Murine neonatal melanocytes exhibit a heightened proliferative response to ultraviolet radiation and migrate to the epidermal basal layer

Melanocytes respond to UVR not only by producing melanin, but also by proliferating. This is essentially a protective response. We have studied the melanocyte proliferative response after a single UVR exposure to neonatal mice. At 3 days post-UVR in wild-type neonates we observed a marked melanocyte activation not seen in adults. Melanocytes migrated to the epidermal basal layer, their numbers peaking at 3-5 days after UVR then diminishing. They appeared to emanate from the hair follicle, migrating to the epidermis via the outer root sheath. In melanoma-prone mice with melanocyte-specific overexpression of Hras(G12V), basal layer melanocytes were increased in size and dendricity compared to UVR-treated wild-type mice. Melanocytes in mice carrying a pRb pathway cell-cycle defect (oncogenic Cdk4(R24C)) did not show an enhanced response to UVR such as those carrying Hras(G12V). The exquisite sensitivity to UVR-induced proliferation and migration that characterizes neonatal mouse melanocytes may partly explain the utility of this form of exposure for inducing melanoma in mice that carry oncogenic mutations.     

Relative responsiveness of cultured human epidermal melanocytes and melanoma cells to selected mitogens

Early cellular events in the malignant transformation of melanocytes to melanoma are virtually unknown. In vitro investigation of this phenomenon has been hampered by the fastidious nature of the human epidermal melanocyte, which has proven difficult to cultivate. The present study compares responsiveness of cultured human epidermal melanocytes and established melanoma cell lines to serum, cholera toxin, and melanocyte growth factor (MGF), three established melanocyte mitogens. Four of four established human melanoma lines were substantially stimulated by fetal bovine serum, as were newborn foreskin-derived epidermal melanocytes. In contrast, none of the four melanoma lines responded to hypothalamic preparations containing MGF that consistently produced an approximately 30-fold increase in newborn melanocyte cell yield over a 2-week period. Cholera toxin, required for successful establishment of primary melanocyte cultures, had small and variable effects on the melanoma lines, with slight stimulation in one case, moderate inhibition in another, and essentially no effect in two others. These data suggest that transformation of epidermal melanocytes to melanoma often involves at least one phenotypic change resulting in escape from MGF regulation and another associated with insensitivity to cyclic AMP modulation; while at least some of the pathways conferring serum dependence are unaltered. Improved culture systems for the human epidermal melanocyte should facilitate further studies into the mechanism of its malignant conversion and may provide useful insights for the prevention and treatment of human melanoma.     

Infrared A radiation promotes survival of human melanocytes carrying ultraviolet radiation‐induced DNA damage

The link between solar radiation and melanoma is still elusive. Although infrared radiation (IR) accounts for over 50% of terrestrial solar energy, its influence on human skin is not well explored. There is increasing evidence that IR influences the expression patterns of several molecules independently of heat. A previous in vivo study revealed that pretreatment with IR might promote the development of UVR‐induced non‐epithelial skin cancer and possibly of melanoma in mice. To expand on this, the aim of the present study was to evaluate the impact of IR on UVR‐induced apoptosis and DNA repair in normal human epidermal melanocytes. The balance between these two effects is a key factor of malignant transformation. Human melanocytes were exposed to physiologic doses of IR and UVR. Compared to cells irradiated with UVR only, simultaneous exposure to IR significantly reduced the apoptotic rate. However, IR did not influence the repair of UVR‐induced DNA damage. IR partly reversed the pro‐apoptotic effects of UVR via modification of the expression and activity of proteins mainly of the extrinsic apoptotic pathway. In conclusion, IR enhances the survival of melanocytes carrying UVR‐induced DNA damage and thereby might contribute to melanomagenesis.     

