Stimulation of Melanoblast Pigmentation by 8-Methoxypsoralen:The Involvement of Microphthalmia-Associated Transcription Factor, the Protein Kinase A Signal Pathway, and Proteasome-Mediated Degradation

Abstract

In this study, we used melb-a melanoblasts as a model to study mechanisms involved in stimulating melanocyte function in vitiliginous skin following exposure to 8-methoxypsoralen (8MOP). Melanin content and tyrosinase activity increased 3- and 7-fold, respectively, in melanoblasts treated with 8MOP for 6 d compared with untreated controls. The intracellular signal pathways involved in 8MOP-induced effects on melanoblasts were investigated, particularly the roles of protein kinase A and protein kinase C. Forskolin, a protein kinase A activator, mimicked and enhanced the 8MOP stimulation of melanoblast pigmentation whereas a protein kinase C activator, 1-oleoyl-2-acetylglycerol, had no effect, indicating that the protein kinase A pathway is involved rather than the protein kinase C pathway. Those observations were confirmed using inhibitors of the protein kinase A or protein kinase C pathways. Western blot and semiquantitative reverse transcriptase polymerase chain reaction were performed to assess the protein and mRNA expression levels of microphthalmia-associated transcription factor and tyrosinase in melanoblasts treated with 8MOP for 3 h, 6 h, 1 d, 3 d, or 6 d. Incubation with 8MOP stimulated microphthalmia-associated transcription factor protein and mRNA levels within 3 h, but, in contrast, tyrosinase mRNA and protein levels did not increase following 8MOP treatment until 1 d after treatment. The proteasome inhibitor lactacystin blocked the proteasome-mediated proteolysis of tyrosinase, and its effect on proteasomal function was enhanced by 8MOP. Taken together, these results show that 8MOP functions by initially stimulating levels of microphthalmia-associated transcription factor expression via activation of the protein kinase A pathway, which thereby stimulates tyrosinase expression and function and eventually leads to dramatic increases in melanin production by melanoblasts.