No / Hair follicle development and its postnatal regeneration are characterized by dramatic changes in its microanatomy and cellular activity, which are controlled by multiple signalling pathways, transcription factors and epigenetic regulators, including microRNAs (miRNAs). miRNAs and their targets form remarkably diverse regulatory networks, playing a key role in the execution of gene expression programmes in the different cell lineages of the hair follicle. This review summarizes the roles of miRNAs in the control of hair follicle development, cycling and hair pigmentation, emphasizes the remaining problems/unanswered questions, and provides future directions in this rapidly growing and exciting area of research / MRC

The hair follicle (HF) is a human mini-organ that autonomously cycles between phases of growth (anagen), regression (catagen) and relative quiescence (telogen). Whilst many molecular controls are now appreciated to influence hair cycle, what ultimately choreographs the switch between each cycle stage is yet to be elucidated. With the increasing link between molecular clock activities in controlling local tissue physiology, we began by studying the hypothesis that the human HF has a functional molecular clock. Utilising human HF organ culture, qRT-PCR and immunofluorescence we found that the HF does indeed have oscillating clock gene expression over 24 and 48 hours in situ, separate from the suprachiasmatic-nucleus. Moreover, core clock proteins BMAL1 and PER1 are expressed in the human HF with PER1 increasing as HFs enter catagen. Next utilising siRNA mediated gene knock-down of either BMAL1 or PER1 in situ, we were able to show that silencing either clock gene leads to anagen prolongation in cultured HFs, demonstrating that the molecular clock modulates the human hair cycle, namely the anagen-catagen switch in situ. As human pigmentation is tightly coupled to the hair cycle and both human HFs and epidermal melanocytes express clock genes/proteins, this led us to investigate the hypothesis that the molecular clock modulates human pigmentation. By silencing BMAL1 or PER1 in HFs an increase in melanin content (Masson-Fontana) was observed in a hair-cycle independent manner. Furthermore, tyrosinase expression/activity as well as TYRP1 and 2 expression, gp100 protein expression, melanocyte dendricity and the number of HF melanocytes were all significantly increased in BMAL1 and/or PER1-silenced HFs. Mechanistically, BMAL1 knockdown reduced PER1 transcription, and PER1 silencing was found to induce phosphorylation of the master regulator of melanogenesis, MITF, thus stimulating human melanogenesis and melanocyte activity. This provides the first evidence that the peripheral molecular clock influences human pigmentation. Finally, the thyroid hormone (T4) has strong links with peripheral clock activity and has been shown to prolong anagen and increase human HF pigmentation. Moreover, T4 is a commonly prescribed treatment for thyroid disorder. As such, we investigated the hypothesis that T4 influences HF clock gene activity. It was observed that transient T4 treatment reduces the amplitude of clock gene oscillations whilst circadian rhythmicity is maintained. Conversely with longer term treatment clock gene activity was significantly increased compared to a scrambled oligo-control. Here we have demonstrated that the human HF has peripheral molecular clock activity which influences the human hair cycle and pigmentation. Finally we were able to uncover a potential novel target, T4, whose pulsatile administration may potentially be used to treat not only hair growth and pigmentation disorders but may be able to modulate circadian activity in peripheral tissues and treat clock-related disease.

MiRNAs control skin homeostasis through post-transcriptional gene repression by binding to their target mRNAs. However, their role in regulation of apoptosis and hair loss in alopecia areata (AA) is largely unknown, which became the aim of this study. In AA mouse model (C3H/HeJ), global miRNA profiling revealed 22 miRNAs with significant changes in their expression in AA affected skin. Amongst these miRNAs, miR-486-5p was dramatically decreased in alopecic skin in both humans and mice, in striking contrast to its prominent expression in the hair follicle (HF) epithelium of healthy anagen skin. Moreover, the expression of both pri-miR-486 and miR-486 is down-regulated in the human anagen HFs and keratinocytes treated with IFN-g, one of the key factors contributing to the immune privilege (IP) collapse in HFs. Intradermal delivery of miR-486-5p mimic into mouse skin affected by AA prevented premature entrance of HFs into catagen phase and reduced the numbers of CD4+ and CD8+ lymphocytes in the peri- and intra-follicular skin compartments. Consistently, subcutaneous administration of miR-486-5p inhibitor delayed anagen progression associated with a higher number of intrafollicular NKG2D+ cells in C3H/HeJ mice. Silencing of miR-486-5p in human anagen HFs ex vivo caused premature catagen development and led to suppression of IP by up-regulating HLA class 1, IRF1, ICAM1 and CADM1 expression of which CADM1 was confirmed to be a direct target of miR-486- 5p. Transcriptome profiling of primary human epidermal keratinocytes overexpressing miR-486-5p revealed damping the signalling pathways associated with inflammatory chemokines, cytokines and interleukins. Taken together, these data suggest that miR-486-5p plays a protective role in the pathogenesis of AA by maintaining anagen phase and preventing the IP collapse. / National Alopecia Areata Foundation

