Expression of E6 and E7 papillomavirus oncogenes in the outer root sheath of hair follicles extends the growth phase and bypasses resting at telogen.

Hair follicle growth cycle proceeds through a series of stages in which strict control of cell proliferation, differentiation, and cell death occurs. Transgenic mice expressing human papillomavirus type 16 E6/E7 papillomavirus oncogenes in the outer root sheath (ORS) display a fur phenotype characterized by lower hair density and the ability to regenerate hair much faster than wild-type mice. Regenerating hair follicles of transgenic mice show a longer growth phase (anagen), and although bulb regression (catagen) occurs, rest at telogen was not observed. No abnormalities were detected during the first cycle of hair follicle growth, but by the second cycle, initiation of catagen was delayed, and rest at telogen was again not attained, even in the presence of estradiol, a telogen resting signal. In conclusion, expression of E6/E7 in the ORS delays entrance to catagen and makes cells of the ORS insensitive to telogen resting signals bearing to a continuous hair follicle cycling in transgenic mice.     

Circadian Clock Genes Contribute to the Regulation of Hair Follicle Cycling

Hair follicles undergo recurrent cycling of controlled growth (anagen), regression (catagen), and relative quiescence (telogen) with a defined periodicity. Taking a genomics approach to study gene expression during synchronized mouse hair follicle cycling, we discovered that, in addition to circadian fluctuation, CLOCK–regulated genes are also modulated in phase with the hair growth cycle. During telogen and early anagen, circadian clock genes are prominently expressed in the secondary hair germ, which contains precursor cells for the growing follicle. Analysis of Clock and Bmal1 mutant mice reveals a delay in anagen progression, and the secondary hair germ cells show decreased levels of phosphorylated Rb and lack mitotic cells, suggesting that circadian clock genes regulate anagen progression via their effect on the cell cycle. Consistent with a block at the G1 phase of the cell cycle, we show a significant upregulation of p21 in Bmal1 mutant skin. While circadian clock mechanisms have been implicated in a variety of diurnal biological processes, our findings indicate that circadian clock genes may be utilized to modulate the progression of non-diurnal cyclic processes.     

Decapeptide with fibroblast growth factor (FGF)-5 partial sequence inhibits hair growth suppressing activity of FGF-5

Earlier studies demonstrated that knock-out of fibroblast growth factor-5 gene (Fgf-5) prolonged anagen VI phase of hair cycle, resulting long hairs in the mice. We showed the activities on hair growth of the two Fgf-5 gene products, one of which, FGF-5 suppressed hair growth by inhibiting anagen proceeding and inducing the transition from anagen to catagen, and FGF-5S, a shorter polypeptide with FGF-5-antagonizing activity translated from alternatively spliced mRNA, suppressed this activity of FGF-5. As the results suggested that FGF-5 antagonist would increase hair growth, we synthesized various peptides having partial sequences of human FGF-5 and FGF-5S and determined their FGF-5 antagonist activity. Among them, a decapeptide designated P3 (95-VGIGFHLQIY-104) that aligns with receptor binding sites of FGF-1 and FGF-2 suppressed FGF-5-induced proliferation of BALB/3T3 A31 and NIH/3T3 murine fibroblasts, and FGF receptor-1c (FGFR-1c)-transfected Ba/F3 cell line (FR-Ba/F3 cells). IC50s of this peptide on these cell proliferations were 64, 28, 146 microM, respectively. On the other hand, IC50 of this peptide on binding of FGF-5 to the FGFR-1(IIIc)/Fc chimera was 483 microM. Examination in dorsal depilated mice revealed that the P3 peptide reduced the activity of FGF-5 to recover hair pigmentation and hair follicle lengths. The classification of histologically observed skin sections showed FGF-5-induced delations of anagen procedure had reduced by the P3 peptide. The anti-Ki67 antibody staining of hair follicles was inhibited by administration of FGF-5, and this inhibition by FGF-5 was recovered by administration of the P3 peptide. The P3 peptide alone did not affect hair follicle length and hair cell proliferation. These results indicate that the decapeptide antagonized FGF-5 activity in vivo, and reduced the inhibition of FGF-5 in hair growth, confirming that FGF-5 inhibitors are promising substances against hair loss and/or for promoting hair growth.     

