- Email: [email protected]
Defining microRNA signatures of hair follicular stem and progenitor cells in healthy and androgenic alopecia patients
Journal Pre-proof Defining microRNA signatures of hair follicular stem and progenitor cells in healthy and androgenic alopecia patients Parvaneh Mohammadi, Mohammad Ali Nilforoushzadeh, Khalil Kass Youssef, Ali Sharifi-Zarchi, Sharif Moradi, Pardis Khosravani, Raheleh Aghdami, Payam Taheri, Ghasem Hosseini-Salekdeh, Hossein Baharvand, Nasser Aghdami
PII:
S0923-1811(20)30351-0
DOI:
https://doi.org/10.1016/j.jdermsci.2020.11.002
Reference:
DESC 3679
To appear in:
Journal of Dermatological Science
Received Date:
30 April 2020
Revised Date:
22 October 2020
Accepted Date:
3 November 2020
Please cite this article as: Mohammadi P, Nilforoushzadeh MA, Youssef KK, Sharifi-Zarchi A, Moradi S, Khosravani P, Aghdami R, Taheri P, Hosseini-Salekdeh G, Baharvand H, Aghdami N, Defining microRNA signatures of hair follicular stem and progenitor cells in healthy and androgenic alopecia patients, Journal of Dermatological Science (2020), doi: https://doi.org/10.1016/j.jdermsci.2020.11.002
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier.
Defining microRNA signatures of hair follicular stem and progenitor cells in healthy and androgenic alopecia patients Parvaneh Mohammadia,b,c, Mohammad Ali Nilforoushzadehd, Khalil Kass Youssefe, Ali Sharifi-Zarchif, Sharif Moradia, Pardis Khosravania, Raheleh Aghdamia,c, Payam Taheria, Ghasem Hosseini-Salekdeha, Hossein Baharvanda,b, Nasser Aghdamic* Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute
of
a.
for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
c.
Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell
ro
b.
-p
Biology and Technology, ACECR, Tehran, Iran.
Skin and Stem Cell Research Center, Tehran University of Medical Science, Tehran, Iran.
e.
Instituto de Neurociencias (CSIC-UMH), Sant Joan d'Alacant, Spain.
f.
Computer Engineering Department, Sharif University of Technology, Tehran, Iran.
re
d.
lP
*Correspondence
Nasser Aghdami: [email protected]
Highlights
ur na
Tel: +98 21 22306485
Introducing a platform for understanding miRNA dynamic regulation in hair follicular cells in baldness
Jo
This study represents that miR-324-3p is depleted in bald stem cells compared to normal hair stem and progenitor cells
MiR-324-3p overexpression impact significantly keratinocytes differentiation program, promotes cell migration and reduces proliferation.
1
Abstract Background The exact pathogenic mechanism causes hair miniaturization during androgenic alopecia (AGA) has not been delineated. Recent evidence has shown a role for non-coding regulatory RNAs, such as microRNAs (miRNAs), in skin and hair disease. There is no reported information about the role of miRNAs in hair epithelial cells of
of
AGA. Objectives
ro
To investigate the roles of miRNAs affecting AGA in normal and patient’s epithelial hair cells.
-p
Methods
Normal follicular stem and progenitor cells, as well as follicular patient’s stem cells, were sorted from hair
re
follicles, and a miRNA q-PCR profiling to compare the expression of 748 miRNA (miRs) in sorted cells were performed. Further, we examined the putative functional implication of the most differentially regulated miRNA
lP
(miR-324-3p) in differentiation, proliferation and migration of cultured keratinocytes by qRT-PCR, immunofluorescence, and scratch assay. To explore the mechanisms underlying the effects of miR-324-3p, we used specific chemical inhibitors targeting pathways influenced by miR-324-3p.
ur na
Result: We provide a comprehensive assessment of the "miRNome" of normal and AGA follicular stem and progenitor cells. Differentially regulated miRNA signatures highlight several miRNA candidates including miRNA-324-3p as mis regulated in patient’s stem cells. We find that miR-324-3p promotes differentiation and migration of cultured keratinocytes likely through the regulation of mitogen-activated protein kinase (MAPK)
Jo
and transforming growth factor (TGF)-β signaling. Importantly, pharmacological inhibition of the TGF-β signaling pathway using Alk5i promotes hair shaft elongation in an organ-culture system. Conclusion: Together, we offer a platform for understanding miRNA dynamic regulation in follicular stem and progenitor cells in baldness and highlight miR-324-3p as a promising target for its treatment.
Keywords: miRNA, androgenic alopecia, hair follicular stem cells, miR-324-3p Abbreviations: AGA: Androgenic Alopecia, miRNAs: microRNAs, NSCs: Normal Stem Cells, NPCs: Normal Progenitor Cells,
2
PSCs: Patient Stem Cells, miRs: miRNA, MAPK: Mitogen-Activated Protein Kinase, TGF-β: transforming growth factor, FACS: Fluorescence-Activated Cell Sorting, KEGG: Kyoto Encyclopedia of Genes and Genomes, HFs: Hair Follicles, APM: Arrector Pili Muscle.
Introduction
Androgenic alopecia (AGA) is the most common type of hair loss [1]. Suggested causes for
of
this gradual hair loss include androgen dependence and genetic predisposition [2]. Recently,
may contribute to a gradual decrease in hair size in AGA [3].
ro
Cotsarelis et al. showed that defects in human hair stem cells conversion to progenitor cells
-p
The hair cycle is regulated by intrinsic and extrinsic factors at the transcriptional and posttranscriptional levels [4]. MicroRNAs (miRNAs), which are ~22-nucleotide non-coding
re
RNAs, regulate the expression of many genes at the post-transcriptional level, thereby
lP
controlling virtually all biological processes and pathways [5]. Pioneering studies by Fuchs and Millar showed that miRNAs have a critical role in mesenchymal-epithelial interaction,
ur na
hair follicle placode invagination, and mouse hair morphogenesis [6, 7]. Exploring the functions of individual miRNAs in skin and hair cells has shown that miRNA-203 acts as a switch between proliferation and differentiation in the skin [8], and miRNA-22 is reported as a critical post-transcriptional regulator of mouse hair cycle [9]. Other studies report
Jo
differential expression of miRNA-31 during mouse hair cycle [10] and upregulation of transforming growth factor (TGF)-2 via downregulation of miRNA-31 by a Hairless gene that causes an abnormal hair cycle in hairpoor mice [11].
3
Another key miRNA, miRNA-125b, has been reported to be responsible for stem cell commitment in the hair follicle [4]; thus, a miRNA network that governs hair follicle development and hair loss is beginning to be identified. Although substantial progress has been made in discovering the significant roles of miRNAs in hair follicle development and hair cycle-associated tissue remodeling, most information
of
comes from investigations in cell cultures, mice, sheep, and goat models [12, 13]. Hence, future studies should emphasize the roles of miRNAs in human hair development and hair
ro
loss. In the present study, we have generated miRNA signatures for three types of cell
-p
populations from hair follicles: normal stem cells (NSCs), normal progenitor cells (NPCs), and patient’s stem cells (PSCs). We observed that miR-324-3p was depleted in PSCs
re
compared to normal NSCs and NPCs and found that the miR-324-3p overexpression impact significantly keratinocytes differentiation program, promotes cell migration and reduces
lP
proliferation. Notably, small molecules that inhibited putative miR-324-3p targets, mitogenactivated protein kinase (MAPK) and transforming growth factor (TGF)-β signaling
ur na
pathways, phenocopied miR-324-3P overexpression in keratinocytes. Finally, the inhibition of the TGF-β signaling pathway by Alk5i in an anagen organ-cultured hair follicle system promoted hair shaft elongation. Taken together, these data provide an important foundation for further analysis of miR-324-3p as a probable candidate to regulate human hair
Jo
keratinocyte differentiation and also new effective therapy to treat AGA.
Materials and Methods
Hair follicle samples 4
Human occipital and frontal samples were taken from the scalp of volunteer donors who suffered from AGA during hair transplantation. The Institutional Review Board and Ethical Committee of Royan Institute and Tehran University approved this study. All tissue donors were males with an average age of 35 years (range: 25-50 years). The normal samples
of
consisted of a scalp strip (1.5 cm × 1.5 cm) taken from the occipital area. The bald samples were cylindrical punches removed from the frontal scalp because of the creation of recipient
ro
sites for donor occipital scalp. A complete description of the methodology used in this study
-p
is provided in the supplementary material.
re
Results
lP
Characterizing human hair stem and progenitor cells in anagen hair
ur na
To evaluate miRNA expression in human hair bulge stem and progenitor cells, we first define markers combination sufficient to purify reliably hair follicle populations. Occipital human hair is divided into five portions : infundibulum, bulge, sub-bulge, suprabulbar, and bulb (Fig. 1a) [14]. Moreover, we divided the sub-bulge region into proximal and distal part and
Jo
suprabulbar area into three parts the proximal, medial, and distal. Therefore, we analyzed by immunofluorescence the expression of CD200, K15, CD34, and CD49f markers, widely used previously to define murine and human stem and progenitor cell populations (Fig. S1-4). CD200, a hair stem cell marker [15], was expressed in bulge area
5
(between the sebaceous gland and the insertion of arrector pili muscle (APM)), with weaker and clearly decaying expression in the proximal part of the sub-bulge area (Fig. S1). K15, another hair stem cell marker [3], was more extended covering the bulge, infundibulum, sub-bulge, and distal part of the suprabulbar area (Fig. S2). CD34, a marker of progenitor cells [15], was expressed specifically in the sub-bulge
of
expanding to the proximal part of the suprabulbar area (Fig. S3). Immunofluorescence data also revealed that CD49f, a basal cell marker [14], was extensively
ro
expressed in all epithelial cells of the hair outer root sheath and basal cells of skin epidermis
-p
(Fig. S4).
Double immunostaining results confirmed co-expression of K15 and CD200 in the bulge and
re
expression of K15 in the sub-bulge and suprabulbar area (Fig. 1b, S5). Also, double immunostaining of CD34 and CD200 showed CD34 expression in the distal part
lP
of sub-bulge and suprabulbar areas, while CD200 expression was limited to the bulge and proximal part of sub-bulge area (Fig. 1c, S6).
ur na
Table 1 summarizes the expression patterns for K15, CD200, CD49f, and CD34 obtained using immunofluorescence staining. Then, to sort bulge live hair stem cells, we used CD200+ CD34- CD49f+, as well as CD200-CD49f+CD34+ cells were gated to purify progenitor cells
Jo
that localized below the bulge area in the outer root sheath.
