Disruption of the hedgehog signaling pathway contributes to the hair follicle cycling deficiency in Vdr knockout mice

Mice null for the Vitamin D receptor (VdrKO) have a disrupted first hair follicle cycle and aborted subsequent hair follicle cycling. We examined the expression of different markers and mediators of hair follicle cycling in the hair follicle of the VdrKO mouse during days 13–22 when the hair follicle normally initiates and completes the first catagen. We compared the expression of those genes in mice with a nonsense mutation in hairless (Rhino), which have a similar alopecia phenotype, and to Cyp27b1 null mice which are deficient in the production of 1,25(OH)2D3, the Vdr ligand, but display normal hair follicle cycling. Our results demonstrate the down regulation of hair follicle markers and the alteration of expression of hedgehog (Hh), Wnt, Fgf, and Tgfβ pathways in VdrKO and Rhino mice, but not in Cyp27b1KO mice. Treatment of VdrKO mice with an agonist to the Hh pathway partially restored hair follicle cycling, suggesting a role of this pathway in the regulation of hair follicle cycling by VDR. These results suggest that Vdr regulates directly or indirectly the expression of genes required for hair follicle cycling, including Hh signaling, independent of 1,25(OH)2D3. J. Cell. Physiol. 225: 482–489, 2010. © 2010 Wiley‐Liss, Inc.     

Interaction of mutant genes Fgf5(go-Y), we, and wal changes the duration of hair growth cycles in mice

Mutant gene wallhaarig (wa) was acting as a modifier of the mutant gene waved alopecia (wal), substantially increasing hair loss rate in mice, as was previously shown in our laboratory. The current paper is devoted to a study of mutant gene angora- Y(Fgf5(go-Y)), which had extended anagen stage of the first and second generations hair growth cycles in triple heterozygotes (Fgf5(go-Y)/Fgf5(go-Y) we/we wal/wal). First generation guard hair in triple homozygotes had their anagen stage 4 days longer than the same stage in double homozygotes (+/+ we/we wal/wal). Hair loss started at a catagen stage in double homozygotes, while it started in triple homozygotes at the end of the same stage or even in a telogen. Such mutant gene interaction in hair follicle morphogenesis led to a partial recovery of a body hair coat in triple homozygotes.     

<Emphasis Type="Italic">Gsdma3</Emphasis> gene is needed for the induction of apoptosis-driven catagen during mouse hair follicle cycle

Gsdm is a newly found gene family, which is restricted in its expression to the gastrointestinal tract and the skin epithelium. As a main member of the Gsdma subfamily, Gsdma3 is expressed specifically in the hair follicle of mouse skin, but its function remains largely unclear. By hematoxylin and eosin staining, we showed that Gsdma3 gene mutation caused an abnormal catagen phase with unshortened length and unshrunk structure of the hair follicle, in which the development of catagen phase was inhibited. TUNEL staining further revealed that the apoptosis of the hair follicle was obviously decreased in mutant mice. Caspase-3 downregulation was also detected by immunofluorescence, Western blot and RT-PCR in the hair follicle of the mutant mice. After intradermal injection of Gsdma3 gene expression plasmid, apoptosis as well as Caspase-3 expression in the hair follicle of mutant mice was enhanced, and so the catagen retardation of Gsdma3-mutant mice was rescued. Our results confirmed that Gsdma3 gene mutation interfered with catagen formation during mouse hair follicle cycle and, by upregulation of Caspase-3 expression and promotion of apoptosis, Gsdma3 gene could play an essential role in normal catagen induction.     

