Melanocyte precursors express elastin binding protein and elastin-derived peptide (VGVAPG) stimulates their melanogenesis and dendrite formation.

BACKGROUND: In congenital or acquired dermal melanocytosis, attachment of melanocyte with elastic fiber was shown in electron microscopy of unknown biological meaning. We hypothesize elastin-derived peptide may play a role in activating dermal melanocyte precursors. OBJECTIVES: This study was designed to determine: (i) whether melanocyte precursors express elastin binding protein (EBP); (ii) ontogenic expression of EBP and elastin in murine embryonic skin; (iii) the effects of elastin-derived peptide (VGVAPG) on melanocyte precursors. METHODS: Using immunohistochemistry or Western blot to identify EBP on murine embryonic sections, neural crest cell (NCC) primary culture explants, or two melanocyte precursor cell lines, NCCmelb4 and NCCmelan5. NCC explants or cells were treated with VGVAPG to compare its effect on proliferation, dendrite formation, melanosome maturation and tyrosinase mRNA expression of melanocyte precursors. RESULTS: EBP was immunostained on c-kit+ melanocyte precursors. 67kDa EBP protein was immunoblotted on NCCmelb4 and NCCmelan5 cells. EBP was expressed early at embryonic day (E) 9.5, but elastin appeared later at E12.5 skin. VGVAPG increased DOPA-positive cell number and enhanced their dendrite formation in NCC explants. Electron microscopy showed advanced melanosome maturation in NCC explants or cells treated with VGVAPG. VGVAPG enhanced tyrosinase mRNA expression in NCCmelan5 cells. CONCLUSIONS: Melanocyte precursors expressed EBP. VGVAPG stimulated their melanogenesis and dendrite formation. In the developmental journey interaction between elastin and EBP-expressed melanocyte precursors in embryos happened mainly since the stage of tertiary melanoblasts. These findings first provide biological evidences for the interaction between melanocyte and elastic fiber.

Involvement of dynein and spectrin with early melanosome transport and melanosomal protein trafficking.

Melanosomes are unique membrane-bound organelles specialized for the synthesis and distribution of melanin. Mechanisms involved in the trafficking of proteins to melanosomes and in the transport of mature pigmented melanosomes to the dendrites of melanocytic cells are being characterized, but details about those processes during early stages of melanosome maturation are not well understood. Early melanosomes must remain in the perinuclear area until critical components are assembled. In this study, we characterized the processing of two distinct melanosomal proteins, tyrosinase (TYR) and Pmel17, to elucidate protein processing in early or late steps of the secretory pathway, respectively, and to determine mechanisms underlying the subcellular localization and transport of early melanosomes. We used immunological, biochemical, and molecular approaches to demonstrate that the movement of early melanosomes in the perinuclear area depends primarily on microtubules but not on actin filaments. In contrast, the trafficking of TYR and Pmel17 depends on cytoplasmic dynein and its interaction with the spectrin/ankyrin system, which is involved with the sorting of cargo from the plasma membrane. These results provide important clues toward understanding the processes involved with early events in melanosome formation and transport.

Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/beta-catenin signaling in keratinocytes.

The epidermis (containing primarily keratinocytes and melanocytes) overlies the dermis (containing primarily fibroblasts) of human skin. We previously reported that dickkopf 1 (DKK1) secreted by fibroblasts in the dermis elicits the hypopigmented phenotype of palmoplantar skin due to suppression of melanocyte function and growth via the regulation of two important signaling factors, microphthalmia-associated transcription factor (MITF) and beta-catenin. We now report that treatment of keratinocytes with DKK1 increases their proliferation and decreases their uptake of melanin and that treatment of reconstructed skin with DKK1 induces a thicker and less pigmented epidermis. DNA microarray analysis revealed many genes regulated by DKK1, and several with critical expression patterns were validated by reverse transcriptase-polymerase chain reaction and Western blotting. DKK1 induced the expression of keratin 9 and alpha-Kelch-like ECT2 interacting protein (alphaKLEIP) but down-regulated the expression of beta-catenin, glycogen synthase kinase 3beta, protein kinase C, and proteinase-activated receptor-2 (PAR-2), which is consistent with the expression patterns of those proteins in human palmoplantar skin. Treatment of reconstructed skin with DKK1 reproduced the expression patterns of those key proteins observed in palmoplantar skin. These findings further elucidate why human skin is thicker and paler on the palms and soles than on the trunk through topographical and site-specific differences in the secretion of DKK1 by dermal fibroblasts that affects the overlying epidermis.