Loss of hair follicle volume is associated with decreased numbers of follicular stem and progenitor cells during hair follicle miniaturization
6
Further, we checked the expression of the above markers’ combination in samples from the occipital (Fig. 2a) and frontal hairs by FACS analysis (Fig. 2b, c). We found two types of hair in the frontal samples, thicker (type 1; Fig. 2b), and miniature or thinner (type 2; Fig. 2c). Thicker hairs were smaller, thinner and less pigmented than hairs which were obtained from occipital scalp while the thinner were small, tiny, and nonpigmented. There was no significant
of
difference detected between the percentage of CD200+ CD49f+CD34- type 1 frontal hairs and occipital hairs (Fig. 2d). In contrast, these cells were strongly diminished in type 2 frontal
ro
hairs (P<0.001) (Fig. 2d). These data indicated that hair follicle stem cells were preserved in
-p
normal and thicker frontal hairs, and diminished in miniature thinner hairs.
We sought to determine if the CD200- CD49f+CD34+ cell populations were depleted in the
re
bald samples. Our results revealed that CD200-CD49f+CD34+ cells decreased approximately 8.3-fold in type 1 frontal hairs versus occipital hairs (Fig. 2b, d). There were no progenitor
lP
cells detected in the miniature hairs (P<0.001) (Fig. 2c, d). These results showed that the
AGA.
ur na
number of progenitor cells was dramatically decreased during miniaturization in men with
To unsure the accuracy of gating strategy, we performed real-time qRT-PCR to check cell population purity [4]. CD200 mRNA levels in follicular stem cells were approximately 24fold higher compared to the progenitor cells. CD34 mRNA levels in progenitor cells were
Jo
approximately 91-fold higher than the follicular stem cells. The expression level of CD49f did not significantly differ between the two cell types (P<0.001) (Fig. 2e). Therefore, these results confirmed the fidelity of our FACS strategy.
Follicular epithelial cell miRNA profiling identified specific miRNA signatures 7
We then wanted to examine if miRNAs were dynamically regulated in the bald scalps of men with AGA. We performed qRT-PCR analysis to explore miRNAs that are preferentially expressed in each FACS-purified fraction. In AGA patients’ occipital hairs are preserved [16], thus, occipital stem cells were called normal stem cells (NSCs) and occipital progenitor cells
cells from frontal type 1 were termed patient stem cells (PSCs).
of
named normal progenitor cells (NPCs). While, frontal hairs are miniaturized [16] and stem
ro
In total, we detected 252 miRNAs in all of the samples. In NSCs, 82 miRNAs were
-p
upregulated, whereas there were 41 miRNAs upregulated in NPCs, and 23 miRNAs that were specifically expressed in PSCs (Fig. 3a).
re
Unsupervised hierarchical clustering based on miRNA relative expression suggested that NSCs, NPCs, and PSCs were differentially expressed discrete miRNAs and showed that
lP
NSCs and NPCs were more similar to each other than to PSCs (Fig. 3b). Next, we determined which miRNAs were differentially expressed in each group (S7a -c,).
ur na
Further we examine the putative functional implication of our most differentially regulated miRNAs by first extracting their target genes using TargetScan 7.1 followed by pathway enrichment analysis using Enrichr [17] (Fig. S8).
Jo
MiR-324-3p promotes keratinocyte differentiation and migration, and inhibits proliferation
Further, we focus our attention on a particular miRNA, miR-324-3p, as it was strongly enriched in normal hair cells but not detected in bald cells (P<0.05) (Fig. 3b, S9). 8
Normally, mature hair has a regenerative cycling system maintained by a tightly regulated balance between keratinocyte proliferation, migration, and terminal differentiation [18, 19]. Impaired differentiation from stem to progenitor cells has been reported, and may contribute to the reduction of hair follicle size, in AGA [1]. We sought to determine whether the absence of miR-324-3p expression in bald follicular stem cells might contribute to these alterations.
of
To do so, we overexpressed miR-324-3p in an HEK001 keratinocyte culture system because bulge stem cell phenotypes are difficult to maintain in vitro[14, 20], and we measured the
ro
expression of differentiation markers K10 and K1, and basal cell markers (K14, Integrin β1).
-p
MiR-324-3p overexpression was confirmed by qRT-PCR (P<0.05) (Fig. S10).
When miR-324-3p was overexpressed in the cells, the expression of K14, integrin-β1, and
re
K10 did not change significantly compared to scramble. While, there was a significant increase in the expression of K1 (P<0.01) (Fig. 4a). Consistent with these results, HEK cells
lP
showed a higher K1 protein expression than cells treated with scramble (P<0.0001) (Fig. 4b, c). These data indicated that miR-324-3p promoted keratinocyte differentiation.
ur na
Then, we investigated the effects that modulating miR-324-3p would have on keratinocyte migration. Linear scratches were made on confluent sheets of HEKs treated with miR-324-3p or control. We observed, after 24 h, miR-324-3p-treated HEKs sealed the wound at a faster rate than control cells (P<0.0001) (Fig. 4d, e)
Jo
In the epidermis and epidermal appendages, proliferation and differentiation are mutually exclusive programs [21]. Therefore, we investigated the ability of miR-324-3p to regulate the switch between proliferative and differentiating keratinocytes. The switch from proliferation to differentiation in keratinocytes is concomitant with induction of the cell-cycle exit [22]. We checked CCNB1 (cyclin B1) and CCND1(cyclin D1) expressions. With the overexpression of 9
miR-324-3p, it was found that CCND1 expression was reduced (P<0.05) (Fig. 4f), suggesting a decrease in cell cycling. Next, we used Ki67 staining to distinguish cells that were in the active cell cycle from those that exited the cell cycle [21]. We found a significant reduction in cell proliferation as seen by the significantly decreased Ki67+ cells in the miR-324-3poverexpressed cells compared to the control group (P<0.01) (Fig. 4g).
of
Collectively, these results point to the miR-324-3p role in supporting keratinocyte cells exit
ro
from the cell cycle to embark on the differentiation and migratory program [8].
-p
MiRNA-324-3p controls keratinocyte differentiation by modulating the activity of
re
MAPK and TGF-β pathways
We next investigated the mechanism underlying miR-324-3p contribution to keratinocyte
lP
differentiation. The possible miR-324-3p targets were predicted by the TargetScan 7.1 prediction algorithm and evaluated by Enrichr software. A search of the database of the
ur na
KEGG showed that miR-324-3p target genes were mainly involved in Ras, phosphatidylinositol, ErbB, mTOR, and MAPK signaling pathways (Fig. 5a). Next, we sought to investigate the expression patterns of the targets of miR-324-3p that might explain the observed differentiation phenotype after its overexpression in vitro. Using qRT-PCR, we
Jo
evaluated the expression of nine targets related to the five most enriched pathways. Our results showed that the expression of MAPK1, MAPK3, REL A, HSPA2, and TGF-β3 was downregulated in cultured keratinocytes in miR-324-3p-overexpressed cells (P<0.05, P<0.01); however, the expression level of REL B was upregulated (P<0.001) (Fig. 5b). These findings confirm MAPK and TGF-β signaling pathways regulation by miR-324-3p in our system. 10
In order to analyze the potential role of MAPK signaling pathways in mediating the differentiating effects of miR-324-3p, we used specific chemical inhibitors that target these pathways. Keratinocytes were separately treated with PD0325901 (inhibitor of MEK), SP600125 (inhibitor of JNK), and SB203580 (inhibitor of p38 MAPK). To inhibit TGF-β, Alk5i and SB431542 were used. In addition to TGF-β3, according to TargetScan 7.1 algoritm,
of
transforming growth factor beta receptor associated protein 1 is one of the targets of miR-3243p.
ro
Next, we analyzed the expressions of K14, Integrinβ1, K10, K1, CCDN1, and CCNB1 by
-p
qRT-PCR. Similar to what we observed with miR-324-3p, HEKs treated with either PD0325901, SB431542, SP600125, or Alk5i small molecules exhibited increased K1
re
expression (P<0.05, P<0.01). Notably, these small molecules increased K10 expression, as an early keratinocyte differentiation marker (P<0.05, P<0.01, P<0.001), whereas SB203580 had
lP
no effect on differentiation (Fig. 5c-g). Blocking MEK, JNK, and TGF-β signaling pathways by PD0325901, SP600125, and Alk5i small molecules increased the expression of K14, a
ur na
basal cell marker (P<0.05, P<0.01, P<0.001) (Fig. 5c, e, f). Moreover, SP600125 increased the expression levels of CCDN1 and CCNB1 while Alk5i decreased their expression levels (P<0.05, P<0.01). These transcripts did not appear significantly altered in the presence of other small molecules (Fig. 5c, d, g). Taken together,
Jo
these findings supported the idea that downregulation of the MEK, JNK, and TGF-β signaling pathways could ultimately accelerate keratinocyte differentiation. Because PD0325901 and Alk5i small molecules had increased K1 expression more than the other small molecules, we checked their effects on human hair in an anagen organ-cultured hair follicle system [23]. Compared with the control, PD0325901 samples, we observed that 11
treatment with Alk5i significantly increased the lengths of the hair shafts after eight days
of
(P<0.01) (Fig. 5h), which indicated a positive effect on the rate of hair elongation.
-p
ro
Discussion
Hair loss can occur with factors that cause abnormalities in HF cycling, decreases in follicular
re
stem cell activity, or failure in regrowth of hair fibers from existing HFs [18]. Recently, Garza et al. reported that in AGA, the lack of an activator or existing inhibitor could lead to
follicle size [3].
lP
problems with follicular stem cell conversion into progenitor cells and result in decreased hair
ur na
On the other hand, a role for miRNAs at the post-transcriptional level in regulating skin and hair stem cell proliferation and differentiation has been reported [4, 8, 24, 25]. In this study, we characterized human hair cells in vivo by immunofluorescence and revealed that CD200+CD49f+CD34- cells were located in the bulge area of normal occipital hair. We
Jo
checked for the presence of these cells in bald samples by evaluating two types of hair in the frontal area, thicker (type 1) and miniature or thinner (type 2). Notably, we observed that type 1 frontal hairs preserved the CD200+CD49f+CD34- cells, similar to the non-bald samples, whereas these cells were not detected in the miniature or type 2 hairs. These results agreed with those reported by Garza et al., who proposed a gradual decrease in the number of stem 12
cells in the miniaturized HF, thereby resulting in a reduction in HF volume during miniaturization and reversible hair loss [3]. CD34+CD49f+CD200- cells, which are found in the sub-bulge and proximal part of the suprabulbar area, are remarkably diminished in the frontal hairs of bald samples. In line with these results, Garza et al. demonstrated that a loss of progenitor cells, but not stem cells, contributed to male pattern baldness in humans [3].
of
Decreased numbers of these cells could potentially result from diminished conversion of hair follicle stem cells to progenitor cells and lead to miniaturization as well as a decreased
ro
amount of anagen follicles in the bald scalp [3].
