol, which has direct access to CYP11A1, is stimulated. Intervention with cycloheximide or a metalloprotease inhibitor pauses the cycle in an active state. Cholesterol metabolism is released by removal of either inhibitor. In this acute stimulation, approximately 400 cholesterol molecules transfer every cycle, suggesting a receptor/channel model for activity [18].

StAR activity at mitochondrial–ER contacts (MAM sites): the impact of fusion changes There is clear evidence for an alternative mitochondrial cholesterol transfer that is more closely associated with the mechanism resolved in COS1 cells. Mitochondria are not static but are constantly involved in the process of fusion, internal reorganization, and fission. This process opens different possibilities for cholesterol transfer. This cycle is mediated by a set of four GTPases: MFN1, MFN2, OPA1, and DRP1 (Graphical Abstract Figure) [2,19]. MFN1 and MFN2 function as dimers on the OMM to promote mitochondrial fusion [20,21]. In MA10 and Y-1 cells, this fusion is stimulated by PKA within 15 min, in parallel with increases in cholesterol metabolism. OPA1 functions on the IMM to redistribute the IMM into a single fused unit. The fission www.sciencedirect.com

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GTPase, DRP1, is a cytosolic protein that is recruited to the OMM by AKAP1 [22]. DRP1 polymerizes to constrict the fused mitochondria, such that separation ensues. Type 2 PKA is also recruited by AKAP1 to the OMM to phosphorylate and inhibit DRP1, while also activating StAR through 195S phosphorylation. Up to 5% of the mitochondrial surface involves contacts with the ER (MAM structures), which functions in both this cycle and StAR-mediated cholesterol transfer (Graphical Abstract Figure). The sigma factor, which links cholesterol-rich caveolin ER rafts to the MAM, is critical to StAR activity at MAM sites [23]. This activity is associated with a 450-kDa multiprotein complex that is trapped by protein cross-linking after approximately 3 h of PKA stimulation [24]. This process is, therefore, much slower than the MFN-induced fusion step. In this complex, the StAR NTD interactions with VDAC1 and VDAC2 occur as key steps in cholesterol transfer [21,23]. Facilitated folding of StAR by Grp78 is also a key step [25]. MFN2 selectively enhances MAM activity, as well as StAR participation in cholesterol metabolism [21,26]. MAM contacts transfer Ca2þ from ER stores to the inner mitochondria. Ca2þ has long been recognized to play a role in IMM cholesterol metabolism [27]. ATAD3, which extends from the MAM structure to the IMM, potentially provides a bridge for both cholesterol and Ca2þ [28]. Another protein, FATE1, attenuates MAM contacts and restricts both Ca2þ transfer and steroid synthesis [29]. StAR activity in MAM sites also depends on specific 232S phosphorylation by OMM-associated Erk kinase [21,26,30]. Thus, PKA-induced StAR expression in MA10 cells is resolved by timing into two distinct processes: 0e2 h, direct transfer to mitochondria, and >3 h, transfer from ER synthesis via MAM structures. The first step dominates the 15-min stimulation in Y-1 cells. These two processes may also serve different functions: an optimal response to a typical pulse of hormonal stimulation that lasts only 30e60 min and a protection against adverse IMM effects of prolonged cholesterol and Ca2þ that can occur with oxygen deprivation. Specialized IMM functions would explain the set of conserved NTD cleavage sites and the selectivity detected in VDAC2 binding [24].

TSPO as a modulator of StAR activity The common OMM component, TSPO, is typically present in mitochondria and appears to modulate StAR activity. TSPO is a general mitochondrial modulator with effects on Ca2þ transfer and membrane potentials that affect both MAM processes and the StAR mechanism. TSPO is an 18-kDa protein that binds benzodiazepine, diazepam, and many other drugs (marker ligand, PK11195) but also has a C-terminal cholesterolbinding domain [31]. TSPO functions through Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205

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interaction with other OMM proteins, particularly VDAC1, Acyl-CoA binding domain 3 (ACBD3), and PKA. TSPO-mediated VDAC1 phosphorylation results in suppressed Ca2þ entry into the mitochondria [32]. Deletion of TSPO in MA10 cells decreases steroid synthesis. However, in mice, several deletion strategies failed to affect either basal or human chorionic gonadotropin hormone (HCG)-stimulated generation of testosterone by the testis or basal adrenal corticosterone levels [31,33,34]. The stimulation by ACTH is, however, attenuated by absence of TSPO [31]. A third TSPO deletion method decreased both the testis and adrenal steroids in aging mice, where mitochondrial functions may be compromised [35]. Decreased activity has also been found after TSPO deletion from rat lines. Perhaps more significantly, a mutation of the cholesterol-binding segment (Ala147/Thr) (CRAC domain) decreases ACTH stimulation of corticosterone, with an accompanying increase in CE formation [36]. This Ala/ Thr mutation, which decreases measured cholesterol binding, also suppressed cortisol in human studies [36]. This CRAC domain may locate TSPO to cholesterolrich membrane sites around MAM sites, where StAR is also active. Unlike StAR removal where suppression is near complete, TSPO deletion either has no effect or has only partial suppression [37]. The rapid ACTH activation of cholesterol transfer in adrenal mitochondria may have different mechanistic features that are more sensitive to TSPO. Removal of TSPO may also select adaptations where local steroid synthesis is sustained.

StAR mRNA regulation by cAMP: roles of 3.5-kb mRNA and salt-inducible kinase/ CRTC modulation StAR is transcribed as 1.6-kb and 3.5-kb mRNA that arise from alternative 30 UTR polyadenylation [38e40]. cAMP activates transcription through PKA stimulation of CREB activity in a 300-bp proximal promoter [41]. This stimulation is supported by SF1 coupled to suppressions by Dax1 [42] and sumoylation [43] by GATA4 linked to MEF2 [44] and by Fos/Jun AP1 complexes [45]. This CREB activity additionally depends on partnership with CRTC2 for recruitment of CBP [14,46]. These complexes function in concert with changes in histone modification [47] and with distal enhancers, including the Bmal/Clock E box [48]. Secondary PKA-induced factors, notably C/EBPb and NR4a1, also contribute [49]. The roles of phosphodiesterases (PDE4 and PDE8), which control cAMP levels [50], and protein phosphatases, which determine phosphorylation of CRTC2 and other regulators (PP1, PP2A, and PP2A) [51], are often overlooked. StAR activation through MEK/Erk signaling, modeled by protein kinase C activators (phorbol esters) [45], Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205

bypasses CRTC2. A second form, CRTC3, which is controlled by salt-inducible kinase (SIK) activity, responds more to this PKC activation [51]. In vivo stimulation of adrenals by ACTH pulses shows rapid effects on DAX1 and SF1 that may extend to CRTC and SIK forms [42]. CRTC2 is released from SIK inhibition by PKA phosphorylation of the repressor kinases, SIK1eS577 and SIK2eS587 [14,46,52]. SIK1 is distinguished by extensive and rapid PKA induction, rapid turnover, and full nuclear localization of the active, unphosphorylated form (S577) (Figure 2a). A vector that expresses PKAresistant SIK1-GFP-S577A completely inhibits PKAinduced StAR expression. Inhibition of SIK forms by low levels of staurosporine fully stimulates StAR expression, without CREB phosphorylation [14]. The 30 UTR of the 3.5-kb mouse StAR has equivalently extended sequences in bovine and human forms but with minimal sequence conservation, except for sets of 2e3 UAUUUAUU elements in the terminal regions [38,53]. These elements bind dimers of the zinc finger protein, Znf36l2/TIS11b (Figure 2b). TIS11b is stimulated by PKA but enhances turnover of the 3.5-kb mRNA [38,53] (Figure 2c). The 3.5-kb mRNA is initially formed almost exclusively, whereas the more stable, the 1.6-kb form slowly predominates [10]. AKAP1, which is attached to the OMM, binds to KH domains adjacent to TIS11b sites on the 30 UTR [54]. AKAP1 may, therefore, facilitate the mitochondrial location of 3.5-kb mRNA [11,55]. AKAP1 also recruits type 2 PKA [54], which phosphorylates the emerging p37 protein [8,56]. Although removal of AKAP1 decreases steroidogenesis, this scaffold protein has multiple mitochondrial functions. Supplementation of StAR expression in MA10 cells using constructs that express StAR devoid of 30 UTR (dUTR-StAR) delivers comparable processed and phosphorylated StAR, but with minimal additional pregnenolone synthesis (Figure 2d) [14]. The 30 UTR is therefore unnecessary for StAR protein phosphorylation and import-linked cleavage and does not contribute to PKA-stimulated cholesterol metabolism. Thus, early cholesterol metabolism may not depend on StAR. Cross-linking of associated mitochondrial proteins in MA10 cells identified aggregates that include CYP11A1, but not StAR [57].

High-resolution imaging of StAR by singlemolecule FISH distinguishes single cells Br-cAMP stimulation of StAR expression in MA10 cells initially produces a linear increase in primary transcription, but exhibits delayed intron splicing to form mRNA (Figure 3a) [40]. Single-molecule (sm) FISH high-resolution fluorescent oligomers identified primary RNA (p-RNA), spliced RNA (sp-RNA), and extended 30 UTR, at the level of single molecules by using www.sciencedirect.com

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Figure 2

cAMP-induced modulators of StAR transcription in testis Leydig MA10 cells. (a) PKA/CREB-induced transcription of StAR depends on CRTC2 and SIK inhibition by PKA. cAMP stimulation of CREB depends on association of CRTC2, which then enhances recruitment of the HAT coactivator, CBP. cAMP-inducible SIK1 and constitutive SIK2 maintain CRTC2 in a phosphorylated state, which is sequestered by 14-3-3. Both SIK forms lose their kinase activity when phosphorylated by PKA (SIK1–S577; SIK2–S587). SF1, GATA4, and AP1 are key participants in transcription. Dephospho-CRTC2 moves to the nucleus and associates with speckles. CRTC2 then slowly colocates to speckles that contain the splicing factor, S35, and may, therefore, mediate the delayed splicing. StAR is initially transcribed predominantly as a 3.5-kb mRNA. The extended 30 UTR contains conserved AU-rich elements that bind TIS11b and an adjacent sequence binds AKAP1, which sequesters type 2 PKA.(b) Conserved elements in mouse and human StAR 30 UTR that bind TIS11b dimers. (c) Effect of 30 UTR on StAR expression. Constructs that express StAR with no 30 UTR (dUTR), 1.6-kb and 3.5-kb forms are transfected into MA10 cells (zero basal StAR). Empty Neo vector and a Neo-TIS11b vector are expressed as indicated, −/+, respectively. The 3.5-kb vector alone expresses less mRNA and protein than the 1.6-kb and dUTR forms. TIS11b supplementation selectively suppresses the 3.5-kb form (modified from Lee et al. [14]). (d) StAR supplementation of MA10 cells by transfected dUTRStAR before addition of Br-cAMP. StAR phosphorylation, import, and mitochondrial cleavage of the NTD are not affected by removal of the 30 UTR (right) when compared with natural expression (left). The additional expression at time zero increases the low basal activity, but not after 30 and 60 min of stimulation. Activities show pregnenolone increases in a 5-min window, when trilostane inhibits rapid conversion to progesterone (contrast linear progesterone kinetics) (Modified from Lee et al. [14]). NTD, Nterminal domain; PKA, protein kinase A; SIK, salt-inducible kinase; StAR, steroidogenesis acute regulator; 30 UTR, 30 -untranslated region.