The effects of dickkopf 1 on gene expression and Wnt signaling by melanocytes: mechanisms underlying its suppression of melanocyte function and proliferation.

Dickkopf 1 (DKK1), which is expressed at high mRNA levels by fibroblasts in the dermis of human skin on the palms and soles, inhibits the function and proliferation of melanocytes in the epidermis of those areas via the suppression of beta-catenin and microphthalmia-associated transcription factor (MITF). In this study, we investigated the protein expression levels of DKK1 between palmoplantar and non-palmoplantar areas and the effects of DKK1 on melanocyte gene expression profiles and on Wnt signaling pathways using DNA microarray technology, reverse transcriptase-PCR, Western blot, 3-dimensional reconstructed skin, immunocytochemistry, and immunohistochemistry. DKK1-responsive genes included those encoding proteins involved in the regulation of melanocyte development, growth, differentiation, and apoptosis (including Kremen 1, G-coupled receptor 51, lipoprotein receptor-related protein 6, low-density lipoprotein receptor, tumor necrosis factor receptor super-family 10, growth arrest and DNA-damage-inducible gene 45beta, and MITF). Of special interest was the rapid decrease in expression of MITF in melanocytes treated with DKK1, which is concurrent with the decreased activities of beta-catenin and of glucose-synthase kinase 3beta via phosphorylation at Ser9 and with the upregulated expression of protein kinase C alpha. These results further clarify the mechanism by which DKK1 suppresses melanocyte density and differentiation, and help explain why DKK1-rich palmoplantar epidermis is paler than non-palmoplantar epidermis via mesenchymal-epithelial interactions.

Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes.

Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.

Sorting of Pmel17 to melanosomes through the plasma membrane by AP1 and AP2: evidence for the polarized nature of melanocytes.

Adaptor proteins (AP) play important roles in the sorting of proteins from the trans-Golgi network, but how they function in the sorting of various melanosome-specific proteins such as Pmel17, an essential structural component of melanosomes, in melanocytes is unknown. We characterized the processing and trafficking of Pmel17 via adaptor protein complexes within melanocytic cells. Proteomics analysis detected Pmel17, AP1 and AP2, but not AP3 or AP4 in early melanosomes. Real-time PCR, immunolabeling and tissue in-situ hybridization confirmed the coexpression of AP1 isoforms mu1A and mu1B (expressed only in polarized cells) in melanocytes and keratinocytes, but expression of mu1B is missing in some melanoma cell lines. Transfection with AP1 isoforms (mu1A or mu1B) showed two distinct distribution patterns that involved Pmel17, and only mu1B was able to restore the sorting of Pmel17 to the plasma membrane in cells lacking mu1B expression. Finally, we established that expression of mu1B is regulated physiologically in melanocytes by UV radiation or DKK1. These results show that Pmel17 is sorted to melanosomes by various intracellular routes, directly or indirectly through the plasma membrane, and the presence of basolateral elements in melanocytes suggests their polarized nature.

Intracellular composition of fatty acid affects the processing and function of tyrosinase through the ubiquitin-proteasome pathway.