Hermansky-Pudlak HPS1/pale ear gene regulates epidermal and dermal melanocyte development

The pale ear (ep) mouse strain is a model for the Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder causing pigmentary dilution, visual disturbances, bleeding diatheses, pulmonary fibrosis, and granulomatous colitis. The ep mice have a coat color very similar to the black-colored parental strain, C57BL/6. However, the ears and tails of ep mice are significantly hypopigmented compared with the control animals, suggesting that the gene mutation in ep mice reveals a differential regulation of melanocyte function in dorsal back skin melanocytes versus tail or ear skin. In this study, we analyzed the mutant phenotype in detail and determined that in the tail, the defective gene causes delayed onset of interfollicular epidermal melanocyte tyrosinase activity, decreased numbers of melanocytes in the interfollicular epidermis and dermis, and severe immaturity of tail epidermal melanosomes, findings not observed in dorsal back follicular melanocytes. These results highlight differences between follicular and interfollicular melanocyte biology and demonstrate that defects in the ep protein not only affect melanosome biogenesis, but also play a developmental role in determining interfollicular epidermal and dermal melanocyte function. The implications of these findings for the mechanisms governing physiologic variation in human pigmentation and for the pathogenesis of vitiligo are discussed.     

4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol)‐induced melanocyte cytotoxicity is enhanced by UVB exposure through generation of oxidative stress

4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, was reported to cause skin depigmentation in some users, which is attributed to its cytotoxicity to melanocyte. It was reported that cytotoxicity to melanocyte is possibly mediated by oxidative stress in a tyrosinase activity‐dependent manner. We examined the effect of UV radiation (UVR) on RD‐induced melanocyte cytotoxicity as an additional aggravating factor. UVR enhanced RD‐induced cytotoxicity in normal human epidermal melanocytes (NHEMs) via the induction of endoplasmic reticulum (ER) stress. Increased generation of intracellular reactive oxygen species (ROS) was detected. Pretreatment with N‐acetyl cysteine (NAC), antioxidant and precursor of glutathione significantly attenuated ER stress‐induced cytotoxicity in NHEMs treated with RD and UVR. Increase in cysteinyl‐RD‐catechol and RD‐pheomelanin in NHEMs treated with RD and UVR suggested that, after UVR excitation, RD or RD metabolites are potent ROS‐generating substances and that the tendency to produce RD‐pheomelanin during melanogenesis amplifies ROS generation in melanocytes. Our results help to elucidate the development mechanisms of RD‐induced leukoderma and provide information for innovation of safe skin‐whitening compounds.     

Melanocyte mitosis in UVB-irradiated mouse skin.

Little is known about the mitotic activity of the epidermal melanocyte population. Vincristine sulphate has now been used in vivo to study the frequency of mitosis of epedermal melanocytes in the ear skin of C57BL mice. It is shown that there is continuous formation of new melanocytes in unstimulated skin due to mitosis of pigment-producing epidermal melanocytes. The increase in this mitotic rate following daily UVB irradiation suggests that this is a major mechanism responsible for the UV-induced increase in the epidermal melanocyte population.     

Physiologic distribution and differentiation of melanocytes in human fetal and neonatal skin equivalents

There is evidence that epidermal keratinocytes play a critical role in melanocyte position and differentiation in the epidermis, although little is known about the molecular mechanisms involved. We have used an in vitro skin equivalent as a model system in which to study keratinocyte/melanocyte interactions in both fetal and neonatal skin. Because the skin equivalent model has been shown to closely simulate the morphologic and biochemical features of differentiated epidermis we hypothesized that the factors that influence melanocyte position and differnetiation would also function in this system. Localization of melanocytes in skin equivalents, using the monoclonal antibody HMB-45, established that melanocytes in fetal skin equivalents are grouped and distributed both basally and suprabasally, whereas melanocytes in neonatal skin equivalents are singly distributed among basal epidermal keratinocytes, similar to the distributions of fetal and neonatal melanocytes, respectively, in vivo. Similarly, in fetal and neonatal skin equivalents the patterns of expression of a number of melanoma/melanocyte-associated antigens closely parallels that seen in vivo. These results suggest that the skin equivalent model is an excellent system in which to study the dynamic factors that regulate melanocyte migration, proliferation, and differentiation during ontogeny and post-natal differentiation of the skin.     

Ultraviolet-B-irradiated mouse epidermis releases mediators that stimulate melanoma cells

We determined whether the exposure of mouse epidermal cells to ultraviolet radiation (UVR) induces the production of factors that can stimulate the in vitro and in vivo growth of syngeneic melanoma cells. Epidermal sheets isolated from the back skin of C3H/HeN mice were placed into sterile medium and exposed to different doses of UVB radiation; 18 h later, culture supernatants were harvested. Supernatants of epidermal sheets not exposed to UVR served as controls. Supernatants from UVR-treated epidermal sheets, but not control supernatants, stimulated the in vitro and in vivo growth of some murine melanomas, but not cells of a fibrosarcoma, a hepatocellular carcinoma, or a squamous carcinoma. Supernatants of UVR-treated epidermal cells injected into the ears of syngeneic mice stimulated epidermal cell proliferation in a short-term assay. We conclude that UVR may contribute to the incidence of cutaneous melanoma by its ability to cause the release from epidermal cells of diffusible factors that promote the outgrowth of small numbers of melanoma cells.     