fluorescent 20mers (Figure 3b). The delay in splicing is unusual because the excision of introns by spliceosomes is typically concerted with Pol2 elongation [58]. Nevertheless, the persistence of introns within nuclear loci was confirmed by smFISH during the, notably, asymmetric Br-cAMP stimulation of the cells and their loci. Most cells express high p-RNA and sp-RNA at two loci after 60 min (Figure 3c), but StAR mRNA is not detectable in the cytoplasm until a subsequent surge that peaks between 120 and 180 min, while p-RNA and then sp-RNA disappear at the loci. These two phases represent, respectively, slow and fast transcription that is distinguished by a shift from uncoupled to coupled splicing. These phases also closely match the fast fusion of mitochondria [21] and www.sciencedirect.com

the delayed integration of StAR into MAM complexes, respectively, [25]. Spatial separation of p-RNA and spRNA in the loci (Figure 3b and c) is attributed to delays in splicing until exon 7 transcription. A further delay in polyadenylation slows the release from the loci and transfer to the cytoplasm, where the 3.5-kb mRNA particles are detected by distinct dual open reading frame (ORF) and extended 30 UTR hybridization (Figure 3d). Spatially separated particles bound by a single sp-RNA likely represent 1.6-kb mRNA. Without an AKAP1 30 UTR target, they may concentrate in ER ribosomes that deliver StAR to MAM sites. These single-cell experiments show that stimulation of StAR expression over 60 min corresponds to a progressive increase in the number of cells that show active Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205

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Figure 3

Slow splicing of primary StAR RNA transcripts and differences in mRNA elongation probed by smFISH in testis (MA10) and adrenal (Y-1) cells.(a)Differences in the kinetics of StAR expression in MA10 cells according to the intron and 30 UTR position, based on DNA block determinations of transcript numbers. Introns 1 (blue) and 6 (purple) respond identically within 5 min, whereas spliced RNA/mRNA (red) and extended 30 UTR (brown) are delayed by 15 min. (b) Fluorescent 20 mers selectively target intron 1 (p-RNA), spliced RNA (ORF/sp-RNA), and extended 30 UTR (30 EU) respectively. (c) Representative Y-1 cells imaged by smFISH (Olympus IX80), with both p-RNA and sp-RNA probe sets (Br-cAMP, 0- to 180-min stimulations). (d) Resolution (NSIM) of 60-min p-RNA and sp-RNA accumulations, resolved at gene loci. p-RNA (green) and sp-RNA (red) are resolved in nuclei (DAPI). Insert: enlargements. (e)Z-stack projections of mRNA particles in the cytoplasm after 3-h stimulation of Y1 cells. Red, sp-RNA/ORF; green, 30 EU. Three different mRNA probe-binding patterns: sp-RNA/ORF and 30 EU bind to distinct sites on the same mRNA (enlarged in the insert); sp-RNA/ORF and 30 EU each bind separately in spatially distinct clusters. The spatial separation of these three clusters indicates that the binding selectivity is determined by distinct species rather than random associations. The single large accumulation represents a locus where there is a complete ORF/30 EU overlap. 30 EU, extended 30 UTR; p-RNA, primary RNA; sm, single-molecule; sp-RNA, spliced RNA; StAR, steroidogenesis acute regulator ; 30 UTR, 30 -untranslated region; DAPI, 40 ,6-diamidino-2-phenylindole; ORF, open reading frame.

expression. MA10 cells require about 30 min of PKA stimulation to reach basal Y-1 expression levels. The latter, however, show remarkable heterogeneity of expression, including appreciable basal cytoplasmic mRNA (Figure 4a). Stimulation of Y-1 cell RNA at loci follows the asymmetric pattern of MA10 activation. Nevertheless, cholesterol metabolism peaks within 15 min, during which time the RNA at loci increases, but not the mRNA. Three-dimensional Z-stack reconstruction of a MA10 cell after 180 min of PKA stimulation shows three zones. These comprise StAR nuclear loci, mitochondria marked by the StAR protein, and separate StAR mRNA (Figure 4b). Detailed examination of the central region Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205

shows that most mitochondria are paired with a single StAR mRNA (Figure 4c). According to Figure 3d, this pairing is similar in both Y-1 and MA10 cells. Such targeting of nuclear mRNA to mitochondria is predominantly determined by binding of the expressed NTD to the TOM40 OMM import channel [59]. Electron cryotomography studies of yeast mitochondria show that relatively few ribosomes bind at a single time. However, arrest of translation with cycloheximide increases their presence [60]. TIS11b potentially clears stalled StAR mRNA by enhancing ribonuclease activity on polyadenylation and 30 UTR. Electron microscopy of adrenal and MA10 mitochondria with MAM sites and LDs provides complementary images, but without visualization of the mRNA [21,24,28,61,62]. www.sciencedirect.com