Proteasomes are multicatalytic proteinase complexes within cells that selectively degrade ubiquitinated proteins. We have recently demonstrated that fatty acids, major components of cell membranes, are able to regulate the proteasomal degradation of tyrosinase, a critical enzyme required for melanin biosynthesis, in contrasting manners by relative increases or decreases in the ubiquitinated tyrosinase. In the present study, we show that altering the intracellular composition of fatty acids affects the post-Golgi degradation of tyrosinase. Incubation with linoleic acid (C18:2) dramatically changed the fatty acid composition of cultured B16 melanoma cells, i.e. the remarkable increase in polyunsaturated fatty acids such as linoleic acid and arachidonic acid (C20:4) was compensated by the decrease in monounsaturated fatty acids such as oleic acid (C18:1) and palmitoleic acid (C16:1), with little effect on the proportion of saturated to unsaturated fatty acid. When the composition of intracellular fatty acids was altered, tyrosinase was rapidly processed to the Golgi apparatus from the ER (endoplasmic reticulum) and the degradation of tyrosinase was increased after its maturation in the Golgi. Retention of tyrosinase in the ER was observed when cells were treated with linoleic acid in the presence of proteasome inhibitors, explaining why melanin synthesis was decreased in cells treated with linoleic acid and a proteasome inhibitor despite the abrogation of tyrosinase degradation. These results suggest that the intracellular composition of fatty acid affects the processing and function of tyrosinase in connection with the ubiquitin-proteasome pathway and suggest that this might be a common physiological approach to regulate protein degradation.

Establishment of a kit-negative cell line of melanocyte precursors from mouse neural crest cells.

We previously established a mouse neural crest cell line named NCCmelb4, which is positive for Kit and negative for tyrosinase. NCCmelb4 cells were useful to study the effects of extrinsic factors such as retinoic acids and vitamin D(3) on melanocyte differentiation, but in order to study the development of melanocytes from multipotent neural crest cells, cell lines of melanocyte progenitors in earlier developmental stages are needed. In the present study, we established an immortal cell line named NCCmelb4M5 that was derived from NCCmelb4 cells. NCCmelb4M5 cells do not express Kit and are immortal and stable in the absence of Kit ligand. They are positive for melanocyte markers such as tyrosinase-related protein 1 and DOPAchrome tautomerase and they contain stage I melanosomes. Interestingly, glial fibrillary acidic protein, which is a marker for glial cells, is also positive in NCCmelb4M5 cells, while NCCmelb4 cells are negative for this protein. Immunostaining and a cell ELISA assay revealed that 12-O-tetradecanoylphorbol 13-acetate (TPA) and cholera toxin (CT) induce Kit expression in NCCmelb4M5 cells. Real-time polymerase chain reaction analysis also demonstrated the induction of Kit mRNA by TPA and CT. Microphthalmia-associated transcription factor mRNA is simultaneously enhanced by the same treatment. Kit induced by TPA/CT in NCCmelb4M5 cells disappeared after the cells were subcultured and incubated without TPA/CT. These findings show that NCCmelb4M5 cells have the potential to differentiate into Kit-positive melanocyte precursors and may be useful to study mechanisms of development and differentiation of melanocytes in mouse neural crest cells.

MART-1 is required for the function of the melanosomal matrix protein PMEL17/GP100 and the maturation of melanosomes.

More than 125 genes that regulate pigmentation have been identified to date. Of those, MART-1 has been widely studied as a melanoma-specific antigen and as a melanosome-specific marker. Whereas the functions of other melanosomal proteins, such as tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, and Pmel17, are known, the function of MART-1 in melanogenesis, is unclear. A role for MART-1 in pigmentation is expected because its expression pattern and subcellular distribution is quite similar to the other melanosomal proteins and usually correlates with melanin content. We investigated the function of MART-1 using a multidisciplinary approach, including the use of siRNA to inhibit MART-1 function and the use of transfection to re-express MART-1 in MART-1-negative cells. We show that MART-1 forms a complex with Pmel17 and affects its expression, stability, trafficking, and the processing which is required for melanosome structure and maturation. We conclude that MART-1 is indispensable for Pmel17 function and thus plays an important role in regulating mammalian pigmentation.

