Ralf Paus

Melanogenesis is coupled to murine anagen: Toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth

Hair is actively pigmented only when it grows: the melanogenic activity of follicular melanocytes (MC) is strictly coupled to the anagen stage of the hair cycle. In catagen, melanin formation is switched off and is absent throughout telogen. The appearance of pigmentation is preceded, and further accompanied by, a time-frame - restricted, differential pattern of tyrosinase transcription, translation, and enzyme activities during the development of anagen follicles. In this speculative review, we argue that signals required for melanin synthesis and pigment transfer to bulb keratinocytes (KC) are intrinsic to the skin, rather than coming from the serum. First, the proopiomelanocortin (POMC) gene is expressed and translated during anagen, but is below the level of detectability in telogen; POMC is a precursor protein for adrenocorticotropin and melanotropins, which are potent regulators of MC proliferation and differentiation. Second, fibroblasts and KC produce factors that affect MC proliferation and differentiation. We suggest that signals regulating follicular MC activity partially derive from cutaneous cells expressing POMC. Vice versa, MC transfer to surrounding KC pigment granules with potent bioregulatory properties. MC also produce and secrete various signal molecules that can regulate mesenchymal and epithelial cell functions. Anagen-associated melanogenesis and the cyclic production of a pigmented hair shaft result from programmed and tightly coordinated epithelial-mesenchymal-neuroectodermal interactions, in which MC may act not only as pigmentary, but also as hair growth-regulatory cells.

Vitiligo pathogenesis: autoimmune disease, genetic defect, excessive reactive oxygen species, calcium imbalance, or what else?

Abstract:u2002 The pathobiology of vitiligo has been hotly disputed for as long as one remembers, and has been a magnet for endless speculation. Evidently, the different schools of thought – ranging, e.g. from the concept that vitiligo essentially is a free‐radical disorder to that of vitiligo being a primary autoimmune disease – imply very different consequences for the best therapeutic strategies that one should adopt. As a more effective therapy for this common, often disfiguring pigmentary disorder is direly needed, we must strive harder to settle the pathogenesis debate definitively – on the basis of sound experimental evidence, rather than by a war of dogmatic theories.

On the potential role of proopiomelanocortin in skin physiology and pathology

Pro-opiomelanocortin (POMC) is the precursor of ACTH, alpha-MSH and beta-endorphin, neuropeptides with multiple regulatory functions. Both the pituitary gland and peripheral tissues such as mammalian skin are capable of generating POMC-derived neuropeptides. Mammalian skin is also a target for POMC products; their possible roles in skin physiology and pathology are discussed in this communication.

Senile hair graying: H2O2-mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair

Senile graying of human hair has been the subject of intense research since ancient times. Reactive oxygen species have been implicated in hair follicle melanocyte apoptosis and DNA damage. Here we show for the first time by FT‐Raman spectroscopy in vivo that human gray/white scalp hair shafts accumulate hydrogen peroxide (H2O2) in millimolar concentrations. Moreover, we demonstrate almost absent catalase and methionine sulfoxide reductase A and B protein expression via immunofluorescence and Western blot in association with a functional loss of methionine sulfoxide (Met‐S=O) repair in the entire gray hair follicle. Accordingly, Met‐S=O formation of Met residues, including Met 374 in the active site of tyrosinase, the key enzyme in melanogenesis, limits enzyme functionality, as evidenced by FT‐Raman spectroscopy, computer simulation, and enzyme kinetics, which leads to gradual loss of hair color. Notably, under in vitro conditions, Met oxidation can be prevented by L‐ methionine. In summary, our data feed the long‐voiced, but insufficiently proven, concept of H2O2‐induced oxidative damage in the entire human hair follicle, inclusive of the hair shaft, as a key element in senile hair graying, which does not exclusively affect follicle melanocytes. This new insight could open new strategies for intervention and reversal of the hair graying process.—Wood, J. M., Decker, H., Hartmann, H., Chavan, B., Rokos, H., Spencer, J. D., Hasse, S., Thornton, M. J., Shalbaf, M., Paus, R., Schallreuter, K. U. Senile hair graying: H2O2‐mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair. FASEB J. 23, 2065–2075 (2009)

