Hair Bulb Keratinocytes Research Articles

Hair-cycle dependent differential expression of ADAM 10 and ADAM 12

ADAM proteases play important roles in processes of development and differentiation. However, no report has been found in the literature addressing the expression and function of ADAM proteases during hair cycling. Cytoplasmic expression pattern of ADAM 10, 12 was similar between normal epidermis and hair infundibulum. In addition, cytoplasmic expression of ADAM 10 was observed in the hair bulb keratinocytes and fibroblasts of dermal papilla in anagen I-III hair follicles. In contrast, decreased ADAM 10 expression was observed in the hair matrix keratinocytes as compared to the hair bulb keratinocytes in anagen I-III hair follicles. Interestingly, ADAM 10 immunoreactivity was expressed weakly in the lower portion of outer root sheath (ORS) of anagen VI hair follicles, and strong ADAM 10 expression was detected in the ORS of catagen and telogen hair follicles. By contrast, ADAM 12 expression was not detected in the hair bulb keratinocytes of anagen I-III hair follicles. ADAM 12 immunoreactivity firstly appeared in the inner root sheath ( IRS ) of anagen IV-V hair follicles and was down-regulated in the IRS and hair cortex and medulla of catagen hair follicles, Strong ADAM 12 immunoreactivity was observed in the ORS of catagen and telogen hair follicles. Samples of normal human skin (n = 30) were used. Immunohistochemical analysis was performed using ADAM 10, 12 specific polyclonal antibodies and a sensitive streptavidin-peroxidase technique. Our study demonstrates a comparable staining pattern of decreased ADAM 10 immunoreactivity in hair matrix keratinocytes and the basal cell layer of normal epidermis and hair infundibulum. Expression of ADAM 10 in dermal papilla cells may imply a role in the induction and development of anagen hair follicles. In addition, expression of ADAM 10 in the ORS and hair bulb assume the involvment of ADAM 10 in the downward migration of anagen hair follicles. Furthermore ADAM 12 expression in the IRS may indicate a role in the differentiation of anagen hair follicles. Downregulation of ADAM 12 upon the onset of catagen hair stage suggests that ADAM 12 may play an important role of ADAM 12 in the apoptosis of hair follicle keratinocytes. In summary our findings suggest that ADAM 10 and 12 may be of importance for the regulation of hair cycling.

Human hair pigmentation – biological aspects

Skin and hair colour contribute significantly to our overall visual appearance and to social/sexual communication. Despite their shared origins in the embryologic neural crest, the hair follicle and epidermal pigmentary units occupy distinct, although open, cutaneous compartments. They can be distinguished principally on the basis of the former's stringent coupling to the hair growth cycle compared with the latter's continuous melanogenesis. The biosynthesis of melanin and its subsequent transfer from melanocyte to hair bulb keratinocytes depend on the availability of melanin precursors and on a raft of signal transduction pathways that are both highly complex and commonly redundant. These signalling pathways can be both dependent and independent of receptors, act through auto-, para- or intracrine mechanisms and can be modified by hormonal signals. Despite many shared features, follicular melanocytes appear to be more sensitive than epidermal melanocytes to ageing influences. This can be seen most dramatically in hair greying/canities and this is likely to reflect significant differences in the epidermal and follicular microenvironments. The hair follicle pigmentary unit may also serve as an important environmental sensor, whereby hair pigment contributes to the rapid excretion of heavy metals, chemicals and toxins from the body by their selective binding to melanin; rendering the hair fibre a useful barometer of exposures. The recent availability of advanced cell culture methodologies for isolated hair follicle melanocytes and for intact anagen hair follicle organ culture should provide the research tools necessary to elucidate the regulatory mechanisms of hair follicle pigmentation. In the longer term, it may be feasible to develop hair colour modifiers of a biological nature to accompany those based on chemicals.

T-oligos as differential modulators of human scalp hair growth and pigmentation: a new “time lapse system” for studying human skin and hair follicle biology in vitro?