-p
. Then, we used a miRNA expression profiling approach to identify miRNAs in hair epithelial cells that might contribute to AGA.
re
To the best of our knowledge, this study represents the first data set of results for miRNA expression profiling from normal and AGA follicular epithelial cells. The remarkable
lP
differences in miRNA expression patterns between normal and patient epithelial hair cells suggested that the miRNAs might serve important functions in the pathogenesis of AGA.
ur na
Interestingly, we found no expression of miR-324-3p in bald follicular stem cells. To date, nothing is known about the function of miR-324-3p in hair epithelial cells. MiR-324-3p is dysregulated in breast, hepatocellular, and pancreatic cancers. Previous reports have shown that miR-324-3p inhibited invasion of nasopharyngeal carcinoma cells by
Jo
targeting WNTB2 [26] and promoted the shift of neuroepithelial-like stem cells from selfrenewal to neuronal differentiation [27]. Overexpression of hsa-miR-324-3p mimics in HEKs promoted expression of a late differentiation marker, K1, but did not affect the expression of the early differentiation marker, K10, and basal markers, K14 and integrin β1. Consistent with this mode of regulation, 13
Fuchs et al. showed that miR-125b inhibited hair lineage differentiation without affecting maintenance or activation of hair follicle stem cells in mice [4]. Treatment of HEKs with hsamiR-324-3p mimic increased the ability of these cells to seal linear scratch wounds after 24 h and reduced keratinocyte proliferation as well as exited them from the cell cycle. Then, we performed in silico target prediction for miR-324-3p to gain additional insight into
of
its effects and finding the pathway(s) by which it works. Among nine predicted targets for miR-324-3p, we noted that REL A, HSP A2, MAPK1,3, and TG-β3 expression was
ro
significantly decreased in cells overexpressing miR-324-3p compared to the control.
-p
Previous studies have shown that RelA acts as an inhibitor of keratinocyte growth [28, 29]. Another study indicated that specific deletion of RelA did not have any effect on epidermal
re
differentiation [30]. HSPA2 knockdown by RNAi in HaCaT cells has been shown to accelerate keratinocyte differentiation [31]. Hashizume et al. observed increased expressions
lP
of HSP 60 and 72 in the cycling hair bulb during mid-anagen VI and an additional increase in catagen development, which might be associated with apoptosis of follicle keratinocytes [32].
ur na
Therefore, absence of miR-324-3p in bald hair keratinocyte cells might increase the level of HSP72 and consequently cause an increase in keratinocyte apoptosis and resting in catagen phase.
Previously, it has been shown that the MAPK cascade was implicated in keratinocyte terminal
Jo
differentiation and epidermal hyper proliferation in psoriasis and wound healing. Forced activation of MAPK signaling in primary human keratinocytes and reconstituted epidermis by retroviral infection caused an increase in growth rate and delayed the onset of keratinocyte differentiation [19]. Similar to this study, we observed that overexpression of miR-324-3p led to downregulation of MAPK1 and MAPK3, and caused a reduction of cyclin D1 and increased 14
K1 expression. Analysis of the potential changes in the signaling pathway activation state in patients treated with 5% minoxidil topical foam has shown downregulation of the MAPK signaling pathway in the vertex and front scalp of responder patients [33]. This suggests that miR-324-3p might contribute to AGA therapies trough modulating MAPK signaling. Dihydrotestosterone (DHT) stimulates synthesis of TGF-β that leads to premature entry of
of
hair follicles into the catagen phase and induces male pattern baldness. TGF-β has three isoforms: TGF-β1, 2, and 3 which are expressed in different parts of hair structure and bound
ro
to type I (Alk5) and receptors type II (Tgfbr2). Soma et al. showed that TGF-β1 and TGF-β2
-p
were involved in catagen induction of the human hair cycle in a human hair organ culture [3437].
re
Collectively, these findings suggested that the differentiation effects of miR-324-3p might be mediated through the MAPK and TGF-β signaling pathways, in addition to downregulation of
lP
HSP 72 and REL A. Among these candidates, we were interested in evaluating the role of the MAPK and TGF-β signaling pathways in keratinocyte differentiation. Thus, specific
ur na
chemicals were used to inhibit these two pathways. Our results showed that PD0325901 and Alk5i could function in a manner similar to miR-324-3p in promoting the expression of cytokeratin1 in keratinocytes more than the other small molecules. Then, we used an anagen organ-cultured hair follicle system to address the role of these
Jo
signaling pathways in human hair follicle growth. Inhibition of TGF-β signaling led to increased hair shaft elongation, raising the possibility that the migration and differentiation effects ascribed to miR-324-3p are also involved in human bulge stem cell differentiation and migration of AGA.
15
In summary, we found gradual decrease in the number of follicular stem cells during hair loss in AGA, might be resulting in a reduction in HF volume during miniaturization and reversible hair loss. Moreover, we have identified miR-324-3p which was strongly enriched in normal follicular epithelial cells in comparison to bald ones. Also, we have shown miR-324-3p as a novel regulator of cell proliferation, differentiation and migration of keratinocytes.
of
Furthermore, miRNA-324-3p controls keratinocyte differentiation likely by modulating the activity of MAPK and TGF-β pathways (Fig. S11), thus, it may be a promising new
ro
therapeutic target for hair loss by topical application of an miR-324-3p mimic. Although these
-p
data are persuasive, the physiological response to miR-324-3p mimic in human scalp affected with AGA needs to be determined in the preclinical and clinical context. Also, further studies
re
are required to determine the regulatory mechanism between miR-324-3p expression and
mechanism in AGA.
lP
TGF-β downregulation, which may provide a better understanding of the hair miniaturization
Moreover, other miRs, such as miR-148b-3p, miR-196a-5p, miR-652-3p, and miR-328-3p
ur na
showing similar expression pattern changes like miR-324-3p, can be remarkable candidates for finding pathogenic mechanisms of hair miniaturization in future investigation.
Jo
Acknowledgments
This study was funded by a grant provided from Royan Institute and Disease Models & Mechanisms Travelling Fellowship by Biologists Company. We express our appreciation to all members of the Skin Program at Royan Institute and Cedric Blanpain lab for their helpful deliberations and consultation during this work. We would like to thank Dr Kiarash 16
Khosrotehrani, Dr Saeid Amini Nik, and Dr Seyedeh Nafiseh Hassani for their critical
Jo
ur na
lP
re
-p
ro
of
comments.
17
References
Jo
ur na
lP
re
-p
ro
of
[1] J. Qi, L.A. Garza, An overview of alopecias, Cold Spring Harb Perspect Med. 4(3) (2014). [2] E.G.Y. Chew, J.H.J. Tan, A.W. Bahta, B.S. Ho, X. Liu, T.C. Lim, Y.Y. Sia, P.L. Bigliardi, S. Heilmann, A.C.A. Wan, M.M. Nothen, M.P. Philpott, A.M. Hillmer, Differential Expression between Human Dermal Papilla Cells from Balding and Non-Balding Scalps Reveals New Candidate Genes for Androgenetic Alopecia, J Invest Dermatol. 136(8) (2016) 1559-1567. [3] L.A. Garza, C.C. Yang, T. Zhao, H.B. Blatt, M. Lee, H. He, D.C. Stanton, L. Carrasco, J.H. Spiegel, J.W. Tobias, G. Cotsarelis, Bald scalp in men with androgenetic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells, J Clin Invest. 121(2) (2011) 613-22. [4] L. Zhang, N. Stokes, L. Polak, E. Fuchs, Specific microRNAs are preferentially expressed by skin stem cells to balance self-renewal and early lineage commitment, Cell Stem Cell. 8(3) (2011) 294-308. [5] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell. 136(2) (2009) 215-33. [6] R. Yi, D. O'Carroll, H.A. Pasolli, Z. Zhang, F.S. Dietrich, A. Tarakhovsky, E. Fuchs, Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs, Nat Genet. 38(3) (2006) 356-62. [7] T. Andl, E.P. Murchison, F. Liu, Y. Zhang, M. Yunta-Gonzalez, J.W. Tobias, C.D. Andl, J.T. Seykora, G.J. Hannon, S.E. Millar, The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles, Curr Biol. 16(10) (2006) 1041-9. [8] R. Yi, M.N. Poy, M. Stoffel, E. Fuchs, A skin microRNA promotes differentiation by repressing 'stemness', Nature. 452(7184) (2008) 225-9. [9] S. Yuan, F. Li, Q. Meng, Y. Zhao, L. Chen, H. Zhang, L. Xue, X. Zhang, C. Lengner, Z. Yu, Post-transcriptional Regulation of Keratinocyte Progenitor Cell Expansion, Differentiation and Hair Follicle Regression by miR-22, PLoS Genet. 11(5) (2015) e1005253. [10] A.N. Mardaryev, M.I. Ahmed, N.V. Vlahov, M.Y. Fessing, J.H. Gill, A.A. Sharov, N.V. Botchkareva, Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle, FASEB J. 24(10) (2010) 3869-81. [11] B.K. Kim, S.K. Yoon, Hairless Up-Regulates Tgf-beta2 Expression via Down-Regulation of miR-31 in the Skin of "Hairpoor" (HrHp) Mice, J Cell Physiol. 230(9) (2015) 2075-85. [12] Z. Wenguang, W. Jianghong, L. Jinquan, M. Yashizawa, A subset of skin-expressed microRNAs with possible roles in goat and sheep hair growth based on expression profiling of mammalian microRNAs, OMICS. 11(4) (2007) 385-96. [13] M.I. Ahmed, M. Alam, V.U. Emelianov, K. Poterlowicz, A. Patel, A.A. Sharov, A.N. Mardaryev, N.V. Botchkareva, MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway, J Cell Biol. 207(4) (2014) 549-67. [14] K. Inoue, N. Aoi, T. Sato, Y. Yamauchi, H. Suga, H. Eto, H. Kato, J. Araki, K. Yoshimura, Differential expression of stem-cell-associated markers in human hair follicle epithelial cells, Lab Invest. 89(8) (2009) 844-56. [15] M. Ohyama, A. Terunuma, C.L. Tock, M.F. Radonovich, C.A. Pise-Masison, S.B. Hopping, J.N. Brady, M.C. Udey, J.C. Vogel, Characterization and isolation of stem cell-enriched human hair follicle bulge cells, J Clin Invest. 116(1) (2006) 249-60. [16] D. Rathnayake, R. Sinclair, Male androgenetic alopecia, Expert Opin Pharmacother. 11(8) (2010) 1295-304. 18
