Human placental lipid induces melanogenesis by increasing the expression of tyrosinase and its related proteins in vitro

Abstract

Lipids, particularly sphingolipids, are emerging as novel regulators of cellular activity. A placental total lipid fraction (PTLF), the total lipid prepared from an hydroalcoholic extract of fresh term human placenta, was previously shown to have a pigment-inducing activity in an animal model. The PTLF contains sphingolipids which stimulate DNA synthesis and melanin formation with marked morphological changes in B16F10 melanoma cells. In order to identify the mechanism underlying the increased melanin synthesis, B16F10 cells were treated with PTLF to assess the catalytic activities of tyrosinase (i.e. tyrosine hydroxylase and DOPA oxidase), the key regulatory enzyme of melanin synthesis. Tyrosine hydroxylase (estimated by the release of (3)H(2)O) as well as DOPA oxidase (measured spectrophotometrically and also in non-denaturing gels), was stimulated significantly by PTLF. Western blot analysis demonstrated an increase in the expression of tyrosinase, tyrosinase related proteins 1 and 2 (TRP1 and TRP2) at the protein level and RT-PCR analysis revealed stimulated transcription of tyrosinase, TRP1 and TRP2 mRNAs in PTLF-treated B16F10 cells. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PTLF-induction of tyrosinase activity with a corresponding decrease in melanogenesis. In all cases, the response to PTLF was similar to that induced by alpha-melanocyte stimulating hormone, a well-known stimulator of melanogenesis. Thus, these results provide the basis of action of PTLF stimulated melanogenesis in B16F10 cells showing that this placental extract is a strong inducer of pigmentation at the transcriptional and translational levels.