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Originally published In Press as doi:10.1074/jbc.M401786200 on April 5, 2004

J. Biol. Chem., Vol. 279, Issue 26, 27774-27780, June 25, 2004
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Down-regulation of Melanogenesis by Phospholipase D2 through Ubiquitin Proteasome-mediated Degradation of Tyrosinase*

Akiko Kageyama{ddagger}, Masahiro Oka{ddagger}§, Taro Okada¶, Shun-ichi Nakamura¶, Takehiko Ueyama||, Naoaki Saito||, Vincent J. Hearing**, Masamitsu Ichihashi{ddagger}, and Chikako Nishigori{ddagger}

From the Divisions of {ddagger}Dermatology, Clinical Molecular Medicine, and Biochemistry, Department of Molecular and Cellular Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan, ||Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan, and **Laboratory of Cell Biology, NCI/National Institutes of Health, Bethesda, Maryland 20892

Received for publication, February 18, 2004 , and in revised form, April 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 characterized. Tyrosinase is first synthesized as a 55-kDa unglycosylated protein (4, 5) and, like other membrane glycoproteins, it is processed in the endoplasmic reticulum (ER)1 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 (6, 7). 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 {alpha}-melanocyte-stimulating hormone ({alpha}-MSH), and is aberrantly modulated in many different types of pigment cell disorders, including albinism, vitiligo, and melanoma (1, 8). 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 (9-11). 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 (11, 12). They utilize phosphatidylcholine as a substrate and require phosphatidylinositol 4,5-bisphosphate for their activation (9-11). On the other hand, it has been demonstrated that these PLD isozymes are regulated in distinct manners. PLD1 has a low basal activity and is activated by protein kinase C (PKC)-{alpha} (13, 14), ADP-ribosylation factor (15, 16), RhoA (17, 18), and a GM2 activator (19). In contrast, PLD2 exhibits a high basal activity and is less responsive to PLD1-activating factors such as PKC{alpha}, ADP-ribosylation factor, and RhoA (9), although it has been reported recently that PLD2 acquires ADP-ribosylation factor responsiveness in the presence of GM2 activator (20). 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 (9, 21). 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 (22-25). The aim of the present study was to investigate the possible involvement of PLD in the TPA-induced inhibition of melanogenesis and to determine whether tyrosinase is a downstream target of PLD. We assessed the involvement of PLD isozymes in the TPA-induced regulation of melanogenesis by over-expressing PLD1 or PLD2 using an adenoviral gene-transfer system. We show that PLD2 but not PLD1 is involved in TPA-induced inhibition of melanogenesis. The molecular mechanism of PLD2-mediated inhibition of melanogenesis is assessed herein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Antibiotics (penicillin, streptomycin, and fungizone) were purchased from BioWhittaker (Walkersville, MD). Phenylmethylsulphonyl fluoride, aprotinin, and leupeptin were obtained from Sigma. Dimethyl sulfoxide (Me2SO) and L-3,4-dihydroxyphenylalanine were obtained from Nacalai Tesque (Kyoto, Japan). L-[ring-3,5-3H]tyrosine (49.0 Ci/mmol) and 1-[1-14C]palmitoyl-2-lyso-sn-glycero-3-phosphocholine (52.0 mCi/mmol) were purchased from Amersham Pharmacia Biotech (Tokyo, Japan). Dowex-50 was purchased from Bio-Rad. The thin layer chromatography plates were from Merck (Darmstadt, Germany). Antibodies against PKC{alpha}, PKC{zeta}, and PLD2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against PKC{delta} and PKC{epsilon} were obtained from Transduction Laboratories (Lexington, KY). Rabbit polyclonal antiserum to mouse tyrosinase ({alpha}PEP-7) was generated as described previously (26). Alkaline phosphatase-conjugated anti-mouse and anti-rabbit antibodies were obtained from Promega (Madison, WI). The proteasome inhibitors MG132 (benzyloxycarbonyl-Leu-Leu-leucinal) and LLnL (N-acetyl-Leu-Leu-norleucinal, also known as ALLN) were purchased from Calbiochem and Sigma, respectively. [Nle4,D-Phe7]-{alpha}-MSH was obtained from Sigma.