Urokinase plasminogen activator (uPA) is a positive regulator of outer root sheath keratinocyte proliferation

Urokinase plasminogen activator (uPA), a serine proteinase, is important in the development and epidermal wound healing, and seems to play a regulatory role in the proliferation of mouse epidermal keratinocytes (KC). In the present study, we found detectable uPA expression in outer root sheath (ORS) KC in the early anagen phase in mouse vibrissa follicles, but not in the late anagen or in the telogen and categen phases. uPA was also detected in ORS KC cultured from neonatal mice vibrissa. Specific exogenous inhibitors of uPA, amiloride and uPA antibody, significantly reduced the proliferation of ORS KC. Thus uPA is consistently elevated in the hyperproliferative hair follicle KC, and inhibition of the enzyme decreases hair follicle KC proliferation. We deduce that uPA is a very important mediator of the hair follicle cycle because its activity correlates with ORS KC proliferation.     

Wnt3a在毛囊及黑素细胞谱系中的表达

目的:探讨毛囊周期中,Wnt3a在毛囊及黑素细胞中的表达变化。方法:以DCT-LacZ转基因小鼠为动物模型,通过X-gal染色技术观察黑素细胞谱系在小鼠皮肤中的分布情况;采用X-gal染色结合免疫组化方法检测Wnt3a在毛囊及黑素细胞谱系中的表达情况;采用RT-PCR方法对小鼠皮肤全层Wnt3a和TYR的mRNA表达进行半定量分析。结果:在生长期毛囊中,Wnt3a蛋白在表皮、毛囊外根鞘Bulge区、内根鞘以及毛球部均有表达,在黑素干细胞与黑素细胞也观察到Wnt3a;在退化期,Wnt3a的表达逐渐减弱,仅在外根鞘有较弱的表达,但黑素干细胞中没有观察到Wnt3a;在静止期,几乎检测不到Wnt3a的表达;TYR mRNA与Wnt3a mRNA在毛囊周期中的表达模式一致,在生长期最强,退化期减弱,静止期最弱。结论:Wnt3a可能对黑素细胞谱系分化起到促进作用。     

Expression of galectin-1 and -3 and of accessible binding sites during murine hair cycle

Although protein-carbohydrate interactions are supposed to play key roles in cell adhesion, signalling and growth control. Their exact role in skin physiology has only recently been investigated. The endogenous lectins galectin-1 and galectin-3 have been identified in skin including hair follicles. Here, we analyzed the expression and distribution of these galectins and their binding sites in C57BL/6 mice during hair cycle. The expression of galectin-1 and galectin-3 binding sites was found to be predominantly hair cycle-dependent showing some overlapping to the expression of galectin-1 and -3. The outer root sheath (ORS) expressed galectin-1 binding sites during anagen IV to VI and in early catagen, whereas galectin-1 was expressed from early anagen to late catagen. The ORS expressed galectin-3 binding sites during catagen transition corresponding to a galectin-3 expression during anagen V and catagen. The innermost layer of the ORS expressed galectin-3 binding sites during anagen VI until catagen VIII, but galectin-3 during anagen III to IV and catagen. The inner root sheath (IRS) expressed galectin-3 binding sites only in anagen IV but missed expression of any of the two galectins. The matrix cells expressed galectin-3 binding sites in catagen II-III as well as galectin-3 during anagen V to catagen IV. The present study provides the first evidence for a cycle-related expression of both galectin-1 and -3 and their binding sites during murine hair cycle.     

