Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells

Melanogenesis is a principal parameter of differentiation in melanocytes and melanoma cells. Our recent study has demonstrated that phospholipase D1 (PLD1) regulates the melanogenic signaling through modulating the expression of tyrosinase, the rate-limiting step enzyme in the melanin biosynthesis. The current study was designed to gain more insight into the involvement of PLD1 in the regulation of melanogenesis. To investigate the role of PLD1, we examined the effect of knockdown of endogenous PLD1 by small interference RNA (siRNA) on melanogenesis in B16 melanoma cells. It was shown that the melanin synthesis was induced in PLD1-knockdowned cells, and also that the level of melanin synthesis was well correlated with increases in expression level of tyrosinase and its related proteins (Tyrp1 and Dct). Furthermore, the reduction of expression levels of PLD1 by siRNA transfection was accompanied by diminution of ribosomal S6 kinase 1 (S6K1) phosphorylation. The activity of mammalian target of rapamycin (mTOR) is essential for phosphorylation of S6K1 and the treatment malanoma cells with rapamycin, a potent inhibitor of mTOR effectively induced melanogenesis. The results obtained here provide possible evidence that PLD1 exerts a negative regulatory role in the melanogenic process through mTOR/S6K1 signaling.     

Mutant Alleles at the Brown Locus Encoding Tyrosinase‐Related Protein‐1 (TRP‐1) Affect Proliferation of Mouse Melanocytes in Culture *

Tyrosinase related protein‐1 (TRP‐1) is a melanocyte‐specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP‐1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1^b (brown) and Tyrp1^b‐cj (cordovan) compared with wild type Tyrp1^B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP‐1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP‐1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

Mutant alleles at the brown locus encoding tyrosinase-related protein-1 (TRP-1) affect proliferation of mouse melanocytes in culture

Tyrosinase related protein-1 (TRP-1) is a melanocyte-specific gene product involved in eumelanin synthesis. Mutation in the Tyrp1 gene is associated with brown pelage in mouse and oculocutaneous albinism Type 3 in humans (OCA3). It has been demonstrated that TRP-1 expresses DHICA oxidase activity in the murine system. However, its actual function in the human system is still unclear. The study was designed to determine the effects of mutation at two Typr1 alleles, namely the Tyrp1b (brown) and Tyrp1b-cj (cordovan) compared with wild type Tyrp1B (black) on melanocyte function and melanin biosynthesis. The most significant finding was that both of the Tyrp1 mutations (i.e. brown expressing a point mutation and cordovan expressing decreased amount of TRP-1 protein) resulted in attenuation of cell proliferation rates. Neither necrosis nor apoptosis was responsible for the observed decrease in cell proliferation rates of the brown and cordovan melanocytes. Ultrastructural evaluation by electron microscopic analysis revealed that both mutations in Tyrp1 affected melanosome maturation without affecting its structure. These observations demonstrate that mutation in Tyrp1 compromised tyrosinase activity within the organelle. DOPA histochemistry revealed differences in melanosomal stages between black and brown melanocytes but not between black and cordovan melanocytes. There were no significant differences in tyrosine hydroxylase activities of tyrosinase and TRP-1 in wild type black, brown and cordovan melanocyte cell lysates. We conclude that mutations in Tyrp1 compromise cell proliferation and melanosomal maturation in mouse melanocyte cultures.     

Degradation of tyrosinase induced by phenylthiourea occurs following Golgi maturation

Tyrosinase, the rate-limiting enzyme of melanin synthesis, is a di-copper metalloprotein that catalyzes the conversion of L-tyrosine to L-DOPAquinone. Phenylthiourea (PTU) is a well-known inhibitor of tyrosinase and melanin synthesis and is known to interact with sweet potato catechol oxidase, an enzyme possessing copper binding domain homology to tyrosinase. While PTU is frequently used to induce hypopigmentation in biological systems, little is known about its effects on tyrosinase and other melanogenic proteins. We have found that PTU induces degradation of tyrosinase but not of other melanogenic proteins including the tyrosinase-related metalloproteins tyrosinase-related protein (Tyrp)1 and Tyrp2. Using pulse-chase analysis coupled with glycosidase digestion, we observed that tyrosinase degradation occurs following complete maturation of the protein and that degradation was reversed by cysteine protease inhibitor E64 but not proteasome inhibitor N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. We conclude that PTU specifically induces tyrosinse degradation following Golgi maturation. Our data suggest that in addition to well-known ER-directed quality control, tyrosinase is also subject to post-Golgi quality control.     

Isolation and developmental expression of tyrosinase family genes in Xenopus laevis

The tyrosinase family of genes in vertebrates consists of three related members encoding melanogenic enzymes, tyrosinase (Tyr), tyrosinase-related protein-1 (TRP-1, Tyrp1) and tyrosinase-related protein-2 (Dct, TRP-2, Tyrp2). These proteins catalyze melanin production in pigment cells and play important roles in determining vertebrate coloration. This is the first report examining melanogenic gene expression in pigment cells during embryonic development of amphibians. Xenopus provides a useful experimental system for analyzing molecular mechanisms of pigment cells. However, in this animal little information is available not only about the developmental expression but also about the isolation of pigmentation genes. In this study, we isolated homologues of Tyr, Tyrp1 and Dct in Xenopus laevis (XlTyr, XlTyrp1, and XlDct). We studied their expression during development using in situ hybridization and found that all of them are expressed in neural crest-derived melanophores, most of which migrate through the medial pathway, and in the developing diencephalon-derived retinal pigment epithelium (RPE). Further, XlDct was expressed earlier than XlTyr and XlTyrp1, which suggests that XlDct is the most suitable marker gene for melanin-producing cells among them. XlDct expression was detected in migratory melanoblasts and in the unpigmented RPE. In addition, the expression of XlDct was detected in the pineal organ. The sum of these studies suggests that expression of the tyrosinase family of genes is conserved in pigment cells of amphibians and that using XlDct as a marker gene for pigment cells will allow further study of the developmental mechanisms of pigment cell differentiation using Xenopus.