Cell Culture—Mouse B16 melanoma cells were cultured in Eagle's minimal essential medium containing 10% fetal bovine serum (HyClone Laboratories, Logan, UT), 100 units/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml fungizone in a humidified atmosphere containing 5% CO2 at 37 °C.

Melanin Determination—The melanin content was determined as described previously (27) with a minor modification. Briefly, cells from a subconfluent monolayer in a 10-cm culture dish were solubilized in 150 µl of 1 N NaOH, incubated at 80 °C for 2 h, and vortexed to solubilize the melanin. The absorbance of the solution was measured at 400 nm and was compared with a standard curve of known concentrations of synthetic melanin (Sigma). The melanin content is expressed as µg/mg protein.

PLD Assay—PLD activity was assayed by monitoring the in vivo transphosphatidylation activity (28). Cells in six-well culture plates were infected with various plaque-forming units (pfu)/cell of adenovirus. One day after infection, cells were incubated with 1-[1-14C]palmitoyl-2-lyso-sn-glycero-3-phosphocholine (0.25 µCi/107 cells) for 16 h at 37 °C and were washed three times with phosphate-buffered saline. Cells were then incubated in the presence of 1% ethanol (in a total volume of 1 ml) in serum-free Eagle's minimal essential medium for 30 min at 37 °C. Where indicated, cells were stimulated with 5 nM TPA in the presence of 1% ethanol for 30 min. During the incubation, the reaction proceeded in a linear fashion. After incubation, the medium was removed by aspiration, and ice-cold methanol (400 µl) was added to each well. The cells were scraped into an Eppendorf tube (1.5 ml) and kept on ice; chloroform (400 µl) was then added to each tube. After mixing, 0.1 N HCl/1 mM EGTA (400 µl) was added, and the solution was mixed vigorously. The tube was centrifuged, and the organic phase was separated. The lipids extracted were analyzed by thin layer chromatography as described previously (29). Radioactivity was quantitated using a Fujix Bio-Imaging Analyzer BAS 2000 (Fuji Photo Film).

Reverse Transcription-PCR—Total RNA (5 µg) was subjected to reverse transcription using Superscript II (Invitrogen) according to the manufacturer's protocol. The following primers were used for mouse PLD1 (30) and mouse PLD2 (31), respectively: mouse PLD1, 5'-A CAC AGG ATA CCA GGT GTG A-3' (sense) and 5'-T AGA CTC TAC TGA TGC TGC C-3' (antisense); mouse PLD2, 5'-CC AAG GCC AGG TAT AAG ACA CC-3' (sense) and 5'-CAA GTA GAC TCG GAA ACA CTG C-3' (antisense). The PCR was carried out in a PCR Thermal Cycler MP (TaKaRa, Tokyo, Japan) in a 50-µl reaction volume using Hot Star Taq (Qiagen, Valencia, CA). Reaction conditions were as follows: 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1.5 min for 35 cycles. An aliquot (10 µl) of each reaction sample was analyzed in a 2% agarose gel and visualized by ethidium bromide fluorescence staining. Molecular weight markers (Promega Corp., Madison, WI) were used to estimate the size of the amplified fragments.

Adenovirus Vectors—The adenovirus vectors for rat FLAG-epitope-tagged PLD1 (32) and rat FLAG-epitope-tagged PLD2 (20) were generated as described previously and were designated AxPLD1 and Ax-PLD2, respectively. To generate lipase activity-negative PLD1 (LNPLD1) and PLD2 (LN-PLD2), site-directed mutagenesis (to create K860R and K758R, respectively) was performed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The mutagenic oligonucleotides 5'-G CTC ATC TAT GTC CAC AGC AGG TTG TTA ATT GCT GAT-3', 5'-C ATC TAT ATC CAC AGC AGG TTG CTC ATT GCA GAT GAC-3' and their reverse complements were used, respectively. The adenovirus vectors for rat FLAG-epitope-tagged LN-PLD1 and LN-PLD2 were generated as described previously (20) and were designated AxLN-PLD1 and AxLN-PLD2, respectively. The adenovirus carrying the {beta}-galactosidase gene (LacZ) from Escherichia coli (33), which was kindly provided by Dr. I. Saito (Tokyo University, Japan), was designated as AxLacZ and was used as a control virus.