Melanocytes adhere to and synthesize laminin-5 in vitro

Melanocytes arise from the neural crest, migrate to the skin, and can be detected in the basal layer of the epidermis in skin biopsies of human fetuses as early as 11 weeks gestational age. During post-natal life, melanocytes reside at the basal layer of the epidermis, but the ligands to which they attach are unknown. Laminin-5 is a component of anchoring filaments of the lamina lucida of the epidermal basement membrane. In this report we show that human melanocytes adhere to purified laminin-5 to a level comparable with normal human keratinocytes. Blocking antibodies to the 165 kDa subunit of laminin-5 significantly inhibited fetal and neonatal melanocyte attachment to the surface of salt-split skin, which exposes laminin-5 on its surface, suggesting that laminin-5 is a ligand for melanocyte attachment to the basement membrane in vivo. Western blotting of concentrated culture supernatant of fetal and neonatal melanocytes with anti-laminin-5 antibodies demonstrated a single immunoreactive band of the expected size of laminin-5. In contrast, 3 human metastatic melanoma cell lines did not produce laminin-5. Immunofluorescence microscopy with antibodies to each of the three chains of laminin-5 confirmed the presence of laminin-5 in a peri-cellular distribution around melanocytes, but not melanoma cells. Our results suggest that laminin-5 may be a ligand for normal human melanocytes in the basement membrane, and that loss of laminin-5 production by melanoma cells may be a marker for malignant transformation.     

Study of CCN3 (NOV) and DDR1 in normal melanocytes and vitiligo skin

We have hypothesised that melanocytes disappear in vitiligo because they are weakly attached to the epidermal basal membrane (melanocytorrhagy). In the epidermis, attachment of melanocytes to collagen IV is mediated through DDR1, which is under the control of CCN3. DDR1 genetic variants have been associated with vitiligo in patients of different ethnic origin. In vitro studies have shown that inhibition of CCN3 induces the detachment of melanocytes. We have studied in parallel the expression of CCN3 and DDR1 in lesional and perilesional skin of patients with vitiligo and the impact of the silencing of CCN3 and DDR1 in normal human melanocytes on their behaviour in epidermal reconstructs. Our in vivo study provides evidence of a dysregulation of the DDR1-CCN3 interaction in vitiligo skin as melanocytes remaining in perilesional skin did not express CCN3. Expression of DDR1 was decreased in lesional versus perilesional vitiligo skin in the majority of patients, and the expression of collagen IV was found decreased in all patients. Silencing of CCN3 in melanocytes induced a significant inhibition of cell adhesion to collagen IV whereas melanocytes transduced with shDDR1 still adhered well on collagen IV and did not increase melanocyte loss in epidermal reconstructs as compared with normal melanocytes. Melanocyte detachment was observed but not in all reconstructs using CCN3 silenced melanocytes. Overall, our study confirms that a downregulation of CCN3 is implicated in melanocyte adhesion in part through DDR1. In vitiligo skin, the interaction of CCN3 with other molecules, such as TGFβ and CCN2, needs to be addressed.     

The effect of ultraviolet radiation and melanocyte-stimulating hormone on tyrosinase activity in epidermal melanocytes of the mouse

The role of melanocyte stimulating hormone (MSH) as a mediator of the melanogenic response to ultraviolet radiation (UVR) was examined in C57 BL/6 mice. While exposure to UVR (250-300 nm) for 7, 14 and 27 days increased tyrosinase activity in epidermal melanocytes of the ear MSH had no effect and failed to alter the response to UVR. Plasma alpha-MSH concentrations were unchanged following UVR. Theophylline, a phosphodiesterase inhibitor, also had no effect on epidermal tyrosinase activity in non-irradiated and UV irradiated mice. Prostaglandin E2 and arachidonic acid were also ineffective in non-irradiated and UV irradiated mice and indomethacin, an inhibitor of prostaglandin synthesis, failed to increase epidermal tyrosinase activity after UVR. On the other hand, 12-0-tetradecanoyl phorbol 13 acetate, an activator of protein kinase C, increased epidermal tyrosinase activity in non-irradiated mice and also enhanced the effect of UVR.     