Jo
ur na
lP
re
-p
ro
of
[17] S. Moradi, A. Sharifi-Zarchi, A. Ahmadi, S. Mollamohammadi, A. Stubenvoll, S. Gunther, G.H. Salekdeh, S. Asgari, T. Braun, H. Baharvand, Small RNA Sequencing Reveals Dlk1-Dio3 Locus-Embedded MicroRNAs as Major Drivers of Ground-State Pluripotency, Stem Cell Reports. 9(6) (2017) 2081-2096. [18] P. Mohammadi, K.K. Youssef, S. Abbasalizadeh, H. Baharvand, N. Aghdami, Human Hair Reconstruction: Close, But Yet So Far, Stem Cells Dev. 25(23) (2016) 1767-1779. [19] I. Haase, R.M. Hobbs, M.R. Romero, S. Broad, F.M. Watt, A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis, J Clin Invest. 108(4) (2001) 527-36. [20] L. Xie, R. Yang, S. Liu, S. Lyle, G. Cotsarelis, L. Xiang, L. Zhang, B. Li, M. Wan, X. Xu, TR3 is preferentially expressed by bulge epithelial stem cells in human hair follicles, Lab Invest. 96(1) (2016) 81-8. [21] I. Amelio, A.M. Lena, G. Viticchiè, R. Shalom-Feuerstein, A. Terrinoni, D. Dinsdale, G. Russo, C. Fortunato, E. Bonanno, L.G. Spagnoli, miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration, J Cell Biol. 199(2) (2012) 347-363. [22] C. Kolly, M.M. Suter, E.J. Muller, Proliferation, cell cycle exit, and onset of terminal differentiation in cultured keratinocytes: pre-programmed pathways in control of C-Myc and Notch1 prevail over extracellular calcium signals, J Invest Dermatol. 124(5) (2005) 1014-25. [23] S. Harel, C.A. Higgins, J.E. Cerise, Z. Dai, J.C. Chen, R. Clynes, A.M. Christiano, Pharmacologic inhibition of JAK-STAT signaling promotes hair growth, Sci Adv. 1(9) (2015) e1500973. [24] L. Zhang, Y. Ge, E. Fuchs, miR-125b can enhance skin tumor initiation and promote malignant progression by repressing differentiation and prolonging cell survival, Genes Dev. 28(22) (2014) 2532-46. [25] D. Aberdam, E. Candi, R.A. Knight, G. Melino, miRNAs, 'stemness' and skin, Trends Biochem Sci. 33(12) (2008) 583-91. [26] C. Liu, G. Li, N. Yang, Z. Su, S. Zhang, T. Deng, S. Ren, S. Lu, Y. Tian, Y. Liu, Y. Qiu, miR324-3p suppresses migration and invasion by targeting WNT2B in nasopharyngeal carcinoma, Cancer Cell Int. 17 (2017) 2. [27] L. Stappert, L. Borghese, B. Roese-Koerner, S. Weinhold, P. Koch, S. Terstegge, M. Uhrberg, P. Wernet, O. Brüstle, MicroRNA-based promotion of human neuronal differentiation and subtype specification, PloS one. 8(3) (2013) e59011. [28] C.S. Seitz, Q. Lin, H. Deng, P.A. Khavari, Alterations in NF-κB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-κB, Proc Natl Acad Sci U S A. 95(5) (1998) 2307-2312. [29] J.Y. Zhang, C.L. Green, S. Tao, P.A. Khavari, NF-κB RelA opposes epidermal proliferation driven by TNFR1 and JNK, Genes Dev. 18(1) (2004) 17-22. [30] Y. Grinberg-Bleyer, T. Dainichi, H. Oh, N. Heise, U. Klein, R.M. Schmid, M.S. Hayden, S. Ghosh, Cutting edge: NF-kappaB p65 and c-Rel control epidermal development and immune homeostasis in the skin, J Immunol. 194(6) (2015) 2472-6. [31] A. Gogler‐Pigłowska, K. Klarzyńska, D.R. Sojka, A. Habryka, M. Głowala‐Kosińska, M. Herok, M. Kryj, M. Halczok, Z. Krawczyk, D. Scieglinska, Novel role for the testis‐enriched HSPA2 protein in regulating epidermal keratinocyte differentiation, J Cell Physiol. 233(3) (2018) 2629-2644. 19
Jo
ur na
lP
re
-p
ro
of
[32] H. Hashizume, Y. Tokura, M. Takigawa, R. Paus, Hair cycle‐dependent expression of heat shock proteins in hair follicle epithelium, Int J Dermatol. 36(8) (1997) 587-592. [33] G.N. Stamatas, J. Wu, A. Pappas, P. Mirmirani, T.S. McCormick, K.D. Cooper, M. Consolo, J. Schastnaya, I.V. Ozerov, A. Aliper, A. Zhavoronkov, An analysis of gene expression data involving examination of signaling pathways activation reveals new insights into the mechanism of action of minoxidil topical foam in men with androgenetic alopecia, Cell Cycle. 16(17) (2017) 1578-1584. [34] T. Soma, M. Ogo, J. Suzuki, T. Takahashi, T. Hibino, Analysis of Apoptotic Cell Death in Human Hair Follicles In Vivo andIn Vitro, J Invest Dermatol. 111(6) (1998) 948-954. [35] T. Soma, Y. Tsuji, T. Hibino, Involvement of transforming growth factor-β2 in catagen induction during the human hair cycle, J Invest Dermatol. 118(6) (2002) 993-997. [36] T. Hibino, T. Nishiyama, Role of TGF-beta2 in the human hair cycle, J Dermatol Sci. 35(1) (2004) 9-18. [37] H.Y. Lin, L.T. Yang, Differential response of epithelial stem cell populations in hair follicles to TGF-beta signaling, Dev Biol. 373(2) (2013) 394-406.
20
lP
re
-p
ro
of
Figure legends:
ur na
Fig. 1 Differential expressions of K15, CD200, CD49f, and CD34 markers in longitudinal section of human anagen hair. a: Occipital human hair is divided into five portions: INF, BU, SBU, SBUL, and BUL. b: Evaluation of CD200 and K15 expression. Higher magnification images indicated by dashed box. Arrows show CD200 and K15 expression. c: Evaluation of CD200 and CD34 expressions. Higher magnification images indicated by dashed box. Arrows show CD200 and CD34 expression. The nuclei were stained with DAPI. INF: Infundibulum BU: Bulge,
Jo
SBU: Sub-bulge, SBUL: Suprabulbar, BUL: Bulb, SG: sebaceous gland, APM: arrector pili muscle, PSBU: Proximal Sub-bulge, DSBU: Distal Sub-bulge, SBUL: Supra-bulbar.
21
of ro -p re lP ur na Jo Fig. 2 Characterization and purification of follicular stem and progenitor cells from occipital and frontal hairs using fluorescence-activated cell sorting. Samples gated by debris exclusion (P1: FSC /SCC) and living cells (P2: PI dye exclusion). a: Occipital samples: NSC: P3-a=CD34-CD49f+, P4-a= CD200+CD49f+CD34- and NPC: P3-b=CD200-
22
CD49f+, P4-b= CD200-CD49f+CD34+. b: Frontal type 1: P3=CD34-CD49f+, P4=CD200+CD49f+CD34-. c: frontal type 2: P3=CD34-CD49f+, P4= CD200+CD49f+CD34-. d: Quantification of the percentage of follicular stem and progenitor cells in occipital and frontal hairs. Mean±SD, ***P<0.001. e: RT-qPCR quantification of CD49f, CD34 and CD200 in sorted NSCs and NPCs compared with total hair cells. GAPDH was used as the reference gene (n=3). Mean ± SE, ***P<0.001. FSC: Forward scatter, SSC: Side scatter, NSC: normal stem cells, NPC: normal
Jo
ur na
lP
re
-p
ro
of
progenitor cells, PSC: patient stem cells, ND: Not detected.
Fig. 3 Differential expression of microRNAs of follicular stem and progenitor cells from normal and bald hairs. a: Venn diagram of all and differentially miRNAs expressed. b: Heat map and unsupervised hierarchical clustering display of miRNA expression profiles of NSC, NPC, and PSC samples. The clustering is performed on all samples and on the top 50 miRNAs with the highest standard deviation. Each row represents one miRNA and each column represents one sample. The miRNA clustering tree is shown on the left. NSC: normal stem cells, NPC: normal progenitor cells, PSC: patient stem cells.
23
of ro -p re lP ur na Jo
Fig. 4 miR-324-3p regulates keratinocyte differentiation, proliferation and migration. a: Real-time qPCR analysis of K10, K1, K14 and ITGβ1 72 h following HEK001 transfection with synthetic mimic miR-324-3p and scramble. b, c: Immunostaining (b) and quantification (c) of K1 in HEK001 cells, 72 h after transfecting. The nuclei were stained by DAPI (Blue). d, e: Phase-contrast micrographs (d) and quantification (e) of transfected HEKs migrating into scratched region. f: qRT-PCR analysis of CCNB1 and CCND1 72 h following HEK001 transfection. g: Flow
24
cytometry analysis of Ki67 expression 24 h after transfecting. Scale bar: 100 μm. GAPDH was used as an internal
ur na
lP
re
-p
ro
of
normalization control. Data are shown as mean ± SE, n=3, *P<0.05, **P<0.01, ****P<0.0001.
Jo
Fig. 5 Effect of miR-324-3p, PD0325901, SB431542, Alk5i, SP600125, and SB203580 on keratinocyte differentiation program in vitro. a: Probable pathways involved by the genes targeted by miR-324-3p. b: qRT-PCR analysis of K14, Integrinβ1, K10, K1, CCDN1, and CCNB1 transcripts 3 days after keratinocytes treatment with miR-324-3p or scramble. Data are shown as mean±SD, n=3. c-g: qRT-PCR analysis of K14, Integrinβ1, K10, K1, CCDN1, and CCNB1 transcripts 3 days following keratinocytes treatment with PD0325901, SB431542, Alk5i, SP600125, and SB203580. Data are shown as mean±SD, n=3. h: Quantification of the length of the hair shaft treated
25
with culture medium, PD0325901 and Alk5i after 8 days. Data are mean±SE, n=5. *P<0.05, **P<0.01,
Jo
ur na
lP
re
-p
ro
of
***P<0.001,0.002. GAPDH was used as an internal normalization control.
26
Table: Table 1: Summary of immunofluorescence staining patterns of the human anagen hair follicles. Table 1 : Summary of immunofluorescence staining patterns in longitudinal sections of human anagen hair follicles Marker/Replicate Epidermis Infudibulum Bulge Sub-bulge Supra bulbar area Distal
Proximal Medial
Distal
B -
S -
ro
-p
re
lP ur na Jo 27
B -
S -
B -
S -
of
Proximal
B S B S B S B S B S CD200 1 ++ +/- +/- 2 ++ +/- +/- 3 + +/- 4 + +/- 5 + + +/- +/- K15 1 + + + + + + 2 + + + + + + 3 + + + + + +/- + +/CD34 1 + 2 +/- +/- + + 3 + 4 +/- +/B : basal cells, S : Supra basal cells - :no staining, +/- : marginally or partially positive, + : positive, ++ : strong positive
-
-
+ -
-
+ +/+
-
+ + + +
+ +
+ -
-
-
-
PII:
S0923-1811(20)30351-0
DOI:
https://doi.org/10.1016/j.jdermsci.2020.11.002
Reference:
DESC 3679
To appear in:
Journal of Dermatological Science
Received Date:
30 April 2020
Revised Date:
22 October 2020
Accepted Date:
3 November 2020
Please cite this article as: Mohammadi P, Nilforoushzadeh MA, Youssef KK, Sharifi-Zarchi A, Moradi S, Khosravani P, Aghdami R, Taheri P, Hosseini-Salekdeh G, Baharvand H, Aghdami N, Defining microRNA signatures of hair follicular stem and progenitor cells in healthy and androgenic alopecia patients, Journal of Dermatological Science (2020), doi: https://doi.org/10.1016/j.jdermsci.2020.11.002
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier.