Immunoprecipitation of FLAG-PLD1 and FLAG-PLD2—Cells plated into 10-cm tissue culture dishes were infected with various doses of adenovirus. One day after infection, cells were washed 3x with phosphate-buffered saline and were lysed in buffer A (20 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 1 mM EGTA, 10 mM 2-mercaptoethanol, 1% (w/v) Triton X-100, 150 mM NaCl, 10 mM NaF, 1 mM sodium orthovanadate, and 1 mM phenylmethylsulphonyl fluoride). The cell lysate was then incubated with anti-FLAG M2 affinity gel (30 µl) (Sigma) for 3 h with constant mixing. The immunoprecipitates were used for immunoblot analysis.

Preparation of Cytosol and Particulate Fractions—Cells plated into 10-cm tissue culture dishes were infected with either AxLacZ or Ax-PLD2. At the indicated time points after infection, cells were washed 3x with phosphate-buffered saline and were disrupted by sonication in buffer A without Triton X-100 and centrifuged at 100,000 x g for 60 min at 4 °C. The resulting supernatants were used as cytosolic fractions. The pellets were solubilized in buffer A and centrifuged at 100,000 x g for 60 min at 4 °C, and the supernatants were used as particulate fractions.

Immunoblot Analysis—For the detection of PLD2 and tyrosinase, the cells were lysed in 0.1 M sodium phosphate buffer (pH 6.8) containing 0.5% (w/v) Triton X-100, 1 µM phenylmethylsulphonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. Each cell lysate was centrifuged at 17,000 x g for 10 min at 4 °C. Aliquots from the cytosolic and particulate fractions or immunoprecipitates of FLAG-PLD1 and FLAG-PLD2 were separated by SDS-7.5% PAGE and transferred to Immobilon P membranes (Millipore Corp., Bedford, MA). After blocking with 5% bovine serum albumin, the membranes were incubated with antibodies against PLD2, tyrosinase ({alpha}PEP-7), FLAG, PKC{alpha}, PKC{delta}, PKC{epsilon}, or PKC{zeta}, and were then incubated with alkaline phosphatase-conjugated anti-goat (for PLD2), anti-rabbit (for tyrosinase and PKC{zeta}), or anti-mouse (for FLAG-PLD1, FLAG-PLD2, PKC{alpha}, PKC{delta}, and PKC{epsilon}) secondary antibodies. The color reaction was carried out using 5-bromo-4-chloro-3-indoyl-phosphate and nitro-blue tetrazolium as substrates, as described previously (34).

Tyrosinase Assay—Tyrosinase activity was measured using a modification of the method of Pomerantz (35). Cells from a subconfluent monolayer in 10-cm tissue culture dishes were solubilized in 250 µl of 0.1 M sodium phosphate buffer (pH 6.8) containing 0.5% (w/v) Triton X-100, 1 µM phenylmethylsulphonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. After sonication for 20 s on ice, the extracts were clarified by centrifugation at 17,000 x g for 10 min at 4 °C. 90 µl of each clarified cell extract was incubated with 10 µl of 0.1 M sodium phosphate buffer (pH 6.8) containing 1 µCi of L-[ring-3,5-3H]tyrosine, 5 µg L-3,4-dihydroxyphenylalanine, and 1% (w/v) Triton X-100 for 60 min at 37 °C. 1 ml of activated charcoal (10% w/v) in 0.1 M citric acid was then added to stop the reaction, and the aliquots were centrifuged at 17,000 x g for 10 min at 4 °C. The supernatants were applied to Dowex-50 columns (0.6 x 4 cm) equilibrated in 0.1 M citric acid, washed with 0.5 ml of 0.1 M citric acid, and the effluents were counted by scintillation spectrometry to determine the extent of formation of 3H2O.

