Quantification of tyrosinase, TRP-1, and TRP-2 transcripts in human melanocytes by reverse transcriptase-competitive multiplex PCR - Regulation by steroid hormones

Abstract

We have introduced a reverse transcriptase polymerase chain reaction based method to measure mRNA levels of the melanogenesis enzymes tyrosinase, tyrosinase-related-protein 1 (TRP-1), and tyrosinase-related-protein 2 (TRP- 2). Expression was determined by reverse transcriptase-competitive multiplex polymerase chain reaction of (i) melanogenesis enzyme transcripts and the 'housekeeping' gene glyceraldehyde-3-phosphate dehydrogenase, and (ii) two internal standards consisting of mutated melanogenesis enzyme cDNA and mutated gene glyceraldehyde-3-phosphate dehydrogenase cDNA. This was investigated on in vitro cultured melanocytes in the presence of three different steroids; one glucocorticoid (betamethasone-17-valerate) and two sex steroids (diethylstilbestrol and estradiol). All three steroids lead to an increase of about 1.5-2.5-fold of tyrosinase transcripts. The amount of TRP-1 transcripts was likewise enhanced, but only moderately (≃1.5-fold). In contrast, TRP-2 transcripts were reduced by ≃40% in number after betamethasone-17-valerate treatment, whereas the two sex steroids, diethylstilbestrol and estradiol, caused an upregulation of about 20-fold of the initial TRP-2 transcript level. We therefore suggest that hyperpigrnentation during pregnancy or under contraceptive treatment is mediated by a direct induction of melanogenesis via sex steroids.