Pigment content of cultured human melanocytes does not correlate with tyrosinase message level

Abstract

Tyrosinase is considered to be the rate-limiting enzyme for the biosynthesis of melanin in epidermal melanocytes, and thus tyrosinase activity is thought to be a major regulatory step in melanogenesis. To determine whether the rate of pigment production was controlled at the level of tyrosinase gene expression, we developed a culture system capable of generating large populations of pure human melanocytes and then measured both melanin content as determined spectrophotometrically by absorption at 475 nm and mRNA levels as detected by hybridization with cloned cDNA Pmel 34, encoding human tyrosinase. We examined the relationship between pigment content and tyrosinase mRNA levels among human melanoma and melanocyte lines with very different levels of basal pigmentation; between two clones of a single human melanoma line, one pigmented and one amelanotic; and sequentially in melanocytes before and after simulation with isobutylmethylxanthine to increase melanin content per cell. Using Northern blot analysis and in-situ hybridization we found no correlation between tyrosinase message levels and melanin content, suggesting that posttranscriptional regulation of tyrosinase and/or other events determine the rate of pigment synthesis in human melanocytes.

Protein Kinase C-β Activates Tyrosinase by Phosphorylating Serine Residues in Its Cytoplasmic Domain

“…For example, after several negative or ambiguous studies, it was finally reported that under defined culture conditions, ␣-MSH can induce tyrosinase mRNA, protein, and activity in human melanocytes (44,45), but its overall contribution to regulation of human melanogenesis is appears nevertheless to be far less than in the murine system. Importantly, posttranslational modification appears to play a more critical role in regulating tyrosinase activity in human than in murine melanocytes, with mRNA and protein levels poorly correlated with the enzyme activity (19,20), and amelanotic human melanoma cells often expressing ample tyrosinase protein (21,22).…”

“…Primary cultures were then established in medium 199 containing 1.8 mM Ca ϩ2 and supplemented with 10 Ϫ9 M triiodothyronine, 10 g/ml insulin, 1.4 ϫ 10 Ϫ6 M hydrocortisone, 10 Ϫ9 M choleragen, 10 ng/ml basic fibroblast growth factor, and 5-10% fetal bovine serum. All postprimary cultures were maintained in a low calcium (0.03 mM) version of this medium known to selectively support melanocyte growth (19). Cells at first to third passages were routinely used.…”

“…Cells at first to third passages were routinely used. The MM4 human melanoma cells were obtained from Dr. U. Stierner (Gothenburg, Sweden) and maintained in DMEM (55%), L15 (27%), fetal bovine serum (15%), nonessential amino acids, 1% glutamine (2 mM), and insulin (10 g/ml) (19).…”

“…Unlike murine melanoma cells, the regulation of tyrosinase activity appears to occur mostly at the posttranslational level. For example, the level of tyrosinase mRNA does not correlate with the total melanin level in cultured human melanocytes (19,20), and human melanoma cells often fail to produce melanin despite having abundant tyrosinase protein (21,22). However, the posttranslational mechanism by which tyrosinase activity is putatively regulated is not well understood.…”

Stopped-flow Fluorescence Studies of Inhibitor Binding to Tyrosinase from Streptomyces antibioticus

“…Physiological Relevance of the Halide Inhibition-It has been proposed that the large variations in the extent of pigmentation in different human skin types originate from variations in the melanosomal pH (63)(64)(65). These pH variations would influence the Ty activity, a lower pH value being associated with a lower Ty activity and, thus, with a lower pigmentation level.…”