Defining microRNA signatures of hair follicular stem and progenitor cells in healthy and androgenic alopecia patients Parvaneh Mohammadia,b,c, Mohammad Ali Nilforoushzadehd, Khalil Kass Youssefe, Ali Sharifi-Zarchif, Sharif Moradia, Pardis Khosravania, Raheleh Aghdamia,c, Payam Taheria, Ghasem Hosseini-Salekdeha, Hossein Baharvanda,b, Nasser Aghdamic* Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute
of
a.
for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Department of Developmental Biology, University of Science and Culture, Tehran, Iran.
c.
Department of Regenerative Medicine, Cell Science Research Center, Royan Institute for Stem Cell
ro
b.
-p
Biology and Technology, ACECR, Tehran, Iran.
Skin and Stem Cell Research Center, Tehran University of Medical Science, Tehran, Iran.
e.
Instituto de Neurociencias (CSIC-UMH), Sant Joan d'Alacant, Spain.
f.
Computer Engineering Department, Sharif University of Technology, Tehran, Iran.
re
d.
lP
*Correspondence
Nasser Aghdami: [email protected]
Highlights
ur na
Tel: +98 21 22306485
Introducing a platform for understanding miRNA dynamic regulation in hair follicular cells in baldness
Jo
This study represents that miR-324-3p is depleted in bald stem cells compared to normal hair stem and progenitor cells
MiR-324-3p overexpression impact significantly keratinocytes differentiation program, promotes cell migration and reduces proliferation.
1
Abstract Background The exact pathogenic mechanism causes hair miniaturization during androgenic alopecia (AGA) has not been delineated. Recent evidence has shown a role for non-coding regulatory RNAs, such as microRNAs (miRNAs), in skin and hair disease. There is no reported information about the role of miRNAs in hair epithelial cells of
of
AGA. Objectives
ro
To investigate the roles of miRNAs affecting AGA in normal and patient’s epithelial hair cells.
-p
Methods
Normal follicular stem and progenitor cells, as well as follicular patient’s stem cells, were sorted from hair
re
follicles, and a miRNA q-PCR profiling to compare the expression of 748 miRNA (miRs) in sorted cells were performed. Further, we examined the putative functional implication of the most differentially regulated miRNA
lP
(miR-324-3p) in differentiation, proliferation and migration of cultured keratinocytes by qRT-PCR, immunofluorescence, and scratch assay. To explore the mechanisms underlying the effects of miR-324-3p, we used specific chemical inhibitors targeting pathways influenced by miR-324-3p.
ur na
Result: We provide a comprehensive assessment of the "miRNome" of normal and AGA follicular stem and progenitor cells. Differentially regulated miRNA signatures highlight several miRNA candidates including miRNA-324-3p as mis regulated in patient’s stem cells. We find that miR-324-3p promotes differentiation and migration of cultured keratinocytes likely through the regulation of mitogen-activated protein kinase (MAPK)
Jo
and transforming growth factor (TGF)-β signaling. Importantly, pharmacological inhibition of the TGF-β signaling pathway using Alk5i promotes hair shaft elongation in an organ-culture system. Conclusion: Together, we offer a platform for understanding miRNA dynamic regulation in follicular stem and progenitor cells in baldness and highlight miR-324-3p as a promising target for its treatment.
Keywords: miRNA, androgenic alopecia, hair follicular stem cells, miR-324-3p Abbreviations: AGA: Androgenic Alopecia, miRNAs: microRNAs, NSCs: Normal Stem Cells, NPCs: Normal Progenitor Cells,
2
PSCs: Patient Stem Cells, miRs: miRNA, MAPK: Mitogen-Activated Protein Kinase, TGF-β: transforming growth factor, FACS: Fluorescence-Activated Cell Sorting, KEGG: Kyoto Encyclopedia of Genes and Genomes, HFs: Hair Follicles, APM: Arrector Pili Muscle.
Introduction
Androgenic alopecia (AGA) is the most common type of hair loss [1]. Suggested causes for
of
this gradual hair loss include androgen dependence and genetic predisposition [2]. Recently,
may contribute to a gradual decrease in hair size in AGA [3].
ro
Cotsarelis et al. showed that defects in human hair stem cells conversion to progenitor cells
-p
The hair cycle is regulated by intrinsic and extrinsic factors at the transcriptional and posttranscriptional levels [4]. MicroRNAs (miRNAs), which are ~22-nucleotide non-coding
re
RNAs, regulate the expression of many genes at the post-transcriptional level, thereby
lP
controlling virtually all biological processes and pathways [5]. Pioneering studies by Fuchs and Millar showed that miRNAs have a critical role in mesenchymal-epithelial interaction,
ur na
hair follicle placode invagination, and mouse hair morphogenesis [6, 7]. Exploring the functions of individual miRNAs in skin and hair cells has shown that miRNA-203 acts as a switch between proliferation and differentiation in the skin [8], and miRNA-22 is reported as a critical post-transcriptional regulator of mouse hair cycle [9]. Other studies report
Jo
differential expression of miRNA-31 during mouse hair cycle [10] and upregulation of transforming growth factor (TGF)-2 via downregulation of miRNA-31 by a Hairless gene that causes an abnormal hair cycle in hairpoor mice [11].
3
Another key miRNA, miRNA-125b, has been reported to be responsible for stem cell commitment in the hair follicle [4]; thus, a miRNA network that governs hair follicle development and hair loss is beginning to be identified. Although substantial progress has been made in discovering the significant roles of miRNAs in hair follicle development and hair cycle-associated tissue remodeling, most information
of
comes from investigations in cell cultures, mice, sheep, and goat models [12, 13]. Hence, future studies should emphasize the roles of miRNAs in human hair development and hair
ro
loss. In the present study, we have generated miRNA signatures for three types of cell
-p
populations from hair follicles: normal stem cells (NSCs), normal progenitor cells (NPCs), and patient’s stem cells (PSCs). We observed that miR-324-3p was depleted in PSCs
re
compared to normal NSCs and NPCs and found that the miR-324-3p overexpression impact significantly keratinocytes differentiation program, promotes cell migration and reduces
lP
proliferation. Notably, small molecules that inhibited putative miR-324-3p targets, mitogenactivated protein kinase (MAPK) and transforming growth factor (TGF)-β signaling
ur na
pathways, phenocopied miR-324-3P overexpression in keratinocytes. Finally, the inhibition of the TGF-β signaling pathway by Alk5i in an anagen organ-cultured hair follicle system promoted hair shaft elongation. Taken together, these data provide an important foundation for further analysis of miR-324-3p as a probable candidate to regulate human hair
Jo
keratinocyte differentiation and also new effective therapy to treat AGA.
Materials and Methods
Hair follicle samples 4
Human occipital and frontal samples were taken from the scalp of volunteer donors who suffered from AGA during hair transplantation. The Institutional Review Board and Ethical Committee of Royan Institute and Tehran University approved this study. All tissue donors were males with an average age of 35 years (range: 25-50 years). The normal samples
of
consisted of a scalp strip (1.5 cm × 1.5 cm) taken from the occipital area. The bald samples were cylindrical punches removed from the frontal scalp because of the creation of recipient
ro
sites for donor occipital scalp. A complete description of the methodology used in this study
-p
is provided in the supplementary material.
re
Results
lP
Characterizing human hair stem and progenitor cells in anagen hair
ur na
To evaluate miRNA expression in human hair bulge stem and progenitor cells, we first define markers combination sufficient to purify reliably hair follicle populations. Occipital human hair is divided into five portions : infundibulum, bulge, sub-bulge, suprabulbar, and bulb (Fig. 1a) [14]. Moreover, we divided the sub-bulge region into proximal and distal part and
Jo
suprabulbar area into three parts the proximal, medial, and distal. Therefore, we analyzed by immunofluorescence the expression of CD200, K15, CD34, and CD49f markers, widely used previously to define murine and human stem and progenitor cell populations (Fig. S1-4). CD200, a hair stem cell marker [15], was expressed in bulge area
5
(between the sebaceous gland and the insertion of arrector pili muscle (APM)), with weaker and clearly decaying expression in the proximal part of the sub-bulge area (Fig. S1). K15, another hair stem cell marker [3], was more extended covering the bulge, infundibulum, sub-bulge, and distal part of the suprabulbar area (Fig. S2). CD34, a marker of progenitor cells [15], was expressed specifically in the sub-bulge
of
expanding to the proximal part of the suprabulbar area (Fig. S3). Immunofluorescence data also revealed that CD49f, a basal cell marker [14], was extensively
ro
expressed in all epithelial cells of the hair outer root sheath and basal cells of skin epidermis
-p
(Fig. S4).
Double immunostaining results confirmed co-expression of K15 and CD200 in the bulge and
re
expression of K15 in the sub-bulge and suprabulbar area (Fig. 1b, S5). Also, double immunostaining of CD34 and CD200 showed CD34 expression in the distal part
lP
of sub-bulge and suprabulbar areas, while CD200 expression was limited to the bulge and proximal part of sub-bulge area (Fig. 1c, S6).
ur na
Table 1 summarizes the expression patterns for K15, CD200, CD49f, and CD34 obtained using immunofluorescence staining. Then, to sort bulge live hair stem cells, we used CD200+ CD34- CD49f+, as well as CD200-CD49f+CD34+ cells were gated to purify progenitor cells
Jo
that localized below the bulge area in the outer root sheath.
Loss of hair follicle volume is associated with decreased numbers of follicular stem and progenitor cells during hair follicle miniaturization
6
Further, we checked the expression of the above markers’ combination in samples from the occipital (Fig. 2a) and frontal hairs by FACS analysis (Fig. 2b, c). We found two types of hair in the frontal samples, thicker (type 1; Fig. 2b), and miniature or thinner (type 2; Fig. 2c). Thicker hairs were smaller, thinner and less pigmented than hairs which were obtained from occipital scalp while the thinner were small, tiny, and nonpigmented. There was no significant
of
difference detected between the percentage of CD200+ CD49f+CD34- type 1 frontal hairs and occipital hairs (Fig. 2d). In contrast, these cells were strongly diminished in type 2 frontal
ro
hairs (P<0.001) (Fig. 2d). These data indicated that hair follicle stem cells were preserved in
-p
normal and thicker frontal hairs, and diminished in miniature thinner hairs.