Small DNA oligonucleotides homologous to the 3' overhang of human telomeres, called T-oligos, stimulate pigmentation in human epidermal melanocytes in vitro and in vivo. They induce UV-mimetic effects in the absence of DNA-damage, however, it is unknown how T-oligos affect human hair follicle keratinocyte and melanocyte functions in situ. Here, we present the first evidence that these oligonucleotides are powerful modulators of pigmentation and growth of microdissected, organ-cultured human scalp hair follicles. Hair follicles were incubated with T-oligo or vehicle control and were then assessed for changes in hair shaft length, follicle morphology, pigmentation, proliferation and apoptosis. After only 48 h, T-oligos induced a fourfold increase in pigmentation of human anagen VI hair bulbs, while hair matrix keratinocyte proliferation was reduced by 65%, without apparent changes in hair bulb cell apoptosis. This corresponded well with a significant inhibition of hair shaft elongation, which was not accompanied by premature catagen induction in anagen VI hair follicles. These diametrically opposed effects of T-oligos on human hair follicle melanocytes (stimulation of melanogenesis) versus human hair bulb keratinocytes (inhibition of proliferation) in situ illustrate that human hair follicle organ culture offers an excellent tool for T-oligo research. They suggest that T-oligos deserve to be further explored for the management of clinical hair growth and pigmentation disorders, and raise the possibility that this model may offer a unique "time lapse system" for studying skin and hair follicle biology and DNA repair strategies under physiologically relevant conditions.

Human Scalp Hair Follicles Are Both a Target and a Source of Prolactin, which Serves as an Autocrine and/or Paracrine Promoter of Apoptosis-Driven Hair Follicle Regression

The prototypic pituitary hormone prolactin (PRL) exerts a wide variety of bioregulatory effects in mammals and is also found in extrapituitary sites, including murine skin. Here, we show by reverse transcriptase-polymerase chain reaction and immunohistology that, contrary to a previous report, human skin and normal human scalp hair follicles (HFs), in particular, express both PRL and PRL receptors (PRL-R) at the mRNA and protein level. PRL and PRL-R immunoreactivity can be detected in the epithelium of human anagen VI HFs, while the HF mesenchyme is negative. During the HF transformation from growth (anagen) to apoptosis-driven regression (catagen), PRL and PRL-R immunoreactivity appear up-regulated. Treatment of organ-cultured human scalp HFs with high-dose PRL (400 ng/ml) results in a significant inhibition of hair shaft elongation and premature catagen development, along with reduced proliferation and increased apoptosis of hair bulb keratinocytes (Ki-67/terminal dUTP nick-end labeling immunohistomorphometry). This shows that PRL receptors, expressed in HFs, are functional and that human skin and human scalp HFs are both direct targets and sources of PRL. Our data suggest that PRL acts as an autocrine hair growth modulator with catagen-promoting functions and that the hair growth-inhibitory effects of PRL demonstrated here may underlie the as yet ill-understood hair loss in patients with hyper-prolactinemia.

Interferon-gamma is a potent inducer of catagen-like changes in cultured human anagen hair follicles