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Figure 4

Heterogeneous StAR expression and spatial refinement within individual cells. (a)Eight basal Y-1 cells: Each shows distinctive distributions and expression levels of spliced StAR RNA at loci and mRNA in the cytoplasm.(b)Three-dimensional Z-Stack reconstruction of StAR expression in an MA10 cell after 180 min of PKA stimulation. The image shows three zones: high zone, StAR loci (red) express sp-RNA above cytoplasmic expression of protein and mRNA; central zone: shows both mRNA (red) and StAR protein/mitochondria (green); and lower zone: enriched in mRNA (red) separate from mitochondria. (c)XY sections of perinuclear central zone: StAR protein accumulates inside the mitochondrial matrix, whereas mRNA is attached to ribosomes outside the OMM. The diameter of mitochondria is approximately 500 nm. Three nuclear sections are shown at 1-um. sp-RNA, spliced RNA; StAR, steroidogenesis acute regulator.

StAR on the OMM surface interacts with hormonesensitive lipase on the LD to accept cholesterol [63]. In vitro reconstitution of LDs with accessory SNARE proteins, adrenal mitochondria, and recombinant StAR exhibited succinate-supported cholesterol metabolism [64]. A revised model of StAR turnover at steroid-producing mitochondria locates labile 3.5-kb transcripts to OMM-LD sites through NTD translation and 30 UTR facilitation by AKAP1, whereas the more stable 1.6-kb mRNA concentrates in the ER and MAM (Graphical Abstract Figure).

The role of StAR as a cell-type selective bridge between cholesterol trafficking, metabolism, and oxidative stress

StAR-KO mice retain <5 percent of normal steroid synthesis [61,62]. When an NTD-deficient StAR transgene is restored into StAR-ko mice (N-47 StAR mice), most adrenal glucocorticoid syntheses returns [61], but generation of testosterone by the testis remains absent. Deletion of TSPO suppresses steroidogenesis more in the adrenal than in the testis [31]. Normal adrenal LDs average about twice the diameter of mitochondria, but many small droplets intersperse between mitochondria, particularly in N-47 mice, where www.sciencedirect.com

they are targeted by N-47 StAR [61]. Their lifetime is prolonged in absence of uptake into mitochondria. In wild-type (WT) mice, large droplets are contacted by several mitochondria, with structural features at the interface that may include partnering SNARE proteins. Enhanced partnership with N-47 StAR is likely to partially compensate for deficient OMM interactions and import. Cholesterol enters adrenal cells from HDL through transfer of esters to SRB1, which then transfers cholesterol through microfilaments to LDs, where esters are reformed by acyl-coenzyme A:cholesterol acyltransferase (ACAT) [65] (Figure 1). STARD4 redistributes cholesterol between organelles and mediates the exit, transfer, and redistribution to the endosome network. Niemann-Pick C1 (NPC1), a transmembrane cholesterol-binding protein, and STARD3 separately transfer cholesterol out of late endosomes, which receive cholesterol from LDL and ApoE [4,66]. The sterol-binding protein, ORP5, partners with NPC1 [67]. In Chinese hamster ovary (CHO) cells, deletion of NPC1 enhances cholesterol transfer into mitochondria, mediated by NPC2 within the endosomes and STARD3, which provides a bridge to the ER [66,68]. This Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205

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cholesterol reaches mitochondria via MAM sites, without any StAR participation. Inhibition of steroid synthesis in MA10 cells by the specific NPC1 ligand, U18666A, implicates this transfer in StAR activity at the mitochondria [69]. Cholesterol bound to NPC1 is also a control point for lipogenesis and protein synthesis, via activation of the arginine transporter, SLC38A9, the TORC1 complex, and sterol regulatory element binding protein (SREBP) forms [70]. In situ CRISPR deletion of StAR in either MA10 or Y1 cells shows a rapid increase in lipids in endosomes adjacent to mitochondria, concomitant within a few hours of StAR removal, consistent with an extensive NPC1/StAR connection [71]. Together with increases in LDs, this cholesterol accumulation far exceeds the loss of cholesterol metabolism but matches changes seen in N-47 adrenals and the testis [61]. CRISPR deletion of TSPO also effects loss of steroids, with less striking lipid increases [72]. StAR provides a bridge between the cholesterol storage hub in the LDs and the synthetic source of the ER. Cholesterol passes through the filter of the mitochondrion, itself a sentinel of the cell’s energy and oxygen status, to deliver organ-selective steroid hormones through CYP11A1 or components of the cholesterolprocessing machinery, via CYP27A1, hydroxycholesterols (HOeCH), and the LXR nuclear receptors [63].

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increase and decrease in PKA activity in 30-min hormonal pulses [42,52]? Does promoter signaling from SF1, AP1, Gata 4, and Bmal/Clock link to the control of CRTC2 by SIK forms (1e3) or to AMPK and their upstream regulator, LKB1 [51]? To what extent is mitochondrial fusion involved in this acute StAR response? The complementary role of TSPO and VDAC complexes supports such participation [24,28]. What are the relative contributions of Niemann-Pick C1 (NPC1) endosome, LDs, and ER/MAM pathways with diverse stimuli and cell types [66,68,69]? The extended residence of StAR in the inner mitochondria suggests a more complex role in other mitochondrial functions, notably protection from oxidative stress [55,68,74].

Our recent work has used new imaging methods to view StAR in single cells; we have seen large local differences in expression and a novel partnering between StAR mRNA and mitochondria. It is evident that the diversity of StAR activity shown in these model systems needs to be further evaluated in more physiological environments. The early experiments that preceded StAR raised questions that can now be tackled with new imaging and single-cell technologies [73].

