title: Down-regulation of melanogenesis by phospholipase D2 through ubiquitin proteasome-mediated degradation of tyrosinase.
abstract: 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 alpha-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.
authors.affiliation: Division of Dermatology, Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Kageyama
authors.forename: Masahiro
authors.initials: M
authors.lastname: Oka
authors.forename: Taro
authors.initials: T
authors.lastname: Okada
authors.forename: Shun-ichi
authors.initials: S
authors.lastname: Nakamura
authors.forename: Takehiko
authors.initials: T
authors.lastname: Ueyama
authors.forename: Naoaki
authors.initials: N
authors.lastname: Saito
authors.forename: Vincent J
authors.initials: VJ
authors.lastname: Hearing
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Ichihashi
authors.forename: Chikako
authors.initials: C
authors.lastname: Nishigori
mesh_terms: D020001:1-Butanol / Q000494:pharmacology; D000818:Animals; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015536:Down-Regulation; D004789:Enzyme Activation / Q000187:drug effects; D007668:Kidney / Q000201:enzymology; D007976:Leupeptins / Q000494:pharmacology; D008168:Lung / Q000201:enzymology; D008543:Melanins / Q000096:biosynthesis*; D008546:Melanoma, Experimental / Q000378:metabolism; D051379:Mice; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000037:antagonists & inhibitors / Q000494:pharmacology; D025801:Ubiquitin / Q000378:metabolism*; D000521:alpha-MSH / Q000037:antagonists & inhibitors / Q000494:pharmacology
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008543:Melanins; D009097:Multienzyme Complexes; D025801:Ubiquitin; D000521:alpha-MSH; D020001:1-Butanol; D014442:Monophenol Monooxygenase; D011493:Protein Kinase C; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D013755:Tetradecanoylphorbol Acetate; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde