Co-regulation of melanin precursors and tyrosinase in human pigment cells: roles of cysteine and glutathione.

Glutathione (GSH) and cysteine (CysH) have both been implicated in the biogenesis of the pheomelanin precursor 5-S-cysteinyldopa (5-S-CD). However, recent studies have shown that only CysH is transported across the membrane of isolated melanosomes, and that the positive regulation of CysH in pigment cells leads to an increased production of 5-S-CD. In the present study, the question was examined as to whether melanin precursors and tyrosinase could be coregulated by cellular thiols. To address this issue, the levels of CysH and GSH were varied in normal melanocytes and melanoma cells using buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis. Treatment with 50-100 microM BSO decreased GSH levels to less than 10% of control, and increased CysH levels between two- and five-fold in both cell types. Concomitant with this, an increase in the ratio of 5-S-CD to DOPA and a decrease in the pigment content of the cells were observed. The decrease in cell pigmentation was associated with strong decreases in tyrosine hydroxylase activity and 14C-melanin production. Only melanoma cells showed a modified tyrosinase isozyme pattern on Western immunoblots in response to BSO, while the mRNA expression of tyrosinase and TRP-1 were unchanged in both cell types. These results suggest that the balance between CysH and GSH, which is partly determined by the rate of utilization of CysH for GSH biosynthesis, regulates not only the levels of 5-S-CD and DOPA but also the melanogenic activity of pigment cells. Since DOPA functions as a cofactor in the monophenolase reaction of tyrosinase, it is proposed that the ratio of 5-S-CD to DOPA may be an important factor in the regulation of tyrosinase activity in situ

Sphingosylphosphorylcholine-induced ERK activation inhibits melanin synthesis in human melanocytes

Sphingosylphosphorylcholine (SPC) is emerging as a potent signaling-lipid mediator. In this study, we investigated the effects of SPC on melanogenesis using cultured human melanocytes. Our results show that SPC significantly inhibits melanin synthesis in a concentration-dependent manner, and further that it reduces the activity of tyrosinase, the rate-limiting melanogenic enzyme. SPC treatment was also found to induce short-thick dendrites in human melanocytes, but not to reduce tyrosinase activity in a cell-free system, whereas kojic acid directly inhibited tyrosinase. These results suggest that SPC reduces pigmentation by indirectly regulating tyrosinase. In further experiments, SPC was found to downregulate microphthalmia-associated transcription factor (MITF) and tyrosinase, and Western blotting showed that SPC induces the activations of extracellular signal-regulated kinase (ERK) and 90 kDa ribosomal S6 kinase (RSK-1). Moreover, the specific ERK pathway inhibitor, PD98059, blocked the hypopigmentation effect of SPC, and abrogated the SPC-mediated downregulation of MITF. These results suggest that the ERK pathway is involved in the melanogenic signaling cascade, and that ERK activation by SPC reduces melanin synthesis via MITF downregulation

Transcriptional activation of tyrosinase gene by human placental sphingolipid

The sphingolipids, a class of complex bioactive lipids, are involved in diverse cellular functions such as proliferation, differentiation, and apoptosis as well as growth inhibition. Recently sphingosylphosphorylcholine (SPC), sphingosine-1-phosphate (S1P), and C2-ceramide (C2-Cer), sphingolipid containing acetic acid are emerging as melanogenic regulators. A bioactive sphingolipid (PSL) was isolated from hydroalcoholic extract of fresh term human placenta and it induced melanogenesis in an in vitro culture of mouse melanoma B16F10 cells. Tyrosinase, the rate-limiting enzyme for melanogenesis, is required to be upregulated for the increased melanin production. The expression of tyrosinase, both at protein as well as mRNA level, was higher in the PSL treated B16F10 cells as evidenced by Western blot and RT-PCR analysis. Actinomycin D and cycloheximide, inhibitors of transcription and translation, respectively, inhibited PSL-induced tyrosinase activity and its protein expression showing decrease in melanogenesis, correspondingly. The activity of GFP coupled tyrosinase promoter was upregulated in transfected B16F10 cells after treating with PSL as determined by fluorescence microscopy, fluorometric analysis, and Western blot. These results, thus, suggested that PSL upregulated tyrosinase gene expression at transcription level through promoter activation to show increased melanogenesis. Therefore, PSL as an inducer of melanogenesis might account for the recovery of pigment in depigmentation disorder