A mouse model of vitiligo induced by monobenzone

The paucity of vitiligo animal models limits the understanding of vitiligo pathogenesis and the development of therapies for the skin disorder. In this study, we developed a new mouse model of vitiligo by topically applying the skin‐depigmenting agent monobenzone on mice. We demonstrated that monobenzone‐induced skin depigmentation on the non‐exposed sites and that the severity of lesions depended on drug dosage. The result of the histological examination of the depigmented skin indicated loss of epidermal melanocytes and perilesional accumulation of CD8⁺ T cells. Furthermore, the monobenzone‐induced depigmentation of the Rag1 gene knockout did not appear on the non‐exposed sites, supporting the involvement of infiltrating CD8⁺ T cells in melanocyte destruction. Resemblance in histological characteristics and pathogenesis between monobenzone‐induced depigmentation and active human vitiligo suggests good potential of our mouse model for use in vitiligo research.     

Ribosomal stress, p53 activation and the tanning response

Melanocytes (MC) sit along the epidermal basal layer, largely quiescent except for constitutive melanin production. They are usually only activated after sun exposure. The recent paper by McGowan et al. (1) describes a novel mechanism by which melanocytes are induced to proliferate upon p53 activation in adjacent keratinocytes (KC). In this study, small subunit ribosomal protein mutations cause a dramatic activation of p53 that we propose mimics important aspects of the skin sunburn response after ultraviolet radiation (UVR) exposure. McGowan et al. show that the phenotype of their hyperpigmented mouse mutants results from p53-dependent upregulation of KITLG, a cytokine that binds to the KIT receptor on melanocytes and influences melanin synthesis, melanocyte proliferation, and dictates MC localization at the dermo-epidermal junction. These findings extend our knowledge about skin stress responses, in particular, how p53 activity in keratinocytes is central to the regulation of melanocyte behaviour.     

Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells

Abstract:  Cholesterol is important for membrane stability and is the key substrate for the synthesis of steroid hormones and vitamin D. Furthermore, it is a major component of the lipid barrier in the stratum corneum of the human epidermis. Considering that steroid hormone synthesis is taking place in epidermal melanocytes, we tested whether downstream oestrogen receptor/cAMP signalling via MITF/tyrosine hydroxylase/tyrosinase/pigmentation could be possibly modulated by cholesterol. For this purpose, we utilized human primary melanocyte cell cultures and human melanoma cells with different pigmentation capacity applying immunofluorescence, RT-PCR, Western blotting and determination of melanin content. Our in situ and in vitro results demonstrated that melanocytes can synthesize cholesterol via HMG-CoA reductase and transport cholesterol via LDL/Apo-B100/LDLR. Moreover, we show that cholesterol increases melanogenesis in these cells and in human melanoma cells of intermediate pigmentation (FM55) in a time- and dose-dependent manner. Cellular cholesterol levels in melanoma cells with different pigmentation patterns, epidermal melanocytes and keratinocytes do not differ except in the amelanotic (FM3) melanoma cell line. This result is in agreement with decreasing cholesterol content versus increasing pigmentation in melanosomes. Cholesterol induces cAMP in a biphasic manner i.e. after 30 min and later after 6 and 24 h, meanwhile protein expression of oestrogen receptor β, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72 h. Taken together, we show that human epidermal melanocytes have the capacity of cholesterol signalling via LDL/Apo-B100/LDL receptor and that cholesterol under in vitro conditions increases melanogenesis.     

