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

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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.