Effects of ultraviolet light on melanocyte differentiation: studies with mouse neural crest cells and neural crest-derived cell lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L-3,4-dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT- or DOPA-positive cells between the UV-irradiated cultures and the non-irradiated cultures. We then examined the effects of UV light on KIT-positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase-positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with alpha-melanocyte-stimulating hormone (alpha-MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase-negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal-regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as alpha-MSH and/or endothelin-1.     

Keratinocyte expression of transgenic hepatocyte growth factor affects melanocyte development, leading to dermal melanocytosis

Using the epidermis-specific cytokeratin 14 promoter to deliver HGF exclusively from epidermal keratinocytes, we have examined the potential of hepatocyte growth factor (HGF) secreted from the normal environment to control morphogenesis. The transgenic mice displayed a significant increase of the number of melanocytes and their precursors in embryos starting not later than 16.5 dpc, and then after birth an explosive increase of dermal melanocytes started within 1 week, and these melanocytes were maintained throughout the entire life of the mice. Thus, HGF acts as a paracrine agent to promote survival, proliferation and differentiation of melanocyte precursors in vivo, and eventually causes melanocytosis. Loss of E-cadherin expression in dermal melanocyte precursors suggests that HGF caused dermal localization of melanocytes and their precursors by down-regulation of E-cadherin molecules.     

Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

Functionally distinct melanocyte populations revealed by reconstitution of hair follicles in mice

Hair follicle reconstitution analysis was used to test the contribution of melanocytes or their precursors to regenerated hair follicles. In this study, we first confirmed the process of chimeric hair follicle regeneration by both hair keratinocytes and follicular melanocytes. Then, as first suggested from the differential growth requirements of epidermal skin melanocytes and non‐cutaneous or dermal melanocytes, we confirmed the inability of the latter to be involved as follicular melanocytes to regenerate hair follicles during the hair reconstitution assay. This clear functional discrimination between non‐cutaneous or dermal melanocytes and epidermal melanocytes suggests the presence of two different melanocyte cell lineages, a finding that might be important in the pathogenesis of melanocyte‐related diseases and melanomas.     

Terminal migration and early differentiation of melanocytes in embryonic chick skin

The microenvironment is thought to play a key role in the control of neural crest cell diversification. To investigate its role in melanocyte differentiation we mapped the temporal and spatial distribution of pigmented melanocytes in embryonic chick skin and determined, by experimental means, the route taken by migrating melanocytes in the skin. We show that the New Hampshire Red/Black Australorp crossbreed exhibits melanization from 5 days of incubation (2 1/2 days earlier than is reported in other breeds). Contrary to previous reports our findings show that melanization is at first predominantly dermal. Both dermal and epidermal melanocyte numbers increase until Day 8, whereafter there is a dramatic decline in dermal melanocytes and by Day 10, melanocytes are almost exclusively located in the epidermis. Using homeotypic and heterotypic combinations of white and red/black dermis and epidermis we have demonstrated that premelanocytes arrive in the dermis of the trunk by Day 3 and begin to move into the epidermis from Day 4 onward. Results from these grafts and from tritium labeling studies strongly suggest that there is little or no reverse migration of premelanocytes from epidermis to dermis. Our findings indicate that overt melanocyte differentiation is not dependent on location in an epidermal environment, and that melanogenesis does not signify the end-stage in the migration process. Further, they suggest that the early dermal mesenchyme plays a key role in controlling melanogenesis.     

Dermal nevus cells from congenital nevi cannot penetrate the dermis in skin reconstructs

