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 ␣-melanocytestimulating hormone-enhanced melanogenesis were almost completely blocked by expressing a lipase activitynegative 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 (1, 2). 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 (2). 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 (3). Tyrosinase is extensively processed by post-translational modifications, and those processes have been well characterize...

Varp Is a Novel Rab32/38-binding Protein That Regulates Tyrp1 Trafficking in Melanocytes

“…Because melanogenic enzymes have recently been suggested to be mistargeted and degraded in Rab32/38-deficient melanocytes (Wasmeier et al, 2006), the most likely function of Varp is to control trafficking of Tyrp1 (e.g., transport from an endoplasmic reticulum [ER] to the Golgi, transport from endosomes or the trans-Golgi network, tethering, docking, and/or fusion of Tyrp1-containing vesicles to melanosomes) together with Rab32/38. In the absence of Varp, Tyrp1 is not correctly targeted to melanosomes, and mistargeted Tyrp1 (or untransported Tyrp1 in the ER) may be ubiquitinated (Ando et al, 2004(Ando et al, , 2007Kageyama et al, 2004) and degraded by proteasomes (e.g., ER-associated degradation), because our preliminary experiments showed that treatment of Varpknockdown cells (or GFP-ANKR1-expressing cells) with a proteasome inhibitor, MG132, but not with lysosomal protease inhibitors (E64d and pepstatin A), restored Tyrp1 staining (Supplemental Figure S7Af). However, treatment of cells with proteasome inhibitors also affected the morphology of the cell (i.e., change into extended and filamentous shape), which precludes us to determine the exact localization of mistargeted proteins by immunofluorescence analysis (Supplemental Figure S7A, e-h).…”

Hypopigmenting agents: an updated review on biological, chemical and clinical aspects

“…Topical application of linolenic, linoleic and oleic acids (efficiency in decreasing order) produce a bleaching effect on guinea pig skin stimulated with UV light (Ando et al, 1998). As the number of melanosomes or the level of tyrosinase mRNA do not seem to be influenced, melanin inhibition by linoleic acid is probably due to a decrease of the amount of active tyrosinase inside the melanocytes because of a stimulation of tyrosinase ubiquitination and degradation by the proteasome (Ando et al, 1999(Ando et al, , 2006Kageyama et al, 2004). On contrary, those unsaturated fatty acids seem to stimulate desquamation of the skin and the stratum corneum turnover.…”

Effects of Quercetin on Mushroom Tyrosinase and B16-F10 Melanoma Cells

“…The melanin content was determined as previously described [36,37] with minor modifications. Cells were washed with PBS, harvested by trypsinization, and centrifuged for 10 minutes at 1500 g. The cell pellets were then dissolved in 1.4 M NaOH containing 14% DMSO during 2 hours incubation at 80 ºC.…”