Conflict of interest statement A selection of unresolved StAR questions Many questions can now be raised concerning StAR that apply beyond endocrinology to the situations where StAR is expressed without steroidogenesis, notably to the CYP27A1/LXR pathway and to protection of the IMM from excess cholesterol. The principles introduced here extend to StAR participation in hepatocytes (bile acids), macrophages (CYP27A1), astrocytes/ microglia [68,73], and mesenchymal repair processes [74]. The function of yeast START proteins in ERe mitochondria contacts has very general functions [68]. The linkage of cholesterol to oxidative stress in mitochondria expands the role of matrix StAR beyond import [55]. In this expanded perspective, the presence or absence of the StAR bridge can redirect cholesterol trafficking from rapid entry to slow entry through MAM contacts [68]. Important unresolved questions include the following: 1. Are there functional differences between the 3.5-kb and 1.6-kb StAR forms, notably relating to involvement of TIS11b, AKAP1, and targeting of labile 3.5kb StAR mRNA at mitochondria [53,56]? 2. Do the rapid changes in the SIK1/CRTC2 cycle, the rapid translation/import cycle of adrenal cells, or the turnover of 3.5-kb StAR mRNA integrate with the Current Opinion in Endocrine and Metabolic Research 2019, 8:195–205

Nothing declared.

Funding This work was supported by the National Institutes of Health (grant number: R01 HD090660).

References Papers of particular interest, published within the period of review, have been highlighted as: * of special interest * * of outstanding interest 1.

Ikonen E: Cellular cholesterol trafficking and compartmentalization. Nat Rev Mol Cell Biol 2008, 9:125–138.

2.

Pernas L, Scorrano L: Mito-morphosis: mitochondrial fusion, fission, and cristae remodeling as key mediators of cellular function. Annu Rev Physiol 2016, 78:505–531.

3. *

Iaea DB, et al.: Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane. Mol Biol Cell 2017, 28:1111–1122. Application of ergosterol as a fluorescent probe to track the rates of sterol movement between plasma membrane and endosomes. Insight into differences between vesicular sterol intermembrane transfer and protein-mediated transfer that is substantially mediated by StARD4. 4. *

Wilhelm LP, et al.: STARD3 mediates endoplasmic reticulumto-endosome cholesterol transport at membrane contact sites. EMBO J 2017, 36:1412–1433. Characterization of the roles of the N-terminal endosome anchoring domain and the VAP ER partner. Evidence that transfer occurs substantially from ER to endosome.

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5.

Jefcoate CR, et al.: The detection of different states of the P450 cytochromes in adrenal mitochondria: changes induced by ACTH. Ann N Y Acad Sci 1973, 212:243–261.

6.

DiBartolomeis MJ, Jefcoate CR: Characterization of the acute stimulation of steroidogenesis in primary bovine adrenal cortical cell cultures. J Biol Chem 1984, 259:10159–10167.

7.

Hall PF, et al.: The role of microfilaments in the response of adrenal tumor cells to adrenocorticotropic hormone. J Biol Chem 1979, 254:9080–9084.

8.

Pon LA, Hartigan JA, Orme-Johnson NR: Acute ACTH regulation of adrenal corticosteroid biosynthesis. Rapid accumulation of a phosphoprotein. J Biol Chem 1986, 261: 13309–13316.

9.

Elliott ME, Goodfriend TL, Jefcoate CR: Bovine adrenal glomerulosa and fasciculata cells exhibit 28.5-kilodalton proteins sensitive to angiotensin, other agonists, and atrial natriuretic peptide. Endocrinology 1993, 133:1669–1677.

10. Clark BJ, et al.: Hormonal and developmental regulation of the steroidogenic acute regulatory protein. Mol Endocrinol 1995, 9:1346–1355. 11. Jefcoate CR, Orme-Johnson WH: Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states. J Biol Chem 1975, 250:4671–4677. 12. Tsujishita Y, Hurley JH: Structure and lipid transport mechanism of a StAR-related domain. Nat Struct Biol 2000, 7: 408–414. 13. Arakane F, et al.: Phosphorylation of steroidogenic acute regulatory protein (StAR) modulates its steroidogenic activity. J Biol Chem 1997, 272:32656–32662. 14. Lee J, et al.: Regulation of StAR by the N-terminal domain and coinduction of SIK1 and TIS11b/Znf36l1 in single cells. Front Endocrinol 2016, 7:107. 15. Bose HS, Lingappa VR, Miller WL: Rapid regulation of steroidogenesis by mitochondrial protein import. Nature 2002, 417:87–91. 16. Miller WL: Disorders in the initial steps of steroid hormone * synthesis. J Steroid Biochem Mol Biol 2017, 165:18–37. Characterization of the clinical symptoms arising from over 40 mutations in the human StAR gene that cause congenital adrenal lipoidal hyperplasia. Presentations of clinical symptoms associated with specific mutations and with conditions arising from other mutations in trafficking genes. 17. Yamazaki T, et al.: Mitochondrial processing of bovine adrenal steroidogenic acute regulatory protein. Biochim Biophys Acta 2006, 1764:1561–1567. 18. Artemenko IP, et al.: Mitochondrial processing of newly synthesized steroidogenic acute regulatory protein (StAR), but not total StAR, mediates cholesterol transfer to cytochrome P450 side chain cleavage enzyme in adrenal cells. J Biol Chem 2001, 276:46583–46596. 19. Tilokani L, et al.: Mitochondrial dynamics: overview of mo* * lecular mechanisms. Essays Biochem 2018, 62:341–360. Excellent contemporary presentation of the fast-moving field of the mitochondrial fusion fission cycle, which links not only to cell energy demands, but also to cAMP stimulation of StAR activity. 20. Helfenberger KE, et al.: Angiotensin II stimulation promotes * * mitochondrial fusion as a novel mechanism involved in protein kinase compartmentalization and cholesterol transport in human adrenocortical cells. J Steroid Biochem Mol Biol 2019, 192:105413. Most recent of a series of papers that establish mitofusin2 (MFN2) and hormone-induced mitochondrial fusion as rapid contributors to StARinduced cholesterol transfer. Here, the range of these activity is extended to Ca2+ and PKC forms that are induced by angiotensin in glomerulosa cells. 21. Duarte A, et al.: Mitochondrial fusion is essential for steroid biosynthesis. PLoS One 2012, 7, e45829. 22. Merrill RA, Strack S: Mitochondria: a kinase anchoring protein * * 1, a signaling platform for mitochondrial form and function. Int J Biochem Cell Biol 2014, 48:92–96. www.sciencedirect.com

