Bone morphogenetic protein signaling inhibits hair follicle anagen induction by restricting epithelial stem/progenitor cell activation and expansion

Epithelial stem cells (EP-SCs) located in the bulge region of a hair follicle (HF) have the potential to give rise to hair follicle stem/progenitor cells that migrate down to regenerate HFs. Bone morphogenetic protein (BMP) signaling has been shown to regulate the HF cycle by inhibiting anagen induction. Here we show that active BMP signaling functions to prevent EP-SC activation and expansion. Dynamic expression of Noggin, a BMP antagonist, releases EP-SCs from BMP-mediated restriction, leading to EP-SC activation and initiation of the anagen phase. Experimentally induced conditional inactivation of the BMP type IA receptor (Bmpr1a) in EP-SCs leads to overproduction of HF stem/progenitor cells and the eventual formation of matricomas. This genetic manipulation of the BMP signaling pathway also reveals unexpected activation of beta-catenin, a major mediator of Wnt signaling. We propose that BMP activity controls the HF cycle by antagonizing Wnt/beta-catenin activity. This is at least partially achieved by BMP-mediated enhancement of transforming growth factor-beta-regulated epithelial cell-specific phosphatase (PTEN) function. Subsequently, PTEN, through phosphatidyl inositol 3-kinase-Akt, inhibits the activity of beta-catenin, the convergence point of the BMP and Wnt signaling pathways.     

Differential expression of cyclin D1 in the human hair follicle

The proliferation of keratinocytes in the hair follicle varies from slowly cycling, intermittently proliferating stem cells in the bulge to rapidly proliferating, transient cells in the bulb. To better understand the biological differences between these two compartments, we sought to identify differentially expressed genes using cDNA macroarray analysis. Cyclin D1 was one of 13 genes increased in the bulge compared to the bulb, and its differential expression was corroborated by quantitative real-time polymerase chain reaction (PCR) on the original samples. Using immunohistochemical staining, laser-capture microdissection (LCM) and quantitative real-time PCR, we localized cyclin D1 to the suprabasal cells of the telogen bulge and anagen outer root sheath (ORS). Surprisingly, cyclin D1, D2, and D3 were not detectable by immunohistochemistry in the rapidly proliferating hair-producing cells of the anagen bulb (matrix cells), while these cells were strongly positive for Ki-67 and retinoblastoma protein. In contrast, pilomatricoma, a tumor thought to be derived from matrix cells, was positive for cyclin D1, D2, and D3. Our results suggest that cyclin D1 may mediate the proliferation of stem cells in the bulge to more differentiated transient amplifying cells in the suprabasal ORS. In contrast, non-cyclin D1-proteins appear to control cell division of the highly proliferative bulb matrix cells. This non-cyclin D1-mediated proliferation may provide a protective mechanism against tumorigenesis, which is overridden in pilomatricomas. Our data also demonstrate that the combination of DNA macroarray, LCM and quantitative real-time PCR is a powerful approach for the study of gene expression in defined cell populations with limited starting material.     

BMP signaling in dermal papilla cells is required for their hair follicle-inductive properties

Hair follicle (HF) formation is initiated when epithelial stem cells receive cues from specialized mesenchymal dermal papilla (DP) cells. In culture, DP cells lose their HF-inducing properties, but during hair growth in vivo, they reside within the HF bulb and instruct surrounding epithelial progenitors to orchestrate the complex hair differentiation program. To gain insights into the molecular program that maintains DP cell fate, we previously purified DP cells and four neighboring populations and defined their cell-type-specific molecular signatures. Here, we exploit this information to show that the bulb microenvironment is rich in bone morphogenetic proteins (BMPs) that act on DP cells to maintain key signature features in vitro and hair-inducing activity in vivo. By employing a novel in vitro/in vivo hybrid knockout assay, we ablate BMP receptor 1a in purified DP cells. When DPs cannot receive BMP signals, they lose signature characteristics in vitro and fail to generate HFs when engrafted with epithelial stem cells in vivo. These results reveal that BMP signaling, in addition to its key role in epithelial stem cell maintenance and progenitor cell differentiation, is essential for DP cell function, and suggest that it is a critical feature of the complex epithelial-mesenchymal cross-talk necessary to make hair.     