Northern Blot Analysis of Tyrosinase—Total RNA (25 µg) was isolated by the guanidinium isothiocyanate method (36), electrophoresed on a 1% agarose-formaldehyde gel, and blotted onto a nitrocellulose membrane (Schleicher & Schüll). After baking for 2 h at 80 °C, the membrane was prehybridized for 1 h at 42 °C in 40% (v/v) formamide, 1x Denhardt's solution (0.02% each of polyvinyl pyrrolidone, bovine serum albumin, and Ficoll 400), 4x sodium citrate/chloride (SSC) buffer (1x SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 7 mM Tris-HCl (pH 7.5), and 20 µg/ml sheared salmon sperm DNA. The blot was then hybridized overnight at 42 °C with randomly primed 32P-labeled mouse tyrosinase cDNA clone Tyrs-J (37) in a solution containing 4x SSC, 40% (v/v) formamide, 10% (w/v) dextran sulfate, 20 mM Tris-HCl (pH 7.5), 1x Denhardt's solution, 250 µg/ml yeast tRNA, and 20 µg/ml salmon sperm DNA. The membrane was washed at 65 °Cin0.1x SSC and 0.5% SDS, dried, and autoradiographed. After boiling for 5 min in 0.1% SDS, the membrane was rehybridized with 32P-labeled mouse {beta}-actin cDNA (Clontech Laboratories) under the conditions described above as an internal loading control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TPA Causes Activation of PLD and Suppresses Melanogenesis—To understand the molecular mechanism underlying the TPA-induced inhibition of melanogenesis, the effects of TPA on PLD activity, a well known downstream target of TPA, as well as the effects upon melanin content were examined. As shown in Fig. 1A, treatment of the cells with 5 nM TPA for 72 h markedly decreased the melanin content. TPA at this concentration caused a marked stimulation of PLD activity (Fig. 1B). These results suggest a potential involvement of PLD in the TPA-induced inhibition of melanogenesis.



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  		FIG. 1.
TPA activates PLD and reduces melanin content. Cells were cultured in medium containing 5 nM TPA or 0.1% Me2SO (control) for 72 h. Cell pellets were photographed (A, upper panel), and the melanin contents (A, lower panel) were measured. B, cells were metabolically labeled with [14C]lysophosphatidylcholine and were assayed for PLD in the absence (0.1% Me2SO) or presence of 5 nM TPA. Data are means ± S.D. of three independent experiments carried out in duplicate.

 
Reversal of TPA-induced Suppression of Melanogenesis by 1-Butanol—To confirm that PLD activation is a necessary downstream event in TPA-induced suppression of melanogenesis, the effects of 1-butanol upon the TPA-induced inhibition of melanogenesis were investigated (Fig. 2). 1-Butanol is a primary alcohol frequently used as a substrate for the PLD transphosphatidylation reaction at the expense of phosphatidic acid production, which inhibits PLD-induced phosphatidic acid-mediated biological responses (38). The TPA-induced suppression of melanogenesis was completely reversed by 1-butanol, whereas it was unaffected by 2-butanol, indicating that PLD activation is necessary for the TPA-induced suppression of melanogenesis.



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  		FIG. 2.
1-Butanol but not 2-butanol abrogates TPA-induced down-regulation of melanogenesis. Cells were incubated in the presence or absence of 0.2% 1-butanol or 2-butanol for 60 min and then stimulated with 5 nM TPA or 0.1% Me2SO. 72 h after stimulation, the melanin contents were measured. Data are means ± S.D. of three independent experiments carried out in duplicate.