Dynamic change of histone H2AX phosphorylation independent of ATM and DNA-PK in mouse skin in situ

Histone H2AX undergoes phosphorylation on Ser 139 (γ-H2AX) rapidly in response to DNA double-strand breaks induced by exogenous stimuli, such as ionizing radiation. However, the endogenous phosphorylation pattern and modifier of H2AX remain unclear. Here we show that H2AX is regulated physically at the level of phosphorylation at Ser139 during a hair cycle in the mouse skin. In anagen hair follicles, γ-H2AX-positive cells were observed in the outer root sheath (ORS) and hair bulb in a cycling inferior region but not in a permanent superficial region. In telogen hair follicles, γ-H2AX-positive cells were only detected around the germ cell cap. In contrast, following X-irradiation, γ-H2AX was observed in various cell types including the ORS cells in the permanent superficial region. Furthermore, γ-H2AX-positive cells were detected in the skin of mice lacking either ATM or DNA-PK, suggesting that these kinases are not essential for phosphorylation in vivo.     

IGF1和IGF1R在小鼠第一个毛囊生长周期皮肤的表达

毛囊生长周期中,真皮乳头和毛基质间的基质 上皮信号调控细胞的增殖和分化。多功能细胞调控因子胰岛素样生长因子1（IGF1)是该信号路径的成员之一。第1个毛囊生长周期决定着毛囊的正常生长和发育,但IGF1在此期的作用未见报道。实时荧光定量PCR结果显示,IGF1在生长期皮肤中的相对表达量最低,在退化期表达量最高,在静止期表达量又降低。与生长初期相比,IGF1在退化期和静止期的表达量呈差异极显著（P＜0.01）;胰岛素样生长因子1受体（IGF1R）在生长期皮肤中的相对表达量最高,在退化期表达量最低,而在静止期表达量又升高。与生长初期相比,IGF1R在退化期和静止期的表达量呈差异极显著（P＜0.01）。Western 印迹结果显示,IGF1和IGF1R蛋白在小鼠皮肤第1个毛囊生长周期各阶段的表达趋势分别与其mRNA的表达趋势一致;免疫组织化学结果表明,IGF1主要分布在小鼠表皮,而IGF1R免疫阳性在小鼠毛囊毛球部、内外根鞘和毛乳头均有分布。以上实验结果揭示,IGF1和IGF1R在小鼠皮肤第1个毛囊生长周期的各阶段的差异性表达,可能在毛囊生长周期各阶段的转化过程中参与了黑色素的形成。然而,IGF1和IGF1R表达趋势不一致,提示IGF1在小鼠皮肤中发挥作用时,并非只与IGF1R结合才能发挥作用。     

Cutaneous application of α-methylspermidine activates the growth of resting hair follicles in mice

Recent studies using transgenic animals have revealed a crucial role for polyamines in the development and the growth of skin and hair follicles. In mammals, the growth of hair is characterized by three main cyclic phases of transformation, including a rapid growth phase (anagen), an apoptosis-driven regression phase (catagen) and a relatively quiescent resting phase (telogen). The polyamine pool during the anagen phase is higher than in telogen and catagen phases. In this study, we used α-methylspermidine, a metabolically stable polyamine analog, to artificially elevate the polyamine pool during telogen. This manipulation was sufficient to induce hair growth in telogen phase mice after 2 weeks of daily topical application. The application site was characterized by typical features of anagen, such as pigmentation, growing hair follicles, proliferation of follicular keratinocytes and upregulation of β-catenin. The analog penetrated the protective epidermal layer of the skin and could be detected in dermis. The natural polyamines were partially replaced by the analog in the application site. However, the combined pool of natural spermidine and α-methylspermidine exceeded the physiological spermidine pool in telogen phase skin. These results highlight the role of polyamines in hair cycle regulation and show that it is possible to control the process of hair growth using physiologically stable polyamine analogs.     

Control of murine hair follicle regression (catagen) by TGF-beta1 in vivo.

