Expression profiling and cellular localization of genes associated with the hair cycle induced by wax depilation

The hair cycle is a highly regulated process controlled by multiple factors. Systematic analysis of gene expression patterns in each stage of the hair cycle would provide information useful for understanding this complicated process. To identify genes associated with the hair cycle, we used DNA microarray hybridization to analyze sequential gene expression patterns in mouse skin following hair cycle synchronization by wax depilation. Messenger RNA levels in mouse skin at various times after depilation were compared with those prior to depilation (resting phase). According to their expression patterns, upregulated genes were categorized into four groups: early anagen, middle anagen, late anagen/early catagen, and middle/late catagen, and processes that take place in each stage were evaluated. We identified 12 new components that are specifically expressed in the hair follicle, 11 genes in anagen including carbonic anhydrase 6, cytokeratin 12, and matrix metalloproteinase-11 in catagen that were confirmed using in situ hybridization. The strategy used here allowed us to identify unknown genes or process previously not suspected to have a role in hair biology. These analyses will contribute to elucidating the mechanisms of hair cycle regulation and should lead to the identification of novel molecular targets for hair growth and/or depilation agents.     

Hair cycle resting phase is regulated by cyclic epithelial FGF18 signaling

Hair follicles repeatedly cycle through growth (anagen), regression (catagen), and resting (telogen) phases. Although the signaling molecules involved in the anagen and anagen-catagen transition have been studied extensively, the signaling that controls telogen is only partly understood. Here we show that fibroblast growth factor (Fgf)18 is expressed in a hair stem cell niche throughout telogen, and that it regulates the hair cycle through the non-growth phases. When the Fgf18 gene is conditionally knocked out in keratin 5-positive epithelial cells in mice, telogen becomes very short, giving rise to a strikingly rapid succession of hair cycles. In wild-type mice, hair follicle growth during anagen is strongly suppressed by local delivery of FGF18 protein. Our results demonstrate that epithelial FGF18 signaling and its reduction in the milieu of hair stem cells are crucial for the maintenance of resting and growth phase, respectively.     

Do human dermal adipocytes switch from lipogenesis in anagen to lipophagy and lipolysis during catagen in the human hair cycle?

In murine skin, dermal white adipose tissue (DWAT) undergoes fluctuations in size across the hair cycle, whereas changes in size, function and metabolism of dermal adipocytes (DAs) during the human scalp hair cycle remain unexplored. Transmission electron microscopy results suggest that during anagen‐catagen transition, human DAs co‐opt the autophagy machinery to undergo lipophagy within their lipid droplets. Whole‐mount staining of hair follicles (HFs) and surrounding DWAT for the autophagy marker LC3B confirms the increased presence of LC3B+ lipid droplets adjacent to catagen HFs; moreover, DWAT around catagen HFs engages in greater glycerol release compared to DWAT surrounding anagen HFs. Thus, we hypothesize that human DAs switch from lipogenesis during anagen to lipophagy together with lipolysis during catagen. We propose various experiments to further prove this hypothesis, whose systematic exploration should help to better characterize the functions of human DWAT and its communication with the HF.     

Hair cycle-specific immunolocalization of retinoic acid synthesizing enzymes Aldh1a2 and Aldh1a3 indicate complex regulation

Retinoic acid has long been known to alter skin and hair growth but an exact mechanism is unclear. This study was performed to examine the sites of endogenous retinoic acid synthesis in the cycling hair follicle to better understand the role retinoic acid plays in this process. Retinal dehydrogenases (Aldh1a1, 2, and 3, formerly Raldh 1, 2, and 3) are the enzymes responsible for the last step in retinoic acid synthesis. Immunohistochemistry was performed on adult C57BL/6J mouse skin sections with antibodies against Aldh1a2 and Aldh1a3. Aldh1a2 expression was seen primarily in the outer root sheath and basal/spinous layer during all stages of the hair cycle, and in the bulge during anagen and early catagen, whereas Aldh1a3 expression was primarily in the dermal papilla, pre-cortex, and hair shaft during mid-late anagen. The expression patterns of these two similar retinoic acid synthesizing enzymes at specific follicular sites suggest that they mediate and are regulated by different epithelial proliferation and differentiation signaling pathways.     