Interferon (IFN)-gamma appears to be an important hair cycle modulator in mice. It is unclear whether it has similar hair growth modulatory functions in human hair follicles. To study whether IFN-gamma can be exploited to modulate the growth, pigmentation and/or cycling of organ-cultured human anagen scalp hair follicles, as an in vitro indicator system for how IFN-gamma affects human hair growth in vivo. This was correlated with the hair follicle expression patterns of IFN-gamma receptors alpha and beta. In addition, we wanted to establish a new, simple tool for the rapid experimental induction of catagen in vitro. Normal human scalp hair follicles in the anagen VI stage of the hair cycle were cultured according to the method of Philpott et al., with or without IFN-gamma (50-1000 IU mL(-1)). Hair shaft elongation and pigmentation changes were measured, complemented by quantitative histomorphometry to assess changes in hair follicle cycling (hair cycle score), proliferation (Ki-67), melanogenesis (Masson-Fontana) and apoptosis (TUNEL). IFN-gamma receptors were also localized by immunofluorescence and EnVision technique. As transforming growth factor (TGF)-beta2 is a recognized key inducer of catagen in human hair follicles, TGF-beta2 expression was investigated by tyramide signal amplification and reverse transcription-polymerase chain reaction in anagen hair follicles treated with vehicle (phosphate-buffered saline) or IFN-gamma. IFN-gamma rapidly inhibited hair elongation in cultured human anagen hair follicles and induced morphological signs of catagen transformation after only 4 days of culture, i.e. faster than with other reported catagen-inducers (e.g. TGF-beta2). Proliferation was inhibited, apoptosis was increased and follicular melanogenesis was switched off in hair bulb keratinocytes treated in situ with IFN-gamma. Anagen hair follicles displayed strong IFN-gamma receptor alpha-like immunoreactivity, while the immunoreactivity for IFN-gamma receptor beta in the hair matrix was only weak. TGF-beta2 immunoreactivity and mRNA transcript levels were enhanced in hair follicles treated with IFN-gamma. These data suggest that IFN-gamma is a potent catagen inducer in normal human scalp hair follicles, which express cognate receptors, and show that IFN-gamma administration offers an excellent tool for experimental catagen induction in organ-cultured human hair follicles. This catagen induction probably occurs at least in part via upregulation of the recognized catagen-stimulatory growth factor TGF-beta2.

A Short Peptide GPIGS Promotes Proliferation of Hair Bulb Keratinocytes and Accelerates Hair Regrowth in Mice

The aim of this study was to discover a novel agent that promotes hair growth. We carried out a screening test in 298 types of conditioned medium (CM) from cultures of bacteria by using a hair bulb keratinocyte (HBK) growth assay. As a result, we found a HBK growth factor in the CM of Bacillus sp. M18. This HBK growth factor was purified by collecting biologically active fractions in three steps, including HP-20 batch processing, LH-20 chromatography and C18 reverse-phase high-pressure liquid chromatography, and identified as a short peptide GPIGS. GPIGS increased Akt phosphorylation in HBKs. Moreover, the GPIGS-stimulated HBK growth was inhibited by the treatment with LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI-3K). These results suggest that GPIGS promotes HBK growth via the PI-3K/Akt pathway. In addition to in vitro tests, GPIGS was found to accelerate hair regrowth in telogen mice. Our results indicate that GPIGS is a potential agent to promote hair growth.

Targeted disruption of the protein kinase SGK3/CISK impairs postnatal hair follicle development.

Members of the serum- and glucocorticoid-regulated kinase (SGK) family are important mediators of growth factor and hormone signaling that, like their close relatives in the Akt family, are regulated by lipid products of phosphatidylinositol-3-kinase. SGK3 has been implicated in the control of cell survival and regulation of ion channel activity in cultured cells. To begin to dissect the in vivo functions of SGK3, we generated and characterized Sgk3 null mice. These mice are viable and fertile, and in contrast to mice lacking SGK1 or Akt2, respectively, display normal sodium handling and glucose tolerance. However, although normal at birth, by postpartum day 4 they have begun to display an unexpected defect in hair follicle morphogenesis. The abnormality in hair follicle development is preceded by a defect in proliferation and nuclear accumulation of beta-catenin in hair bulb keratinocytes. Furthermore, in cultured keratinocytes, heterologous expression of SGK3 potently modulates activation of beta-catenin/Lef-1-mediated gene transcription. These data establish a role for SGK3 in normal postnatal hair follicle development, possibly involving effects on beta-catenin/Lef-1-mediated gene transcription.

Expression of the TGF-beta coreceptor endoglin in epidermal keratinocytes and its dual role in multistage mouse skin carcinogenesis.