Gene angora as a modifier of the mouse hairless gene

The interactions between mouse angora-Y (Fgf5go-Y) and hairless (hr) genes have been studied. Homozygous mutant gene Fgf5go-Y increases hair length starting on day 14 after birth. We obtained mice with genotypes +/+ hr/hr F2, +/Fgf5go-Y hr/hr and Fgf5go-Y/Fgf5go-Y hr/hr. Both +/Fgf5go-Y hr/hr and +/+ hr/hr mice began to loose hair from their heads on day 14. This further extended on the whole body. On day 21 the mice were completely deprived of hair. Therefore a single dose of gene Fgf5go-Y does not affect alopecia mice homozygous for hr. However in double homozygotes Fgf5go-Y/Fgf5gO-hr/hr alopecia started 4 days later, namely on day 18. It usually finished 10-12 days after detection of first bald patches. On days 28-30 double homozygotes have lost all the hair. Hair loss in double homozygous mice was 1,5-fold slower than in +/+ hr/hr mice. This resulted from a significant extension of anagen phase induced by a mutant homozygous gene Fgf5go-Y in morphogenesis of the hair follicle. In contrast, hr gene was expressed only at the transmission phase from anagen to catagen. Our data shows that the angora gene is a modifier of the hairless gene and this results in a strong repression of alopecia progression in double homozygous mice compared to +/+ hr/hr animals.     

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。     

Involvement of hepatocyte growth factor/scatter factor and met receptor signaling in hair follicle morphogenesis and cycling.

HGF/SF and its receptor (Met) are principal mediators of mesenchymal-epithelial interactions in several different systems and have recently been implicated in the control of hair follicle (HF) growth. We have studied their expression patterns during HF morphogenesis and cycling in C57BL/6 mice, whereas functional hair growth effects of HGF/SF were assessed in vivo by analysis of transgenic mice and in skin organ culture. In normal mouse skin, follicular expression of HGF/SF and Met was strikingly localized: HGF/SF was found only in the HF mesenchyme (dermal papilla fibroblasts) and Met in the neighboring hair bulb keratinocytes. Both HGF/SF and Met expression peaked during the initial phases of HF morphogenesis, the stage of active hair growth (early and mid anagen), and during the apoptosis-driven HF regression (catagen). Met+ cells in the regressing epithelial strand appeared to be protected from undergoing apoptosis. Compared to wild-type controls, transgenic mice overexpressing HGF/SF under the control of the MT-1 promoter had twice as many developing HF and displayed accelerated HF development on postnatal day 3. They also showed significant catagen retardation on P17. In organ culture and in vivo, HGF/SF i.c. resulted in a significant catagen retardation. These results demonstrate an important role of HGF/SF and Met in murine hair growth control and suggest that Met-mediated signaling might be exploited for therapeutic manipulation of human hair growth disorders.-Lindner, G., Menrad, A., Gherardi, E., Merlino, G., Welker, P., Handjiski, B., Roloff, B., Paus, R. Involvement of hepatocyte growth factor/scatter factor and Met receptor signaling in hair follicle morphogenesis and cycling.     

The TGF-beta2 isoform is both a required and sufficient inducer of murine hair follicle morphogenesis.

Hair follicle development serves as an excellent model to study control of organ morphogenesis. Three specific isoforms of TGF-beta exist which exhibit a distinct pattern of expression during hair follicle morphogenesis. To clarify the still elusive role of these factors in hair follicle development, we have used a combined genetic and functional approach: analysis of hair follicle development in mice with disruptions of the TGF-beta1, 2, and 3 genes was coupled with a direct functional test of the effect of added purified factors on fetal hair follicle development in skin organ cultures. TGF-beta2 null mice exhibited a profound delay of hair follicle morphogenesis, with a 50% reduced number of hair follicles. In contrast to hair follicle development, growth and differentiation of interfollicular keratinocytes proceeded unimpaired. Unlike TGF-beta2-/- mice, mice with a disruption of the TGF-beta1 gene showed slightly advanced hair follicle formation, while lack of the TGF-beta3 gene did not have any effects. Treatment of wild-type, embryonic skin explants (E14.5 or E15.5) with TGF-beta2 protein in either soluble form or slow release beads induced hair follicle development and epidermal hyperplasia, while similar TGF-beta1 treatment exerted suppressive effects. Thus, the TGF-beta2 isoform plays a specific role, not shared by the other TGF-beta isoforms, as an inducer of hair follicle morphogenesis and is both required and sufficient to promote this process.     

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.     