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Reviews how the signaling platform, AKAP1, can integrate three features of StAR activity: C-Translational Type2 PKA phosphorylation of StAR; Recruitment of 3.5 kb mRNA to mitochondria and inhibition of mitochondrial fission by DRP1. 23. Marriott KS, et al.: sigma-1 receptor at the mitochondrialassociated endoplasmic reticulum membrane is responsible for mitochondrial metabolic regulation. J Pharmacol Exp Ther 2012, 343:578–586. 24. Prasad M, et al.: Mitochondria-associated endoplasmic reticulum membrane (MAM) regulates steroidogenic activity via steroidogenic acute regulatory protein (StAR)-voltagedependent anion channel 2 (VDAC2) interaction. J Biol Chem 2015, 290:2604–2616. 25. Prasad M, et al.: Mitochondrial metabolic regulation by * * GRP78. Sci Adv 2017, 3, e1602038. Most recent in a series of papers that characterize the contributions of MAM constituents to StAR cholesterol transfer activity and import. Here, the authors establish a key contribution of GRP78 to StAR activity, derived from folding of protein translated from mRNA bound to ER ribosomes. 26. Castillo AF, et al.: The role of mitochondrial fusion and StAR phosphorylation in the regulation of StAR activity and steroidogenesis. Mol Cell Endocrinol 2015, 408:73–79. 27. Kowluru R, et al.: Metabolism of exogenous cholesterol by rat adrenal mitochondria is stimulated equally by physiological levels of free Ca2+ and by GTP. Mol Cell Endocrinol 1995, 107: 181–188. 28. Issop L, et al.: Mitochondria-associated membrane formation in hormone-stimulated Leydig cell steroidogenesis: role of ATAD3. Endocrinology 2015, 156:334–345. 29. Doghman-Bouguerra M, et al.: FATE1 antagonizes calciumand drug-induced apoptosis by uncoupling ER and mitochondria. EMBO Rep 2016, 17:1264–1280. 30. Paz C, et al.: Role of protein phosphorylation and tyrosine phosphatases in the adrenal regulation of steroid synthesis and mitochondrial function. Front Endocrinol 2016, 7:60. 31. Fan J, et al.: Conditional steroidogenic cell-targeted deletion of TSPO unveils a crucial role in viability and hormonedependent steroid formation. Proc Natl Acad Sci U S A 2015, 112:7261–7266. 32. Gatliff J, et al.: A role for TSPO in mitochondrial Ca(2+) homeostasis and redox stress signaling. Cell Death Dis 2017, 8, e2896. 33. Tu LN, et al.: Peripheral benzodiazepine receptor/translocator protein global knock-out mice are viable with no effects on steroid hormone biosynthesis. J Biol Chem 2014, 289: 27444–27454. 34. Selvaraj V, Tu LN, Stocco DM: Crucial role reported for TSPO in viability and steroidogenesis is a misconception. Commentary: conditional steroidogenic cell-targeted deletion of TSPO unveils a crucial role in viability and hormonedependent steroid formation. Front Endocrinol 2016, 7:91. 35. Barron AM, et al.: Steroidogenic abnormalities in translocator * * protein knockout mice and significance in the aging male. Biochem J 2018, 475:75–85. Excellent paper that uses a new method (Zn-finger nucleases; ZFN) to generate Rat lines that compare a general TSPO disruption with a specific mutation that decreases cholesterol binding in the TSPO CRAC domain. Human subjects with this mutation, like the rats, lose cortisol synthesis. 36. Owen DR, et al.: TSPO mutations in rats and a human polymorphism impair the rate of steroid synthesis. Biochem J 2017, 474:3985–3999. 37. Costa B, Da Pozzo E, Martini C: Translocator protein and ste* * roidogenesis. Biochem J 2018, 475:901–904. Valuable independent commentary on the diversity of effects of TSPO deletion. The authors minimize controversy by emphasizing the diversity within the context of each model. 38. Duan H, Jefcoate CR: The predominant cAMP-stimulated 3 x 5 kb StAR mRNA contains specific sequence elements in the