Congenital nevi are composed of pigment cells bearing common features with melanocytes but showing altered differentiation which leads to nesting and dermal involvement. Using a dead de-epidermized dermis seeded with a combination of keratinocytes and various sources of pigment cells (normal melanocytes, dermal nevus cells from congenital nevi, Bowes melanoma cells), we have studied the formation of nests and the dermal migration of pigment cells together with their secretion profiles of matrix metalloproteinases (MMP). Dermal fibroblasts were also used as control cells in epidermal reconstructs. Besides their morphologic features, the absence of pigment donation to keratinocytes was the major characteristic of dermal nevus cells. A positive correlation was established between the increasing percentage of seeded nevus cells and the patchy pigmentation of reconstructs, as well as the clustering of cells in junctional nests. However, the presence of nevus cells in the dermis of reconstructs was never detected, whereas melanoma cells and dermal fibroblasts could invade the dermis during the time span of the experiments. MMP9 was never expressed in congenital dermal nevus cells but pro-MMP2 was constitutively expressed by all strains of congenital nevus cells and dermal fibroblasts. Melanocytes produced comparable amounts of both pro-MMP2 and pro-MMP9, and Bowes melanoma cells secreted a marginal level of pro-MMP2. In view of their three-dimensional behaviour and secretion of MMPs, we propose that dermal congenital nevus cells correspond to an intermediate status of differentiation between normal melanocytes and melanoma cells. Activation of MMPs by a cofactor or the activation of another signalling pathway seems necessary to induce the dermal passage of nevus cells.     

THE FINE STRUCTURE OF NORMAL AND NEOPLASTIC MELANOCYTES IN THE SYRIAN HAMSTER, WITH PARTICULAR REFERENCE TO CARCINOGEN-INDUCED MELANOTIC TUMORS

The dermal melanocyte system of the Syrian hamster is particularly responsive to the melanogenetic and tumor-inducing effects of 7,12-dimethylbenz(a)anthracene (DMBA). The melanocytes of the hair follicles appear to be susceptible to the melanogenetic effect of DMBA but not to its tumor-inducing effect. The epidermal melanocytes are non-pigmented and are unresponsive to both melanogenetic and carcinogenic effects of DMBA. The pigmented granules of the dermal melanocytes of both the golden and the white hamster have an identical substructure and pattern of melanization which occurs in an orderly fashion on a delicate fibrillar component. The hair melanocytes have larger pigment granules with a more complicated fibrillar substructure. The epidermal melanocytes do not possess pigment granules but are recognized by their dendritic shape, the absence of desmosomes and tonofilaments, and the presence of racket-shaped or rod-shaped organelles. The melanin granules in neoplastic melanocytes of the golden hamster differ from corresponding normal melanocytes only in their larger size. In the white hamster, however, the melanin granules in tumors produced under identical experimental conditions are so bizarre and atypical that consideration was given to the possibility that a genetic difference in the melanization pattern between the two varieties becomes apparent in carcinogen-induced melanotic tumors. No definite conclusions could be reached as to the precise origin of the melanin granules in either normal or neoplastic melanocytes.     

Development of N,O-(carboxymethyl)chitosan/collagen matrixes as a wound dressing

In an attempt to accelerate wound healing by stimulating the recruitment of fibroblasts and improve the mechanical properties of collagen matrixes, N,O-(carboxymethyl)chitosan (NOCC) was incorporated into the backbone of a collagen (COL) matrix without or with chondroitin sulfate (CS) or an acellular dermal matrix (ADM). The result of a cell migration study demonstrated that the migration of fibroblasts was significantly enhanced by NOCC in a concentration-dependent manner. In the analysis with a dynamic mechanical analyzer, NOCC/CS/COL matrixes presented higher tensile strengths than did NOCC/ADM/COL matrixes. Skin fibroblasts cultured on the matrixes containing NOCC showed increased proliferation and secretion of three kinds of cytokines compared with the control. Results of the in vivo wound healing study showed that matrixes incorporating NOCC showed markedly enhanced wound healing compared with the control. Therefore, the above results clearly suggest that NOCC/COL matrixes containing CS or ADM can be potential wound dressings for clinical applications.     