Expression of a candidate marker for progenitor cells, Musashi-1, in the proliferative regions of human antrum and its decreased expression in intestinal metaplasia

AIM: Reliable makers for progenitor cells in the human stomach have not been elucidated. The aim of the present study was to clarify whether Musashi-1 (Msi-1), which has recently been proposed as a stem cell marker in mouse intestine, serves as a marker for progenitor cells in human stomach. METHODS AND RESULTS: Immunohistochemistry revealed that Msi-1+ cells were detected especially in the isthmus/neck region (the putative position of stem cells) of the adult antrum, but were limited to the basal regions of fetal pyloric glands during the early stages of development. These results suggest that Msi-1 expression occurs specifically in the stem cell-containing regions. Msi-1+ cells were intermingled with proliferating cell nuclear antigen (PCNA)+ cells in the isthmus/neck region of the adult antrum, but did not coexpress PCNA or Ki 67. Msi-1 expression overlapped partly with expression of MUC 5 AC and MUC 6, indicating that Msi-1+ cells retain some features of both foveolar and pyloric gland cell differentiation phenotypes. In contrast, Msi-1 expression in gastric glands showing intestinal metaplasia (IM) became weaker than that in the glands without IM. CONCLUSION: The specific expression of Msi-1 within the proliferative regions suggests that Msi-1 is a marker of cells with progenitor characteristics before active proliferation in human antrum.     

Expression of CD1d in human scalp skin and hair follicles: hair cycle related alterations

BACKGROUND: CD1d belongs to a family of antigen presenting molecules that are structurally and distantly related to the classic major histocompatibility complex class I (MHC I) proteins. However, unlike MHC I molecules, which bind protein antigens, CD1d binds to lipid and glycolipid antigens. CD1d is expressed by cells of lymphoid and myeloid origin, and by cells outside of the lymphoid and myeloid lineages, such as human keratinocytes of psoriatic skin.AIMS: To investigate whether CD1d is also expressed in sun exposed skin and in the immuno-privileged anagen hair follicle.Materials/METHODS: CD1d immunoreactivity was studied in human scalp skin and hair follicles of healthy women in situ by immunofluorescent and light microscopic immunohistology. Skin biopsies were obtained from normal human scalp containing mainly anagen VI hair follicles from women (age, 53-57 years) undergoing elective plastic surgery.RESULTS: CD1d showed strong immunostaining in human scalp skin epidermis, pilosebaceous units, and eccrine glands. In the epidermis, CD1d was strongly expressed by basal and granular keratinocytes. In hair follicles, CD1d was expressed in the epithelial compartment and showed hair cycle related alterations, with an increase in the anagen and a reduction in the catagen and telogen phases.CONCLUSIONS: These results suggest that CD1d plays a role in human scalp skin immunology and protection against lipid antigen rich infectious microbes. They also raise the question of whether keratinocytes of the immuno-privileged anagen hair follicle can present lipid antigens to natural killer T cells. These data could help provide new strategies for the manipulation of hair related disorders.     

Distinct population of hair cell progenitors can be isolated from the postnatal mouse cochlea using side population analysis.

In mammals, the permanence of hearing loss is due mostly to the incapacity of the cochlea to replace lost mechano-receptor cells (i.e., hair cells [HCs]). The generation of new HCs from a renewable source of progenitors is a principal requirement for developing a cell therapy within this sensory organ. A subset of stem cells, termed side population (SP), has been identified in several tissues of mammals. The ATP-binding cassette transporter Abcg2/Bcrp1 contributes to the specification of the SP phenotype and is proposed as a universal marker for stem/progenitor cells. A defining character of these SP cells is a high efflux capacity for Hoechst dye. Here, we demonstrate that Abcg2 transporter is expressed with two other stem/progenitor cell markers (i.e., Nestin and Musashi1) in distinct and overlapping domains of the supporting cells within the postnatal cochlea. We have developed and describe a fluorescence-activated cell sorting (FACS) technique that enables the purification of a discrete subpopulation of SP-supporting cells from the early postnatal mouse cochlea based on their ability to exclude Hoechst dye. These FACS-isolated cells can divide and express markers of stem/progenitor cells such as Abcg2, a determinant of the SP phenotype, and Musashi1, a neural stem/progenitor cell marker. These markers can differentiate cells expressing markers of HCs and supporting cells in vitro. Our observation that these SP cells are capable of differentiating into HC-like cells implies a possible use for such cells (i.e., the replacement of lost auditory HCs within damaged cochlea).     