Hair follicle expression of 1,25‐dihydroxyvitamin D3 receptors during the murine hair cycle

Because the hair follicle is a highly hormone‐sensitive miniorgan, the role of hormones produced locally in the skin in the control of hair growth deserves systematic analysis. It has been shown previously that the potent steroid hormone 1,25‐dihydroxyvitamin D3 (1,25‐D3) modulates growth and differentiation of keratinocytes via binding to a high‐affinity nuclear vitamin D receptor (VDR). In this study, we have examined the in situ expression of VDR during the murine hair cycle. VDR expression was detected immunohistochemically. To obtain defined stages of the murine hair cycle, hair growth was induced by depilation in C57 BL‐6 mice. In addition to the recognized VDR expression of outer root sheath keratinocytes, we detected VDR immunoreactive cells in the dermal papilla, the mesenchymal key structure of the hair follicle. Furthermore, VDR immunoreactivity in the nuclei of outer root sheath keratinocytes and in dermal papilla cells was stronger during anagen IV‐VI and catagen than during telogen and anagen I‐III. This suggests hair cycle‐associated changes in the expression of VDR, and points to a potential role for 1,25‐D3 in hair follicle biology. Selected follicular cell populations may display hair cycle‐dependent sensitivity to 1,25‐D3 stimulation.

Distribution and changing density of gamma-delta T cells in murine skin during the induced hair cycle.

Summary Gamma‐delta T cells (gdTC) are recognized as the predominant intraepidermal T‐cell population in murine skin, although their physiological functions are still unclear. Little is known of the exact distribution of gdTC in the other epithelial skin compartments of normal mice. Using selective gdTC‐receptor antibodies in immunohistology (alkaline phosphatase technique), the distribution and density of gdTC was analysed morphometrically in cryostat sections of full‐thickness back skin of normal, adolescent C57 BL‐6 mice in all the different stages of the depilation‐induced hair cycle. We found that, during the entire hair cycle, V gamma 3‐TCR‐bearing lymphocytes are restricted to the epidermis, and to the epithelial hair bulb in, and distal to, the bulge area. No gdTC were seen in the sebaceous glands. During early anagen development, the number of pan‐gdTC receptor‐positive cells increased significantly (P<0·005) in the interfollicular epidermis and the supainfundibular portion of the hair bulb, whereas the number decreased in the infrainfundibular region (P≤0.005). As gdTC are thought to migrate into the skin only during embryogenesis, this finding suggests hair cycle‐dependent, differential intraepithelial proliferation of gdTC in murine skin. We advocate employing only skin of defined hair cycle stages in immunological studies on murine skin, and discuss the value of the C57 BL‐6 model for assessing the functions of gdTC in skin and hair biology.

Nerve growth factor modulates keratinocyte proliferation in murine skin organ culture.

Summary Despite the fact that several cell types residing in or travelling through the skin are targets and/or sources of nerve growth factor (NGF), little is known about the role of NGF in skin development, physiology and disease. Employing a previously defined skin organ culture assay for studying the proliferation of murine keratinocytes in their natural tissue environment, we have assessed the effect of murine NGF (7S) on keratinocyte proliferation in intact skin derived from two defined stages of the murine hair cycle. We found that 10–200 ng/ml NGF stimulated epidermal keratinocyte proliferation in organ‐cultured C57 BL‐6 mouse skin in the telogen phase of the hair cycle. Follicle keratinocyte proliferation was stimulated by 100 ng/ml NGF in telogen skin organ culture, but this concentration of NGF inhibited both epidermal and follicle keratinocyte proliferation in organ culture of anagen skin. The latter inhibitory effect of NGF was abrogated by co‐incubation with neutralizing anti‐NGF antibodies or with the protein kinase C inhibitor staurosporine. The proliferation‐modulatory effects of NGF were associated with the induction of significant mast cell degranulation, and were inhibited by cromoglycate co‐administration. This is the first report of a modulatory, hair cycle‐dependent effect of NGF on keratinocyte proliferation in situ, which may require the presence of mast cells. Our study supports the notion of auto‐ and paracrine functions of NGF in murine skin physiology, which can be further assessed in the physiologically relevant mouse model delineated here.