Effects of gamma-radiation on the differentiation of mouse melanocytes in the hair follicles

Pregnant mice were whole-body irradiated with a single acute dose of gamma-rays (60Co) to investigate the effect of gamma-radiation on embryonic melanoblasts. The effect was studied by scoring changes in the differentiation of melanocytes in the hair follicles of mice heterozygous for the recessive coat color mutation pink-eyed dilution (p). Abnormal round melanocytes were found in the hair matrix and the dermal papilla of F1 offspring 3.5 days after birth. However, these round melanocytes possessed a melanin deposition of similar intensity to normal hair follicular melanocytes. The frequency of the abnormal hair follicles increased in a dose-dependent manner. Moreover, higher frequencies were found in the animals irradiated at earlier stages of embryonic development. These results indicate that gamma-radiation affects dendritogenesis and the location of mouse melanocytes in the hair follicles, with greater effects seen at the earlier stages of development.     

Dermal melanocytosis in Japanese monkeys (Macaca fuscata)

The skin of Japanese monkeys (Macaca fuscata) shows diffuse discolorations resembling human dermal melanocytosis. Very few laboratory animals have melanocytes in the dermis. The purpose of this study was to clarify the dermatologic characteristics of Japanese monkeys in terms of gross appearance, skin color, and histopathologic findings. A colorimeter was used to record the skin colors of pigmented and nonpigmented sites. Tissue specimens obtained from both types of sites were examined histopathologically. All animals examined had pigmented sites on their bodies, and the discolorations extended over 25% to 33% of the body surface. The colorimeter could detect differences in skin color due to dermal melanocytosis. All parameters of the colorimetric systems used (Yxy, L*a*b*, and L*C*h* systems) demonstrated significant differences between pigmented and nonpigmented sites. In pigmented sites, the epidermis lacked melanocytes, but the dermis had numerous melanocytes with abundant melanin. Activated melanocytes with well-developed dendrites were distributed throughout the upper part of the dermal layer. Melanocytes were not arranged in clusters, and elastic and collagen fibers in the dermis showed no histological abnormalities. Nonpigmented sites lacked melanin granules in both the epidermis and dermis. This study revealed that gross dermal melanocytosis correlated well with colorimetric results and histopathologic findings. These findings suggest that the pigmentation of Japanese monkeys is equivalent to dermal melanocytosis in humans, to the end that Japanese monkeys may be a useful animal model for investigating dermal melanogenesis.     

Characterization of melanocyte-specific inducible Cre recombinase transgenic mice

Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.     

Melanogenesis in dermal melanocytes of Japanese Silky chicken embryos

The Japanese Silky chicken (SK) shows dermal and visceral hyperpigmentation. This study characterizes ultrastructurally the melanin granules developing in dermal melanocytes of the dorsal skin of SK, in an attempt to better understand the processes of melanogenesis in these permanently ectopic cells. The steps of melanogenesis are similar to those described for epidermal melanocytes, with melanosomes going from stage I to IV but, in SK, the maturation occurs in the cell body, as well as in the cytoplasmic processes. At stage III, the deposition of melanin is cumulative and can aggregate in rounded structures, which combine to turn into the mature granule. The final destiny of mature melanosomes is still unclear, although it was observed that dermal macrophages can accumulate melanin granules in their phagosomes. Even with the close proximity between melanocytes and other dermal cells, the transference of melanosomes was not observed. Our findings indicate that melanogenesis in dermal melanocytes in SK has the same morphological characteristics found in epidermal melanocytes, but the functional aspect still remains to be elucidated.     

Deposition of Basic Fibroblast Growth Factor on Surface of Epidermal Melanocytes Suggesting the Stromal Control of Epidermal Pigmentation

Scanning electron microscopy with immunogold labeling was used to demonstrate the in vivo distribution of molecules of basic fibroblast growth factor (bFGF) that were expressed and/or present on the surface of the cells of the normal epidermis and dermal connective tissue of humans. We found that molecules of bFGF, seen as deposits of gold particles, were present densely on the surfaces of the melanocytes but not the epidermal keratinocytes. In connective tissue, these molecules were present exclusively on the surfaces of the fibroblasts, macrophages, vascular endothelial cells, and the basement membrane surrounding the endothelial tube. The selective deposition of bFGF molecules on the melanocytes suggests that the dermal connective tissue may be involved in controlling the proliferation of melanocytes by means of bFGF molecules in vivo, since these melanocytes require bFGF to proliferate in vitro. The latter is synthesized and stored exclusively in the connective tissue.     

