Analysis of apoptosis during hair follicle regression (catagen)

Keratinocyte apoptosis is a central element in the regulation of hair follicle regression (catagen), yet the exact location and the control of follicular keratinocyte apoptosis remain obscure. To generate an "apoptomap" of the hair follicle, we have studied selected apoptosis-associated parameters in the C57BL/6 mouse model for hair research during normal and pharmacologically manipulated, pathological catagen development. As assessed by terminal deoxynucleotide transferase dUTP fluorescein nick end-labeling (TUNEL) stain, apoptotic cells not only appeared in the regressing proximal follicle epithelium but, surprisingly, were also seen in the central inner root sheath, in the bulge/isthmus region, and in the secondary germ, but never in the dermal papilla. These apoptosis hot spots during catagen development correlated largely with a down-regulation of the Bcl-2/Bax ratio but only poorly with the expression patterns of interleukin-1beta converting enzyme, p55TNFR, and Fas/Apo-1 immunoreactivity. Instead, a higher correlation was found with p75NTR expression. During cyclophosphamide-induced follicle dystrophy and alopecia, massive keratinocyte apoptosis occurred in the entire proximal hair bulb, except in the dermal papilla, despite a strong up-regulation of Bax and p75NTR immunoreactivity. Selected receptors of the tumor necrosis factor/nerve growth factor family and members of the Bcl-2 family may also play a key role in the control of follicular keratinocyte apoptosis in situ.     

Short tail with skin lesion phenotype occurs in transgenic mice with keratin-14 promoter-directed expression of mutant CXCR2

CXCR2 plays an important role during cutaneous wound healing. Transgenic mice were generated using the keratin-14 promoter/enhancer to direct expression of wild-type human CXCR2 (K14hCXCR2 WT) or mutant CXCR2, in which the carboxyl-terminal domain (CTD) was truncated at Ser 331 and the dileucine AP-2 binding motif was mutated to alanine (K14hCXCR2 331T/LL/AA/IL/AA). Our results indicate that K14hCXCR2WT transgenic mice exhibited a normal phenotype, while K14hCXCR2 331T/LL/AA/IL/AA transgenic mice were born with tails of normal length, but three to eight days after birth their tails degenerated, leaving only a short tail stub. The tissue degeneration in the tail started between caudal somites with degeneration of bone and connective tissue distal to the constriction, which was replaced with stromal tissue heavily infiltrated with inflammatory cells. The tail lesion site revealed coagulation in enlarged vessels and marked edema that eventually led to loss of the distal tail. Moreover, 66% of the mice exhibited focal skin blemishes and inflammation that exhibited an increase in the number of sebaceous glands and blood vessels, enlargement of the hair follicles due to increased number of keratinocytes, reduction in the connective tissue content, and a thickening of the epidermis. Furthermore, immunohistochemical staining of the epidermis from tail tissue in the transgenic mice indicated a loss of the cell adhesion markers E-cadherin and desmoplakin. These data suggest that keratinocyte expression of a CTD mutant of CXCR2 has effects on homeostasis of the connective tissue in the tail, as well as the maintenance of the epidermis and its appendages.     

Epidermal expression of the truncated prelamin A causing Hutchinson-Gilford progeria syndrome: effects on keratinocytes, hair and skin

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by point mutation in LMNA encoding A-type nuclear lamins. The mutations in LMNA activate a cryptic splice donor site, resulting in expression of a truncated, prenylated prelamin A called progerin. Expression of progerin leads to alterations in nuclear morphology, which may underlie pathology in HGPS. We generated transgenic mice expressing progerin in epidermis under control of a keratin 14 promoter. The mice had severe abnormalities in morphology of skin keratinocyte nuclei, including nuclear envelope lobulation and decreased nuclear circularity not present in transgenic mice expressing wild-type human lamin A. Primary keratinocytes isolated from these mice had a higher frequency of nuclei with abnormal shape compared to those from transgenic mice expressing wild-type human lamin A. Treatment with a farnesyltransferase inhibitor significantly improved nuclear shape abnormalities and induced the formation of intranuclear foci in the primary keratinocytes expressing progerin. Similarly, spontaneous immortalization of progerin-expressing cultured keratinocytes selected for cells with normal nuclear morphology. Despite morphological alterations in keratinocyte nuclei, mice expressing progerin in epidermis had normal hair grown and wound healing. Hair and skin thickness were normal even after crossing to Lmna null mice to reduce or eliminate expression of normal A-type lamins. Although progerin induces significant alterations in keratinocyte nuclear morphology that are reversed by inhibition of farnesyltransferasae, epidermal expression does not lead to alopecia or other skin abnormalities typically seen in human subjects with HGPS.     

