Analysis of SOX10 function in neural crest-derived melanocyte development: SOX10-dependent transcriptional control of dopachrome tautomerase.

SOX10 is a high-mobility-group transcription factor that plays a critical role in the development of neural crest-derived melanocytes. At E11.5, mouse embryos homozygous for the Sox10(Dom) mutation entirely lack neural crest-derived cells expressing the lineage marker KIT, MITF, or DCT. Moreover, neural crest cell cultures derived from homozygous embryos do not give rise to pigmented cells. In contrast, in Sox10(Dom) heterozygous embryos, melanoblasts expressing KIT and MITF do occur, albeit in reduced numbers, and pigmented cells eventually develop in nearly normal numbers both in culture and in vivo. Intriguingly, however, Sox10(Dom)/+ melanoblasts transiently lack Dct expression both in culture and in vivo, suggesting that during a critical developmental period SOX10 may serve as a transcriptional activator of Dct. Indeed, we found that SOX10 and DCT colocalized in early melanoblasts and that SOX10 is capable of transactivating the Dct promoter in vitro. Our data suggest that during early melanoblast development SOX10 acts as a critical transactivator of Dct, that MITF, on its own, is insufficient to stimulate Dct expression, and that delayed onset of Dct expression is not deleterious to the melanocyte lineage.

Involvement of ITF2 in the transcriptional regulation of melanogenic genes.

In response to agouti signal protein, melanocytes switch from producing eumelanin to pheomelanin concomitant with the down-regulation of melanogenic gene transcription. We previously reported that a ubiquitous basic helix-loop-helix transcription factor, known as ITF2, is up-regulated during this switch, and we now report that treatment of melanocytes with melanocyte-stimulating hormone down-regulates expression of ITF2. To more fully characterize the involvement of ITF2 in regulating melanogenic gene transcription, ITF2 sense or antisense constructs were introduced into melan-a melanocytes. Gene and protein expression analyses and luciferase reporter assays using promoters from melanogenic genes showed that up-regulation of ITF2 suppressed melanogenic gene expression as well as the expression of Mitf, a melanocyte-specific transcription factor. In addition, stable ITF2 sense transfectants had significant reductions in pigmentation and a less dendritic phenotype compared with mock transfectants. In contrast, ITF2 antisense-transfected melanocytes were more pigmented and more dendritic. These results demonstrate that up-regulation of ITF2 during the pheomelanin switch is functionally significant and reveal that differential expression of a ubiquitous basic helix-loop-helix transcription factor can modulate expression of melanogenic genes and the differentiation of melanocytes.

Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF expression by SOX10 and PAX3.

Waardenburg syndrome (WS) is associated with neural crest-derived melanocyte deficiency caused by mutations in either one of three transcription factors: MITF, PAX3, and SOX10. However, the hierarchical relationship of these transcription factors is largely unknown. We show that SOX10 is capable of transactivating the MITF promoter 100-fold, and that this transactivation is further stimulated by PAX3. Promoter deletion and mutational analyses indicate that SOX10 can activate MITF expression through binding to a region that is evolutionarily conserved between the mouse and human MITF promoters. A SOX10 mutant that models C-terminal truncations in WS can reduce wild-type SOX10 induction of MITF, suggesting these mutations may act in a dominant-negative fashion. Our data support a model in which the hypopigmentation in WS, of which these factors have been implicated, results from a disruption in function of the central melanocyte transcription factor MITF.

Cysteine transport in melanosomes from murine melanocytes.

The synthesis of pheomelanin requires the incorporation of thiol-containing compound(s) during the process of mammalian melanogenesis. Since melanins are produced only in specialized, membrane-bound organelles, known as melanosomes, such thiol donor(s) must cross the membrane barrier from the cytosol to the melanosome interior. Cysteine and/or glutathione (GSH) were proposed as suitable thiol donors, although uptake of these compounds into melanosomes was not previously characterized. In this study, we show that cysteine is transported, in a temperature- and concentration-dependent manner, across membranes of melanosomes derived from murine melanocytes. Additional proof that cysteine uptake results from a carrier-mediated process and is not due to simple diffusion or to a membrane channel, was obtained in countertransport experiments, in which melanosomes preloaded with cysteine methyl ester took up significantly more [35S]cysteine than did unloaded controls. In contrast, we were unable to detect any significant uptake of [35S]GSH over a wide concentration range, in the presence or in the absence of reducing agent. This study is the first demonstration of melanosomal membrane transport of cysteine, and it strongly suggests that free cysteine is the thiol source utilized for pheomelanin synthesis in mammalian melanocytes.

Normal tyrosine transport and abnormal tyrosinase routing in pink-eyed dilution melanocytes.