Hair cycle dynamics: the case of the human hair follicle

The existence of a growth and regeneration cycle makes the hair follicle a true paradigm of tissue homeostasis. Analysis of about 9000 cycles led us to propose a stochastic model of human hair dynamics. The existence of hair cycles implies that stem cells must be cyclically activated and hair melanin unit has to be renewed. Using different markers, we were able to identify two distinct epithelial stem cell reservoirs, located in the upper and lower thirds of the anagen hair follicle outer root sheath. These two reservoirs fuse during the regression phase and individualize again in the new forming anagen hair follicle. Using a set of antibodies specific of melanocyte lineage and melanogenesis, pigmentation unit turnover was followed throughout the entire hair cycle. In the terminal anagen hair, active melanocytes were localized on top of the dermal papilla, while amelanotic melanocytes were identified in the upper third of the outer root sheath (ORS). Those amelanotic melanocytes located in upper ORS probably represented a melanocyte reservoir for successive hair generation, since at the induction of anagen phase, some melanocytes were committed to cell division and melanogenesis was turned on, but only in the nascent hair bulb, close to the dermal papilla.     

The Proteolytic Potential of Normal Human Melanocytes: Comparison With Other Skin Cells and Melanoma Cell Lines

To understand the contribution of epidermal melanocytes in the proteolytic potential of human skin, we have studied melanocytes grown in a low-serum medium deprived of phorbol esters, cholera toxin, and other non-physiological supplements. We focused on the plasminogen activation system and certain matrix metalloproteinases (gelatinases). Supposing that the proteolytic activity of cells can influence binding to collagen matrix and its reorganization, we have analyzed these parameters as well. We found that human melanocytes secreted tissue-type plasminogen activator and utilised it to generate cell-bound plasmin. No urokinase-type plasminogen activator was detected in the cultures but its receptor was found in cell extracts. Both the 72 kDa and 92 kDa gelatinases were secreted by the cells and in equal amounts. In addition, melanocytes secreted the wide-spectrum proteinase inhibitor alpha-2-macroglobulin. Melanocytes cast into collagen matrices retained a rounded morphology, did not extend processes, and were unable to contract collagen lattices. As a control, these parameters were investigated in parallel in cultures of human keratinocytes, dermal fibroblasts, and two melanoma cell lines. The obtained characteristics suggest that normal human melanocytes are proteolytically active cells. This function may pertain to skin physiology and pathophysiology.     

Melanocytes: morphological basis for an exteroceptive sensory system for monitoring ultraviolet radiation

From the conventional medical perspective, melanocytes have been traditionally viewed as epidermal responders/reactors to ultraviolet radiation. In this paper we have begun an analysis of the functional significance of melanocytes as monitors for ultraviolet radiation with their neuronal, endocrinological and immunological intercalations. This preliminary study was performed using as a human model unilateral sural nerve sensory blockade before and after ultraviolet exposure to both feet while the unblocked foot acted as the control. Full thickness surgical skin biopsies were taken of (I), normal pre-exposure skin, in the center of the two centimeter exposure area of both the sural nerve blocked area (II-III) and control (IV-V) one centimeter outside the exposed area in both the sural nerve blocked foot and the control or non-blocked foot. The objective clinical, blinded morphological and immuno-histochemical data from this experiment support the initial conclusion that neuronal connection is necessary for the normal ultraviolet exposure dermal reaction. Based upon this study, we further propose the existence of an exteroceptive sensory system in which melanocytes, with direct nervous system connection initiate an ultraviolet radiation reactive response that mobilizes both conventional endocrine and immune pathways.     