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extended 30 UTR that confer high basal instability. J Mol Endocrinol 2007, 38:159–179. 39. Ariyoshi N, et al.: Characterization of the rat Star gene that encodes the predominant 3.5-kilobase pair mRNA. ACTH stimulation of adrenal steroids in vivo precedes elevation of Star mRNA and protein. J Biol Chem 1998, 273:7610–7619. 40. Lee J, et al.: Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: Application to hormonal stimulation of StAR transcription. Mol Cell Endocrinol 2016, 429:93–105. 41. Clem BF, Hudson EA, Clark BJ: Cyclic adenosine 30 ,50 -monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter. Endocrinology 2005, 146:1348–1356. 42. Spiga F, et al.: Dynamic responses of the adrenal steroido* * genic regulatory network. Proc Natl Acad Sci U S A 2017, 114: E6466–E6474. Examination of the kinetics of adrenal gene responses to short in vivo pulses of ACTH of 30- to 60-min duration. Immediate and substantial primary StAR RNA increases precede a slow rise in spliced RNA. SF1 mRNA and protein are elevated, while Dax1 mRNA and protein decline. This model provides the first representation of a typical in vivo stimulation. A kinetic modeling provides a good representation of the data. 43. Yang WH, et al.: SUMOylation inhibits SF-1 activity by reducing CDK7-mediated serine 203 phosphorylation. Mol Cell Biol 2009, 29:613–625. 44. Daems C, et al.: MEF2 cooperates with forskolin/cAMP and GATA4 to regulate star gene expression in mouse MA-10 leydig cells. Endocrinology 2015, 156:2693–2703. 45. Manna PR, Dyson MT, Stocco DM: Regulation of the steroidogenic acute regulatory protein gene expression: present and future perspectives. Mol Hum Reprod 2009, 15:321–333. 46. Takemori H, Okamoto M: Regulation of CREB-mediated gene expression by salt inducible kinase. J Steroid Biochem Mol Biol 2008, 108:287–291.

zinc finger protein ZFP36L1/TIS11b. Mol Endocrinol 2009, 23: 497–509. 54. Dyson MT, et al.: Mitochondrial A-kinase anchoring protein 121 binds type II protein kinase A and enhances steroidogenic acute regulatory protein-mediated steroidogenesis in MA-10 mouse leydig tumor cells. Biol Reprod 2008, 78: 267–277. 55. Solsona-Vilarrasa E, et al.: Cholesterol enrichment in liver * mitochondria impairs oxidative phosphorylation and disrupts the assembly of respiratory supercomplexes. Redox Biol 2019, 24:101214. Cholesterol-rich diet causes oxidative stress in liver mitochondria, which is analyzed in terms of changes in respiratory electron transfer.Other recent publications discussed in [68] show that StAR can either mediate or prevent liver mitochondrial stress depending on model. 56. Grozdanov PN, Stocco DM: Short RNA molecules with high binding affinity to the KH motif of A-kinase anchoring protein 1 (AKAP1): implications for the regulation of steroidogenesis. Mol Endocrinol 2012, 26:2104–2117. 57. Rone MB, et al.: Identification of a dynamic mitochondrial protein complex driving cholesterol import, trafficking, and metabolism to steroid hormones. Mol Endocrinol 2012, 26: 1868–1882. 58. Bentley DL: Coupling mRNA processing with transcription in time and space. Nat Rev Genet 2014, 15:163–175. 59. Bottinger L, Ellenrieder L, Becker T: How lipids modulate mitochondrial protein import. J Bioenerg Biomembr 2016, 48: 125–135. 60. Gold VA, et al.: Visualization of cytosolic ribosomes on the * * surface of mitochondria by electron cryo-tomography. EMBO Rep 2017, 18:1786–1800. High resolution electron cryo-tomography images of single mRNA bound by ribosomes attached to the outer mitochondrial membrane, via insertion of N-terminal translated sequence into the TOM40 import assemblage. Insight is provided into basis of single StAR 3.5 kb mRNA complexes on individual mitochondria.

47. Hiroi H, Christenson LK, Strauss 3rd JF: Regulation of transcription of the steroidogenic acute regulatory protein (StAR) gene: temporal and spatial changes in transcription factor binding and histone modification. Mol Cell Endocrinol 2004, 215:119–126.

61. Sasaki G, et al.: Complex role of the mitochondrial targeting signal in the function of steroidogenic acute regulatory protein revealed by bacterial artificial chromosome transgenesis in vivo. Mol Endocrinol 2008, 22:951–964.

48. Son GH, et al.: Adrenal peripheral clock controls the autonomous circadian rhythm of glucocorticoid by causing rhythmic steroid production. Proc Natl Acad Sci U S A 2008, 105: 20970–20975.

62. Ishii T, et al.: The roles of circulating high-density lipoproteins and trophic hormones in the phenotype of knockout mice lacking the steroidogenic acute regulatory protein. Mol Endocrinol 2002, 16:2297–2309.

49. Abdou HS, Robert NM, Tremblay JJ: Calcium-dependent Nr4a1 expression in mouse Leydig cells requires distinct AP1/CRE and MEF2 elements. J Mol Endocrinol 2016, 56:151–161.

63. Manna PR, et al.: Mechanisms of action of hormone-sensitive lipase in mouse Leydig cells: its role in the regulation of the steroidogenic acute regulatory protein. J Biol Chem 2013, 288: 8505–8518.

50. Golkowski M, et al.: Studying mechanisms of cAMP and cyclic * * nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics. Cell Signal 2016, 28:764–778. Comprehensive examination of how PDE4 and 8 each play key roles in cAMP-induced endocrine regulation. The value of specific PDE inhibitors, Rolipram (PDE4) and PF-04959325 (PDE8), in selectively affecting steroidogenic responses is shown. The paper reports the selective effects on LCMS/MS analyses of phosphor–protein profiles in MA10 cells.