Expression of classical and non‐classical MHC class I antigens in murine hair follicles

Not all keratinocytes in human and rat hair follicles express MHC class I antigens (MHC I). In the present study, we report the first immunohistological profile of classical and non‐classical MHC I expression in the skin of adolescent C57 BL‐6 mice during the induced hair cycle. MHC I immunoreactivity (H‐2b, H‐2Db) is absent in the matrix and inner root sheath of growing (=anagen) hair follicles, and the dermal papillae are H‐2b negative during catagen and telogen. This lack of normal MHC I expression may serve to sequester potentially damaging autoantigens from immune recognition. In addition, we present the first evidence of non‐classical MHC class I antigen expression in normal mammalian skin: during the entire hair cycle, the distal hair follicle shows strong Qa‐2 immunoreactivity, which appears to be restricted to an epithelial follicle compartment densely populated by gamma‐delta T cells with which Qa‐2 molecules may interact as part of a primitive antibacterial defense system of the follicle. The murine hair cycle is an attractive model for dissecting the functional roles of H‐2b and Qa‐2 molecules in hair biology and in related tissue‐interaction systems.

Substance P stimulates murine epidermal keratinocyte proliferation and dermal mast cell degranulation in situ

The growth modulatory and immunomodulatory tachykinin, substance P (SP), is a key neuropeptide in neurogenic inflammation [1]. Its release from cutaneous sensory nerves may play a role in inflammatory hyperproliferative skin diseases like psoriasis and atopic dermatitis [2, 3]. In this pilot study, we have addressed three controversial issues: (1) whether SP stimulates normal epidermal keratinocyte (KC) proliferation; (2) if so, how this is mediated (role of mast cells, MC [1-3]); (3) which SP concentrations induce KC proliferation and/or MC degranulation. Here we show that murine skin organ culture provides a useful system for studying these issues. Previously, we had demonstrated the suitability of murine skin organ culture for studying KC proliferation in situ [4-6], hair growth induction by SP in mice [7] and the involvement of MC degranulation in murine hair growth [8]. Female, 6-8-week-old C57BL/6 mice (Charles River, Sulzfeld, Germany) with all hair follicles in the telogen stage of the hair cycle were sacrificed by CO2 inhalation, and their back skin was shaved, washed twice with 70% ethanol, followed by three rinses with saline. Full-thickness back skin was dissected at the level of the subcutis under sterile conditions, and was placed in ice-cold DME containing 10% fetal bovine serum, 2 • antibiotic/antimycotic mixture and 50 gg/ml gentamicin. Within 30-45 min of dissection, 4-mm punch biopsies of telogen skin were prepared under sterile conditions, as described previously