Role of the vitamin D receptor in hair follicle biology

The vitamin D receptor (VDR) is expressed in numerous cells and tissues, including the skin. The critical requirement for cutaneous expression of the VDR has been proven by investigations in mice and humans lacking functional receptors. These studies demonstrate that absence of the VDR leads to the development of alopecia. The hair follicle is formed by reciprocal interactions between an epidermal placode, which gives rise to the hair follicle keratinocytes and the underlying mesoderm which gives rise to the dermal papilla. Hair follicle morphogenesis ends the second week of life in mice. Studies in VDR null mice have failed to demonstrate a cutaneous abnormality during this period of hair follicle morphogenesis. However, VDR null mice are unable to initiate a new hair cycle after the period of morphogenesis is complete, therefore, do not grow new hair. Investigations in transgenic mice have demonstrated that restricted expression of the VDR to keratinocytes is capable of preventing alopecia in the VDR null mice, thus demonstrating that the epidermal component of the hair follicle requires VDR expression to maintain normal hair follicle homeostasis. Studies were then performed to determine which regions of the VDR were required for these actions. Investigations in mice lacking the first zinc finger of the VDR have demonstrated that they express a truncated receptor containing an intact ligand binding and AF2 domain. These mice are a phenocopy of mice lacking the VDR, thus demonstrate the critical requirement of the DNA binding domain for hair follicle homeostasis. Transgenic mice expressing VDRs with mutations in either the ligand-binding domain or the AF2 domain were generated. These investigations demonstrated that mutant VDRs incapable of ligand-dependent transactivation were able to prevent alopecia. Investigations are currently underway to define the mechanism by which the unliganded VDR maintains hair follicle homeostasis.     

Pathobiology of androgenetic alopecia

Human hair morphogenesis is a dynamic process caused by the remodelling of the skin. Hair growth is cyclic in mammals consisting of three distinct stages: an active stage (anagen), a regressive stage (catagen), and a resting stage (telogen). One disorder in this process is gradual balding of the scalp called androgenetic alopecia. Little is known about the cell biological or molecular mechanisms involved and thus very little treatment is currently available. In this review we focus on the most significant parameters affecting hair growth which participate in baldness.     

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).     

Molecular biology of hair morphogenesis: development and cycling

In mammals, hair follicles produce hairs that fulfill a number of functions including thermoregulation, collecting sensory information, protection against environmental trauma, social communication, and mimicry. Hair follicles develop as a result of epithelial-mesenchymal interactions between epidermal keratinocytes committed to hair-specific differentiation and cluster of dermal fibroblasts that form follicular papilla. During postnatal life, hair follicles show patterns of cyclic activity with periods of active growth and hair production (anagen), apoptosis-driven involution (catagen), and relative resting (telogen). During last decade, substantial progress has been achieved in delineating molecular mechanisms that control hair follicle development and cyclic activity. In this review, we summarize the data demonstrating that regulation of hair follicle development in the embryo and control of hair follicle growth during postnatal life are highly conserved and both require involvement of similar molecular mechanisms. Since many of the molecules that control hair follicle development and cycling are also involved in regulating morphogenesis and postnatal biology of other ectodermal derivatives, such as teeth, feathers, and mammary glands, basic principles and molecular mechanisms that govern hair follicle development and growth may also be applicable for other developmental systems.     

Histophysiology of hair follicles: current concept

Hair follicle histophysiology importance isn't limited by hair role in psychosocial consequences. More scientists consider the hair follicle as an attractive system for studying major biological phenomena because the hair follicle is a regenerating system. In this review we revisit the current information about histophysiology and control of hair follicle cycling. All mature follicles undergo a growth cycle consisting of following phases: growth (anagen), regression (catagen) and rest (telogen). We attempt to integrate the morphology with the physiology and molecular biology. Hair follicles are influenced by environmental, systemic and local factors. The most interesting point of this problem is discussed--an integral regulation of hair follicle cycle by systemic, intertissue and intercellular interactions.     

A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression.

To examine the mechanisms that underlie the neurotrophin-induced, apoptosis-driven hair follicle involution (catagen), the expression and function of p75 neurotrophin receptor (p75NTR), which is implicated in apoptosis control, were studied during spontaneous catagen development in murine skin. By RT-PCR, high steady-state p75NTR mRNA skin levels were found during the anagen-catagen transition of the hair follicle. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL+/Bcl2- keratinocytes of the regressing outer root sheath, but both p75NTR and TrkB and/or TrkC were expressed by the nonregressing TUNEL-/Bcl2+ secondary hair germ keratinocytes. To determine whether p75NTR is functionally involved in catagen control, spontaneous catagen development was compared in vivo between p75NTR knockout (-/-) and wild-type mice. There was significant catagen retardation in p75NTR knockout mice as compared to wild-type controls (P<0.05). Instead, transgenic mice-overexpressing NGF (promoter: K14) showed substantial acceleration of catagen (P<0.001). Although NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT-3) accelerated catagen in the organ-cultured skin of C57BL/6 mice, these neurotrophins failed to promote catagen development in the organ-cultured p75NTR null skin. These findings suggest that p75NTR signaling is involved in the control of kerotinocyte apoptosis during catagen and that pharmacological manipulation of p75NTR signaling may prove useful for the treatment of hair disorders that display premature entry into catagen.     

Deletion of hypoxia-inducible factor prolyl 4-hydroxylase 2 in FoxD1-lineage mesenchymal cells leads to congenital truncal alopecia

Hypoxia-inducible factors (HIFs) induce numerous genes regulating oxygen homeostasis. As oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs) regulate the stability of HIFs in an oxygen-dependent manner. During hair follicle (HF) morphogenesis and cycling, the location of dermal papilla (DP) alternates between the dermis and hypodermis and results in varying oxygen levels for the DP cells. These cells are known to express hypoxia-inducible genes, but the role of the hypoxia response pathway in HF development and homeostasis has not been studied. Using conditional gene targeting and analysis of hair morphogenesis, we show here that lack of Hif-p4h-2 in Forkhead box D1 (FoxD1)-lineage mesodermal cells interferes with the normal HF development in mice. FoxD1-lineage cells were found to be mainly mesenchymal cells located in the dermis of truncal skin, including those cells composing the DP of HFs. We found that upon Hif-p4h-2 inactivation, HF development was disturbed during the first catagen leading to formation of epithelial-lined HF cysts filled by unorganized keratins, which eventually manifested as truncal alopecia. Furthermore, the depletion of Hif-p4h-2 led to HIF stabilization and dysregulation of multiple genes involved in keratin formation, HF differentiation, and HIF, transforming growth factor β (TGF-β), and Notch signaling. We hypothesize that the failure of HF cycling is likely to be mechanistically caused by disruption of the interplay of the HIF, TGF-β, and Notch pathways. In summary, we show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs.     

NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo

NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.     

Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and pertubation of hair follicle cycling.

Lysosomal cysteine proteinases of the papain family are involved in lysosomal bulk proteolysis, major histocompatibility complex class II mediated antigen presentation, prohormone processing, and extracellular matrix remodeling. Cathepsin L (CTSL) is a ubiquitously expressed major representative of the papain-like family of cysteine proteinases. To investigate CTSL in vivo functions, the gene was inactivated by gene targeting in embryonic stem cells. CTSL-deficient mice develop periodic hair loss and epidermal hyperplasia, acanthosis, and hyperkeratosis. The hair loss is due to alterations of hair follicle morphogenesis and cycling, dilatation of hair follicle canals, and disturbed club hair formation. Hyperproliferation of hair follicle epithelial cells and basal epidermal keratinocytes-both of ectodermal origin-are the primary characteristics underlying the mutant phenotype. Pathological inflammatory responses have been excluded as a putative cause of the skin and hair disorder. The phenotype of CTSL-deficient mice is reminiscent of the spontaneous mouse mutant furless (fs). Analyses of the ctsl gene of fs mice revealed a G149R mutation inactivating the proteinase activity. CTSL is the first lysosomal proteinase shown to be essential for epidermal homeostasis and regular hair follicle morphogenesis and cycling.