Keratinocytes and dermal factors activate CRABP-I in melanocytes

Abstract: Recognition that cellular retinoic acid binding protein (CRABP)-I and CRABP-II are found in different cell types has provided additional support for the presumably divergent roles of these two proteins in mediating retinoic acid (RA) effects in human skin. CRABP-II is expressed in fibroblasts and keratinocytes, and CRABP-I in as yet unidentified cells, possibly epidermal melanocytes. Recently, we demonstrated that each of these RA-binding proteins in human skin possesses two classes of binding sites, possibly related to the state of phosphorylation of the proteins. We now characterize the cutaneous origin of CRABP-I further using an anion-exchange HPLC assay that allows effective separation of the two proteins in human skin, and a fluorescent in situ hybridization technique. We report that CRABP-I is expressed in isolated melanocytes at the mRNA level, although under these circumstances the protein has minimal RA-binding activity, and that keratinocytic and dermal influences are required for CRABP-I activity in melanocytes. This melanocyte origin for CRABP-I and the improvement by RA of the irregular hyperpigmentation associated with photoaging led us to examine the effects of RA using various cellular associations, from conventional pure cultures of melanocytes grown on plastic dishes to a pigmented skin equivalent consisting of melanocytes and keratinocytes grown on a dermal equivalent. We established that the inhibitory effects of RA on melanogenesis do not result from a direct effect on melanocytes alone but also involve keratinocytes and dermal influence. These data expand our understanding of cell-to-cell signaling in cutaneous pigmentation, and strongly suggest a role for CRABP-I in mediating RA effects on melanogenesis.     

Ageing and photoageing of keratinocytes and melanocytes

An overview of keratinocyte and melanocyte function is provided. The processes of cutaneous ageing and photoageing are defined, and age-associated modulations in gene expression are described. The changes in keratinocytes and melanocytes that occur with skin ageing and photoageing and the characteristics of chronologically aged vs. photoaged skin are delineated. Mutations that are found in malignant and premalignant tumors of epidermal origin are described.     

Abnormal wound healing in UV-irradiated skin of Sencar mice

Sencar mice demonstrate an unusual sensitivity to epidermal carcinogenesis by initiation-promotion or single high-dose exposure to ultraviolet radiation (UVR). These mice exhibited an exaggerated and persistent epidermal hyperplasia in response to tissue damage caused by UVR. The persistent hyperplasia was not present in similarly treated BALB/c mice, a strain that is relatively resistant to skin carcinogenesis by initiation-promotion or single-exposure UVR. Epithelial cell proliferation and migration were examined by autoradiography to determine the cellular basis for the persistence of hyperplasia in Sencar mouse skin. Twelve weeks after irradiation, the rate of epidermal basal cell proliferation was approximately 4 times greater in Sencar mice than in BALB/c mice, whereas epidermal cell transit times were similar in the two strains. This result indicated that persistent hyperplasia was due to sustained epithelial cell division rather than delayed cell maturation. Surgical incision of Sencar skin did not cause abnormal hyperplasia, nor did this procedure enhance the induction of tumors by UVR. These findings suggest that Sencar mice may possess a heritable defect that mediates both tissue regeneration and tumorigenesis in UV-irradiated skin.     

Cross-reactive tumor antigens in the skin of mice exposed to subcarcinogenic doses of ultraviolet radiation

This study suggests that cross-reactive tumor-associated antigens (TAA) are induced in the epidermis of mice exposed to subcarcinogenic doses of ultraviolet radiation (UVR). Foot-pad immunization of C3H mice with viable epidermal cells from syngeneic UVR-exposed mice induced cytotoxic cells in the draining lymph nodes (DLN). These cells were capable of lysing a battery of UVR-induced tumor targets in a short-term chromium release assay. In contrast, the DLN cells of mice immunized with epidermal cells from normal non-UVR-exposed mice did not mature into effector cells with antitumor activity. The spectrum of tumor recognition of cytotoxic cells induced by immunization with UVR-exposed epidermal cells was identical to that of cytotoxic T cells obtained from animals that were foot-pad immunized with UVR-induced tumor cells. Both cytotoxic cell populations were shown to lyse tumors of diverse origin, including syngeneic UVR- and methylcholanthrene-induced tumors, as well as allogeneic UVR-induced tumors. These cells displayed minimal lytic activity against YAC-1 lymphoma cells, peritoneal exudate cells, and concanavalin A-stimulated splenocytes. The results from this study demonstrate that antigens are expressed on UVR-exposed mouse epidermal cells prior to the emergence of skin tumors, and these antigens can induce cytotoxic cells with specificity for TAA. We conclude that the early antigenic changes observed in UVR-exposed epidermis and their effect on the host's immune system may influence the emergence and progression of UVR-induced skin cancers.