Keratin 17 modulates hair follicle cycling in a TNFalpha-dependent fashion

Mammalian hair follicles cycle between stages of rapid growth (anagen) and metabolic quiescence (telogen) throughout life. Transition from anagen to telogen involves an intermediate stage, catagen, consisting of a swift, apoptosis-driven involution of the lower half of the follicle. How catagen is coordinated, and spares the progenitor cells needed for anagen re-entry, is poorly understood. Keratin 17 (K17)-null mice develop alopecia in the first week post-birth, correlating with hair shaft fragility and untimely apoptosis in the hair bulb. Here we show that this abnormal apoptosis reflects premature entry into catagen. Of the proapoptotic challenges tested, K17-null skin keratinocytes in primary culture are selectively more sensitive to TNFalpha. K17 interacts with TNF receptor 1 (TNFR1)-associated death domain protein (TRADD), a death adaptor essential for TNFR1-dependent signal relay, suggesting a functional link between this keratin and TNFalpha signaling. The activity of NF-kappaB, a downstream target of TNFalpha, is increased in K17-null skin. We also find that TNFalpha is required for a timely anagen-catagen transition in mouse pelage follicles, and that its ablation partially rescues the hair cycling defect of K17-null mice. These findings identify K17 and TNFalpha as two novel and interdependent regulators of hair cycling.     

p53-Regulated Apoptosis Is Differentiation Dependent in Ultraviolet B-Irradiated Mouse Keratinocytes

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (−/−) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53−/− mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m² UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53−/− cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53−/− cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.     

Bone morphogenetic protein signaling regulates postnatal hair follicle differentiation and cycling

Hair follicle morphogenesis and cycling were examined in transgenic mice that overexpress the bone morphogenetic protein (BMP) inhibitor Noggin under the control of the neuron-specific enolase promoter. The Noggin transgene was misexpressed in the proximal portion of the hair follicle, primarily the matrix cells, apart from the usual expression in neurons. Transgene expression appeared only after induction of both the primary (tylotrich) and secondary (nontylotrich) pelage hair follicles had already occurred, thus allowing examination of the role of BMP signaling in follicles that had been induced normally in the presence of BMPs. The overexpression of Noggin in these animals resulted in a dramatic loss of hair postnatally. There was an apparently normal, but shortened period of postnatal hair follicle morphogenesis, followed by premature initiation of hair follicle cycling via entry into the first catagen transformation. This resulted in a complete loss of hair shafts from the nontylotrich hair follicles in these mice while the tylotrich hair follicles were normal. The onset of anagen of the first postnatal hair follicle cycle was also accelerated in the transgenic mice. Our results show that BMP signaling is specifically required for proper proliferation and differentiation during late morphogenesis of nontylotrich hair follicles and that inhibition of this signaling pathway may be one of the triggers for the onset of catagen when the follicles are in anagen and the onset of anagen when the follicles are in telogen. Ectopic sebocyte differentiation was another hallmark of the phenotype of these transgenic mice suggesting that BMP signaling may be an important determinant of lineage selection by common progenitor cells in the skin. BMPs likely promote a hair follicle-type differentiation pathway of keratinocytes while suppressing the sebaceous differentiation pathway of skin epithelium.     

Expression of apoptosis-related genes in the mouse skin during the first postnatal catagen stage,focused on localization of Bnip3L and caspase-12

Hair follicles undergo repetitive stages of cell proliferation and programmed cell death. The catagen stage of physiological apoptosis is connected with dynamic changes in morphology and alterations in gene expression. However, hair follicle apoptosis must be in balance with events in surrounding tissues, such as keratinocyte cornification, to maintain complex skin homeostasis. Several pro- and anti-apoptotic molecules in the skin have been reported but mainly in pathological states. In this investigation, apoptosis-related gene expression was examined during the first catagen stage of mouse hair follicle development by PCR arrays under physiological conditions. Postnatal stages P15 and P17, representing early and late catagen stages, were evaluated relatively to stage P6, representing the hair follicle growing phase, to demonstrate dynamics of gene activation during the catagen. Several statistically significant alterations were observed at P15 and particularly at P17. Bnip3L and caspase-12 identified by the PCR arrays at both catagen stages were additionally localized using immunofluorescence and were reported in physiological hair development for the first time.     