The regression phase of the hair cycle (catagen) is an apoptosis-driven process accompanied by terminal differentiation, proteolysis, and matrix remodeling. As an inhibitor of keratinocyte proliferation and inductor of keratinocyte apoptosis, transforming growth factor beta1 (TGF-beta1) has been proposed to play an important role in catagen regulation. This is suggested, for example, by maximal expression of TGF-beta1 and its receptors during late anagen and the onset of catagen of the hair cycle. We examined the potential involvement of TGF-beta1 in catagen control. We compared the first spontaneous entry of hair follicles into catagen between TGF-beta1 null mice and age-matched wild-type littermates, and assessed the effects of TGF-beta1 injection on murine anagen hair follicles in vivo. At day 18 p.p., hair follicles in TGF-beta1 -/- mice were still in early catagen, whereas hair follicles of +/+ littermates had already entered the subsequent resting phase (telogen). TGF-beta1-/- mice displayed more Ki-67-positive cells and fewer apoptotic cells than comparable catagen follicles from +/+ mice. In contrast, injection of TGF-beta1 into the back skin of mice induced premature catagen development. In addition, the number of proliferating follicle keratinocytes was reduced and the number of TUNEL + cells was increased in the TGF-beta1-treated mice compared to controls. Double visualization of TGF-beta type II receptor (TGFRII) and TUNEL reactivity revealed colocalization of apoptotic nuclei and TGFRII in catagen follicles. These data strongly support that TGF-beta1 ranks among the elusive endogenous regulators of catagen induction in vivo, possibly via the inhibition of keratinocyte proliferation and induction of apoptosis. Thus, TGF-betaRII agonists and antagonists may provide useful therapeutic tools for human hair growth disorders based on premature or retarded catagen development (effluvium, alopecia, hirsutism).     

Hair cycle dynamics: the case of the human hair follicle

The existence of a growth and regeneration cycle makes the hair follicle a true paradigm of tissue homeostasis. Analysis of about 9000 cycles led us to propose a stochastic model of human hair dynamics. The existence of hair cycles implies that stem cells must be cyclically activated and hair melanin unit has to be renewed. Using different markers, we were able to identify two distinct epithelial stem cell reservoirs, located in the upper and lower thirds of the anagen hair follicle outer root sheath. These two reservoirs fuse during the regression phase and individualize again in the new forming anagen hair follicle. Using a set of antibodies specific of melanocyte lineage and melanogenesis, pigmentation unit turnover was followed throughout the entire hair cycle. In the terminal anagen hair, active melanocytes were localized on top of the dermal papilla, while amelanotic melanocytes were identified in the upper third of the outer root sheath (ORS). Those amelanotic melanocytes located in upper ORS probably represented a melanocyte reservoir for successive hair generation, since at the induction of anagen phase, some melanocytes were committed to cell division and melanogenesis was turned on, but only in the nascent hair bulb, close to the dermal papilla.     

Telogen elongation in the hair cycle of ob/ob mice

Alopecia impairs the physical and mental health of patients. We have previously shown that 8-week-old ob/ob mice have no reactivity to depilation, which is a stimulus that induces anagen transition in normal mice, while no hair cycle abnormalities have been reported in other studies until mice reach 7 weeks of age. Therefore, we hypothesized that ob/ob mice have abnormalities in hair cycle progression beyond 7 weeks of age. We examined 6- to 24-week-old ob/ob and 6- to 10-week-old normal mice. After acclimation, the dorsal skin was harvested and the hair cycle phase was identified histologically and immunohistochemically. Normal mice showed catagen–telogen and telogen–anagen transitions at 6 and 8–9 weeks old, respectively. In contrast, the anagen–catagen transition was observed in 7-week-old mice and the telogen phase was maintained from 10 to 24 weeks in most ob/ob mice. These results suggests that ob/ob mice are a possible model animal for telogen effluvium.     

