Human placental lipid induces melanogenesis through p38 MAPK in B16F10 mouse melanoma

Melanogenesis is one of the characteristic functional activities of melanocyte/melanoma and is regulated via mitogen-activated protein kinase (MAPK) and Akt/protein kinase B (PKB) pathways. Placental total lipid fraction (PTLF), prepared from a hydroalcoholic extract of fresh term human placenta contains sphingolipids and was recently shown to stimulate melanogenesis via up-regulation of the key enzyme tyrosinase in B16F10 mouse melanoma cells. How such lipids mediate their effects on pigmentation and tyrosinase expression is a particularly important aspect of melanogenesis. To study the signaling that leads to tyrosinase expression, we have investigated the roles of the MAPK and Akt/PKB pathways in B16F10 melanoma cells in melanogenesis in response to PTLF. Treatment of cells with PTLF led to the time dependent phosphorylation of p38 MAPK. SB203580, a p38 MAPK inhibitor, completely blocked the PTLF-induced melanogenesis by inhibiting promoter activity and subsequent expression of tyrosinase. Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 a blocker of the Akt signaling pathway, or an inhibitor of MEK (MAPK/ERK Kinase), PD98059 when included along with PTLF was found to potentiate PTLF-induced phosphorylation of p38 MAPK together with tyrosinase expression and melanogenesis. The results suggest that the activation of p38 MAPK plays a crucial role in PTLF-induced B16F10 melanogenesis by up-regulating tyrosinase expression.     

p38 Regulates Pigmentation via Proteasomal Degradation of Tyrosinase

The synthesis of melanin pigments, or melanogenesis, is regulated by the balance of a variety of signal transduction pathways. Among these pathways, p38 MAPK signaling was found to be involved in stress-induced melanogenesis and to be activated by α-melanocyte-stimulating hormone (α-MSH) and ultraviolet irradiation. Previous studies have shown that α-MSH-stimulated melanogenesis can be inhibited by blocking p38 MAPK activity with SB203580, a pyridinyl imidazole compound. Consistent with this, we observed that pyridinyl imidazoles (SB203580 and SB202190) inhibited both basal and α-MSH-induced melanogenesis in B16 melanoma cells. However, SB202474, which has no ability to inhibit p38 MAPK activity and is usually used as a negative control compound in p38 MAPK studies, also suppressed melanin synthesis induction. Furthermore, the independence of the p38 kinase pathway from the repression of melanogenesis by pyridinyl imidazole compounds was also confirmed by small interfering RNA experiments. Interfering with p38 MAPK expression surprisingly stimulated melanogenesis and tyrosinase family protein expression. Although the molecular mechanism(s) by which p38 promotes the degradation of melanogenic enzymes remain to be determined, the involvement of the ubiquitin-proteasome pathway was demonstrated by co-treatment with the proteasome-specific inhibitor MG132 and the relative decrease in the ubiquitination of tyrosinase in cells transfected with p38-specific small interfering RNA.     

Stimulation of melanogenesis in murine melanoma cells by 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB)

The effects of 2-mercapto-1-(beta-4-pyridethyl) benzimidazole (MPB), one of the benzimidazole derivatives designed for a nucleic acid analogue, on melanogenesis of murine B16-F10 melanoma cell lines were investigated. MPB (40 microM) induced a striking dendricity in B16 melanoma cells within 12 h and maximal dendricity between 48 and 72 h. The stimulation of melanin synthesis was observed after only 2 days of treatment together with a dose-dependent growth inhibition. Moreover, MPB increased the activity of tyrosinase through the expression of tyrosinase mRNA without increasing the intracellular cyclic AMP content. MPB-induced melanogenesis was inhibited by novel protein kinase A inhibitors, KT-5720 and H-85. These findings indicate that MPB stimulated B16 cells to terminally differentiate and may be a useful drug in studying the regulation of melanogenesis.     

Involvement of calpain in melanogenesis of mouse B16 melanoma cells

In the current study, the involvement of calpain, a cysteine proteinase in the regulation of melanogenesis was examined using mouse B16 melanoma cells. In response to α-melanocyte-stimulating hormone (α-MSH), B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. The total calapain activity was decreased within 2 h following α-MSH-treatment, and restored to the initial level in 6–12 h. To further investigate the involvement of calpain in the regulation of melanogenesis, the effect of calpain inhibitors on α-MSH-induced melanogenesis was examined. Inhibition of calpain by either N-acetyl-Leu-Leu-norleucinal (ALLN) or calpastatin (CS) peptide blocked α-MSH-induced melanogenesis. The magnitude of inhibition of melanin biosynthesis was well correlated with a decrease in the activity of tyrosinase, a key regulatory enzyme in melanogenesis. Treatment of B16 cells with ALLN caused marked decrease in both tyrosinase protein and mRNA levels. These results indicate that calpain would be involved in the melanogenic signaling by modulating the expression of tyrosinase in mouse B16melanoma cells.     