Skin histoculture assay for studying the hair cycle

Dear Editor: The poor understanding of the basic molecular mechanisms governing the growth, loss and pigmentation of hair is partly due to the paucity of relevant in vitro models for studying these phenomena. Elucidation of the biological clock that governs the cyclic activity of the hair follicle (telogen-anagen-catagen-telogen) would be a major breakthrough in hair research. Specifically, effective pharmacological manipulation of hair growth would greatly be facilitated by knowledge of the signals that initiate, drive and terminate anagen. Yet, there is currently no assay available that allows the in vitrostudy of the total growth phase, let alone cycling of adult hair follicles, or anagen-associated pigment production over an extended period of time. Though the growth of non-embryonic mouse, rat and human anagen hair follicles in vitro has been reported in various culture systems (cf. 11,18-20), none of these assays has utilized homogeneous populations of mature follicles of a well-defined stage of the growth cycle. Also, all assays were associated with considerable tissue traumatization by enzymatic digestion or mechanical manipulation and/or loss of the physiological follicular tissue environment, thus eliminating the intercellular communication between follicle cells on the one hand, and e.g. epidermal keratinocytes, fibroblasts, macrophages and mast cells on the other. This could be a serious limitation, considering that the epidermis (13,14), mast cells (15), macrophages (12,25) or other localimmunological factors (16) may contribute to the regulation of hair growth. Previously, we have used the C 57 B1-6 mouse for hair growth studies in vivo and in skin organ culture on metal grids (13,14,16,17). In this assay, anagen is induced in telogen mice pharmacologically (16) or by depilation (13). In mice, all truncal melanocytes are confined to the hair follicles, melanogenesis is strictly coupled to the anagen phase of the hair cycle, and follicular growth patterns are synchronized (1,22). Thus, the development of anagen can easily be recognized by observing the gradual change in skin color from white/pink (telogen) to grey (mid anagen) and finally to black (late anagen) (1,13,16,22). This assay provides large populations of homogeneous, mature mouse follicles of defined hair cycle stages that can readily be studied in skin organ culture by incubating biopsies of whole skin under in vivo-like conditions at the air-liquid interphase on metal grids (13,17). However, keratinocyte viability is rather limited under these conditions so that it is not possible to follow follicle development and the formation and pigmentation of anagen hair shafts over an extended period of time. Thus, we have turned to a simple, yet effective histoculture technique which uses collagen-containing sponge-gel supports to mainrain tissue viability (5,8), Most recently, we observed hair shaft growth in mouse skin histocultured with this technique at approximately the in vivo rate, though we could not tell what stage of the hair cycle the follicles had been in at the beginning of tissue culture, since white-mouse skin was used (9,27). We have also observed hair growth from human scalp (10) where the follicles are not synchronous which makes it difficult to study the hair cycle. Combining the C 57 BI-6 mouse model and the sponge-gel histoculture technique, we demonstrate here that follicles of the earliest anagen stage develop into full anagen follicles and produce pigmented hair shafts in vitro. C 57 BI-6 mice (female, syngeneic, 6 -8 weeks old) were purchased from Charles River, Kingston, NY, housed in community cages in the Albany Medical College Facility, AntiCancer Inc. and University Hospital R. Virchow, Freie Universitaet Berlin with 12hour light periods and fed ad libitum with water and rat/mouse chow 3000 (Agway, Syracuse). Anagen was induced in telogen mice (recognizable by their pink, skin color) by depilation with a wax/rosin mixture under anaesthesia as previously described (13,14,17,22). Two days and 14 days after anagen-induction, back skin was harvested under sterile conditions from mice that had been sacrificed by cervical dislocation under ether narcosis. Before dissecting the skin at the level of the subcntis, it was flushed twice with 70% ethanol. Harvested skin was then incubated in Hams F-10 medium at +4 ° C for overnight shipment from Albany, NY to San Diego, CA or back skin was prepared for histoculture immediately in San Diego as described below. For some experiments, animals were also anagen-indueed in San Diego or Berlin, and their skin used directly within 2 hours after harvesting for histoculture. Skin was harvested at 2-day and 14-day post anagen-induetion from five C 57 B1-6 mice for each experiment. The skin was put in histocuhure under sterile conditions according to the method developed earlier by Hoffman et al. (5,8), based on the work by Leighton (7). The 2-day-post anagen-induced skin was non-pigmented and showed neither macroscopic nor microscopic evidence for hair shaft formation (Fig. 1 A and 2 A). The 14-day-post-anagen-induced skin was light grey and showed no-hair out growth from shafts which were shaved before histoculture. Briefly, after dissecting the subcutis proximal to the parniculus carnosus, small pieces of skin were cut out with a 4 mm biopsy punch or re-cut into 2 equal fragments in Eagles MEM medium. They were then placed dermis-down onto 1 X 1 X 1 cm pieces of collagen-containing gel (Gelfoam gelatin sponge, Upjohn Co., Kalamazoo, MI) that had been prehydrated for at least 4 hours with culture medium (MEM + 10% FBS + 50 #g/ml gentamycin). From each mouse, 24 skin fragments were studied, i.e. 4 per sponge-gel, distributed between 6 gels placed individually in 6 well-plates with 2 ml medium added per well such that the gels were not covered and the skin was above the liquid medium. Cultures were maintained for up to 16 days at 37 ° C, 100% humidity and gassed with a mixture of 95% air and 5% CO 2. Tissue fixed in 10% buffered formalin was processed for routine histology and stained with hematoxylin/eosin or Giemsa according to standard proeedures. The length of hair shafts was measured under a dissection microscope with a ruler. Viable cells were selectively labeled with 15 #M of the dye 2,7-