In vivo cytokine and receptor gene expression during the rat hair growth cycle

Abstract A number of cytokines have previously been localised within the developing and adult hair follicle, however, the role they play in producing a mature hair follicle remains unknown. In an attempt to identify dermal papilla specific cytokines and thus those that may have an important controlling role, cytokine gene expression profiles, obtained by reverse transcriplase-polymerase chain reaction (RT-PCR), were compared between whole anagen rat hair follicles, passage 2 dermal papillae (a cell type with hair inductive capacity), and footpad fibroblasts (a non-hair inducing cell type). Based on this qualitative data, we were unable to identify a dermal papilla specific gene. The analysis of the pattern and timing of cytokine gene expression during the hair cycle is likely to be more informative. A semi-quantitative RT-PCR technique was therefore developed for studying trends in the level of in vivo expression of the following cytokines and their receptors from early anagen to early catagen in the rat hair growth cycle: insulin-like growth factor I, transforming growth factor β1, tumour necrosis factor, and basic fibroblast growth factor. These genes were found to be differentially expressed and this was correlated with their possible functions in controlling the hair growth cycle, providing valuable insights into the role of cytokines in regulating the hair growth process.     

Hair cycle control by leptin as a new anagen inducer

Our purpose is to clarify the physiological role of leptin in hair cycle as leptin reportedly causes activation of Stat3, which is indispensable for hair cycling. While hair follicles in dorsal skin of 5‐week‐old C57/BL6 mice had progressed to late anagen phase, those in dorsal skin of 5‐week‐old leptin receptor deficient db/db mice remained in the first telogen and later entered the anagen at postnatal day 40, indicating that deficiency in leptin receptor signalling delayed the second hair cycle progression. Next, we shaved dorsal hairs on wild‐type mice at postnatal 7 weeks and injected skin with mouse leptin or a mock. After 20 days, although mock injection showed no effect, hair growth occurred around leptin injection area. Human leptin fragment (aa22–56) had similar effects. Although the hair cycle of ob/ob mice was similar to that of wild‐type mice, injection of mouse leptin on ob/ob mice at postnatal 7 weeks induced anagen transition. Immunohistochemically, leptin is expressed in hair follicles from catagen to early anagen in wild‐type mice, suggesting that leptin is an anagen inducer in vivo. Phosphorylation of Erk, Jak2 and Stat3 in human keratinocytes was stimulated by leptin and leptin fragment. In addition, RT‐PCR and ELISA showed that the production of leptin by human dermal papilla cells increased under hypoxic condition, suggesting that hypoxia in catagen/telogen phase promotes leptin production, preparing for entry into the next anagen. In conclusion, leptin, a well‐known adipokine, acts as an anagen inducer and represents a new player in hair biology.     

Dermal glycosaminoglycan concentration throughout hair growth cycle of rats.

The dermal glycosaminoglycan (GAG) content of rats was measured quantitatively after plucking hair from the backs of the animals, which reinitiates their hair cycle. Mast cells present around the hair follicles were counted at each stage of the hair cycle and compared with the amount of GAG in the skin. GAG reached its peak on the 6th day of the anagen stage and decreased during the catagen stage. In the telogen stage, it was about the same as it had been in the catagen stage. The same pattern was noted for the number of mast cells. The 0.5 M fraction in Dowex-1 column chromatography proved to be hyaluronic acid and its concentration throughout the hair growth cycle showed strong parallels to that of total dermal GAG.     

The biology of hair

Hair is a product of small pits in the skin known as hair follicles. The most important feature of hair follicles is that their activity is intermittent; each active phase or anagen is succeeded by a transitional phase (catagen) and a resting phase (telogen), during which the fully formed "club hair" is retained for a period and then shed. The growth of facial, body, axillary, and pubic hair depends on androgens. Facial hair and body hair require high levels of testosterone and its conversion to 5-alpha-dihydrotestosterone. Pubic and axillary hair follicles require much lower levels of hormone, and 5-alpha-reduction appears to be unnecessary. Paradoxically, male pattern alopecia and its female equivalent also require androgen for their manifestation. The differing lengths of hair in the various regions of the body result largely from differences in the duration of anagen and only to a small extent from differences in the rates of growth. Some hair loss from the scalp can be characterized in terms of the hair growth cycle, and some involves long-term changes in the follicular architecture. Thus postfebrile and postpartum alopecias are telogen effluvia that involve shedding of club hairs, whereas drug-induced alopecia and alopecia areata involve shearing and loss of growing hairs. Male pattern baldness and female diffuse alopecia involve gradual shortening of the periods of anagen and shrinkage of the hair follicles over a long term.     