 
Expression of Both PLD Isozymes in B16 Cells—The expression of PLD isoforms in B16 cells was estimated by reverse transcription-PCR (Fig. 3A). As it has been previously reported that PLD1 and PLD2 are abundantly expressed in kidney and lung tissues, respectively (30), these tissues were used as positive controls for each PLD isozyme. The specific primers to mouse PLD1 and PLD2 amplified signals of 863 and 332 bp, respectively, in B16 cells, and these bands exactly matched the estimated sizes and signals from the positive controls. The control experiments in the absence of template DNA are not shown, but no PCR products were detectable. As it has been reported previously that the expression of PLD2 is very low in B16 cells (39), its expression was further confirmed by immunoblot analysis. As shown in Fig. 3B, PLD2 was clearly detected by an anti-PLD2 antibody at the same molecular position as the positive control. These results indicate that B16 cells express both PLD1 and PLD2.



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  		FIG. 3.
PLD1 and PLD2 isozymes are expressed in B16 cells. A, total RNA prepared from B16 cells (lanes 1 and 3), mouse kidney (lane 2), and mouse lung (lane 4) was subjected to reverse transcription-PCR using primers specific for mouse PLD1 (lanes 1 and 2) and mouse PLD2 (lanes 3 and 4). The size of the molecular weight markers is shown in bp. The positions of the amplified fragments are indicated by arrows. Mouse kidney and lung were used as positive controls for PLD1 and PLD2, respectively. B, cell lysates from B16 cells infected without (lane 1) or with 25 pfu/cell of AxPLD2 (lane 2) were subjected to immunoblot analysis using an antibody against PLD2.

 
Activation of Both PLD1 and PLD2 by TPA—To examine whether both PLD1 and PLD2 are under the control of TPA in B16 cells, the effects of TPA on PLD activation in the cells over-expressing either PLD1 or PLD2 were examined (Fig. 4). Both basal and TPA-stimulated PLD activities were enhanced in PLD1- or PLD2-over-expressing cells. Upon stimulation by TPA, PLD1 and PLD2 activity increased ~3-fold and 2-fold over basal activity, respectively. Neither basal nor TPA-stimulated PLD activity was affected by the control viral infection. These results indicate that both PLD1 and PLD2 activities are under the control of TPA in B16 cells.



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  		FIG. 4.
Both PLD1 and PLD2 isozymes are activated by TPA in B16 cells. Cells were infected with 5 pfu/cell of AxPLD1, AxPLD2, or AxLacZ and assayed for PLD in the presence of 5 nM TPA (black bars) or 0.1% Me2SO (white bars). Data are means ± S.D. of three independent experiments carried out in duplicate.

 
Effects of Over-expression of PLD1 and PLD2 on PLD Activity and Melanin Content—Because both PLD1 and PLD2 are expressed in B16 cells, it is necessary to assess whether either or both PLD isozymes are involved in the regulation of melanogenesis. To get the isozyme-specific effects of PLD, we established a high basal PLD activity, which is comparable with TPA-stimulated PLD activity, by infecting B16 cells with high doses of AxPLD1 or AxPLD2. Infection of cells with either AxPLD1 or AxPLD2 enhanced basal PLD activity in a viral dose-dependent manner (Fig. 5). The basal PLD activity at each pfu of AxPLD2 was ~2-fold higher than that of AxPLD1 at the corresponding pfu, presumably because PLD2 has a high basal activity in cells (11, 21, 40). Importantly, there was a marked decrease in melanin content in the cells over-expressing PLD2. In cells over-expressing PLD1, there was a small decrease in melanin content, which is consistent with a recent report that PLD1 stably expressed in B16 cells caused a decrease in melanin content (39). Infection of cells with the control AxLacZ did not affect either basal PLD activity or melanin content. These results indicate that PLD2 is mainly involved in down-regulating melanogenesis.



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  		FIG. 5.
Over-expression of PLD2 but not PLD1 inhibits melanogenesis. Cells were infected with various doses of AxPLD1, Ax-PLD2, or AxLacZ. 72 h after infection, the melanin contents (upper panel) and PLD activities (lower panel) were measured. The expression of FLAG-PLD1 and FLAG-PLD2 was examined by immunoblot analyses after immunoprecipitation using an anti-FLAG antibody. Data are means ± S.D. of three independent experiments carried out in duplicate.