Bcl-2 reduced and fas activated by the inhibition of stem cell factor/KIT signaling in murine melanocyte precursors.

Stem cell factor (SCF) and its receptor, KIT, are essential to the migration and differentiation of melanocytes during embryogenesis. We previously demonstrated that apoptosis is induced by blocking survival function of the SCF/KIT interaction in a mouse neural crest cell (NCC) primary culture. Using the NCCmelb4 cell line, we investigated the occurrence of apoptosis in the cultured cells when KIT receptors were blocked by the monoclonal anti-KIT antibody (ACK2). Apoptosis following treatment with ACK2 was detected by DNA fragmentation assay, in situ apoptosis detection, and electron microscopy. We noted a decrease in extracellular signal-related kinase (ERK) and ribosomal S6 kinase (RSK) protein expression following ACK2 incubation. Western blot analysis and real-time quantitative RT-PCR revealed an apparent time-dependent reduction in Bcl-2 protein levels with respect to ACK2 within the NCCmelb4 cells. In terms of Bax expression, a difference was not found. Fas and caspase8 proteins increased time-dependently in proportion to ACK2 incubation. We noted apoptotic cell death upon addition of ACK2, with evidence of possible involvement of Bcl-2 and Fas in the induction of apoptosis. In contrast, no significant correlation between Fas ligand (Fas-L) expression and ACK2 was found. Fas activation appears to occur independent of Fas-L during ACK2-induced cell death. Therefore, we propose that Fas-L expression in NCCmelb4 cells does not play a major role in facilitating apoptosis. Furthermore, we hypothesize that these molecules combined with SCF/KIT play an important role in regulating the induction of vertebrate NCC apoptosis during embryogenesis.

Isolation of melanosomes.

The methods used to purify early and late melanosomes are detailed. These methods include the use of highly pigmented cells to maximize recovery and the use of various sucrose density gradients to separate melanosome fractions based on their density (which is determined in large part by the amount of dense melanin pigment that they contain). Early melanosomes lacking pigment must be further purified using free-flow electrophoresis.

Establishment by an original single-cell cloning method and characterization of an immortal mouse melanoblast cell line (NCCmelb4).

We devised a unique new single-cell cloning method which uses microscope cover glasses and established a melanoblast cell line derived from mouse neural crest cells. A microscope cover glass was nicked and broken into small pieces and put on a dish. Culture medium and a suspension of 20-30 cells/ml were dropped in the dish. After 1-3 d, a piece of glass to which only one cell was adhered was picked up and transferred to another dish containing culture medium. The greatest advantage of this method is that the derivation of a colony from a single cell can be directly confirmed by microscopy and there is no risk of migratory cells being contaminated by other colonies. Using this single-cell cloning method, in this study we established a cell line derived from a neural crest cell line (NCC-S4.1) and designated it as NCCmelb4. When the culture medium was supplemented with stem cell factor (SCF) alone, NCCmelb4 cells were KIT-positive and tyrosinase-negative melanocyte precursors; they remained at an immature and undifferentiated stage. When the medium was supplemented with phorbol 12-o-tetradecanoyl-13-acetate (TPA) + cholera toxin (CT), the cell morphology changed and became L-3,4-dihydroxyphenylalanine (DOPA)-positive. This observation indicates that the NCCmelb4 cells are capable of further differentiation with suitable stimulation. NCCmelb4 cells derived from the mouse neural crest has characteristics of melanocyte precursors (melanoblasts), and is a cell line which can be utilized to study differentiation-inducing factors and growth factors without the effects of feeder cells.

Mesenchymal-epithelial interactions in the skin: increased expression of dickkopf1 by palmoplantar fibroblasts inhibits melanocyte growth and differentiation.