Melanocyte subpopulation turnover during the human hair cycle: an immunohistochemical study

The human hair cycle is characterized by successive phases of growth and involution that imply tissue regression and regeneration. As a consequence, the hair melanin unit has to be renewed in a cyclic manner. Actually, the behavior of human hair follicle melanocytes throughout the hair cycle has been poorly studied. Thus, the origin of melanocytes present in the bulb after human hair regeneration is still not clarified, and neither are the events that control the melanin biosynthesis activity in the human hair bulb. In this study, we showed at the cellular level that in human pigmented hair follicles, the expression of tyrosinase and tyrosinase-related protein-1 (TRP-1) was detectable during the anagen phases III/IV through VI, only in those melanocytes which were located in the bulb. During the catagen phase, the two evaluated melanogenic enzymes were detectable no more, although melanocytes were still present in the preceding bulbar area. The epithelial column of catagen follicles and the capsule of telogen follicles also contained inactive melanocytes as evidenced by pMel-17 labeling. At the induction of a new anagen hair follicle, some melanocytes were committed to cell division, but only when located in the nascent bulb close to the dermal papilla. Our results emphasize the close relationship between melanogenesis and the hair cycle and suggest that in humans, melanogenesis is restricted to anagen hair follicles not because of the regulation of tyrosinase activity, but because of melanogenic enzyme expression, e.g., tyrosinase and TRP-1. Furthermore, the fact that in the newly developing anagen hair follicles, cell-division commitment and tyrosinase and TRP-1 expression were observed in melanocytes only when located in the nascent bulb suggests a highly regio-specific melanocyte stimulation in early the anagen phase.     

Cellular and hormonal regulation of pigmentation in human ocular melanocytes

The purpose of this study was to examine some of the factors that may be relevant to regulating pigmentation in the human eye, specifically whether choroidal and iridial melanocytes are sensitive to regulation by epithelial and stromal cells and alpha-melanocyte stimulating hormone (alpha-MSH). Human choroidal and iridial melanocytes were established in culture and co-cultured with epithelial cells and stromal cells derived both from skin and from eye in order to determine their influence on choroidal and iridial melanocyte dopa oxidase activity. In all cases, co-culture of melanocytes with either epithelial cells or fibroblasts led to an increase in dopa oxidase activity during 5 days of co-culture. The extent of the increase ranged from 60% (non-significant) to as much as 185% when both fibroblasts and keratinocytes were present. The optimal ratio of fibroblasts to melanocytes was 1:10 (for dermal fibroblasts) or 1:2 (for iridial fibroblasts) and 1:1 for all epithelial cells to melanocytes. Both choroidal (three out of three cultures) and iridial (two out of three cultures) melanocytes showed increases in dopa oxidase activity to alpha-MSH when cultured in Green's media but the same cells cultured in MCDB153 were unresponsive to alpha-MSH. These in vitro studies suggest that ocular melanocytes have the capacity to be influenced by adjacent epithelial and stromal cells with respect to pigmentation.     

Retinoid-X-Receptors (α/β) in Melanocytes Modulate Innate Immune Responses and Differentially Regulate Cell Survival following UV Irradiation

Understanding the molecular mechanisms of ultraviolet (UV) induced melanoma formation is becoming crucial with more reported cases each year. Expression of type II nuclear receptor Retinoid-X-Receptor α (RXRα) is lost during melanoma progression in humans. Here, we observed that in mice with melanocyte-specific ablation of RXRα and RXRβ, melanocytes attract fewer IFN-γ secreting immune cells than in wild-type mice following acute UVR exposure, via altered expression of several chemoattractive and chemorepulsive chemokines/cytokines. Reduced IFN-γ in the microenvironment alters UVR-induced apoptosis, and due to this, the survival of surrounding dermal fibroblasts is significantly decreased in mice lacking RXRα/β. Interestingly, post-UVR survival of the melanocytes themselves is enhanced in the absence of RXRα/β. Loss of RXRs α/β specifically in the melanocytes results in an endogenous shift in homeostasis of pro- and anti-apoptotic genes in these cells and enhances their survival compared to the wild type melanocytes. Therefore, RXRs modulate post-UVR survival of dermal fibroblasts in a “non-cell autonomous” manner, underscoring their role in immune surveillance, while independently mediating post-UVR melanocyte survival in a “cell autonomous” manner. Our results emphasize a novel immunomodulatory role of melanocytes in controlling survival of neighboring cell types besides controlling their own, and identifies RXRs as potential targets for therapy against UV induced melanoma.