 
Effects of Over-expression PLD2 on Intracellular Distribution of PKC Isoforms in B16 Cells—To assess whether activation of PKC is involved in the PLD2-induced down-regulation of melanogenesis, the effects of over-expression of PLD2 by the adenovirus vector upon the intracellular distribution and the level of expression of PKC isoforms were examined. We have previously reported that B16 cells express PKC{alpha}, PKC{delta}, PKC{epsilon}, and PKC{zeta} (41). As shown in Fig. 6, overexpression of PLD2 did not induce any detectable changes in the levels of expression and intracellular distribution of each PKC isoform at any time intervals examined after the viral infection. These results indicate that activation of PKC is not involved in the PLD2-induced down-regulation of melanogenesis.



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  		FIG. 6.
Over-expression of PLD2 does not affect the intracellular distribution or level of expression of PKC isoforms in B16 cells. Cells were infected with 25 pfu/cell of AxPLD2 or AxLacZ. The cytosol (C) and particulate (P) fractions were subjected to immunoblot analyses using antibodies against PKC{alpha}, PKC{delta}, PKC{epsilon}, and PKC{zeta}.

 
Inhibitory Effects of PLD1 and PLD2 on {alpha}-MSH-enhanced Melanogenesis—Experiments were then performed to study the effect of PLD isozyme expression on the regulation of {alpha}-MSH-stimulated melanogenesis. As reported previously (39, 42), melanogenesis was greatly up-regulated by {alpha}-MSH (Fig. 7). The {alpha}-MSH-enhanced melanogenesis was almost completely inhibited by over-expressing PLD2, whereas similar levels of PLD1 over-expression (Fig. 5) caused a weak but significant inhibition of melanogenesis, which was consistent with a previous report (39). These results indicate that {alpha}-MSH-induced enhancement of melanogenesis is inhibited mainly by PLD2 and, to a lesser extent, by PLD1.



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  		FIG. 7.
PLD2 strongly inhibits {alpha}-MSH-induced enhancement of melanogenesis. Cells were infected with AxPLD1, AxPLD2, or Ax-LacZ. Immediately after infection, 10 nM {alpha}-MSH was added to the culture medium. 72 h after infection, the melanin contents were measured. Data are means ± S.D. of three independent experiments carried out in duplicate.

 
TPA-induced Down-regulation of Melanogenesis Was Completely Overcome by a Dominant-negative Mutant, LN-PLD2, but Not by LN-PLD1—The observation that over-expressed PLD2 strongly inhibits both basal (Fig. 5) and {alpha}-MSH-stimulated melanogenesis (Fig. 7) prompted us to ask whether endogenous PLD isozymes play similar roles in the regulation of melanogenesis. To assess this issue, we constructed adenovirus-carrying lipase activity-negative mutants of PLD1 or PLD2, which are known to act in a dominant-negative fashion to suppress each endogenous PLD isozyme (43-45). As shown previously (Figs. 5 and 7), TPA down-regulated both basal level and {alpha}-MSH-stimulated melanogenesis (Fig. 8). When a dominant-negative mutant LN-PLD1 was over-expressed, an inhibitory effect of TPA on both the basal and {alpha}-MSH-stimulated melanogenesis was unaffected by this mutant expression. However, when LN-PLD2 was over-expressed, this catalytically inactive mutant almost completely blocked the inhibitory action of TPA on both basal level and {alpha}-MSH-stimulated melanogenesis, suggesting strongly that TPA-induced down-regulation of melanogenesis is mediated by PLD2 in a physiological manner.



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  		FIG. 8.
TPA-induced down-regulation of melanogenesis is almost completely blocked by a dominant-negative mutant LN-PLD2 but not by LN-PLD1. Cells were infected with AxLN-PLD1, AxLN-PLD2, or AxLacZ. After infection, 5 nM TPA or 10 nM {alpha}-MSH was added to the culture medium. 72 h after infection, the melanin contents were measured. Data are means ± S.D. of three independent experiments carried out in duplicate. *, p < 0.05 versus AxLacZ-infected, TPA-stimulated cells; #, p < 0.05 versus AxLacZ-infected {alpha}-MSH and TPA-stimulated cells (Student's t test).