Activin controls skin morphogenesis and wound repair predominantly via stromal cells and in a concentration-dependent manner via keratinocytes

The transforming growth factor-beta family member activin is a potent regulator of skin morphogenesis and repair. Transgenic mice overexpressing activin in keratinocytes display epidermal hyper-thickening and dermal fibrosis in normal skin and enhanced granulation tissue formation after wounding. Mice overexpressing the secreted activin antagonist follistatin, however, have the opposite wound-healing phenotype. To determine whether activin affects skin morphogenesis and repair via activation of keratinocytes and/or stromal cells, we generated transgenic mice expressing a dominant-negative activin receptor IB mutant (dnActRIB) in keratinocytes. The architecture of adult skin was unaltered in these mice, but delays were observed in postnatal pelage hair follicle morphogenesis and in the first catagen-telogen transformation of hair follicles. Although dnActRIB-transgenic mice showed slightly delayed wound re-epithelialization after skin injury, the strong inhibition of granulation tissue formation seen in follistatin-transgenic mice was not observed. Therefore, although endogenous activin appeared to affect skin morphogenesis and repair predominantly via stromal cells, overexpressed activin strongly affected the epidermis. The epidermal phenotype of activin-overexpressing mice was partially rescued by breeding these animals with dnActRIB-transgenic mice. These results demonstrate that activin affects both stromal cells and keratinocytes in normal and wounded skin and that the effect on keratinocytes is dose-dependent in vivo.     

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.     

Functional link between retinoblastoma family of proteins and the Wnt signaling pathway in mouse epidermis.

The retinoblastoma family of proteins (pRb, p107, and p130) modulates cell cycle progression and differentiation of several tissues. We have demonstrated recently that p107 and p130 regulate keratinocyte terminal differentiation and hair follicle morphogenesis and development in vivo. This last aspect appears to be mediated by defective signaling from the mesenchyme and is associated with altered bone morphogenetic protein-4 (BMP4) -dependent signaling. However, many alterations were also found in the epithelial compartment. Given the importance of betacatenin in hair biology and in BMP signaling, we studied its expression in p107/p130-deficient skin. Although normal expression of betacatenin was found in p107/p130-deficient hair follicles, we found increased nuclear accumulation of betacatenin in the basal keratinocytes of the p107/p130-deficient mice skin. Biochemical analysis revealed that such an increase in betacatenin was due to the disruption of Axin/GSK3beta/betacatenin complexes promoted by the increased expression of Frat, the mouse homologue of GSK3betabinding protein (GBP), in epidermis, precluding the degradation of betacatenin. Collectively, these data represent the first evidence that retinoblastoma family and Wnt signaling pathways might be interconnected by functional links in skin.     

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.     

The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice

The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.     

Embryonic development of hair follicle pluripotent stem (hfPS) cells

Our laboratory discovered nestin-expressing hair follicle stem cells and demonstrated their pluripotency. We have shown that nestin-positive and K15-negative multipotent hair follicle stem cells are located above the hair follicle bulge, and we termed these cells hair follicle pluripotent stem (hfPS) cells. We have previously shown that hair follicle stem cells can regenerate peripheral nerve and spinal cord. In the present study, we describe the embryonic development of the hair follicle stem cell area (hfPSCA), which is located above the bulge and below the sebaceous glands in the adult mouse. At embryonic day 16.5 (E16.5) of nestindriven GFP (ND-GFP) transgenic mice, which express nestin in hfPS cells, the ND-GFP hair follicle stem cells are located in mesenchymal condensates. At postnatal day 0 (P0), the ND-GFP-expressing cells are migrating to the upper part of the hair follicle from the dermal papilla. At P3, keratin 15 (K15)-positive cells, derived from ND-GFP dermal papilla cells, are located in the outer-root sheath and basal layer of the epidermis. By P10, the ND-GFP have formed the K15-positive outer-root sheath as well as the ND-GFP hfPSA. These results suggest that ND-GFP hfPS cells in the dermal papilla form nestin-expressing hair follicle stem cells in the first hair cycle. These observations provide new insight into the origins of hfPS cells and the hfPSCA.     

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.