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.

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.

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 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.