 
Down-regulation of Tyrosinase by Over-expression of PLD2—To examine the mechanism of PLD2-induced suppression of melanogenesis, the effects of over-expression of PLD2 on tyrosinase protein levels as well as catalytic activity were examined. Over-expression of PLD2 caused a decrease in both tyrosinase content and in its activity in a viral dose-dependent manner (Fig. 9). To further study the mechanism underlying the PLD2-mediated down-regulation of tyrosinase, Northern blot analysis for tyrosinase mRNA was carried out. As shown in Fig. 10, tyrosinase mRNA levels were not altered at any time they were examined after infection with AxPLD2. These results indicate that the decrease in tyrosinase protein levels in PLD2-over-expressing B16 cells is not due to the reduction of tyrosinase mRNA levels.



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  		FIG. 9.
Over-expression of PLD2 suppresses the protein level and activity of tyrosinase. Cells were infected with various doses of AxPLD2 or AxLacZ. 72 h after infection, cell pellets were photographed (upper panel), measured for their tyrosinase content by immunoblot analysis (middle panel), and assayed for tyrosinase activity (lower panel). Data are means ± S.D. of three independent experiments carried out in duplicate.

 



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  		FIG. 10.
Over-expression of PLD2 does not change tyrosinase mRNA levels. Cells were infected with 25 pfu/cell of either AxLacZ (LacZ) or AxPLD2 (PLD2), and total RNA was isolated at 0, 24, 36, and 72 h after infection. Northern blot analysis was carried out using 32P-labeled probes of tyrosinase or {beta}-actin.

 
Involvement of PLD2 in Ubiquitin Proteasome-mediated Degradation of Tyrosinase—To investigate the mechanism of the reduced tyrosinase protein after PLD2 over-expression, cells were treated with the proteasome inhibitor MG132 immediately after their infection with AxPLD2. As shown in Fig. 11, the PLD2-induced decrease in melanin content was completely reversed by treatment with MG132. The TPA-induced down-regulation of melanogenesis was also significantly counteracted by this inhibitor. A similar reversal effect was also observed with another proteasomal inhibitor, LLnL. These results strongly suggest that PLD2 promotes the ubiquitin proteasome-mediated degradation of tyrosinase.



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  		FIG. 11.
Proteasome inhibitors suppress the PLD2-induced down-regulation of melanogenesis. Cells were infected with 25 pfu/cell of either AxPLD2 or AxLacZ. After infection, cells were treated with or without 0.05% Me2SO, 5 nM TPA, and/or the proteasome inhibitors (100 nM MG132 or 5 µM LLnL), as indicated. 72 h after infection, the melanin contents were measured (upper panel), and the expression of tyrosinase was determined (lower panel). Data are means ± S.D. of three independent experiments carried out in duplicate. #, p < 0.05 versus AxLacZ-infected, TPA-stimulated cells; *, p < 0.05 versus AxPLD2-infected, untreated cells (Student's t test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we characterized the downstream signaling events of TPA-induced suppression of melanogenesis. In many cell types, including B16 cells, TPA treatment causes a potent activation of PLD through the PKC-mediated pathway, although it is still controversial whether PLD activation by PKC involves phosphorylation (13, 14). The observation that treatment of B16 cells with 1-butanol almost completely abolished the TPA-induced suppression of melanogenesis (Fig. 2) indicates the involvement of PLD downstream of the TPA effect. To dissect and identify a specific signaling event among the numerous TPA-induced cellular effects, we utilized an adenoviral gene-transfer system to over-express various PLDs and bypass the TPA-mediated signal. By using this approach, we have demonstrated the following: (i) judging from the results of dominant-negative experiments (Fig. 8) as well as over-expression systems (Figs. 5 and 7), the TPA-induced suppression of melanogenesis is mediated by PLD2; (ii) PLD2 down-regulates tyrosinase by enhancing ubiquitin proteasome-mediated degradation of this enzyme (Fig. 11) but not by inhibiting tyrosinase transcription (Fig. 10). We have recently observed a similar mechanism of down-regulation of tyrosinase: fatty acids regulate pigmentation by means of ubiquitin proteasomal degradation of tyrosinase (46). This post-translational modification may function as one of the general mechanisms of regulation of melanogenesis under certain circumstances, including TPA stimulation.