Regulation of melanogenesis in B16 mouse melanoma cells by protein kinase C

Melanogenesis is regulated by a variety of environmental and hormonal factors. In this study, we showed that protein kinase C (PKC) plays a major role in regulating melanogenesis in B16 mouse melanoma cells. Chronic treatment of B16 cells with phorbol dibutyrate resulted in a concentration-dependent loss of density-dependent induction of tyrosinase activity, which correlated positively with a concentration-dependent loss of PKC enzyme activity. In contrast, B16 clones overexpressing PKCα had increased tyrosinase activity. Different phorbol derivatives inhibited tyrosinase activity and depleted cellular PKCα in a manner that reflected their reported tumor-promoting activity. Western blotting analysis showed that phorbol dibutyrate decreased the amount of the brown locus gene product (TRP-1) by 50% and lowered the amount of the albino locus gene product (tyrosinase) to undetectable levels. None of the phorbol derivatives affected the level of the slaty locus protein (TRP-2). The decrease in tyrosinase and TRP-1 protein levels was found to be due to a decrease in the mRNA encoded by these genes. In addition to inhibiting the density-dependent increase in tyrosinase activity, phorbol dibutyrate inhibited some, but not all, of the 8-bromocyclic AMP-induced increase in tyrosinase activity. This was accompanied by a decrease in the amount of tyrosinase protein induced by 8-bromocyclic AMP. Although 8-bromocyclic AMP did not change the level of TRP-1, it did reverse the decrease in the amount of this protein induced by phorbol dibutyrate. The amount of TRP-2 was not altered by any of these agents. These data suggest that PKC regulates melanogenesis primarily by controlling the constitutive expression of tyrosinase and, to a lesser extent, TRP-1. © 1996 Wiley-Liss, Inc.     

Melanogenesis stimulation in B16-F10 melanoma cells induces cell cycle alterations, increased ROS levels and a differential expression of proteins as revealed by proteomic analysis

Considering that stimulation of melanogenesis may lead to alterations of cellular responses, besides melanin production, our main goal was to study the cellular effects of melanogenesis stimulation of B16-F10 melanoma cells. Our results show increased levels of the reactive oxygen species after 15 h of melanogenesis stimulation. Following 48 h of melanogenesis stimulation, proliferation was inhibited (by induction of cell cycle arrest in the G1 phase) and the expression levels of p21 mRNA were increased. In addition, melanogenesis stimulation did not induce cellular senescence. Proteomic analysis demonstrated the involvement of proteins from other pathways besides those related to the cell cycle, including protein disulfide isomerase A3, heat-shock protein 70, and fructose biphosphate aldolase A (all up-regulated), and lactate dehydrogenase (down-regulated). In RT-qPCR experiments, the levels of pyruvate kinase M2 mRNA dropped, whereas the levels of ATP synthase (beta-F1) mRNA increased. These data indicate that melanogenesis stimulation of B16-F10 cells leads to alterations in metabolism and cell cycle progression that may contribute to an induction of cell quiescence, which may provide a mechanism of resistance against cellular injury promoted by melanin synthesis.     

Involvement of phospholipase D1 in melanogenesis of mouse B16 melanoma cells

In response to alpha-melanocyte-stimulating hormone (alpha-MSH) or cAMP-elevating agents (forskolin and isobutylmethylxanthine), mouse B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. However, the mechanism(s) underlying the regulation of melanogenesis during differentiation has not yet been clearly understood. Phospholipase D (PLD) has been reported to be involved in differentiation. This enzyme cleaves phosphatidylcholine upon stimulation with stimuli to generate phosphatidic acid. In the current study, the involvement of PLD in the regulation of melanogenesis characteristic of differentiation was examined using mouse B16 melanoma cells. Treatment of B16 cells with alpha-MSH was found to cause marked decreases in the PLD1 activity concurrent with its reduced protein level. Moreover, treatment of exogenous bacterial PLD also inhibited alpha-MSH-induced melanogenesis. To further investigate the role of PLD1 in the regulation of melanogenesis, we examined the effects of overexpression of PLD1 on melanogenesis in B16 melanoma cells. The B16 cells overexpressing PLD were prepared by transfection with the vector containing the cDNA encoding PLD1. The melanin contents in PLD1-overexpressing cells (B16/PLD1) were observed to be lower compared with those in the vector control cells (B16/Vec), concomitant with the decreases in both activity and protein level of tyrosinase, a key regulatory enzyme in melanogenesis. Moreover, overexpression of PLD1 resulted in a marked inhibition of melanogenesis induced by alpha-MSH. The inhibition of melanogenesis was well correlated with the decrease in the tyrosinase activity associated with its expression. These results indicated that PLD1 negatively regulated the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells.     

Melanogenesis inhibition by an oolong tea extract in b16 mouse melanoma cells and UV-induced skin pigmentation in brownish guinea pigs

To investigate the new physiological functions of oolong tea, the effects on melanogenesis were studied. An oolong tea extract inhibited melanogenesis without affecting cell growth in B16 mouse melanoma cells. However, the oolong tea extract hardly showed any inhibitory effect on mushroom tyrosinase in a cell-free system. The effects of an oolong tea extract on the intracellular tyrosinase level in B16 cells were therefore studied. All the levels of activity, protein and mRNA were decreased in the oolong tea extract-treated cells. We also investigated the inhibitory effects of oolong tea on the pigmentation induced by ultraviolet B (UVB) by using brownish guinea pigs in vivo. The number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes increased by UVB was repressed by an oral administration of oolong tea. These results imply that oolong tea might be effective in whitening and that its inhibitory effect on melanogenesis was involved in the decrease of intracellular tyrosinase at the mRNA level.