64. Kraemer FB, Shen WJ, Azhar S: SNAREs and cholesterol * * movement for steroidogenesis. Mol Cell Endocrinol 2017, 441: 17–21. Review of lipid droplet structure and the contribution of SNARE proteins. Description of an effective model for reconstitution of StAR with mitochondria in vitro and the use of this model to assess the contributions of SNARE proteins to the mitochondrial cholesterol transfer and CYP11A1 activity.

51. Sonntag T, et al.: Mitogenic signals stimulate the CREB * * coactivator CRTC3 through PP2A recruitment. iScience 2019, 11:134–145. Excellent presentation of novel dissection of CRTC2 and CRTC3 differences with respect to their kinase activation and phosphatase inactivation. The key role of the LKB-SIK/AMPK-CRTC intervention in cAMP endocrine control, although presented here, is typically overlooked in cAMP effects on transcription.

65. Shen WJ, et al.: Scavenger receptor B type 1: expression, * * molecular regulation, and cholesterol transport function. J Lipid Res 2018, 59:1114–1131. Comprehensive review of the key role of SRB1 in the uptake of cholesterol esters from HDL and their onward transport as free cholesterol, via microfilaments, to lipid droplets where they are stored as esters. The regulation of expression and post translational modifications are examined, including how these changes integrate into cholesterol trafficking, including StAR activity.

52. Liu Y, et al.: Transcriptional regulation of episodic glucocorticoid secretion. Mol Cell Endocrinol 2013, 371:62–70. 53. Duan H, et al.: cAMP-dependent posttranscriptional regulation of steroidogenic acute regulatory (STAR) protein by the

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66. Kennedy BE, Charman M, Karten B: Niemann-Pick Type C2 protein contributes to the transport of endosomal cholesterol to mitochondria without interacting with NPC1. J Lipid Res 2012, 53:2632–2642.

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67. Du X, et al.: A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. J Cell Biol 2011, 192:121–135. 68. Elustondo P, Martin LA, Karten B: Mitochondrial cholesterol * * import. Biochim Biophys Acta Mol Cell Biol Lipids 2017, 1862: 90–101. Comprehensive and insightful review of cholesterol trafficking to mitochondria from LDL and HDL, with and without StAR, including the interventions by StARD3, NPN1 and NPC2 in late endosomes. Provides an introduction to equivalent yeast mechanisms. 69. Venugopal S, et al.: Plasma membrane origin of the steroidogenic pool of cholesterol used in hormone-induced acute steroid formation in leydig cells. J Biol Chem 2016, 291: 26109–26125. 70. Castellano BM, et al.: Lysosomal cholesterol activates * * mTORC1 via an SLC38A9-Niemann-Pick C1 signaling complex. Science 2017, 355:1306–1311. SLC38a9 is a key mediator that connects protein turnover in lysosomes/late endosomes to lipogenesis via TORC1 activity. LDL/ApoE delivers cholesterol to NPC1, which then activates Slc38a3, TORC1 and then SREBP forms. Cholesterol is also removed from NPC1 by donation externally to StAR. LDL cholesterol is, thus, linked to both nutrition circuitry and to either steroid synthesis or LXR stimulation of cholesterol export via the ABCA1 pump and HDL. 71. Lee J, Jefcoate C: Monitoring of dual CRISPR/Cas9-Mediated * * steroidogenic acute regulatory protein gene deletion and cholesterol accumulation using high-resolution fluorescence in situ hybridization in a single cell. Front Endocrinol 2017, 8: 289. smFISH imaging of acute StAR RNA stimulation at loci is used to directly measure the effect of CRISPR 1.8 kb DNA excision from the gene. Loss of StAR produced rapid increases in lipid in endosomal

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vesicles adjacent to mitochondria, consistent with direct StAR impact on the endosomal cholesterol trafficking. This in situ approach minimizes random off target effects and cell adaptation during clonal expansion. 72. Fan J, et al.: CRISPR/Cas9Mediated tspo gene mutations lead * to reduced mitochondrial membrane potential and steroid formation in MA-10 mouse tumor leydig cells. Endocrinology 2018, 159:1130–1146. The contribution of TSPO to MA10 cells, defined by generation of lines completely deficient in TSPO by use of CRISPR Cas9. Changes in mitochondrial redox characteristics and large decreases in cAMPinduced progesterone synthesis are seen despite robust p37 StAR. 73. Jefcoate CR, Lee J: Cholesterol signaling in single cells: les* * sons from STAR and sm-FISH. J Mol Endocrinol 2018, 60: R213–R235. Review of how single cell RNA imaging and sequencing provides a novel perspective on cholesterol trafficking in steroidogenic tissues, macrophage and the brain.The unusual single cell regulation of StAR expression in MA10 cells and Y-1 cells is compared with respect to the changes in gene loci and mRNA expression at mitochondria. 74. Anuka E, et al.: Infarct-induced steroidogenic acute regulatory protein: a survival role in cardiac fibroblasts. Mol Endocrinol 2013, 27:1502–1517. 75. Ferguson Jr JJ: Protein synthesis and adrenocorticotropin responsiveness. J Biol Chem 1963, 238:2754–2759. 76. Garren LD, Gill GN, Masui H, Walton GM: On the mechanism of action of ACTH. Recent Prog Horm Res 1971, 27:433–478. 77. Simpson ER, Boyd GS: The cholesterol side-chain cleavage system of bovine adrenal cortex. Eur J Biochem 1967, 2: 275–285.

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