Epidermal growth factor as a biologic switch in hair growth cycle

The hair growth cycle consists of three stages known as the anagen (growing), catagen (involution), and telogen (resting) phases. This cyclical growth of hair is regulated by a diversity of growth factors. Although normal expression of both epidermal growth factor and its receptor (EGFR) in the outer root sheath is down-regulated with the completion of follicular growth, here we show that continuous expression of epidermal growth factor in hair follicles of transgenic mice arrested follicular development at the final stage of morphogenesis. Data from immunoprecipitation and immunoblotting showed that epidermal growth factor signals through EGFR/ErbB2 heterodimers in skin. Furthermore, topical application of tyrphostin AG1478 or AG825, specific inhibitors of EGFR and ErbB2, respectively, completely inhibited new hair growth in wild type mice but not in transgenic mice. When the transgenic mice were crossed with waved-2 mice, which possess a lower kinase activity of EGFR, the hair phenotype was rescued in the offspring. Taken together, these data suggest that EGFR signaling is indispensable for the initiation of hair growth. On the other hand, continuous expression of epidermal growth factor prevents entry into the catagen phase. We propose that epidermal growth factor functions as a biologic switch that is turned on and off in hair follicles at the beginning and end of the anagen phase of the hair cycle, guarding the entry to and exit from the anagen phase.     

Estrogen leads to reversible hair cycle retardation through inducing premature catagen and maintaining telogen

Estrogen dysregulation causes hair disorder. Clinical observations have demonstrated that estrogen raises the telogen/anagen ratio and inhibits hair shaft elongation of female scalp hair follicles. In spite of these clinical insights, the properties of estrogen on hair follicles are poorly dissected. In the present study, we show that estrogen induced apoptosis of precortex cells and caused premature catagen by up-regulation of TGF β2. Immediately after the premature catagen, the expression of anagen chalone BMP4 increased. The up-regulation of BMP4 may further function to prevent anagen transition and maintain telogen. Interestingly, the hair follicle stem cell niche was not destructed during these drastic structural changes caused by estrogen. Additionally, dermal papilla cells, the estrogen target cells in hair follicles, kept their signature gene expressions as well as their hair inductive potential after estrogen treatment. Retention of the characteristics of both hair follicle stem cells and dermal papilla cells determined the reversibility of the hair cycle suppression. These results indicated that estrogen causes reversible hair cycle retardation by inducing premature catagen and maintaining telogen.     

Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen

The lowermost portion of the resting (telogen) follicle consists of the bulge and secondary hair germ. We previously showed that the progeny of stem cells in the bulge form the lower follicle and hair, but the relationship of the bulge cells with the secondary hair germ cells, which are also involved in the generation of the new hair at the onset of the hair growth cycle (anagen), remains unclear. Here we address whether secondary hair germ cells are derived directly from epithelial stem cells in the adjacent bulge or whether they arise from cells within the lower follicle that survive the degenerative phase of the hair cycle (catagen). We use 5-bromo-2'-deoxyuridine to label bulge cells at anagen onset, and demonstrate that the lowermost portion of the bulge collapses around the hair and forms the secondary hair germ during late catagen. During the first six days of anagen onset bulge cells proliferate and self-renew. Bulge cell proliferation at this time also generates cells that form the future secondary germ. As bulge cells form the secondary germ cells at the end of catagen, they lose expression of a biochemical marker, S100A6. Remarkably, however, following injury of bulge cells by hair depilation, progenitor cells in the secondary hair germ repopulate the bulge and re-express bulge cell markers. These findings support the notion that keratinocytes can "dedifferentiate" to a stem cell state in response to wounding, perhaps related to signals from the stem cell niche. Finally, we also present evidence that quiescent bulge cells undergo apoptosis during follicle remodeling in catagen, indicating that a subpopulation of bulge cells is not permanent.     