We sought to determine if the CD200- CD49f+CD34+ cell populations were depleted in the
re
bald samples. Our results revealed that CD200-CD49f+CD34+ cells decreased approximately 8.3-fold in type 1 frontal hairs versus occipital hairs (Fig. 2b, d). There were no progenitor
lP
cells detected in the miniature hairs (P<0.001) (Fig. 2c, d). These results showed that the
AGA.
ur na
number of progenitor cells was dramatically decreased during miniaturization in men with
To unsure the accuracy of gating strategy, we performed real-time qRT-PCR to check cell population purity [4]. CD200 mRNA levels in follicular stem cells were approximately 24fold higher compared to the progenitor cells. CD34 mRNA levels in progenitor cells were
Jo
approximately 91-fold higher than the follicular stem cells. The expression level of CD49f did not significantly differ between the two cell types (P<0.001) (Fig. 2e). Therefore, these results confirmed the fidelity of our FACS strategy.
Follicular epithelial cell miRNA profiling identified specific miRNA signatures 7
We then wanted to examine if miRNAs were dynamically regulated in the bald scalps of men with AGA. We performed qRT-PCR analysis to explore miRNAs that are preferentially expressed in each FACS-purified fraction. In AGA patients’ occipital hairs are preserved [16], thus, occipital stem cells were called normal stem cells (NSCs) and occipital progenitor cells
cells from frontal type 1 were termed patient stem cells (PSCs).
of
named normal progenitor cells (NPCs). While, frontal hairs are miniaturized [16] and stem
ro
In total, we detected 252 miRNAs in all of the samples. In NSCs, 82 miRNAs were
-p
upregulated, whereas there were 41 miRNAs upregulated in NPCs, and 23 miRNAs that were specifically expressed in PSCs (Fig. 3a).
re
Unsupervised hierarchical clustering based on miRNA relative expression suggested that NSCs, NPCs, and PSCs were differentially expressed discrete miRNAs and showed that
lP
NSCs and NPCs were more similar to each other than to PSCs (Fig. 3b). Next, we determined which miRNAs were differentially expressed in each group (S7a -c,).
ur na
Further we examine the putative functional implication of our most differentially regulated miRNAs by first extracting their target genes using TargetScan 7.1 followed by pathway enrichment analysis using Enrichr [17] (Fig. S8).
Jo
MiR-324-3p promotes keratinocyte differentiation and migration, and inhibits proliferation
Further, we focus our attention on a particular miRNA, miR-324-3p, as it was strongly enriched in normal hair cells but not detected in bald cells (P<0.05) (Fig. 3b, S9). 8
Normally, mature hair has a regenerative cycling system maintained by a tightly regulated balance between keratinocyte proliferation, migration, and terminal differentiation [18, 19]. Impaired differentiation from stem to progenitor cells has been reported, and may contribute to the reduction of hair follicle size, in AGA [1]. We sought to determine whether the absence of miR-324-3p expression in bald follicular stem cells might contribute to these alterations.
of
To do so, we overexpressed miR-324-3p in an HEK001 keratinocyte culture system because bulge stem cell phenotypes are difficult to maintain in vitro[14, 20], and we measured the
ro
expression of differentiation markers K10 and K1, and basal cell markers (K14, Integrin β1).
-p
MiR-324-3p overexpression was confirmed by qRT-PCR (P<0.05) (Fig. S10).
When miR-324-3p was overexpressed in the cells, the expression of K14, integrin-β1, and
re
K10 did not change significantly compared to scramble. While, there was a significant increase in the expression of K1 (P<0.01) (Fig. 4a). Consistent with these results, HEK cells
lP
showed a higher K1 protein expression than cells treated with scramble (P<0.0001) (Fig. 4b, c). These data indicated that miR-324-3p promoted keratinocyte differentiation.
ur na
Then, we investigated the effects that modulating miR-324-3p would have on keratinocyte migration. Linear scratches were made on confluent sheets of HEKs treated with miR-324-3p or control. We observed, after 24 h, miR-324-3p-treated HEKs sealed the wound at a faster rate than control cells (P<0.0001) (Fig. 4d, e)
Jo
In the epidermis and epidermal appendages, proliferation and differentiation are mutually exclusive programs [21]. Therefore, we investigated the ability of miR-324-3p to regulate the switch between proliferative and differentiating keratinocytes. The switch from proliferation to differentiation in keratinocytes is concomitant with induction of the cell-cycle exit [22]. We checked CCNB1 (cyclin B1) and CCND1(cyclin D1) expressions. With the overexpression of 9
miR-324-3p, it was found that CCND1 expression was reduced (P<0.05) (Fig. 4f), suggesting a decrease in cell cycling. Next, we used Ki67 staining to distinguish cells that were in the active cell cycle from those that exited the cell cycle [21]. We found a significant reduction in cell proliferation as seen by the significantly decreased Ki67+ cells in the miR-324-3poverexpressed cells compared to the control group (P<0.01) (Fig. 4g).
of
Collectively, these results point to the miR-324-3p role in supporting keratinocyte cells exit
ro
from the cell cycle to embark on the differentiation and migratory program [8].
-p
MiRNA-324-3p controls keratinocyte differentiation by modulating the activity of
re
MAPK and TGF-β pathways
We next investigated the mechanism underlying miR-324-3p contribution to keratinocyte
lP
differentiation. The possible miR-324-3p targets were predicted by the TargetScan 7.1 prediction algorithm and evaluated by Enrichr software. A search of the database of the
ur na
KEGG showed that miR-324-3p target genes were mainly involved in Ras, phosphatidylinositol, ErbB, mTOR, and MAPK signaling pathways (Fig. 5a). Next, we sought to investigate the expression patterns of the targets of miR-324-3p that might explain the observed differentiation phenotype after its overexpression in vitro. Using qRT-PCR, we
Jo
evaluated the expression of nine targets related to the five most enriched pathways. Our results showed that the expression of MAPK1, MAPK3, REL A, HSPA2, and TGF-β3 was downregulated in cultured keratinocytes in miR-324-3p-overexpressed cells (P<0.05, P<0.01); however, the expression level of REL B was upregulated (P<0.001) (Fig. 5b). These findings confirm MAPK and TGF-β signaling pathways regulation by miR-324-3p in our system. 10
In order to analyze the potential role of MAPK signaling pathways in mediating the differentiating effects of miR-324-3p, we used specific chemical inhibitors that target these pathways. Keratinocytes were separately treated with PD0325901 (inhibitor of MEK), SP600125 (inhibitor of JNK), and SB203580 (inhibitor of p38 MAPK). To inhibit TGF-β, Alk5i and SB431542 were used. In addition to TGF-β3, according to TargetScan 7.1 algoritm,
of
transforming growth factor beta receptor associated protein 1 is one of the targets of miR-3243p.
ro
Next, we analyzed the expressions of K14, Integrinβ1, K10, K1, CCDN1, and CCNB1 by
-p
qRT-PCR. Similar to what we observed with miR-324-3p, HEKs treated with either PD0325901, SB431542, SP600125, or Alk5i small molecules exhibited increased K1
re
expression (P<0.05, P<0.01). Notably, these small molecules increased K10 expression, as an early keratinocyte differentiation marker (P<0.05, P<0.01, P<0.001), whereas SB203580 had
lP
no effect on differentiation (Fig. 5c-g). Blocking MEK, JNK, and TGF-β signaling pathways by PD0325901, SP600125, and Alk5i small molecules increased the expression of K14, a
ur na
basal cell marker (P<0.05, P<0.01, P<0.001) (Fig. 5c, e, f). Moreover, SP600125 increased the expression levels of CCDN1 and CCNB1 while Alk5i decreased their expression levels (P<0.05, P<0.01). These transcripts did not appear significantly altered in the presence of other small molecules (Fig. 5c, d, g). Taken together,
Jo
these findings supported the idea that downregulation of the MEK, JNK, and TGF-β signaling pathways could ultimately accelerate keratinocyte differentiation. Because PD0325901 and Alk5i small molecules had increased K1 expression more than the other small molecules, we checked their effects on human hair in an anagen organ-cultured hair follicle system [23]. Compared with the control, PD0325901 samples, we observed that 11
treatment with Alk5i significantly increased the lengths of the hair shafts after eight days
of
(P<0.01) (Fig. 5h), which indicated a positive effect on the rate of hair elongation.
-p
ro
Discussion
Hair loss can occur with factors that cause abnormalities in HF cycling, decreases in follicular
re
stem cell activity, or failure in regrowth of hair fibers from existing HFs [18]. Recently, Garza et al. reported that in AGA, the lack of an activator or existing inhibitor could lead to
follicle size [3].
lP
problems with follicular stem cell conversion into progenitor cells and result in decreased hair
ur na
On the other hand, a role for miRNAs at the post-transcriptional level in regulating skin and hair stem cell proliferation and differentiation has been reported [4, 8, 24, 25]. In this study, we characterized human hair cells in vivo by immunofluorescence and revealed that CD200+CD49f+CD34- cells were located in the bulge area of normal occipital hair. We
Jo
checked for the presence of these cells in bald samples by evaluating two types of hair in the frontal area, thicker (type 1) and miniature or thinner (type 2). Notably, we observed that type 1 frontal hairs preserved the CD200+CD49f+CD34- cells, similar to the non-bald samples, whereas these cells were not detected in the miniature or type 2 hairs. These results agreed with those reported by Garza et al., who proposed a gradual decrease in the number of stem 12
cells in the miniaturized HF, thereby resulting in a reduction in HF volume during miniaturization and reversible hair loss [3]. CD34+CD49f+CD200- cells, which are found in the sub-bulge and proximal part of the suprabulbar area, are remarkably diminished in the frontal hairs of bald samples. In line with these results, Garza et al. demonstrated that a loss of progenitor cells, but not stem cells, contributed to male pattern baldness in humans [3].
of
Decreased numbers of these cells could potentially result from diminished conversion of hair follicle stem cells to progenitor cells and lead to miniaturization as well as a decreased
ro
amount of anagen follicles in the bald scalp [3].
