An immortal line of melanocytes, melan-a, were a kind gift of Dr D.C. Bennett (21). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Gibco, USA), 2 mM L-glutamine, 200 nM 12-o-tetradecanoyl phorbol-13-acetate (Sigma, USA), 100 IU/ml penicillin, 50 μg/ml streptomycin, and grown in a humidified atmosphere containing 10% CO₂ in air at 37°C.

The adenoviruses expressing green fluorescent protein (AdGFP), Wnt3a protein (AdWnt3a, also expressing GFP), and SimMITF (AdSimMITF, a small interfering RNA of MITF mediated by adenovirus, also expressing RFP) were kindly provided by Dr T.C. He (Chicago University). The adenoviruses were propagated and purified as previously described (22). Briefly, the adenoviruses were propagated in HEK293 cells, which were collected upon detection of viral cytopathic effect-successful infections were verified by observable GFP incorporation. Cell pellets were resuspended in PBS and lysed by four freeze-thaw-vortex cycles. After being purified by cesium chloride (Amresco, USA) density gradient centrifugation, adenoviruses were dialyzed into storage buffer, then their titers were determined and diluted with storage buffer to the ultimate titer of 10⁸ plaque-forming unit (PFU)/ml. For infection, melan-a cells were plated onto 6- or 24-well plates at a density of 2×10⁴ cells/cm² in the growth medium for 12 h, the cells were then grown in medium supplemented with adenoviruses for 72 h.

Total cellular RNA was isolated at the indicated time-points using TRIzol reagent (Invitrogen, USA). Single-stranded cDNA was synthesized by using ReverTra Ace reverse transcriptase (Toyobo, Japan) and oligo(dt) primers according to the manufacturer’s protocol. Semi-quantitative PCR was performed using primers for Fzd1-10, LRP5, LRP6, MITF, tyrosinase-related protein (TRP)1, TRP2, tyrosinase (Table I lists the primer sequences and amplicon size). PCR reaction was performed by using a touchdown protocol previously described (23). Briefly, touchdown PCR was performed with the following program: 1 cycle at 94°C for 2 min, 12 cycles at 92°C for 20 sec, 68°C for 30 sec, and 70°C for 45 sec with a decrease of one degree per cycle, and 25 cycles at 92°C for 20 sec, 55°C for 30 sec, and 70°C for 45 sec. PCR products were analyzed by gel electrophoresis and stained with ethidium bromide.

Melan-a cells were seeded onto 24-well plates overnight and subsequently infected with AdWnt3a or AdGFP. After 12 h, the cells were transfected with TOP/FOP-flash luciferase reporter plasmid. For each well, 3 μg TOP- or FOP-Flash Firefly Luciferase reporter plasmid, which contain several TCF4-binding elements (TOP) or mutant sequences (FOP), respectively, were transfected together with 0.03 μg phRG-TK Renilla Luciferase standard plasmid (Promega, USA). The transfection reagent, Lipofectamine 2000 (Invitrogen), was used according to the standard protocol. Cell lysates were harvested 24 h after transfection and the Dual-Luciferase Reporter Assay System (E1910; Promega) was used to detect luciferase activity according to the manufacturer’s protocol. The experiments were performed in triplicate.

To study the effect of Wnt3a on melan-a proliferation, 2×10⁴ melan-a cells were plated onto 24-well plates overnight and grown in culture medium supplemented with AdWnt3a at various doses (1, 2, 3 μl) or 2 μl AdGFP as the vehicle control. After 72 h, the cells were detached with trypsin and counted in a hemocytometer. For the MTT assay, melan-a cells were plated onto 96-well plates and treated with AdWnt3a at various doses (0.2, 0.5, 0.8 μl) or 0.5 μl AdGFP for 72 h. Then MTT (Sigma) was added, and the cells were incubated at 37°C for 4 h. The medium was removed and dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. The absorbance was then measured at 490 nm with a common ELISA reader.

Melan-a cells were plated onto glass coverslips and were treated with AdWnt3a or AdGFP. After 72 h, 5-bromodeoxyuridine (BrdU, Sigma; stock of 10 mM in PBS) was added at 10 μM. The cells were then incubated at 37°C for 2 h and fixed in cold acetone for 10 min. After washing three times in PBS, the cells were incubated in 2 M HCl for 45 min to denature the DNA and then neutralized with 0.1 M Na₂B₄O₇ (pH 8.5) for 30 min. The detection of BrdU was performed with a mouse anti-BrdU antibody (1:100; Zhongshan, China) at 4°C overnight. Then, cells were incubated with goat anti-mouse FITC-conjugated secondary antibody (1:100; Zhongshan) at 37°C for 1 h. After washing, the cells were stained with DAPI for 10 min at room temperature. Six areas/well were randomly selected and counted with an upright microscope BH2 (Olympus, Japan).