Le follicule pileux (FP) est un appendice cutané animé par un cycle régénératif dynamique provoquant des modifications de son microenvironnement. Les cellules de Langerhans (CLs), sentinelles de l’épiderme, sont en partie localisées à proximité du FP. Cette association spatiale nous a conduit à explorer le possible impact du cycle pileux sur l’homéostasie des CLs. Durant mon doctorat, nous avons mis en évidence (1) une augmentation de la prolifération des CLs au cours de l’anagène (phase de pousse du poil), (2) le mécanisme moléculaire sous-jacent impliquant une variation d’expression folliculaire de l’IL-34, une cytokine cruciale dans l’homéostasie des CLs et (3) un départ accru des CLs vers les ganglions lymphatiques en catagène (phase de régression du FP) concomitant avec le recrutement de cellules susceptibles d’être des précurseurs des CLs.Par ailleurs, la structure de la peau ainsi que la densité et le type de FP peuvent varier selon la région corporelle considérée. Nous avons émis l’hypothèse de variations locales dans la composition du système immunitaire cutané. Notre étude, focalisée sur les cellules dendritiques cutanées, a démontré l’existence d’une hétérogénéité de ces cellules en fonction de la zone de peau considérée. / The hair follicle (HF) is a skin appendage endowed with a dynamic regenerating cycle. This renewal remodels the HF microenvironment. Langerhans cells (LCs) are epidermal immune sentinels, a part of which localizes close to the HF. This spatial association led us to explore whether the HF cycle could impact on LC homeostasis. During my doctorate, we uncovered an anagen (HF growing phase)-associated burst of LC proliferation with dividing cells associated with the HF. Using mouse models of HF loss and hair cycle manipulation, we showed that HFs are dispensable for initial formation of the LC network but critical for the proliferation burst. We correlated it to a cyclic variation of IL-34 expression, a crucial cytokine for LC homeostasis, by a specific subset of HF cells. In addition, catagen (HF regression phase) is characterized by the departure of LCs to draining lymph nodes and the concomitant recruitment of a potential LC precursor.The skin structure as well as the density and type of HFs vary across body areas. This observation led us to assess the possibility of local variations in skin immune cells composition. Our study, focused on cutaneous dendritic cells, highlighted an heterogeneity in those cells according to the skin area considered.

The hair cycle represents a dynamic process during which a complex mini- organ, the hair follicle, rhythmically regresses and regenerates. The control mechanism that governs the hair cycle ('hair cycle clock') is thought to be an autonomous oscillator system, however, its exact nature is not known. This thesis aims to understand the human hair cycle as a systems biology problem using theoretical and experimental techniques in three distinct study approaches. Using mathematical modelling, a simple two-compartment model of the human hair cycle was developed. The model concentrates on the growth control of matrix keratinocytes, a key cell population responsible for hair growth, and bi-directional communication between these cells and the inductive fibroblasts of the dermal papilla. A bistable switch and feedback inhibition produces key characteristics of human hair cycle dynamics. This study represents the first mathematically formulated theory of the 'hair cycle clock'.A second chronobiological approach was adopted to explore the molecular control of the human hair follicle by a peripheral clock mechanism. The hypothesis was tested that selected circadian clock genes regulate the human hair cycle, namely the clinically crucial follicle transformation from organ growth (anagen) to organ regression (catagen). This revealed that intra- follicular expression of core clock and clock-controlled genes display a circadian rhythm and is hair cycle-dependent. Knock-down of Period1 and Clock promotes anagen maintenance, hair matrix keratinocyte proliferation and stimulates hair follicle pigmentation. This provides the first evidence that peripheral Period1 and Clock gene activity is a component of the human 'hair cycle clock' mechanism. Lastly, an unbiased gene expression profiling approach was adopted to establish important genes and signalling pathways that regulate the human hair cycle. This revealed that similar genes and pathways previously shown to control the murine hair cycle in vivo, such as Sgk3, Msx2 and the BMP pathway, are also differentially regulated during the anagen-catagen transformation of human hair follicles. In summary, by using a three-pronged systems biology approach, the thesis has shed new light on the control of human hair follicle cycling and has generated clinically relevant information: a) The hair cycle model may predict how hair cycle modulatory agents alter human hair growth. b) Period1 and Clock are new therapeutic targets for human hair growth manipulation. c) Gene expression profiling points to additional key players in human hair cycle control with potential for future therapeutic targets.