-p
. Then, we used a miRNA expression profiling approach to identify miRNAs in hair epithelial cells that might contribute to AGA.
re
To the best of our knowledge, this study represents the first data set of results for miRNA expression profiling from normal and AGA follicular epithelial cells. The remarkable
lP
differences in miRNA expression patterns between normal and patient epithelial hair cells suggested that the miRNAs might serve important functions in the pathogenesis of AGA.
ur na
Interestingly, we found no expression of miR-324-3p in bald follicular stem cells. To date, nothing is known about the function of miR-324-3p in hair epithelial cells. MiR-324-3p is dysregulated in breast, hepatocellular, and pancreatic cancers. Previous reports have shown that miR-324-3p inhibited invasion of nasopharyngeal carcinoma cells by
Jo
targeting WNTB2 [26] and promoted the shift of neuroepithelial-like stem cells from selfrenewal to neuronal differentiation [27]. Overexpression of hsa-miR-324-3p mimics in HEKs promoted expression of a late differentiation marker, K1, but did not affect the expression of the early differentiation marker, K10, and basal markers, K14 and integrin β1. Consistent with this mode of regulation, 13
Fuchs et al. showed that miR-125b inhibited hair lineage differentiation without affecting maintenance or activation of hair follicle stem cells in mice [4]. Treatment of HEKs with hsamiR-324-3p mimic increased the ability of these cells to seal linear scratch wounds after 24 h and reduced keratinocyte proliferation as well as exited them from the cell cycle. Then, we performed in silico target prediction for miR-324-3p to gain additional insight into
of
its effects and finding the pathway(s) by which it works. Among nine predicted targets for miR-324-3p, we noted that REL A, HSP A2, MAPK1,3, and TG-β3 expression was
ro
significantly decreased in cells overexpressing miR-324-3p compared to the control.
-p
Previous studies have shown that RelA acts as an inhibitor of keratinocyte growth [28, 29]. Another study indicated that specific deletion of RelA did not have any effect on epidermal
re
differentiation [30]. HSPA2 knockdown by RNAi in HaCaT cells has been shown to accelerate keratinocyte differentiation [31]. Hashizume et al. observed increased expressions
lP
of HSP 60 and 72 in the cycling hair bulb during mid-anagen VI and an additional increase in catagen development, which might be associated with apoptosis of follicle keratinocytes [32].
ur na
Therefore, absence of miR-324-3p in bald hair keratinocyte cells might increase the level of HSP72 and consequently cause an increase in keratinocyte apoptosis and resting in catagen phase.
Previously, it has been shown that the MAPK cascade was implicated in keratinocyte terminal
Jo
differentiation and epidermal hyper proliferation in psoriasis and wound healing. Forced activation of MAPK signaling in primary human keratinocytes and reconstituted epidermis by retroviral infection caused an increase in growth rate and delayed the onset of keratinocyte differentiation [19]. Similar to this study, we observed that overexpression of miR-324-3p led to downregulation of MAPK1 and MAPK3, and caused a reduction of cyclin D1 and increased 14
K1 expression. Analysis of the potential changes in the signaling pathway activation state in patients treated with 5% minoxidil topical foam has shown downregulation of the MAPK signaling pathway in the vertex and front scalp of responder patients [33]. This suggests that miR-324-3p might contribute to AGA therapies trough modulating MAPK signaling. Dihydrotestosterone (DHT) stimulates synthesis of TGF-β that leads to premature entry of
of
hair follicles into the catagen phase and induces male pattern baldness. TGF-β has three isoforms: TGF-β1, 2, and 3 which are expressed in different parts of hair structure and bound
ro
to type I (Alk5) and receptors type II (Tgfbr2). Soma et al. showed that TGF-β1 and TGF-β2
-p
were involved in catagen induction of the human hair cycle in a human hair organ culture [3437].
re
Collectively, these findings suggested that the differentiation effects of miR-324-3p might be mediated through the MAPK and TGF-β signaling pathways, in addition to downregulation of
lP
HSP 72 and REL A. Among these candidates, we were interested in evaluating the role of the MAPK and TGF-β signaling pathways in keratinocyte differentiation. Thus, specific
ur na
chemicals were used to inhibit these two pathways. Our results showed that PD0325901 and Alk5i could function in a manner similar to miR-324-3p in promoting the expression of cytokeratin1 in keratinocytes more than the other small molecules. Then, we used an anagen organ-cultured hair follicle system to address the role of these
Jo
signaling pathways in human hair follicle growth. Inhibition of TGF-β signaling led to increased hair shaft elongation, raising the possibility that the migration and differentiation effects ascribed to miR-324-3p are also involved in human bulge stem cell differentiation and migration of AGA.
15
In summary, we found gradual decrease in the number of follicular stem cells during hair loss in AGA, might be resulting in a reduction in HF volume during miniaturization and reversible hair loss. Moreover, we have identified miR-324-3p which was strongly enriched in normal follicular epithelial cells in comparison to bald ones. Also, we have shown miR-324-3p as a novel regulator of cell proliferation, differentiation and migration of keratinocytes.
of
Furthermore, miRNA-324-3p controls keratinocyte differentiation likely by modulating the activity of MAPK and TGF-β pathways (Fig. S11), thus, it may be a promising new
ro
therapeutic target for hair loss by topical application of an miR-324-3p mimic. Although these
-p
data are persuasive, the physiological response to miR-324-3p mimic in human scalp affected with AGA needs to be determined in the preclinical and clinical context. Also, further studies
re
are required to determine the regulatory mechanism between miR-324-3p expression and
mechanism in AGA.
lP
TGF-β downregulation, which may provide a better understanding of the hair miniaturization
Moreover, other miRs, such as miR-148b-3p, miR-196a-5p, miR-652-3p, and miR-328-3p
ur na
showing similar expression pattern changes like miR-324-3p, can be remarkable candidates for finding pathogenic mechanisms of hair miniaturization in future investigation.
Jo
Acknowledgments
This study was funded by a grant provided from Royan Institute and Disease Models & Mechanisms Travelling Fellowship by Biologists Company. We express our appreciation to all members of the Skin Program at Royan Institute and Cedric Blanpain lab for their helpful deliberations and consultation during this work. We would like to thank Dr Kiarash 16
Khosrotehrani, Dr Saeid Amini Nik, and Dr Seyedeh Nafiseh Hassani for their critical
Jo
ur na
lP
re
-p
ro
of
comments.
17
References
Jo
ur na
lP
re
-p
ro
of
[1] J. Qi, L.A. Garza, An overview of alopecias, Cold Spring Harb Perspect Med. 4(3) (2014). [2] E.G.Y. Chew, J.H.J. Tan, A.W. Bahta, B.S. Ho, X. Liu, T.C. Lim, Y.Y. Sia, P.L. Bigliardi, S. Heilmann, A.C.A. Wan, M.M. Nothen, M.P. Philpott, A.M. Hillmer, Differential Expression between Human Dermal Papilla Cells from Balding and Non-Balding Scalps Reveals New Candidate Genes for Androgenetic Alopecia, J Invest Dermatol. 136(8) (2016) 1559-1567. [3] L.A. Garza, C.C. Yang, T. Zhao, H.B. Blatt, M. Lee, H. He, D.C. Stanton, L. Carrasco, J.H. Spiegel, J.W. Tobias, G. Cotsarelis, Bald scalp in men with androgenetic alopecia retains hair follicle stem cells but lacks CD200-rich and CD34-positive hair follicle progenitor cells, J Clin Invest. 121(2) (2011) 613-22. [4] L. Zhang, N. Stokes, L. Polak, E. Fuchs, Specific microRNAs are preferentially expressed by skin stem cells to balance self-renewal and early lineage commitment, Cell Stem Cell. 8(3) (2011) 294-308. [5] D.P. Bartel, MicroRNAs: target recognition and regulatory functions, Cell. 136(2) (2009) 215-33. [6] R. Yi, D. O'Carroll, H.A. Pasolli, Z. Zhang, F.S. Dietrich, A. Tarakhovsky, E. Fuchs, Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs, Nat Genet. 38(3) (2006) 356-62. [7] T. Andl, E.P. Murchison, F. Liu, Y. Zhang, M. Yunta-Gonzalez, J.W. Tobias, C.D. Andl, J.T. Seykora, G.J. Hannon, S.E. Millar, The miRNA-processing enzyme dicer is essential for the morphogenesis and maintenance of hair follicles, Curr Biol. 16(10) (2006) 1041-9. [8] R. Yi, M.N. Poy, M. Stoffel, E. Fuchs, A skin microRNA promotes differentiation by repressing 'stemness', Nature. 452(7184) (2008) 225-9. [9] S. Yuan, F. Li, Q. Meng, Y. Zhao, L. Chen, H. Zhang, L. Xue, X. Zhang, C. Lengner, Z. Yu, Post-transcriptional Regulation of Keratinocyte Progenitor Cell Expansion, Differentiation and Hair Follicle Regression by miR-22, PLoS Genet. 11(5) (2015) e1005253. [10] A.N. Mardaryev, M.I. Ahmed, N.V. Vlahov, M.Y. Fessing, J.H. Gill, A.A. Sharov, N.V. Botchkareva, Micro-RNA-31 controls hair cycle-associated changes in gene expression programs of the skin and hair follicle, FASEB J. 24(10) (2010) 3869-81. [11] B.K. Kim, S.K. Yoon, Hairless Up-Regulates Tgf-beta2 Expression via Down-Regulation of miR-31 in the Skin of "Hairpoor" (HrHp) Mice, J Cell Physiol. 230(9) (2015) 2075-85. [12] Z. Wenguang, W. Jianghong, L. Jinquan, M. Yashizawa, A subset of skin-expressed microRNAs with possible roles in goat and sheep hair growth based on expression profiling of mammalian microRNAs, OMICS. 11(4) (2007) 385-96. [13] M.I. Ahmed, M. Alam, V.U. Emelianov, K. Poterlowicz, A. Patel, A.A. Sharov, A.N. Mardaryev, N.V. Botchkareva, MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway, J Cell Biol. 207(4) (2014) 549-67. [14] K. Inoue, N. Aoi, T. Sato, Y. Yamauchi, H. Suga, H. Eto, H. Kato, J. Araki, K. Yoshimura, Differential expression of stem-cell-associated markers in human hair follicle epithelial cells, Lab Invest. 89(8) (2009) 844-56. [15] M. Ohyama, A. Terunuma, C.L. Tock, M.F. Radonovich, C.A. Pise-Masison, S.B. Hopping, J.N. Brady, M.C. Udey, J.C. Vogel, Characterization and isolation of stem cell-enriched human hair follicle bulge cells, J Clin Invest. 116(1) (2006) 249-60. [16] D. Rathnayake, R. Sinclair, Male androgenetic alopecia, Expert Opin Pharmacother. 11(8) (2010) 1295-304. 18
