All dipeptides were supplied by Beadtech Inc. (Seoul, Korea). PS and VS were dissolved in dimethyl sulfoxide (DMSO) and stored at 4℃ as stock solutions (10 mg/ml). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA), while 12-O-tetradecanoylphorbol-13-acetate (TPA), cholera toxin (CT), 3,4-dihydroxy-L-phenylalanine (L-DOPA), and mushroom tyrosinase were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), antibiotic-antimycotic mix (penicillin, streptomycin), and trypsin-EDTA were purchased from WelGENE (Dalseogu, Daegu, South Korea). Antibodies specific for phospho-ERK1/2 (Thr202/Tyr204, #9101S) and phospho-CREB (Ser133, #9198) were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific for tyrosinase (C-19) and actin (I-19), and for microphthalmia Ab-1 (C5, MS-771-P0) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA and NeoMarkers (Fremont, CA, USA), respectively. Secondary antibodies (anti-goat IgG (PI-9500), anti-mouse IgG (PI-2000) and anti-rabbit IgG (PI-1000)) were purchased from Vector Laboratories (Burlingame, CA, USA).

The Mel-Ab cell line is a spontaneously immortalized murine melanocyte line that produces large quantities of melanin [17]. Mel-Ab cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 nM TPA, 1 nM CT, 50 µg/ml streptomycin, and 50 µg/ml of penicillin in 5% CO₂ at 37℃.

Cell viability was determined by crystal violet assay [17]. After incubating cells with PS or VS at 1~50 µg/ml for 24 hours, the medium was removed and the cells were stained with 0.1% crystal violet in 10% ethanol for 5 minutes at room temperature. The cells were then rinsed four times with distilled water, and any crystal violet retained by adherent cells was extracted using 95% ethanol. Absorbance was measured at 590 nm using an ELISA plate reader (VersaMax; Molecular Devices, Sunnyvale, CA, USA).

After incubation with PS or VS at 1~20 µg/ml for 4 days, Mel-Ab cells were visualized by phase contrast microscopy (Olympus Optical Co., Tokyo, Japan) and photographed using a DCM300 digital camera (Scopetek, Inc., Hangzhou, China). The melanin content of the cells was quantified as previously described [18] with some modifications. Briefly, the cell pellets were dissolved in 1 ml of 1 N NaOH at 100℃ for 30 minutes, then centrifuged for 20 minutes at 16,000× g. The relative optical densities (OD) of the resulting supernatants were then measured at 400 nm using an ELISA reader. Triplicate samples of synthetic melanin (0~300 µg/ml) were used to prepare standard curves for each experiment.

Tyrosinase activity was assayed to determine DOPA oxidase activity. Mel-Ab cells were incubated for 4 days in six-well plates (2×10⁵ cells/well) containing PS in DMEM. The cells were then washed with PBS, lysed with lysis buffer (0.1 M phosphate buffer [pH 6.8] containing 1% Triton X-100), and disrupted by freeze-thawing. The lysates were clarified by centrifugation at 15,000 rpm for 30 min. After protein quantification using a protein assay kit (Bio-Rad, Hercules, CA, USA), the cell lysates were adjusted with lysis buffer so that all contained equal amounts of protein. Next, 90 µl of each lysate and 10 µl of 10 mM L-DOPA were pipetted into the wells of a 96-well plate. Control wells containing 90 µl of lysis buffer and 10 µl of 10 mM L-DOPA were also prepared. After incubation at 37℃ for 20 minutes, dopachrome formation was quantified from the absorbance at 475 nm using an ELISA reader.

The direct effect of PS on tyrosinase activity was tested in a cell-free assay system. Briefly, 70 µl of phosphate buffer containing PS was mixed with 20 µl of mushroom tyrosinase (stock solution 53.7 units/ml) and 10 µl of 10 mM L-DOPA. Following incubation at 37℃ for 20 min, the absorbance was measured at 475 nm.

Mel-Ab cells were lysed in a cell lysis buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, 5% β-mercaptoethanol, 2 mM phenylmethylsulfonyl fluoride, and protease inhibitors [Complete™; Roche, Mannheim, Germany], 1 mM Na₃VO₄, 50 mM NaF and 10 mM EDTA). Proteins in the total cell lysates were separated by SDS-polyacrylamide gel electrophoresis using 20 µg of protein per lane, blotted onto polyvinylidene fluoride (PVDF) membranes, and blocked with 5% dried milk in Tris-buffered saline containing 0.5% Tween 20. Blots were then incubated with the appropriate primary antibodies at a dilution of 1:1,000, and then re-incubated with horseradish peroxidase-conjugated secondary antibody. Bound antibody was identified using an enhanced chemiluminescence testing kit (Thermo Scientific Inc., Bremen, Germany). All images of the immunoblots were obtained using an LAS-1000 lumino-image analyzer (Fuji Film, Tokyo, Japan).

The statistical significance of the intergroup differences was assessed by analysis of variance (ANOVA) followed by Student's t-test. All p-values <0.01 were considered significant.

At concentrations between 1 and 50 µg/ml in 24-hr incubations, PS and VS showed no cytotoxicity (Fig. 1). Therefore, dipeptides at concentrations from 1 µg/ml to 20 µg/ml were used for the subsequent experiments.

Mel-Ab cells were treated with PS or VS at 1~20 µg/ml for 4 days. Before measuring the melanin content, the cells were observed under a phase contrast microscope. Melanin pigmentation decreased dose-dependently in PS-treated cells (Fig. 2A). Similarly, cell melanin content was reduced in a dose-dependent manner after treatment with either PS or VS (Fig. 2B, C). Accordingly, the capacity of PS and VS to inhibit tyrosinase directly in a cell-free system was examined, and neither dipeptide showed such an effect (Fig. 2D, E).

We determined the levels of MITF and tyrosinase proteins by western blot analysis during culture of cells with PS or VS (20 µg/ml) for 4 days. MITF protein levels were reduced after 48 to 72 hours of treatment with PS, and tyrosinase levels declined in parallel with the reduction in MITF (Fig. 3A). Treatment with VS also decreased levels of MITF and tyrosinase proteins at 24 hours. Levels of both proteins recovered gradually and returned to the normal range by 72 hours (Fig. 3B). These results provide evidence that PS and VS may decrease melanogenesis by down-regulating MITF and tyrosinase.

Emerging data suggest that ERK phosphorylation leads to MITF degradation by phosphorylation of MITF at serine 73 [6,19]. Therefore, we investigated whether PS- or VS-induced hypopigmentation was related to ERK phosphorylation. Cells were treated with PS or VS (20 µg/ml) for 0~360 min and the whole cell lysates were subjected to western blot analysis. Cellular levels of phospho-ERK increased for at least 30 min after treatment with either PS or VS (Fig. 4). We also measured the phosphorylation of cyclic AMP response element-binding protein (CREB), a transcription factor that promotes melanogenesis through MITF regulation [20,21]. However, neither PS nor VS affected CREB phosphorylation. These results suggest that PS and VS modulate melanogenesis by promoting ERK phosphorylation.