Down-regulation of Melanogenesis by Phospholipase D2 through Ubiquitin Proteasome-mediated Degradation of Tyrosinase

The involvement of phospholipase D (PLD) in the regulation of melanogenesis was examined. Treatment of B16 mouse melanoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the activation of PLD and a decrease in melanin content. 1-Butanol, but not 2-butanol, completely blocked the TPA-induced inhibition of melanogenesis, suggesting the involvement of PLD in this event. Reverse transcription-PCR and immunoblot analyses revealed the existence of both PLD isozymes, PLD1 and PLD2, in B16 cells. When PLD1 or PLD2 was introduced into those cells by an adenoviral gene-transfer technique, both PLD1 and PLD2 were activated by TPA. When PLD1 and PLD2 were overexpressed, PLD2 potently caused a decrease in melanin content, whereas the effect of PLD1 expression on melanin content was minimal. Over-expression of PLD2 itself did not affect protein kinase C activity, as assessed by the intracellular distribution and levels of expression of each isoform expressed in B16 cells. The effects of TPA on the down-regulation of basal or α-melanocyte-stimulating hormone-enhanced melanogenesis were almost completely blocked by expressing a lipase activity-negative mutant, LN-PLD2, but not by LN-PLD1. Further, the PLD2-induced decrease in melanin content was accompanied by a decrease in the amount and activity of tyrosinase, a key enzyme in melanogenesis, whereas the mRNA level of tyrosinase was unchanged by the over-expression of PLD2. Moreover, treatment with proteasome inhibitors completely blocked the PLD2-induced down-regulation of melanogenesis. Taken together, the present results indicate that the TPA-induced down-regulation of melanogenesis is mediated by PLD2 but not by PLD1 through the ubiquitin proteasome-mediated degradation of tyrosinase. This suggests that PLD2 may play an important role in regulating pigmentation in vivo. The involvement of phospholipase D (PLD) in the regulation of melanogenesis was examined. Treatment of B16 mouse melanoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in the activation of PLD and a decrease in melanin content. 1-Butanol, but not 2-butanol, completely blocked the TPA-induced inhibition of melanogenesis, suggesting the involvement of PLD in this event. Reverse transcription-PCR and immunoblot analyses revealed the existence of both PLD isozymes, PLD1 and PLD2, in B16 cells. When PLD1 or PLD2 was introduced into those cells by an adenoviral gene-transfer technique, both PLD1 and PLD2 were activated by TPA. When PLD1 and PLD2 were overexpressed, PLD2 potently caused a decrease in melanin content, whereas the effect of PLD1 expression on melanin content was minimal. Over-expression of PLD2 itself did not affect protein kinase C activity, as assessed by the intracellular distribution and levels of expression of each isoform expressed in B16 cells. The effects of TPA on the down-regulation of basal or α-melanocyte-stimulating hormone-enhanced melanogenesis were almost completely blocked by expressing a lipase activity-negative mutant, LN-PLD2, but not by LN-PLD1. Further, the PLD2-induced decrease in melanin content was accompanied by a decrease in the amount and activity of tyrosinase, a key enzyme in melanogenesis, whereas the mRNA level of tyrosinase was unchanged by the over-expression of PLD2. Moreover, treatment with proteasome inhibitors completely blocked the PLD2-induced down-regulation of melanogenesis. Taken together, the present results indicate that the TPA-induced down-regulation of melanogenesis is mediated by PLD2 but not by PLD1 through the ubiquitin proteasome-mediated degradation of tyrosinase. This suggests that PLD2 may play an important role in regulating pigmentation in vivo. Melanin is a mixture of pigmented biopolymers specifically synthesized within pigment cells. It plays a number of important roles, including photoprotection, the determination of phenotypic appearance, and the absorption of toxic drugs and chemicals (1Quevedo Jr., W.C. Fitzpatrick T.B. Szabó G. Jimbow K. Fitzpatrick T.B. Eisen A.Z. Wolff K. Freedberg I.M. Austen K.F. 3rd Ed. in Biology of Melanocytes: Dermatology in General Medicine. McGraw-Hill, New York1987: 224-251Google Scholar, 2Hearing V.J. J. Invest. Dermatol. Symp. Proc. 1999; 4: 24-28Abstract Full Text PDF PubMed Scopus (192) Google Scholar). Melanin is synthesized in specialized organelles, termed melanosomes, which are only observed in pigment cells. In melanosomes, melanogenesis is carried out by means of a specific enzymatic pathway initiated by tyrosinase, an