Hair follicles (HF) undergo precisely regulated recurrent cycles of growth, cessation, and rest. The transitions from anagen (growth), to catagen (regression), to telogen (rest) involve a physiological involution of the HF. This process is likely coordinated by a variety of mechanisms including apoptosis and loss of growth factor signaling. However, the precise molecular mechanisms underlying follicle involution after hair keratinocyte differentiation and hair shaft assembly remain poorly understood. Here we demonstrate that a highly conserved microRNA, miR-22 is markedly upregulated during catagen and peaks in telogen. Using gain- and loss-of-function approaches in vivo, we find that miR-22 overexpression leads to hair loss by promoting anagen-to-catagen transition of the HF, and that deletion of miR-22 delays entry to catagen and accelerates the transition from telogen to anagen. Ectopic activation of miR-22 results in hair loss due to the repression a hair keratinocyte differentiation program and keratinocyte progenitor expansion, as well as promotion of apoptosis. At the molecular level, we demonstrate that miR-22 directly represses numerous transcription factors upstream of phenotypic keratin genes, including Dlx3, Foxn1, and Hoxc13. We conclude that miR-22 is a critical post-transcriptional regulator of the hair cycle and may represent a novel target for therapeutic modulation of hair growth.

Up to 60% people suffer from hair loss throughout their lifetime. Hair growth undergoes recurrent cycling of growth, regression, and resting phases with a defined periodicity. The main cause of human hair loss is due to the premature transition from growth to regression. Understanding of the molecular basis underlying hair regression is important to elucidate the mechanisms of hair loss. Here, we demonstrated that miR-22, a highly conserved microRNA, is critical for the transition from growth to regression of the hair follicle. Importantly, miR-22 could be a novel target for therapeutic therapy of hair loss disorders.

Funding: This work was supported by National Natural Science Foundation of China (NSFC, 31271584), the National Transgenic Research Project (2011ZX08009-001-003), the National Basic Research Program of China (973 program-2011CB944103), 2010SKLAB03-01, 2014SKLAB4-2 and 2015SKLAB6-16. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2015 Yuan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

The function of miR-22 in hair follicles is of particular interest, as microRNA profiling revealed that miR-22 expression markedly increases during the catagen stage, reaching peak expression at the transition to telogen [ 24 ], suggesting a potential role for miR-22 in hair follicle involution. Here we utilize miR-22 gain- and loss-of-function mouse models to investigate the physiological role of miR-22 in hair cycling. We demonstrate that miR-22 is an important post-transcriptional regulator that governs exit from anagen and maintenance of the hair follicle in telogen through inhibition of a transcriptional program driving keratinocyte proliferation, differentiation, and hair shaft assembly.

MicroRNAs play important roles in many biological processes including hair follicle development [ 22 ], and hundreds of microRNAs are expressed in the skin [ 23 , 24 ]. Global ablation of microRNA activity through genetic deletion of microRNA processing enzymes, DGCR8, Drosha, and Dicer demonstrated that microRNAs are critical for both embryonic and adult hair follicle development [ 23 , 25 , 26 ]. Conditional deletion of Drosha and Dicer during anagen causes failure of catagen and follicular degradation [ 25 ], indicating a critical role of microRNAs in the transition of anagen to catagen. However, the specific microRNAs involved in this process are unknown. Recently, numerous reports have documented the functional contribution of several specific microRNAs to hair development, including miR-24 [ 27 ], miR-125b [ 28 ], miR-31 [ 24 ], and miR-205 [ 29 ], and thus a microRNA network governing hair follicle development is beginning to emerge.

Hair follicles undergo recurrent cycles of growth (anagen), regression (catagen), and resting (telogen) phases with a defined periodicity [ 1 , 2 ]. Abnormal regulation of genes associated with hair cycling may cause several types of human hair growth disorders [ 3 ]. For example, male pattern baldness is due to the premature transition from anagen-to-catagen induced by androgens [ 4 ]. The hair follicle contains epithelial cells of the outer root sheath (ORS), matrix, the inner root sheath and hair shaft, and mesenchymal cells of dermal papilla [ 1 , 5 ]. The interaction between epithelial and mesenchymal cells is required for proper hair development and follicle cycling [ 2 ]. At the onset of growth (anagen), the dermal papilla is at the proximal end of the follicle, in close proximity to the hair stem cell niche known as the follicle bulge [ 6 ]. Signals from the condensed dermal papilla induce proliferation of hair bulge stem cells, resulting in the downward extension of the hair germ where it ultimately envelops the dermal papilla and triggers hair matrix formation. Transit-amplifying matrix cells proliferate rapidly in response to signals from the dermal papilla after which they terminally differentiate to form the inner root sheath and hair shaft. Several important signaling pathways including Wnt and BMP play an important role in hair differentiation [ 7 ]. Further, a host of transcription factors promote hair differentiation during anagen including Gata3 [ 8 ], Cutl1 [ 9 ], Lef1 [ 10 ], Dlx3 [ 11 ], Msx2 [ 12 ], Foxn1 [ 13 ], TCF3 [ 14 ] and Hoxc13 [ 15 ]. In contrast to anagen, catagen is the physiological involution of the hair follicle. The hair follicle rapidly degenerates and shortens until it is again adjacent to the hair follicle stem cell reservoir in the bulge region. This process may be triggered by a variety of stimuli, including apoptosis and loss of supportive growth factor signaling needed to maintain cell proliferation and differentiation during anagen [ 16 ]. While some apoptotic triggers promoting catagen have been defined, including Bcl2/Bax [ 17 ], p53 [ 18 ], p57 [ 19 ], and the transforming growth factors, TGF-β1 and TGF-β2 [ 20 , 21 ], little is known about how the stimulatory signals that drive anagen are terminated upon entry to catagen and maintained during telogen.