Endoglin is an integral membrane glycoprotein primarily expressed in the vascular endothelium, but also found on macrophages and stromal cells. It binds several members of the transforming growth factor (TGF)-beta family of growth factors and modulates TGF-beta(1)-dependent cellular responses. However, it lacks cytoplasmic signaling motifs and is considered as an auxiliary receptor for TGF-beta. We show here that endoglin is expressed in mouse and human epidermis and in skin appendages, such as hair follicles and sweat glands, as determined by immunohistochemistry. In normal interfollicular epidermis, endoglin was restricted to basal keratinocytes and absent in differentiating cells of suprabasal layers. Follicular expression of endoglin was high in hair bulb keratinocytes, but decreased in parts distal from the bulb. To address the role of endoglin in skin carcinogenesis in vivo, Endoglin heterozygous mice were subjected to long-term chemical carcinogenesis treatment. Reduction in endoglin had a dual effect during multistage carcinogenesis, by inhibiting the early appearance of benign papillomas, but increasing malignant progression to highly undifferentiated carcinomas. Our results are strikingly similar to those previously reported for transgenic mice overexpressing TGF-beta(1) in the epidermis. These data suggest that endoglin might attenuate TGF-beta(1) signaling in normal epidermis and interfere with progression of skin carcinogenesis.

Prolactin and Its Receptor Are Expressed in Murine Hair Follicle Epithelium, Show Hair Cycle-Dependent Expression, and Induce Catagen

Here, we provide the first study of prolactin (PRL) and prolactin receptor (PRLR) expression during the nonseasonal murine hair cycle, which is, in contrast to sheep, comparable with the human scalp and report that both PRL and PRLR are stringently restricted to the hair follicle epithelium and are strongly hair cycle-dependent. In addition we show that PRL exerts functional effects on anagen hair follicles in murine skin organ culture by down-regulation of proliferation in follicular keratinocytes. In telogen follicles, PRL-like immunoreactivity was detected in outer root sheath (ORS) keratinocytes. During early anagen (III to IV), the developing inner root sheath (IRS) and the surrounding ORS were positive for PRL. In later anagen stages, PRL could be detected in the proximal IRS and the inner layer of the ORS. The regressing (catagen) follicle showed a strong expression of PRL in the proximal ORS. In early anagen, PRLR immunoreactivity occurred in the distal part of the ORS around the developing IRS, and subsequently to a restricted area of the more distal ORS during later anagen stages and during early catagen. The dermal papilla (DP) stayed negative for both PRL and PRLR throughout the cycle. Telogen follicles showed only a very weak PRLR staining of ORS keratinocytes. The long-form PRLR transcript was shown by real-time polymerase chain reaction to be transiently down-regulated during early anagen, whereas PRL transcripts were up-regulated during mid anagen. Addition of PRL (400 ng/ml) to anagen hair follicles in murine skin organ culture for 72 hours induced premature catagen development in vitro along with a decline in the number of proliferating hair bulb keratinocytes. These data support the intriguing concept that PRL is generated locally in the hair follicle epithelium and acts directly in an autocrine or paracrine manner to modulate the hair cycle.

High-dose proinflammatory cytokines induce apoptosis of hair bulb keratinocytes in vivo.

Hair loss following skin inflammation may in part be mediated by keratinocyte (KC) apoptosis. While the effects of different cytokines or other apoptosis stimulating agents such as interferon (IFN)-gamma or tumour necrosis factor (TNF)-alpha on KC apoptosis in vitro have been addressed in several studies, little is known about the effects of proinflammatory cytokines on KC apoptosis in vivo. To study the effects of intradermally injected TNF-alpha, interleukin (IL)-1beta and IFN-gamma on KC apoptosis in the back skin of C57BL/6 mice. Apoptosis in epidermal and hair bulb KCs was analysed by immunohistology using TUNEL staining. Injection of TNF-alpha induced a significantly higher number of apoptotic cells within the epidermis than vehicle; all three proinflammatory cytokines together further increased their number. Intrafollicular hair bulb KCs were much more susceptible to apoptosis induction by TNF-alpha or IL-1beta; their injection significantly upregulated apoptosis after 6 h, which was further increased after 24 h. The combination of all cytokines together accelerated intrafollicular apoptosis after 6 h by doubling the number of apoptotic cells per hair bulb, compared with the effects of TNF-alpha or IL-1beta alone. These data suggest that programmed cell death of proliferating KCs in vivo can be induced by proinflammatory cytokines.