PDGF isoforms induce and maintain anagen phase of murine hair follicles

BACKGROUND: It is known that platelet-derived growth factor (PDGF) receptors are expressed in hair follicle (HF) epithelium. OBJECTIVES: The aim of the present study was to clarify the effects of PDGF-AA and -BB on the cyclic growth of HFs. METHODS: PDGF-AA or -BB was injected into the dorsal skin of C3H mice during the second telogen phase once daily for five consecutive days, or PDGF-AA or -BB dissolved in hyaluronic acid was injected only once. In order to confirm the effects of different PDGF isoforms, anti-PDGF-AA antibody or anti-PDGF-BB antibody was injected just after each injection of PDGF-AA or -BB. In addition, anti-PDGF antibodies were injected into the skin of C3H mice during the second anagen phase once daily for 5 days. We studied expression of signaling molecules in the skin where anagen phase had been induced by PDGF injection by real-time RT-PCR. RESULTS: Both PDGF-AA and -BB injection experiments immediately induced the anagen phase of the hair growth cycle at the injection sites. The induction of anagen was interfered by anti-PDGF antibody treatment. Real-time RT-PCR using extracted RNA from the PDGF injected sites of skin samples showed upregulated expression of HF differentiation-related key signaling molecules, Sonic hedgehog (Shh), Lef-1 and Wnt5a. CONCLUSIONS: These results indicate that both PDGF-AA and -BB are involved in the induction and maintenance of the anagen phase in the mouse hair cycle. Local application of PDGF-AA and -BB might therefore prove to be an effective treatment option for alopecia associated with early catagen induction and elongated telogen phase.     

Hair cycle-specific expression of versican in human hair follicles

BACKGROUND: Versican, a large chondroitin sulfate proteoglycan molecule, is implicated in the induction of hair morphogenesis, the initiation of hair regeneration, and the maintenance of hair growth in mouse species. In contrast, in human hair follicles, the distribution and the roles of versican remains obscure. OBJECTIVES: To elucidate the implication of versican in normal human hair growth. METHODS: Versican expression was examined by in situ hybridization (mRNA) and immunohistochemistry (protein). RESULTS: The results clearly showed specific versican gene expression in the dermal papilla of anagen, which apparently decreased in the dermal papilla of catagen hair follicles. No specific signal was detectable in telogen hair follicles. Consistent with ISH results, versican immunoreactivity was extended over the dermal papilla of anagen hair follicles, and again, this staining diminished in the catagen phase of human hair follicles. Interestingly, versican proteins were deposited outside K15-positive epithelial cells in the bulge throughout the hair cycle. Versican immunoreactivity in the dermal papilla was almost lost in vellus-like hair follicles affected by male pattern baldness. CONCLUSION: Specific expression of versican in the anagen hair follicles suggests its importance to maintain the normal growing phase of human as well as mouse.     

Overexpression of skin protein kinase C-alpha in anagen hair follicles during induced growth of mouse hair

A role for protein kinase C (PKC)-alpha has been implicated in the growth of mouse hair. Topical application of PKC activators, hair plucking, allergic contact dermatitis and skin irritation can all enhance growth of mouse hair, and a significant increase in PKC-alpha level in whole mouse skin in mature anagen has been demonstrated in these processes. Overexpression of PKC-alpha in anagen hair follicles has also been reported in natural growth of mouse hair. It is known that overexpression of PKC-alpha is associated with the acceleration of cell growth. Therefore, we postulated that overexpression of PKC-alpha in mature anagen may relate to enhancement of hair growth. The distribution of PKC-alpha in hair follicles during induced growth of mouse hair has not previously been studied. In this study, hair growth in C57BL/6 mice was induced by plucking the telogen hairs on one side of the back. The undepilated contralateral side served as a control. Expression of PKC-alpha in hair follicles during the hair growth cycle induced was evaluated by immunohistochemistry using cryosections and a specific polyclonal anti-PKC-alpha immunoglobulin G (IgG) antibody. No PKC-alpha was detected in telogen hair follicles or in the hair follicles at 1 day post-depilation, when the induced hair cycle was in early anagen. At 4 days after plucking, when the induced hair cycle was in mid-anagen, intense staining for PKC-alpha was found in hair papillae. At 10 and 17 days after depilation, when the induced hair cycle was in mature anagen and early catagen, respectively, all outer root sheath (ORS) cells and outer connective sheaths of hair follicles were stained positive. Because no PKC-alpha was detected in telogen hair follicles in this study, down-regulation of PKC-alpha in early anagen could not be observed. However, consistent with our previous findings, overexpression of PKC-alpha was found in mid-anagen and mature anagen. As overexpression of PKC-alpha has been shown to be associated with acceleration of cell growth, our results support the notion that PKC-alpha may play an important role in growth of hair follicle cells in induced growth of hair. As PKC levels are known to increase in hyperglycaemia, overexpressed PKC-alpha in mature anagen hair follicles may be related to the putative function of the ORS in mobilizing glycogen stores for anagen growth.     