Le follicule pileux est un micro-organe spécifique des mammifères responsable de la formation des poils. Au cours de la vie postnatale, le follicule pileux subit des phases récurrentes de croissance (anagène), régression (catagène) et repos (télogène). Les mécanismes cellulaires et moléculaires qui régulent le cycle pilaire rappellent certains des évènements qui ont lieu durant la morphogénèse. Bien qu’il y ait eu des avancées significatives dans la connaissance de la biologie du follicule pileux ; les mécanismes qui régulent le passage de la phase anagène à la phase catagène restent mystérieux. Fgf5, un membre de la famille des facteurs de croissance des fibroblastes, a été identifié comme un régulateur clé de la transition anagène-catagène. Les souris qui ne produisent pas de protéine Fgf5 active présentent un phénotype angora (go/go) caractérisé par une phase anagène plus longue et de longs poils. Cependant, les follicules pileux n’ayant pas Fgf5, entrent quand même dans la phase catagène, ce qui suggère que d’autres mécanismes contribuent au control du cycle pilaire. Des précédents résultats obtenus dans notre laboratoire ont établis une relation très proche entre le déclenchement de la phase catagène et le diamètre du poil. En utilisant le follicule de vibrisse comme modèle, nous avons confirmé ces résultats en démontrant, par in hybridation in situ, que l’expression du gène Fgf5 s’active dans les cellules de la gaine épithéliale externe localisées dans la région supra-bulbaire, progressivement l’expression de Fgf5 s’étend jusqu’à l’extrémité inférieur de la gaine épithéliale externe et s’éteint quelques jours avant le début de la phase catagène, de nouveau dans les cellules de la gaine épithéliale externe localisées dans la partie supra-bulbaire. Nous avons également démontré que le nombre de couche cellulaires dans la région du cortex du poil, augmente progressivement au cours du temps jusqu’à atteindre exactement le même nombre de couche, quelques jours avant la fin de la phase de croissance, chez la souris sauvage et la souris Fgf5LacZ/LacZ. Ces résultats confirment notre hypothèse établissant que Fgf5 ne déclenche pas de façon direct la phase catagène. Ensuite, nous avons démontré pour la première fois que les cellules progénitrices du cortex peuvent se diviser symétriquement. Ces divisions symétriques très rares se traduisent, quelques jours après, en la formation d’une nouvelle couche cellulaire dans le cortex du poil. Ces résultats appuient notre hypothèse qu’une boucle de régulation complexe impliquant, la gaine épithéliale externe, la papille dermique (qui exprime Fgfr1, le récepteur de Fgf5), la matrice et la région supra-bulbaire ; est indispensable au control du cycle pilaire. Nous avons ensuite démontré par qRT-PCR et des marquages immunologiques que plusieurs canaux mécano-sensitifs sont exprimés de façon spécifique dans ces régions d’intérêts. De plus, plusieurs gènes importants pour la signalisation, sont également exprimés dans ces régions. Tout cela mis ensemble nos résultats soutiennent l’hypothèse provocatrice que la croissance progressive de la largeur du poil induit une pression mécanique qui entraine l’activation de canaux mécano-sensitifs, qui vont à leur tour activer des voies de signalisation pour finalement contrôler l’expression de Fgf5 dans la région supra-bulbaire et ainsi contrôler le cycle pilaire. / The hair follicle is a skin micro-organ specific to mammals and responsible for the formation of the hair. During postnatal life, the hair follicle undergoes recurrent phases of growth (anagen), regression (catagen) and rest (telogen) termed the hair cycle. The cellular and molecular mechanisms that regulate the hair cycle recapitulate some of the events occurring during morphogenesis. Despite significant advances in the understanding of biology of the hair follicle, the mechanisms regulating the switch from anagen to catagen remain mysterious. Fgf5, a member of the fibroblast growth factor family, has been proposed as a key regulator of the transition between anagen and catagen. Mice that do not produce active Fgf5 have an angora (go/go) phenotype characterized by an extended anagen phase and long hairs. Nevertheless, Fgf5 null hair follicles still enter catagen, suggesting that other mechanisms contribute to the control of the hair cycle. Previous work in the laboratory using Fgf5Lacz/LacZ null mice has unraveled a close connection between the onset of catagen and the diameter of the hair. Using the whisker follicle as a model system, we have confirmed these results and demonstrated by in situ hybridization that the expression of the Fgf5 gene is switched-on in the supra-bulbar region of the outer root sheath, progressively extends towards the lower extremity of the outer root sheath and is switched-off in the supra-bulbar region of the outer root sheath several days before the onset of catagen. We have also demonstrated that the number of cell layers in the hair cortex progressively increases with time to reach the exact same number a few days before the end of anagen in both wild-type and Fgf5 null follicles confirming our working hypothesis that Fgf5 does not directly trigger catagen. Next, we have demonstrated for the first time that the basal cortex-forming cells could divide symmetrically. These rare symmetrical divisions result in the formation of additional cell layers in the cortex. These results support our working hypothesis that a complex regulatory loop involving the outer sheath, the dermal papilla (that express Fgfr1, the Fgf5 receptor), the cortical matrix and the supra bulbar region is critical in controlling whisker growth. We have then demonstrated by q-RTPCR and immunostaining that several mechanosensitive channels are specifically expressed in the regions of interest. Moreover, several genes important for signaling are also expressed in these regions. Altogether, our results support the provocative hypothesis that the progressive increase in the width of the hair induces a mechanical pressure that leads to the activation of mechanosensitive channels, which in turn activate specific signaling pathways and ultimately result in the control of the expression of the Fgf5 gene in the supra-bulbar region of the outer root sheath and then in the control of the hair cycle.