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.

MHC Class I Expression in Murine Skin: Developmentally Controlled and Strikingly Restricted Intraepithelial Expression During Hair Follicle Morphogenesis and Cycling, and Response to Cytokine Treatment In Vivo

Hair bulb keratinocytes generate one of the few "immune privileged" tissue compartments of the mammalian organism by suppressing classical MHC class I (MHC Ia) antigens. Expression of non-classical MHC class I (MHC Ib) antigens in the follicle has been found, but only in its distal epithelium. Here, we have defined when during murine hair follicle morphogenesis these peculiar MHC Ia and Ib expression patterns are established, how they change during the murine hair cycle, and how different MHC I modulatory agents alter follicular MHC Ia and Ib expression in vivo. During neonatal hair follicle morphogenesis in C57BL/6 mice, distal follicle keratinocytes began to express MHC Ia (H2b) only late in development. The MHC Ib antigens, Qa-1 and Qa-2, did not become visible until the initiation of follicle cycling, with Qa-1 expression being more widespread than that of Qa-2. H2b, Qa-1, and TAP-1 immunoreactivity on previously negative keratinocytes of the proximal anagen hair bulb was upregulated by intradermal injection of the proinflammatory cytokine interferon-gamma, but not by tumor necrosis factor-alpha or interleukin-1beta. Injection of the reportedly MHC class I downregulating agents interleukin-10, insulin-like growth factor-1, transforming growth factor-beta, alpha-melanocyte stimulating hormone, or dexamethasone, however, all failed to downregulate constitutive or interferon-gamma-induced follicular MHC Ia expression. This shows that the hair follicle is a previously unrecognized site of Qa-1 expression and that interferon-gamma is a key regulator of follicular MHC I expression in vivo. It also suggests that the developmental and immunologic controls of MHC I expression by follicle keratinocytes differ from those of other epithelial cells.

Hair cycle-dependent expression of heat shock proteins in hair follicle epithelium.

Heat shock proteins (HSPs) have a physiologic function in unstressed cells, which is believed to include a role as a "molecular chaperone." The hair cycle is characterized by rhythmic tissue remodelling processes, and is an intriguing model for studying the relation between keratinocyte differentiation and HSP expression under physiologic circumstances. We have therefore studied, by immunofluorescence, the expression of selected HSPs during the murine hair cycle. The association between hair follicle cycling and the expression of three selected HSPs (HSPs 27, 60, and 72) was examined by immunofluorescence, using the depilation-induced hair cycle of C57BL/6 mice. HSP expression was absent from telogen follicles, and was restricted predominantly to keratinocytes in the bulge and the cycling epithelial portion of the hair follicle during anagen and catagen. Immunoreactivity for HSPs 27, 60, and 72 in the hair bulb increased significantly during anagen VI and the catagen transformation of the follicle, and decreased again abruptly with completion of the catagen-telogen transformation. The expression pattern of HSPs 60 and 72 in situ was cytoplasmic, whereas that of HSP 27 was both cytoplasmic and nuclear. These observations suggest that the synthesis of HSPs by hair bulb keratinocytes is related to the anagen-catagen transformation of the follicle, possibly reflecting keratinocyte apoptosis and/or terminal differentiation in the regressing, cycling portion of the follicle. In addition, the rather proximal localization of HSP expression makes it unlikely that the HSPs examined interact with the more distally located intrafollicular gamma/delta T-cell receptor-positive lymphocytes.