Dissecting the impact of chemotherapy on the human hair follicle: a pragmatic in vitro assay for studying the pathogenesis and potential management of hair follicle dystrophy

Chemotherapy-induced alopecia represents one of the major unresolved problems of clinical oncology. The underlying molecular pathogenesis in humans is virtually unknown because of the lack of adequate research models. Therefore, we have explored whether microdissected, organ-cultured, human scalp hair follicles (HFs) in anagen VI can be exploited for dissecting and manipulating the impact of chemotherapy on human HFs. Here, we show that these organ-cultured HFs respond to a key cyclophosphamide metabolite, 4-hydroperoxycyclophosphamide (4-HC), in a manner that resembles chemotherapy-induced HF dystrophy as it occurs in vivo: namely, 4-HC induced melanin clumping and melanin incontinence, down-regulated keratinocyte proliferation, massively up-regulated apoptosis of hair matrix keratinocytes, prematurely induced catagen, and up-regulated p53. In addition, 4-HC induced DNA oxidation and the mitochondrial DNA common deletion. The organ culture system facilitated the identification of new molecular targets for chemotherapy-induced HF damage by microarray technology (eg, interleukin-8, fibroblast growth factor-18, and glypican 6). It was also used to explore candidate chemotherapy protectants, for which we used the cytoprotective cytokine keratinocyte growth factor as exemplary pilot agent. Thus, this novel system serves as a powerful yet pragmatic tool for dissecting and manipulating the impact of chemotherapy on the human HF.     

Increased expression of the adult stem cell marker Musashi-1 in endometriosis and endometrial carcinoma

Adult stem cells are thought to be responsible for the high regenerative capacity of the human endometrium, and have been implicated in the pathology of endometriosis and endometrial carcinoma. The RNA-binding protein Musashi-1 is associated with maintenance and asymmetric cell division of neural and epithelial progenitor cells. We investigated expression and localization of Musashi-1 in endometrial, endometriotic and endometrial carcinoma tissue specimens of 46 patients. qPCR revealed significantly increased Musashi-1 mRNA expression in the endometrium compared to the myometrium. Musashi-1 protein expression presented as nuclear or cytoplasmic immunohistochemical staining of single cells in endometrial glands, and of single cells and cell groups in the endometrial stroma. Immunofluorescence microscopy revealed colocalization of Musashi-1 with its molecular target Notch-1 and telomerase. In proliferative endometrium, the proportion of Musashi-1-positive cells in the basalis layer was significantly increased 1.5-fold in the stroma, and three-fold in endometrial glands compared to the functionalis. The number of Musashi-1 expressing cell groups was significantly increased (four-fold) in proliferative compared to secretory endometrium. Musashi-1 expressing stromal cell and cell group numbers were significantly increased (five-fold) in both endometriotic and endometrial carcinoma tissue compared to secretory endometrium. A weak to moderate, diffuse cytoplasmic glandular staining was observed in 50% of the endometriosis cases and in 75% of the endometrioid carcinomas compared to complete absence in normal endometrial samples. Our results emphasize the role of Musashi-1-expressing endometrial progenitor cells in proliferating endometrium, endometriosis and endometrioid uterine carcinoma, and support the concept of a stem cell origin of endometriosis and endometrial carcinoma.     