We investigated whether or not the topographic regulation of melanocyte differentiation is determined by mesenchymal-epithelial interactions via fibroblast-derived factors. The melanocyte density in palmoplantar human skin (i.e., skin on the palms and the soles) is five times lower than that found in nonpalmoplantar sites. Palmoplantar fibroblasts significantly suppressed the growth and pigmentation of melanocytes compared with nonpalmoplantar fibroblasts. Using cDNA microarray analysis, fibroblasts derived from palmoplantar skin expressed high levels of dickkopf 1 (DKK1; an inhibitor of the canonical Wnt signaling pathway), whereas nonpalmoplantar fibroblasts expressed higher levels of DKK3. Transfection studies revealed that DKK1 decreased melanocyte function, probably through beta-catenin-mediated regulation of microphthalmia-associated transcription factor activity, which in turn modulates the growth and differentiation of melanocytes. Thus, our results provide a basis to explain why skin on the palms and the soles is generally hypopigmented compared with other areas of the body, and might explain why melanocytes stop migrating in the palmoplantar area during human embryogenesis.

Epitope mapping of the melanosomal matrix protein gp100 (PMEL17): rapid processing in the endoplasmic reticulum and glycosylation in the early Golgi compartment.

Melanosomes, specific organelles produced only by melanocytes, undergo a unique maturation process that involves their transition form amorphous rounded vesicles to fibrillar ellipsoid organelles, during which they move from the perinuclear to the distal areas of the cells. This depends upon the trafficking and processing of gp100 (also known as Pmel17 and the silver protein), a protein of great interest, because it elicits immune responses in melanoma patients but in which specific function(s) remains elusive. In this study, we have used biochemical and immunochemical approaches to more critically assess the synthesis, processing, glycosylation, and trafficking of gp100. We now report that gp100 is processed and sorted in a manner distinct from other melanosomal proteins (such as tyrosinase, Tyrp1 and Dct) and is predominantly delivered directly to immature melanosomes following its rapid processing in the endoplasmic reticulum and cis-Golgi. Following its arrival, gp100 is cleaved at the amino and at the carboxyl termini in a series of specific steps that result in the reorganization of immature melanosomes to the fibrillar mature melanosomes. Once this structural reorganization occurs, melanogenic enzymes begin to be targeted to the melanosomes, which are then competent to synthesize melanin pigment.

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.

Fatty acids regulate pigmentation via proteasomal degradation of tyrosinase: a new aspect of ubiquitin-proteasome function.

Fatty acids are common components of biological membranes that are known to play important roles in intracellular signaling. We report here a novel mechanism by which fatty acids regulate the degradation of tyrosinase, a critical enzyme associated with melanin biosynthesis in melanocytes and melanoma cells. Linoleic acid (unsaturated fatty acid, C18:2) accelerated the spontaneous degradation of tyrosinase, whereas palmitic acid (saturated fatty acid, C16:0) retarded the proteolysis. The linoleic acid-induced acceleration of tyrosinase degradation could be abrogated by inhibitors of proteasomes, the multicatalytic proteinase complexes that selectively degrade intracellular ubiquitinated proteins. Linoleic acid increased the ubiquitination of many cellular proteins, whereas palmitic acid decreased such ubiquitination, as compared with untreated controls, when a proteasome inhibitor was used to stabilize ubiquitinated proteins. Immunoprecipitation analysis also revealed that treatment with fatty acids modulated the ubiquitination of tyrosinase, i.e. linoleic acid increased the amount of ubiquitinated tyrosinase whereas, in contrast, palmitic acid decreased it. Furthermore, confocal immunomicroscopy showed that the colocalization of ubiquitin and tyrosinase was facilitated by linoleic acid and diminished by palmitic acid. Taken together, these data support the view that fatty acids regulate the ubiquitination of tyrosinase and are responsible for modulating the proteasomal degradation of tyrosinase. In broader terms, the function of the ubiquitin-proteasome pathway might be regulated physiologically, at least in part, by fatty acids within cellular membranes.