enzyme that catalyzes the initial rate-limiting reaction of this process, the hydroxylation of tyrosine to dopaquinone via the intermediate 3,4-dihydroxyphenylalanine (2Hearing V.J. J. Invest. Dermatol. Symp. Proc. 1999; 4: 24-28Abstract Full Text PDF PubMed Scopus (192) Google Scholar). Melanin is a mixture of heterogeneous biopolymers formed from various intermediate products derived from dopaquinone. Because tyrosinase controls the first and rate-limiting step of melanogenesis, it is considered to be the key enzyme of this cascade and is the target of different effectors that regulate melanin synthesis. Mammalian tyrosinase is a type I membrane glycoprotein, which contains six putative N-glycosylation sites (3Müller G. Ruppert S. Schmid E. Schütz G. EMBO J. 1988; 7: 2723-2730Crossref PubMed Scopus (229) Google Scholar). Tyrosinase is extensively processed by post-translational modifications, and those processes have been well characterized. Tyrosinase is first synthesized as a 55-kDa unglycosylated protein (4Hearing V.J. Jiménez M. Int. J. Biochem. 1987; 19: 1141-1147Crossref PubMed Scopus (357) Google Scholar, 5del Marmol V. Beermann F. FEBS Lett. 1996; 381: 165-168Crossref PubMed Scopus (359) Google Scholar) and, like other membrane glycoproteins, it is processed in the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; LN, lipase activity-negative; α-MSH, α-melanocyte-stimulating hormone; pfu, plaque-forming unit; PKC, protein kinase C; PLD, phospholipase D; TPA, 12-O-tetradecanoylphorbol-13-acetate. by resident chaperones and enzymes. The 55-kDa tyrosinase protein is modified in the ER by the co-translational addition of multiple N-linked glycans. Complex sugar modifications in the Golgi apparatus further increase the molecular mass of tyrosinase to 80 kDa, the size of the mature protein (6Halaban R. Pomerantz S.H. Marshall S. Lambert D.T. Lerner A.B. J. Cell Biol. 1983; 97: 480-488Crossref PubMed Scopus (253) Google Scholar, 7Halaban R. Pomerantz S.H. Marshall S. Lerner A.B. Arch. Biochem. Biophys. 1984; 230: 383-387Crossref PubMed Scopus (93) Google Scholar). The 80-kDa tyrosinase protein is eventually transferred from the trans-Golgi network to melanosomes, the site of melanin synthesis, by vesicle transport. Melanogenesis is regulated by a variety of environmental and hormonal factors, such as ultraviolet radiation and α-melanocyte-stimulating hormone (α-MSH), and is aberrantly modulated in many different types of pigment cell disorders, including albinism, vitiligo, and melanoma (1Quevedo Jr., W.C. Fitzpatrick T.B. Szabó G. Jimbow K. Fitzpatrick T.B. Eisen A.Z. Wolff K. Freedberg I.M. Austen K.F. 3rd Ed. in Biology of Melanocytes: Dermatology in General Medicine. McGraw-Hill, New York1987: 224-251Google Scholar, 8Mosher D.B. Fitzpatrick T.B. Ortonne J.-P. Hori Y. Fitzpatrick T.B. Eisen A.Z. Wolff K. Freedberg I.M. Austen K.F. 3rd Ed. in Disorders of Pigmentation: Dermatology in General Medicine. McGraw-Hill, New York1987: 794-876Google Scholar). The regulatory mechanisms of melanogenesis, however, are not yet fully understood. Phospholipase D (PLD) is an enzyme that hydrolyzes phosphatidylcholine to generate an important lipid mediator, phosphatidic acid. Although the precise physiological relevance of PLD remains to be elucidated, receptor-stimulated PLD activity has been implicated in a broad range of physiological responses including secretion, superoxide generation, proliferation, differentiation, and immune responses (9Exton J.H. Biochim. Biophys. Acta. 1999; 1439: 121-133Crossref PubMed Scopus (337) Google Scholar, 10Frohman M.A. Sung T.-C. Morris A.J. Biochim. Biophys. Acta. 1999; 1439: 175-186Crossref PubMed Scopus (277) Google Scholar, 11Liscovitch M. Czarny M. Fiucci G. Tang X. Biochem. J. 2000; 345: 401-415Crossref PubMed Scopus (461) Google Scholar). In mammals, two PLD isozymes, PLD1 and PLD2, have been identified. These phospholipases share several similar properties. They have about 50% amino acid similarity and show a broad tissue distribution (11Liscovitch M. Czarny M. Fiucci G. Tang X. 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In contrast, PLD2 exhibits a high basal activity and is less responsive to PLD1-activating factors such as PKCα, ADP-ribosylation factor, and RhoA (9Exton J.H. Biochim. Biophys. Acta. 1999; 1439: 121-133Crossref PubMed Scopus (337) Google Scholar), although it has been reported recently that PLD2 acquires ADP-ribosylation factor responsiveness in the presence of GM2 activator (20Sarkar S. Miwa N. Kominami H. Igarashi N. Hayashi S. Okada T. Jahangeer S. Nakamura S. Biochem. J. 2001; 359: 599-604Crossref PubMed Google Scholar). Despite the quite distinct regulatory features of these isozymes in vitro, it has been demonstrated that both isozyme activities are under the control of a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in intact cells (9Exton J.H. Biochim. Biophys. Acta. 1999; 1439: 121-133Crossref PubMed Scopus (337) Google Scholar, 21Xie Z. Ho W.