Results

miR-22 induction causes hair loss in vivo To determine the functional consequences of miR-22 activity in hair follicles, we generated mice in which miR-22 overexpression could be induced in the ORS of hair follicles with temporal specificity. MiR-22 was placed under the control of a tetracycline regulatory element (TRE) (Fig 2A) and TRE-miR-22 transgenic mice were generated. TRE-miR-22 mice were crossed with K14-rtTA transgenic mice, in which the Keratin 14 (K14) promoter that is active in the basal layer of the epidermis and the ORS of the hair follicle controls expression of the reverse tetracycline transactivator (rtTA). After induction with Doxycycline (Dox), K14-rtTA/TRE-miR-22 double transgenic (DTG) mice exhibited robust miR-22 expression in the basal layer of the epidermis and ORS of the hair follicle (S1B Fig). Expression analysis demonstrated strong miR-22 induction in DTG mice, compared to TRE-miR22 and K14-rtTA controls (Fig 2B). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 2. miR-22 overexpression results in hair loss. (A) Schematic maps of constructs used to generate K14-rtTA/TRE-miR-22 double transgenic mice (DTG). (B) qPCR analysis for miR-22 showing that miR-22 is strongly induced in the Dox-treated DTG mice. The DTG mice were treated with Dox at P21. Samples were collected at P28. ** p < 0.01. (C) The Dox-treated DTG mice exhibit hair loss at 35 days. Both control and DTG mice were treated by oral administration of 2 mg/mL Dox in the drinking water at P1. After removing Dox at P35, the hair loss phenotype recovers by P46. (DTG n = 3; control n = 3). (D) Hair loss in DTG mice at P49 when treated with Dox at P1. (E) External hair regrowth is delayed in the DTG mice after depilation. Both control and DTG mice were treated with Dox at P18. Hairs were plucked at P21. Photographed at P31 and P40. (DTG n = 21; control n = 16). https://doi.org/10.1371/journal.pgen.1005253.g002 We initially induced miR-22 expression at postnatal day 1 during embryonic anagen stage. Strikingly, Dox-treated DTG mice began to exhibit premature hair loss 35 days after induction (Fig 2C). Within 11 days after Dox withdrawal at P35, there was a full recovery from the hair loss phenotype (Fig 2C), demonstrating that the effect of miR-22 induction on hair growth is reversible. When DTG mice were maintained on Dox, hair loss became more pronounced at P49 during the telogen phase, while hair growth of the control mice was normal and morphologically indistinguishable from wildtype littermates (Fig 2D). The hair loss persisted as long as the mice were maintained on Dox. In addition to the hair loss phenotype, we also found that the DTG mice were smaller in size compared to the control littermates (Fig 2C and 2D). Because the K14 promoter is also active in the esophagus and stomach, the reduction in body size is most likely due to digestion defects caused by miR-22 induction. To further explore the effect of miR-22 on hair morphorgenesis, we induced miR-22 expression at embryonic day 15 when the hair placode begins to form. miR-22 induction resulted in obvious thinning of the hair coat in DTG pups at P12 (S2A Fig), indicating that miR-22 induction represses hair morphorgenesis. Surprisingly, the DTG pups died at around P17. We next initiated a synchronous hair growth cycle at P21 by depilating the dorsal hair concomitant to miR-22 induction. External hair regrowth was observed in littermate controls 10 days post-depilation, but was absent in the depilated DTG skin (Fig 2E). Similar findings were observed at P65 when both control and DTG mice were treated with Dox at P53 and depilated at P56 (S2B Fig). Taken together, our data reveal that miR-22 overexpression is sufficient to repress hair morphorgenesis and development in vivo.

miR-22 is required for proper anagen-to-catagen transition The TRE-miR-22 mouse model demonstrates the sufficiency of miR-22 in promoting the anagen-to-catagen transition, but does not address a physiological requirement in this process. We thus examined histology of miR-22 knockout (KO) hair follicles at successive time points. No significant difference was found in the KO follicles at P1 (Fig 3D), however by P9 hair follicle length in the KO was significantly longer than in the controls, although KO follicles appeared morphologically normal (Fig 3D and S2G Fig). By P18, the defects in KO follicles became more pronounced. Control follicles had entered late catagen at this time point, however KO follicles were delayed in catagen entry, evidenced by a large hair bulb (Fig 3D and S2F Fig). By P21, control follicles uniformly had entered telogen, while up to 15% late catagen-stage follicles remained in the KO backskin, indicating a delay in hair development (Fig 3D and S2G Fig). By P26 both control and KO hair follicles entered a new anagen, however the penetrance of downward growth was increased in the KO follicles (Fig 3D and S2G Fig). Overall, the miR-22 KO phenotype is in direct opposition to that of miR-22 gain of function. Taken together, these data point to an important physiological role for miR-22 in promoting the anagen-to-catagen transition and in proper telogen maintenance in hair follicles.