Comprehensive analysis of FGF and FGFR expression in skin: FGF18 is highly expressed in hair follicles and capable of inducing anagen from telogen stage hair follicles

We quantified the mRNA expression of all 22 fibroblast growth factor family members (FGF) and their four receptors (FGFR) in adult mouse full-thickness skin at various stages of the hair growth cycle. We found that in addition to mRNA encoding FGF previously identified in skin (FGF1, 2, 5, 7, 10, 13, and 22), FGF18 mRNA was also strongly expressed. Expression of these FGF varied throughout hair growth cycle: mRNA expression of FGF18 and 13 peaked at telogen; FGF7 and 10 at anagen V; and FGF5 and 22 at anagen VI. In situ hybridization revealed that FGF18 mRNA is mainly expressed in the anagen inner root sheath and telogen bulge of hair follicles. In culture, FGF18 stimulated DNA synthesis in human dermal fibroblasts, dermal papilla cells, epidermal keratinocytes and vascular endothelial cells. When FGF18 was administered subcutaneously to mice in a uniform telogen state, anagen hair growth was observed. Our findings suggest that FGF18 is important for the regulation of hair growth and the maintenance of skin in adult mice.     

Expression of Ras homologous B protein in the human scalp skin and hair follicles: hair follicle cycle stages‐associated changes

Background: RhoB belongs to the Ras homologous (Rho) subfamily which consists of low molecular weight mass GTP‐binding proteins. Rho proteins are regulatory molecules that mediate changes in cell shape, contractility, motility and gene expression. Aim: To test the hypothesis that ‘RhoB protein is expressed in the human skin and its expression undergoes hair follicle cycle dependent changes'. To test this hypothesis, we examined the expression of RhoB in the normal human skin and hair follicles (HFs) using immunohistochemical methods. Methods: A total of 50 normal human scalp skin specimens were obtained from 50 females (age: 53–57 years) undergoing elective cosmetic plastic surgery. The specimens were obtained from both frontal and temporal regions of the scalp. A total of 50 HF, (35 anagen, 10 catagen and 5 telogen) were examined in each case using immunohistologic staining methods. Semiquantitative analysis was done. Results: RhoB protein was strongly expressed in the various elements of the human scalp skin and hair follicles. In the epidermis, a moderate RhoB immunoreactivity was found in all layers except stratum corneum where RhoB protein was completely absent. In sebaceous glands, a strong RhoB immunoreactivity was detected in all sebaceocytes. In the hair follicles, the expression of RhoB protein showed hair follicle cycle stages‐associated changes, i.e. strong expression during anagen, but weak and completely absent expressions during catagen and telogen phases, respectively. Semiquantitative analysis revealed statistically significant high expression values (staining intensity, percentage of positive cells and immunoreactivity scores) in the anagen VI hair follicles compared to either cantagen or telogen ones (p < 0.05). Similarly, RhoB protein expression was significantly high in the stratum basale, stratum spinosum and sebaceous glands compared to stratum granulosum (p < 0.05). Conclusions: Here we report, for the first time, the distribution of RhoB protein in the human scalp skin and hair follicles. We also provide the first indication that there are variations in the expression of this protein in the different stages of the hair cycle. Adly M.A, Assaf H.A, Hussein M.R.A. Expression of Ras homologous B protein in the human scalp skin and hair follicles: hair follicle cycle stages‐associated changes     

Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture

Please cite this paper as: Methods in hair research: how to objectively distinguish between anagen and catagen in human hair follicle organ culture. Experimental Dermatology 2010; 19: 305–312. Abstract: The organ culture of human scalp hair follicles (HFs) is the best currently available assay for hair research in the human system. In order to determine the hair growth‐modulatory effects of agents in this assay, one critical read‐out parameter is the assessment of whether the test agent has prolonged anagen duration or induced catagen in vitro. However, objective criteria to distinguish between anagen VI HFs and early catagen in human HF organ culture, two hair cycle stages with a deceptively similar morphology, remain to be established. Here, we develop, document and test an objective classification system that allows to distinguish between anagen VI and early catagen in organ‐cultured human HFs, using both qualitative and quantitative parameters that can be generated by light microscopy or immunofluorescence. Seven qualitative classification criteria are defined that are based on assessing the morphology of the hair matrix, the dermal papilla and the distribution of pigmentary markers (melanin, gp100). These are complemented by ten quantitative parameters. We have tested this classification system by employing the clinically used topical hair growth inhibitor, eflornithine, and show that eflornithine indeed produces the expected premature catagen induction, as identified by the novel classification criteria reported here. Therefore, this classification system offers a standardized, objective and reproducible new experimental method to reliably distinguish between human anagen VI and early catagen HFs in organ culture.