-T. Exton J.H. Biochim. Biophys. Acta. 2002; 1580: 9-21Crossref PubMed Scopus (21) Google Scholar). The isozyme-specific function(s) of PLD as well as the molecular mechanism(s) of their regulation remain to be clarified. It has been reported previously that treatment of B16 mouse melanoma cells with TPA resulted in the strong inhibition of melanogenesis (22Fuller B.B. Niekrasz I. Hoganson G.E. Mol. Cell. Endocrinol. 1990; 72: 81-87Crossref PubMed Scopus (36) Google Scholar, 23Kuzumaki T. Matsuda A. Wakamatsu K. Ito S. Ishikawa K. Exp. Cell Res. 1993; 207: 33-40Crossref PubMed Scopus (74) Google Scholar, 24Ando H. Itoh A. Mishima Y. Ichihashi M. J. Cell. Physiol. 1995; 163: 608-614Crossref PubMed Scopus (65) Google Scholar, 25Mahalingam H. Vaughn J. Novotny J. Gruber J.R. Niles R.M. J. Cell. Physiol. 1996; 168: 549-558Crossref PubMed Scopus Google Scholar). The of the present was to the involvement of PLD in the TPA-induced inhibition of melanogenesis and to tyrosinase is a target of assessed the involvement of PLD isozymes in the TPA-induced regulation of melanogenesis by PLD1 or PLD2 an adenoviral gene-transfer show that PLD2 but not PLD1 is in TPA-induced inhibition of melanogenesis. The molecular of inhibition of melanogenesis is assessed and were from and were from and were from and were from was from The were from PKCα, and PLD2 were from and were from to mouse tyrosinase was as previously M. K. V.J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and were from The proteasome inhibitors and as were from and was from Cell B16 melanoma cells were in and in a Melanin melanin content was as previously M. H. H. M. M. K. T. M. K. H. Kikkawa U. Ichihashi M. J. Invest. Dermatol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) with a cells from a in a were in of 80 for and to the The of the was and was with a of of melanin The melanin content is expressed as PLD activity was by the in activity M. A. New Scholar). in were with various plaque-forming of cells were with for and were with were in the presence of a of in for cells were with TPA in the presence of for the the reaction in a the was by and was to each The cells were into an and on was to each was and the was The was and the was The were by as previously S. K. Akisue T. Jinnai H. Hitomi T. Y. C. Yoshida K. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). was a Reverse was to to the The were used for mouse PLD1 W.C. D.J. M.A. Biochem. J. PubMed Scopus Google Scholar) and mouse PLD2 W.C. Sung T.-C. R. J. S.M. Y. Morris A.J. M.A. Biol. 7: Full Text Full Text PDF PubMed Scopus Google Scholar), mouse and mouse PLD2, and The was carried out in a in a reaction were as for for and for for of each reaction was in a and by were used to the size of the for PLD1 M. Hitomi T. Okada T. Nakamura S. H. M. T. Kikkawa U. Ichihashi M. Biochem. Biophys. Res. 2002; PubMed Scopus Google Scholar) and PLD2 (20Sarkar S. Miwa N. Kominami H. Igarashi N. Hayashi S. Okada T. Jahangeer S. Nakamura S. Biochem. J. 2001; 359: 599-604Crossref PubMed Google Scholar) were as previously and were and generate lipase activity-negative PLD1 and PLD2 and was a The and their were The for and were as previously (20Sarkar S. Miwa N. Kominami H. Igarashi N. Hayashi S. Okada T. Jahangeer S. Nakamura S. Biochem. J. 2001; 359: 599-604Crossref PubMed Google Scholar) and were and The the from Y. G. Y. M. M. T. S. I. Res. 1995; PubMed Scopus Google Scholar), which was by I. was as and was used as a control of and into tissue were with various of cells were with and were in and The cell was with for with The were used for immunoblot of and into tissue were with or the cells were with and were by in and for The were used as The were in and for and the were used as the of PLD2 and tyrosinase, the cells were in and cell was for from the and or of and were by and transferred to with the were with PLD2, tyrosinase PKCα, or and were with tyrosinase and or PKCα, and The reaction was carried out and as as previously H. M. Y. H. Y. Kikkawa U. Nishizuka Y. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Tyrosinase activity was a of the of Pomerantz S.H. Science. PubMed Scopus Google Scholar). from a in tissue were in of and for on the were by for of each cell was with of of and for of activated in acid was to the and the were for The were to in with of and the were by to the of of of was by the PubMed Scopus Google Scholar), on a and a membrane for 80 the membrane was for in each of and and The was with mouse tyrosinase H. S. T. C. T. J. PubMed Scopus Google Scholar) in a and The membrane was and and for in the membrane was with mouse under the as an TPA of PLD and the molecular the TPA-induced inhibition of melanogenesis, the effects of TPA on PLD activity, a well target of TPA, as well as the effects melanin content were examined. in treatment of the cells with TPA for the melanin content. TPA this caused a of PLD activity These results a involvement of PLD in the TPA-induced inhibition of melanogenesis. of TPA-induced of Melanogenesis by that PLD activation is a in TPA-induced of melanogenesis, the effects of the TPA-induced inhibition of melanogenesis were is a used as a substrate for the PLD reaction the of phosphatidic acid which phosphatidic responses K. Biol. 7: Full Text Full Text PDF PubMed Scopus Google Scholar). The TPA-induced of melanogenesis was completely by whereas it was by 2-butanol, that PLD activation is for the TPA-induced of melanogenesis. of PLD in B16 expression of PLD in B16 cells was by transcription-PCR it has been previously reported that PLD1 and PLD2 are expressed in and W.C. D.J. M.A. Biochem. J. PubMed Scopus Google Scholar), these were used as controls for each PLD The specific to mouse PLD1 and PLD2 of and in B16 and these the and from the The control in the of are not but products were it has been reported previously that the expression of PLD2 is low in B16 cells K. Y. Y. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), expression was further by immunoblot in PLD2 was by an the molecular as the These results indicate that B16 cells both PLD1 and PLD2. of PLD1 and PLD2 by both PLD1 and PLD2 are under the control of TPA in B16 the effects of TPA on PLD activation in the cells PLD1 or PLD2 were basal and PLD activities were in or cells. by TPA, PLD1 and PLD2 activity and basal activity, basal PLD activity was by the control These results indicate that both PLD1 and PLD2 activities are under the control of TPA in B16 cells. of Over-expression of PLD1 and PLD2 on PLD and Melanin both PLD1 and PLD2 are expressed in B16 it is to or both PLD isozymes are in the regulation of melanogenesis. the isozyme-specific effects of PLD, a high basal PLD activity, which is with PLD activity, by B16 cells with high of or of cells with or basal PLD activity in a The basal PLD activity each of was that of the pfu, PLD2 has a high basal activity in cells (11Liscovitch M. Czarny M. Fiucci G. Tang X. Biochem. J. 2000; 345: 401-415Crossref PubMed Scopus (461) Google Scholar, 21Xie Z. Ho W.-T. Exton J.H. Biochim. Biophys. Acta. 2002; 1580: 9-21Crossref PubMed Scopus (21) Google Scholar, G.E. Biochim. Biophys. Acta. 2000; PubMed Scopus Google Scholar). was a decrease in melanin content in the cells PLD2. In cells was a decrease in melanin content, which is with a that PLD1 expressed in B16 cells caused a decrease in melanin content K. Y. Y. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of cells with the control did not affect basal PLD activity or melanin content. These results indicate that PLD2 is in melanogenesis. of Over-expression PLD2 on of in B16 activation of is in the PLD2-induced down-regulation of melanogenesis, the effects of over-expression of PLD2 by the the intracellular distribution and the level of expression of were examined. have previously reported that B16 cells PKCα, and M. K. N. Mishima Y. J. Invest. Dermatol. 1993; Full Text PDF PubMed Scopus (19) Google Scholar). in of PLD2 did not in the levels of expression and intracellular distribution of each isoform the These results indicate that activation of is not in the PLD2-induced down-regulation of melanogenesis. of PLD1 and PLD2 on were to the effect of PLD isozyme expression on the regulation of melanogenesis. reported previously K. Y. Y. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. M. Ortonne J.-P. R. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), melanogenesis was by The melanogenesis was almost completely by PLD2, whereas similar levels of PLD1 over-expression caused a but inhibition of melanogenesis, which was with a K. Y. Y. Y. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These results indicate that of melanogenesis is by PLD2 and, to a by TPA-induced of Melanogenesis by a LN-PLD2, but by that PLD2 both basal and melanogenesis to PLD isozymes play similar in the regulation of melanogenesis. this lipase activity-negative of PLD1 or PLD2, which are to in a to each PLD isozyme T.C. Y. R. S.M. Morris A.J. B. J. M.A. EMBO J. PubMed Scopus Google Scholar, Y. H. M.A. Brown H.A. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, G. M.A. Mol. Biol. Cell. PubMed Scopus Google Scholar). previously and TPA both basal level and melanogenesis When a was an effect of TPA on both the basal and melanogenesis was by this was this almost completely blocked the of TPA on both basal level and melanogenesis, suggesting that TPA-induced down-regulation of melanogenesis is mediated by PLD2 in a physiological of Tyrosinase by Over-expression of the of PLD2-induced of melanogenesis, the effects of over-expression of PLD2 on tyrosinase protein levels as well as activity were examined. Over-expression of PLD2 caused a decrease in both tyrosinase content and in activity in a further the the down-regulation of tyrosinase, for tyrosinase mRNA was carried in tyrosinase mRNA levels were not were with These results indicate that the decrease in tyrosinase protein levels in B16 cells is not to the of tyrosinase mRNA of PLD2 not tyrosinase mRNA were with of or and was and was carried out of tyrosinase or of PLD2 in of the of the tyrosinase protein PLD2 cells were with the proteasome their with in the PLD2-induced decrease in melanin content was completely by treatment with The TPA-induced down-regulation of melanogenesis was by this similar effect was observed with These results that PLD2 the ubiquitin proteasome-mediated degradation of tyrosinase. In this the of TPA-induced of melanogenesis. In many cell including B16 TPA treatment a activation of PLD through the although it is PLD activation by (13Conricode K.M. Brewer K.A. Exton J.H. J. Biol. Chem. 1992; 267: 7199-7202Abstract Full Text PDF PubMed Google Scholar, 14Singer W.D. Brown H.A. Jiang X. Sternweis P.C. J. Biol. Chem. 1996; 271: 4504-4510Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). The that treatment of B16 cells with almost completely the TPA-induced of melanogenesis the involvement of PLD of the TPA and a specific the TPA-induced an adenoviral gene-transfer to various and the this have demonstrated the from the results of as well as over-expression and the TPA-induced of melanogenesis is mediated by PLD2 tyrosinase by ubiquitin proteasome-mediated degradation of this enzyme but not by tyrosinase have recently observed a similar of down-regulation of regulate pigmentation by means of ubiquitin degradation of tyrosinase H. H. K. M. Y. M. Ichihashi M. V.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This post-translational may as of the mechanisms of regulation of melanogenesis under including TPA It has been reported that the inhibition of melanogenesis by TPA results from activation and of to the of the tyrosinase promoter C. K. Ortonne J.-P. R. 1998; PubMed Scopus Google Scholar). physiological TPA utilize different from both of it utilize inhibition of tyrosinase mediated by a isozyme and of degradation of tyrosinase mediated by PLD2. The molecular of PLD2 activation by remains to be clarified. It has been reported recently that PLD1 is in the inhibition of melanogenesis in B16 cells. have observed a similar of PLD1 in B16 cells with less with PLD2 in of TPA-induced that PLD2 is the isozyme of TPA to melanogenesis for the PLD2 is present in B16 which is by both immunoblot analyses as well as analyses PLD2 activity is under the control of TPA PLD2 was in the of melanogenesis was PLD1 the activity was and the TPA-induced of melanogenesis was completely blocked only by the of PLD2 but not by the of PLD1 PLD1 may be in the regulation of melanogenesis or cell regulation by other TPA. the isozyme-specific of PLD is This is used as for the reaction which the of phosphatidic acid both isozyme-specific and ADP-ribosylation factor or in intact cells both isozymes, although show enzymatic activity of these isozymes in the of each PLD many have to the intracellular of each PLD isozyme by In PLD1 is in the whereas PLD2 is the membrane and in S. Cell. Mol. Sci. 2001; PubMed Scopus Google Scholar). PLD isozymes were in B16 these isozymes quite distinct PLD1 was in a in the whereas PLD2 was the membrane and, to a as a in the not It has recently been demonstrated that PLD isozymes show different intracellular distribution from those of PLD1 is in the Golgi whereas of the PLD2 in the Golgi apparatus is the of that Z. S. Mol. Biol. Cell. 2002; PubMed Scopus Google Scholar). are to the distribution of PLD isozymes with specific to the physiological relevance of PLD2 in the control of melanogenesis. I. of for the