The hair follicle is a cyclic biological system that progresses through stages of growth, regression, and quiescence, which involves dynamic changes in a program of gene regulation. Micro-RNAs (miRNAs) are critically important for the control of gene expression and silencing. Here, we show that global miRNA expression in the skin markedly changes during distinct stages of the hair cycle in mice. Furthermore, we show that expression of miR-31 markedly increases during anagen and decreases during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and midanagen phases of the hair cycle results in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signaling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes, both in vitro and in vivo. Using luciferase reporter assay, we show that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. Thus, by targeting a number of growth regulatory molecules and cytoskeletal proteins, miR-31 is involved in establishing an optimal balance of gene expression in the hair follicle required for its proper growth and hair fiber formation.

Yes / Background and Objective: Though devices for hair growth based on low levels of light have shown encouraging results, further improvements of their efficacy is impeded by a lack of knowledge on the exact molecular targets that mediate physiological response in skin and hair follicle. The aim of this study was to investigate the expression of selected light-sensitive receptors in the human hair follicle and to study the impact of UV-free blue light on hair growth ex vivo. Material and Methods: The expression of Opsin receptors in human skin and hair follicles has been characterised using RT-qPCR and immunofluorescence approaches. The functional significance of Opsin 3 was assessed by silencing its expression in the hair follicle cells followed by a transcriptomic profiling. Proprietary LED-based devices emitting two discrete visible wavelengths were used to access the effects of selected optical parameters on hair growth ex vivo and outer root sheath cells in vitro. Results: The expression of OPN2 (Rhodopsin) and OPN3 (Panopsin, Encephalopsin) was detected in the distinct compartments of skin and anagen hair follicle. Treatment with 3.2 J/cm2 of blue light with 453 nm central wavelength significantly prolonged anagen phase in hair follicles ex vivo that was correlated with sustained proliferation in the light-treated samples. In contrast, hair follicle treatment with 3.2 J/cm2 of 689 nm light (red light) did not significantly affect hair growth ex vivo. Silencing of OPN3 in the hair follicle outer root sheath cells resulted in the altered expression of genes involved in the control of proliferation and apoptosis, and abrogated stimulatory effects of blue light (3.2 J/cm2; 453 nm) on proliferation in the outer root sheath cells. Conclusions: We provide the first evidence that 1) OPN2 and OPN3 are expressed in human hair follicle, and 2) 453 nm blue light at low radiant exposure exerts a positive effect on hair growth ex vivo, potentially via interaction with OPN3. / This study was supported by the European Marie-Curie Actions Programme, Grant agreement no.: 607886