常见毛囊细胞角蛋白在毛囊周期中的表达研究

目的探讨常见毛囊细胞角蛋白在毛囊周期中的表达特征。 方法取毛囊发育期、生长期启动、生长期、退化期和静止期的小鼠皮肤,石蜡切片后通过免疫荧光的方法,检测细胞角蛋白Krt5、Krt6、Krt10、Krt14、Krt15和Krt19的表达情况。 结果Krt5在静止期和生长期启动表达于所有毛囊上皮细胞,在其他时期表达不一致;Krt6表达于所有时期的外根鞘细胞和内根鞘细胞;Krt10表达于生长期和退化期的毛母质和内根鞘细胞,在其他时期表达不一致;Krt14在生长期和退化期表达于所有毛囊上皮细胞,在其他时期表达不一致;Krt15和Krt19表达于毛囊发育期、生长期启动和静止期的毛囊隆突区细胞,在生长期和退化期表达不一致。 结论角蛋白作为毛囊结构或毛囊干细胞标记物仅适用于特定的毛囊周期。研究者在使用毛囊角蛋白作为标记物时,应首先明确其在毛囊周期中的表达情况。     

Resting no more: re‐defining telogen,the maintenance stage of the hair growth cycle

The hair follicle (HF) represents a prototypic ectodermal–mesodermal interaction system in which central questions of modern biology can be studied. A unique feature of these stem‐cell‐rich mini‐organs is that they undergo life‐long, cyclic transformations between stages of active regeneration (anagen), apoptotic involution (catagen), and relative proliferative quiescence (telogen). Due to the low proliferation rate and small size of the HF during telogen, this stage was conventionally thought of as a stage of dormancy. However, multiple lines of newly emerging evidence show that HFs during telogen are anything but dormant. Here, we emphasize that telogen is a highly energy‐efficient default state of the mammalian coat, whose function centres around maintenance of the hair fibre and prompt responses to its loss. While actively retaining hair fibres with minimal energy expenditure, telogen HFs can launch a new regeneration cycle in response to a variety of stimuli originating in their autonomous micro‐environment (including its stem cell niche) as well as in their external tissue macro‐environment. Regenerative responses of telogen HFs change as a function of time and can be divided into two sub‐stages: early ‘refractory’ and late ‘competent’ telogen. These changing activities are reflected in hundreds of dynamically regulated genes in telogen skin, possibly aimed at establishing a fast response‐signalling environment to trauma and other disturbances of skin homeostasis. Furthermore, telogen is an interpreter of circadian output in the timing of anagen initiation and the key stage during which the subsequent organ regeneration (anagen) is actively prepared by suppressing molecular brakes on hair growth while activating pro‐regenerative signals. Thus, telogen may serve as an excellent model system for dissecting signalling and cellular interactions that precede the active ‘regenerative mode’ of tissue remodeling. This revised understanding of telogen biology also points to intriguing new therapeutic avenues in the management of common human hair growth disorders.     

Analysis of the expression pattern of involucrin in human scalp skin and hair follicles: hair cycle-associated alterations

Involucrin is a structural component of the keratinocyte cornified envelope that is expressed early in the keratinocyte differentiation process. It is a component of the initial envelope scaffolding and considered as a marker for keratinocyte terminal differentiation. The expression pattern of involucrin in human scalp skin and hair follicle cycle stages is not fully explored. This study addresses this issue and tests the hypothesis that "the expression of involucrin undergoes hair follicle cycle-dependent changes". A total of 50 normal human scalp skin biopsies were examined (healthy females, 51-62?years) using immunofluorescence staining methods and real-time PCR analysis. In each case, 50 hair follicles were analyzed (35, 10 and 5 follicles in anagen, catagen and telogen, respectively). Involucrin was prominently expressed in the human scalp skin and hair follicles, on both gene and protein levels. The protein expression showed hair follicle cycle-associated changes i.e. a very strong expression during early and mature anagen, intermediate to strong expression during catagen and prominent decline in the telogen phase. The expression value of involucrin in both anagen and catagen was statistically significantly higher than that of telogen hair follicles (p?相似文献