It has been reported that the inhibition of melanogenesis induced by TPA results from PKC activation and subsequent diminution of microphthalmia binding to the M-box of the tyrosinase promoter (47). Under physiological conditions, TPA might utilize signaling events different from both of these; i.e. it might utilize inhibition of tyrosinase transcription mediated by a conventional PKC isozyme and enhancement of degradation of tyrosinase mediated by PLD2. The molecular mechanism of PLD2 activation by PKC remains to be clarified. It has been reported recently that PLD1 is involved in the inhibition of melanogenesis in B16 cells. We have also observed a similar inhibitory potential of PLD1 in {alpha}-MSH-treated B16 cells with much less potency compared with PLD2 (Fig. 7). However, in terms of TPA-induced actions, we conclude that PLD2 is the isozyme capable of transducing TPA signals to suppress melanogenesis for the following reasons. First, PLD2 is present in B16 cells, which is confirmed by both immunoblot analyses as well as PCR analyses (Fig. 3). Second, PLD2 activity is under the control of TPA (Fig. 4). Third, PLD2 was much more potent in the suppression of melanogenesis than was PLD1 when the activity was normalized (Fig. 5). Last, and most importantly, the TPA-induced suppression of melanogenesis was completely blocked only by the dominant-negative mutant of PLD2 (LNPLD2) but not by the mutant of PLD1 (LN-PLD1) (Fig. 8). PLD1 may be involved in the regulation of melanogenesis during {alpha}-MSH-induced differentiation or during cell regulation by agonists other than TPA.

Information concerning the isozyme-specific functions of PLD is still limited. This is partly because primary alcohols, commonly used as substrates for the transphosphatidylation reaction which inhibits PLD-mediated functions at the expense of phosphatidic acid formation, inhibit both isozyme-specific events, and partly because ADP-ribosylation factor or PKC in intact cells stimulates both isozymes, although they show clear differences toward enzymatic activity of these isozymes when studied in vitro. To understand the function of each PLD isozyme, many studies have attempted to elucidate the intracellular localization of each PLD isozyme by microscopy using over-expressed, tagged PLDs. In most cases, PLD1 is localized in the endosomal/lysosomal compartment, whereas PLD2 is found at the plasma membrane and also in submembranous vesicular structures (48). Similarly, when green fluorescent protein-tagged PLD isozymes were over-expressed in B16 cells, these isozymes showed quite distinct localization patterns: PLD1 was localized in a punctate pattern in the cytoplasm, whereas PLD2 was localized mainly at the plasma membrane and, to a lesser degree, as a reticular pattern in the cytoplasm (data not shown). It has recently been demonstrated that endogenous PLD isozymes show different intracellular distribution patterns from those of over-expressed, tagged PLD: endogenous PLD1 is predominantly localized in the Golgi cisternae, whereas most of the endogenous PLD2 in the Golgi apparatus is localized at the rims of that organelle (49). Further studies are necessary to elucidate the distribution patterns of endogenous PLD isozymes along with specific melanosomal markers to reveal the physiological relevance of PLD2 in the control of melanogenesis.


   FOOTNOTES
 
* This study was supported in part by research grants from the Scientific Research Funds of the Ministry of Education, Culture, Sports, Science and Technology of Japan (to M. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel.: 81-78-382-6134; Fax: 81-78-382-6149; E-mail: oka{at}med.kobe-u.ac.jp.

1 The abbreviations used are: ER, endoplasmic reticulum; LN, lipase activity-negative; {alpha}-MSH, {alpha}-melanocyte-stimulating hormone; pfu, plaque-forming unit; PKC, protein kinase C; PLD, phospholipase D; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back


   ACKNOWLEDGMENTS
 
We thank Dr. I. Saito (University of Tokyo, Japan) for the adenovirus-carrying LacZ.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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