A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control

The vanilloid receptor-1 (VR1, or transient receptor potential vanilloid-1 receptor, TRPV1) is activated by capsaicin, the key ingredient of hot peppers. TRPV1 was originally described on sensory neurons as a central integrator of various nociceptive stimuli. However, several human skin cell populations are also now recognized to express TRPV1, but with unknown function. Exploiting the human hair follicle (HF) as a prototypic epithelial-mesenchymal interaction system, we have characterized the HF expression of TRPV1 in situ and have examined TRPV1 signaling in organ-cultured human scalp HF and outer root sheath (ORS) keratinocytes in vitro. TRPV1 immunoreactivity was confined to distinct epithelial compartments of the human HF, mainly to the ORS and hair matrix. In organ culture, TRPV1 activation by capsaicin resulted in a dose-dependent and TRPV1-specific inhibition of hair shaft elongation, suppression of proliferation, induction of apoptosis, premature HF regression (catagen), and up-regulation of intrafollicular transforming growth factor-beta(2). Cultured human ORS keratinocytes also expressed functional TRPV1, whose stimulation inhibited proliferation, induced apoptosis, elevated intracellular calcium concentration, up-regulated known endogenous hair growth inhibitors (interleukin-1beta, transforming growth factor-beta(2)), and down-regulated known hair growth promoters (hepatocyte growth factor, insulin-like growth factor-I, stem cell factor). These findings strongly support TRPV1 as a significant novel player in human hair growth control, underscore the physiological importance of TRPV1 in human skin beyond nociception, and identify TRPV1 as a promising, novel target for pharmacological manipulations of epithelial growth disorders.     

Immunohistochemical study of hair follicle stem cells in regenerated hair follicles induced by Wnt10b

The regulation of the periodic regeneration of hair follicles is complicated. Although Wnt10b has been reported to induce hair follicle regeneration, the characteristics of induced hair follicles, especially the target cells of Wnt10b, have not yet been clearly elucidated. Thus, we systematically evaluated the expression and proliferation patterns of Wnt10b-induced hair follicles. We found that Wnt10b promoted the proliferation of hair follicle stem cells from 24 hours after AdWnt10b injection. Seventy-two hours after AdWnt10b injection, cells outside of bulge area began to proliferate. When the induced hair follicle entered full anagen, although the hair follicle stem cells were normal, canonical Wnt signaling was maintained in the hair precortex cells. Our results reveal that the target cells that overexpressed Wnt10b included hair follicle stem cells, hair precortex cells, and matrix cells.     

The incidental pore: CaV1.2 and stem cell activation in quiescent hair follicles

The hair follicle undergoes a lifelong developmental cycle that depends on the integration between activating and inhibitory signals acting to regulate and guide the proliferation and differentiation of pluripotent epithelial stem cells. The effectors and mechanisms responsible for re-entry of quiescent telogen hair follicles into the hair-producing anagen stage in mature skin remain incompletely understood. In the June 1, 2013, issue of Genes & Development, Yucel and colleagues (pp. 1217–1222) reported the unexpected finding that Ca_V1.2, the pore-forming subunit in a well-characterized voltage-gated, L-type calcium channel, is expressed in hair follicle stem cells and contributes to anagen re-entry but does so in a calcium flux-independent fashion.     

Histochemistry of the human hair follicle with consideration of anagen phases I to VI.

The hair follicle is composed of different epithelial layers under participation of mesenchymal and nerval factors. The present study is an attempt to localize differentiation and functional markers in the human hair follicle during anagen phases I to VI. Monoclonal antibody K 8.12 against keratins 13 and 16 showed an increasing reactivity with certain types of the follicle epithelia during anagen I to VI. Ki67 was expressed within the innermost layer of the outer hair root sheath. Scattered Ki67-positive matrix cells could be additionally identified during anagen V and VI but were absent in anagen I to IV. During anagen Merkel cells became more abundant in the bulbar area. Neuropeptide-like immunoreactivity expressed by bulbar (especially matrix) cells were evenly seen during the early anagen I and II. The findings are in favour of a neurohumoral modulatory role during anagen phases accompanied by an increase of expression of certain proliferation-associated antigens like keratin 16 and Ki67 among the complex epithelia of human hair follicles.