The pink-eyed dilution phenotype in mice arises from mutations in the p gene; in humans, analogous mutations in the P gene result in oculocutaneous albinism type 2. Although the molecular mechanisms which underlie this phenotype remain obscure, it has been postulated that mutations in p result in defective tyrosine transport into murine melanosomes, resulting in hypopigmentation and diminished coat color. However, we previously reported no difference in melanosomal tyrosine transport in unpigmented, melanoblast-like pink-eyed dilution (pcp/pcp), and in pigmented (melan-a) murine melanocytes. In this study, we utilized melan-p1 cells, more differentiated pink-eyed dilution (pcp/p25H) melanocytes which can be induced to produce melanin, to characterize the melanogenic lesion(s) more definitively. Uptake of [3H]tyrosine into melan-a melanosomes did not differ significantly from uptake into melanosomes derived from melan-p1 melanocytes, further arguing against its critical role as a tyrosine transporter. Pink-eyed dilution melanocytes incubated in high tyrosine concentrations became extremely pigmented as they became confluent and secreted large amounts of black material into the medium. Total cellular tyrosinase activity in melan-p1 melanocytes was significantly higher than that in melan-a melanocytes (which are wild-type at the p locus), but the localization of tyrosinase to melanosomes was impaired in melan-p1 melanocytes compared to melan-a melanocytes. These results indicate that mechanisms other than deficient tyrosine transport are involved in the pink-eyed dilution phenotype and that this protein may serve a chaperone-like or stabilizing function in melanocytes.

Tyrosine transport into melanosomes is increased following stimulation of melanocyte differentiation.

A variety of physiological factors can stimulate differentiation of melanocytes to increase pigmentation, and critical to this process is the transport of the melanogenic substrate (tyrosine) into melanosomes. In this study, we examined whether stimulation of melanogenesis affects melanosomal tyrosine transport. Tyrosine uptake increased almost 2-fold in melanosomes derived from melanocytes treated with melanocyte-stimulating hormone (MSH), which acts to increase intracellular cAMP levels, resulting in the up-regulation of many genes involved in melanogenesis. Stimulation of melanoma cells with dibutyryl cAMP increased melanosomal tyrosine transport 2- to 3-fold after 24 to 48 hrs, with peak levels occurring after 3 to 5 days of treatment, suggesting that de novo gene expression may be required. The cAMP-induced increase in melanosomal tyrosine transport could be effectively competed with phenylalanine or tryptophan, but not with dopamine or proline, suggesting either that a pool of transporters with greater tyrosine transporting ability pre-exists, or that a greater number of tyrosine transporters reside within the melanosomal membrane. These results illustrate a rare example of hormonal plasma membrane stimulation which transduces a signal for increased vesicular transport of an amino acid.

Characterization of genes modulated during pheomelanogenesis using differential display.

Molecular and biochemical mechanisms that modulate the production of eumelanin or pheomelanin pigments involve the opposing effects of two intercellular signaling molecules, alpha-melanocyte stimulating hormone (MSH) and agouti signal protein (ASP). ASP is an antagonist of MSH signaling through the melanocyte-specific MSH receptor, although its mechanism(s) of action is controversial. We previously have reported significant down-regulation of all known melanogenic genes during the eumelanin to pheomelanin switch in murine hair follicle melanocytes and in cultured melanocytes treated with recombinant ASP. To identify factors that might be involved in the switch to pheomelanogenesis, we screened ASP-treated melanocytes by using differential display and identified three up-regulated genes: a DNA replication control protein, a basic helix-loop-helix transcription factor, and a novel gene. We have simultaneously identified six down-regulated genes in ASP-treated melanocytes; two of those encode tyrosinase and TRP2, melanogenic genes known to be down-regulated during pheomelanogenesis, which provide good internal controls for this approach. These results suggest that there are complex mechanisms involved in the switch to pheomelanin production, and that these modulated genes might be involved in the pleiotropic changes seen in yellow mice, including the change in coat color.

Characterization of a melanosomal transport system in murine melanocytes mediating entry of the melanogenic substrate tyrosine.

In this study, we identify a transport system for tyrosine, the initial precursor of melanin synthesis, in the melanosomes of murine melanocytes. Melanosomes preloaded with tyrosine demonstrated countertransport of 10 microM [3H]tyrosine, indicating carrier-mediated transport. Melanosomal tyrosine transport was saturable, with an apparent Km for tyrosine transport of 54 microM and a maximal velocity of 15 pmol of tyrosine/unit of hexosaminidase/min. Transport was temperature-dependent (Ea = 7.5 kcal/mol) and showed stereospecificity for the l-isomer of tyrosine. Aromatic, neutral hydrophobic compounds (such as tryptophan and phenylalanine), as well as the small, bulky neutral amino acids (such as leucine, isoleucine, and methionine) competed for tyrosine transport. Tyrosine transport was inhibited by the classical system L analogue, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and by monoiodotyrosine, but not by cystine, lysine, glutamic acid, or 2-(methylamino)-isobutyric acid. Tyrosine transport showed no dependence on Na+ or K+, and did not require an acidic environment or the availability of free thiols. These results demonstrate the existence of a neutral amino acid carrier in murine melanocyte melanosomes which resembles the rat thyroid FRTL-5 lysosomal system h. This transport system is critical to the function of the melanosome since tyrosine is the essential substrate required for the synthesis of the pigment melanin.