Jo
ur na
lP
re
-p
ro
of
[17] S. Moradi, A. Sharifi-Zarchi, A. Ahmadi, S. Mollamohammadi, A. Stubenvoll, S. Gunther, G.H. Salekdeh, S. Asgari, T. Braun, H. Baharvand, Small RNA Sequencing Reveals Dlk1-Dio3 Locus-Embedded MicroRNAs as Major Drivers of Ground-State Pluripotency, Stem Cell Reports. 9(6) (2017) 2081-2096. [18] P. Mohammadi, K.K. Youssef, S. Abbasalizadeh, H. Baharvand, N. Aghdami, Human Hair Reconstruction: Close, But Yet So Far, Stem Cells Dev. 25(23) (2016) 1767-1779. [19] I. Haase, R.M. Hobbs, M.R. Romero, S. Broad, F.M. Watt, A role for mitogen-activated protein kinase activation by integrins in the pathogenesis of psoriasis, J Clin Invest. 108(4) (2001) 527-36. [20] L. Xie, R. Yang, S. Liu, S. Lyle, G. Cotsarelis, L. Xiang, L. Zhang, B. Li, M. Wan, X. Xu, TR3 is preferentially expressed by bulge epithelial stem cells in human hair follicles, Lab Invest. 96(1) (2016) 81-8. [21] I. Amelio, A.M. Lena, G. Viticchiè, R. Shalom-Feuerstein, A. Terrinoni, D. Dinsdale, G. Russo, C. Fortunato, E. Bonanno, L.G. Spagnoli, miR-24 triggers epidermal differentiation by controlling actin adhesion and cell migration, J Cell Biol. 199(2) (2012) 347-363. [22] C. Kolly, M.M. Suter, E.J. Muller, Proliferation, cell cycle exit, and onset of terminal differentiation in cultured keratinocytes: pre-programmed pathways in control of C-Myc and Notch1 prevail over extracellular calcium signals, J Invest Dermatol. 124(5) (2005) 1014-25. [23] S. Harel, C.A. Higgins, J.E. Cerise, Z. Dai, J.C. Chen, R. Clynes, A.M. Christiano, Pharmacologic inhibition of JAK-STAT signaling promotes hair growth, Sci Adv. 1(9) (2015) e1500973. [24] L. Zhang, Y. Ge, E. Fuchs, miR-125b can enhance skin tumor initiation and promote malignant progression by repressing differentiation and prolonging cell survival, Genes Dev. 28(22) (2014) 2532-46. [25] D. Aberdam, E. Candi, R.A. Knight, G. Melino, miRNAs, 'stemness' and skin, Trends Biochem Sci. 33(12) (2008) 583-91. [26] C. Liu, G. Li, N. Yang, Z. Su, S. Zhang, T. Deng, S. Ren, S. Lu, Y. Tian, Y. Liu, Y. Qiu, miR324-3p suppresses migration and invasion by targeting WNT2B in nasopharyngeal carcinoma, Cancer Cell Int. 17 (2017) 2. [27] L. Stappert, L. Borghese, B. Roese-Koerner, S. Weinhold, P. Koch, S. Terstegge, M. Uhrberg, P. Wernet, O. Brüstle, MicroRNA-based promotion of human neuronal differentiation and subtype specification, PloS one. 8(3) (2013) e59011. [28] C.S. Seitz, Q. Lin, H. Deng, P.A. Khavari, Alterations in NF-κB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-κB, Proc Natl Acad Sci U S A. 95(5) (1998) 2307-2312. [29] J.Y. Zhang, C.L. Green, S. Tao, P.A. Khavari, NF-κB RelA opposes epidermal proliferation driven by TNFR1 and JNK, Genes Dev. 18(1) (2004) 17-22. [30] Y. Grinberg-Bleyer, T. Dainichi, H. Oh, N. Heise, U. Klein, R.M. Schmid, M.S. Hayden, S. Ghosh, Cutting edge: NF-kappaB p65 and c-Rel control epidermal development and immune homeostasis in the skin, J Immunol. 194(6) (2015) 2472-6. [31] A. Gogler‐Pigłowska, K. Klarzyńska, D.R. Sojka, A. Habryka, M. Głowala‐Kosińska, M. Herok, M. Kryj, M. Halczok, Z. Krawczyk, D. Scieglinska, Novel role for the testis‐enriched HSPA2 protein in regulating epidermal keratinocyte differentiation, J Cell Physiol. 233(3) (2018) 2629-2644. 19
Jo
ur na
lP
re
-p
ro
of
[32] H. Hashizume, Y. Tokura, M. Takigawa, R. Paus, Hair cycle‐dependent expression of heat shock proteins in hair follicle epithelium, Int J Dermatol. 36(8) (1997) 587-592. [33] G.N. Stamatas, J. Wu, A. Pappas, P. Mirmirani, T.S. McCormick, K.D. Cooper, M. Consolo, J. Schastnaya, I.V. Ozerov, A. Aliper, A. Zhavoronkov, An analysis of gene expression data involving examination of signaling pathways activation reveals new insights into the mechanism of action of minoxidil topical foam in men with androgenetic alopecia, Cell Cycle. 16(17) (2017) 1578-1584. [34] T. Soma, M. Ogo, J. Suzuki, T. Takahashi, T. Hibino, Analysis of Apoptotic Cell Death in Human Hair Follicles In Vivo andIn Vitro, J Invest Dermatol. 111(6) (1998) 948-954. [35] T. Soma, Y. Tsuji, T. Hibino, Involvement of transforming growth factor-β2 in catagen induction during the human hair cycle, J Invest Dermatol. 118(6) (2002) 993-997. [36] T. Hibino, T. Nishiyama, Role of TGF-beta2 in the human hair cycle, J Dermatol Sci. 35(1) (2004) 9-18. [37] H.Y. Lin, L.T. Yang, Differential response of epithelial stem cell populations in hair follicles to TGF-beta signaling, Dev Biol. 373(2) (2013) 394-406.
20
lP
re
-p
ro
of
Figure legends:
ur na
Fig. 1 Differential expressions of K15, CD200, CD49f, and CD34 markers in longitudinal section of human anagen hair. a: Occipital human hair is divided into five portions: INF, BU, SBU, SBUL, and BUL. b: Evaluation of CD200 and K15 expression. Higher magnification images indicated by dashed box. Arrows show CD200 and K15 expression. c: Evaluation of CD200 and CD34 expressions. Higher magnification images indicated by dashed box. Arrows show CD200 and CD34 expression. The nuclei were stained with DAPI. INF: Infundibulum BU: Bulge,
Jo
SBU: Sub-bulge, SBUL: Suprabulbar, BUL: Bulb, SG: sebaceous gland, APM: arrector pili muscle, PSBU: Proximal Sub-bulge, DSBU: Distal Sub-bulge, SBUL: Supra-bulbar.
21
of ro -p re lP ur na Jo Fig. 2 Characterization and purification of follicular stem and progenitor cells from occipital and frontal hairs using fluorescence-activated cell sorting. Samples gated by debris exclusion (P1: FSC /SCC) and living cells (P2: PI dye exclusion). a: Occipital samples: NSC: P3-a=CD34-CD49f+, P4-a= CD200+CD49f+CD34- and NPC: P3-b=CD200-
22
CD49f+, P4-b= CD200-CD49f+CD34+. b: Frontal type 1: P3=CD34-CD49f+, P4=CD200+CD49f+CD34-. c: frontal type 2: P3=CD34-CD49f+, P4= CD200+CD49f+CD34-. d: Quantification of the percentage of follicular stem and progenitor cells in occipital and frontal hairs. Mean±SD, ***P<0.001. e: RT-qPCR quantification of CD49f, CD34 and CD200 in sorted NSCs and NPCs compared with total hair cells. GAPDH was used as the reference gene (n=3). Mean ± SE, ***P<0.001. FSC: Forward scatter, SSC: Side scatter, NSC: normal stem cells, NPC: normal
Jo
ur na
lP
re
-p
ro
of
progenitor cells, PSC: patient stem cells, ND: Not detected.
Fig. 3 Differential expression of microRNAs of follicular stem and progenitor cells from normal and bald hairs. a: Venn diagram of all and differentially miRNAs expressed. b: Heat map and unsupervised hierarchical clustering display of miRNA expression profiles of NSC, NPC, and PSC samples. The clustering is performed on all samples and on the top 50 miRNAs with the highest standard deviation. Each row represents one miRNA and each column represents one sample. The miRNA clustering tree is shown on the left. NSC: normal stem cells, NPC: normal progenitor cells, PSC: patient stem cells.
23
of ro -p re lP ur na Jo
Fig. 4 miR-324-3p regulates keratinocyte differentiation, proliferation and migration. a: Real-time qPCR analysis of K10, K1, K14 and ITGβ1 72 h following HEK001 transfection with synthetic mimic miR-324-3p and scramble. b, c: Immunostaining (b) and quantification (c) of K1 in HEK001 cells, 72 h after transfecting. The nuclei were stained by DAPI (Blue). d, e: Phase-contrast micrographs (d) and quantification (e) of transfected HEKs migrating into scratched region. f: qRT-PCR analysis of CCNB1 and CCND1 72 h following HEK001 transfection. g: Flow
24
cytometry analysis of Ki67 expression 24 h after transfecting. Scale bar: 100 μm. GAPDH was used as an internal
ur na
lP
re
-p
ro
of
normalization control. Data are shown as mean ± SE, n=3, *P<0.05, **P<0.01, ****P<0.0001.
Jo
Fig. 5 Effect of miR-324-3p, PD0325901, SB431542, Alk5i, SP600125, and SB203580 on keratinocyte differentiation program in vitro. a: Probable pathways involved by the genes targeted by miR-324-3p. b: qRT-PCR analysis of K14, Integrinβ1, K10, K1, CCDN1, and CCNB1 transcripts 3 days after keratinocytes treatment with miR-324-3p or scramble. Data are shown as mean±SD, n=3. c-g: qRT-PCR analysis of K14, Integrinβ1, K10, K1, CCDN1, and CCNB1 transcripts 3 days following keratinocytes treatment with PD0325901, SB431542, Alk5i, SP600125, and SB203580. Data are shown as mean±SD, n=3. h: Quantification of the length of the hair shaft treated
25
with culture medium, PD0325901 and Alk5i after 8 days. Data are mean±SE, n=5. *P<0.05, **P<0.01,
Jo
ur na
lP
re
-p
ro
of
***P<0.001,0.002. GAPDH was used as an internal normalization control.
26
Table: Table 1: Summary of immunofluorescence staining patterns of the human anagen hair follicles. Table 1 : Summary of immunofluorescence staining patterns in longitudinal sections of human anagen hair follicles Marker/Replicate Epidermis Infudibulum Bulge Sub-bulge Supra bulbar area Distal
Proximal Medial
Distal
B -
S -
ro
-p
re
lP ur na Jo 27
B -
S -
B -
S -
of
Proximal
B S B S B S B S B S CD200 1 ++ +/- +/- 2 ++ +/- +/- 3 + +/- 4 + +/- 5 + + +/- +/- K15 1 + + + + + + 2 + + + + + + 3 + + + + + +/- + +/CD34 1 + 2 +/- +/- + + 3 + 4 +/- +/B : basal cells, S : Supra basal cells - :no staining, +/- : marginally or partially positive, + : positive, ++ : strong positive
-
-
+ -
-
+ +/+
-
+ + + +
+ +
+ -
-
-
-