Melan-a cells were plated onto 6-well plates and treated with 2 μl AdWnt3a or 2 μl AdGFP for 72 h. Afterward, cells were dissociated with trypsin, washed in PBS, and fixed in 1 ml of 70% methanol at 4°C for at least 24 h. The fixed cells were washed twice in PBS, treated with 100 μg/ml RNase and 50 μg/ml propidium iodide (PI) in PBS for 30 min at 37°C, and the cell cycle was detected with a flow cytometer. All proliferation assays were performed in triplicate.

Tyrosinase activity assays were performed according to the method previously reported (24). Melan-a cells in 6-well plates were infected with AdWnt3a or AdGFP for 72 h, then trypsinized and counted. Cells (1×10⁵) were treated with 200 μl of 1% Triton X-100/PBS at −70°C for 30 min and thawed at 37°C. Then, the extracts were clarified by centrifugation, 50 μl of the supernatant were transferred into 96-well plates and 10 μl of 2 mg/ml L-DOPA (Sigma) were added. After incubation for 2 h at 37°C, absorbance was measured at 490 nm. The experiments were performed at least three times.

Cells were lysed in RIPA lysis buffer (Beyontime, China), determined by the Enhanced BCA Protein Assay kit (Beyontime) and denatured by boiling. Protein of 80 μg per lane was loaded on 10% SDS-PAGE and then transferred onto a PVDF membrane. Membranes were blocked with 5% fat-free milk in Tris-buffered saline-Tween-20 (TBST; 20 mM Tris/HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20) for 2 h, then membranes were probed with rabbit anti-Wnt3a antibody (1:1,000; Abcam, USA), goat anti-TRP1 antibody (1:1,000), rabbit anti-TRP2 antibody (1:1,000), goat anti-tyrosinase antibody (1:1,000; Santa Cruz Biotechnology, Inc., USA), and mouse anti-MITF antibody (1:500, Millipore, USA) at 4°C overnight. Blots were then incubated with HRP-conjugated secondary antibody. Peroxidase activity on the membrane was visualized on X-ray film by means of the ECL western blotting detection system.

Data were presented as means ± SD for the three independent experiments. Statistical differences were evaluated by the t-test, and P<0.05 was considered to be statistically significant.

To explore the potential role of Wnt3a in mouse mature melanocytes, we used a mouse melanocyte cell line, melan-a (21), as an in vitro cell model. DOPA staining was used to confirm that the cells were indeed melanocytes (Fig. 1A). RT-PCR analysis showed that the Wnt receptors Fzd1 to Fzd10 and the co-receptors LRP5 and LRP6 were all expressed in melan-a cells, indicating that melan-a cells could receive and transduce Wnt signals (Fig. 1E). We infected melan-a cells with AdWnt3a at a predetermined optimal titer. As observed in Fig. 1B, AdWnt3a mediated efficient gene transfer in melan-a cells. The expression of Wnt3a was confirmed by western blot analysis in AdWnt3a-infected cells but not in AdGFP-infected cells (Fig. 1C).

To evaluate the ability of AdWnt3a to activate Wnt/β-catenin signaling, western blot analysis and the TOP/FOP-flash luciferase reporter assay were performed. As shown in Fig. 1C, AdWnt3a upregulated the expression of β-catenin at the protein level. We infected melan-a cells with AdWnt3a or AdGFP and co-transfected with either the β-catenin responsive TCF4 reporter plasmid (TOP-flash) or with negative control reporter plasmids (FOP-flash). The luciferase activities showed that AdWnt3a-infected cells displayed higher ratios of TOP-flash/FOP-flash compared to AdGFP-infected cells (Fig. 1D). The data demonstrate that AdWnt3a efficiently activates Wnt/β-catenin signaling in melan-a cells.

To analyze the effect of Wnt3a on melan-a cell proliferation, we infected melan-a cells with different doses of AdWnt3a or AdGFP as control. After 72 h, the MTT cell proliferation assay and manual cell count both showed that Wnt3a inhibited the proliferation of melan-a cell in a dose-dependent manner compared to GFP (Fig. 2C and D).

BrdU is a synthetic thymidine analog that gets incorporated into a DNA cell when the cell is dividing, so it is commonly used for the detection of proliferating cells. We measured the BrdU incorporation of AdWnt3a-infected melan-a cells and AdGFP-infected cells. The incorporated BrdU was detected by using the anti-BrdU antibody (Fig. 2A). The percentage of BrdU positive cells in AdWnt3a-infected cells was decreased by 14.9% (from 32.7±4.45 to 17.8±1.32%, P<0.05) (Fig. 2B), which implied that Wnt3a inhibited the proliferation of melan-a cells.

We also performed cell cycle analysis and found that AdWnt3a-infected cells exhibited an increased population in the G₁ phase and a decreased population in the S phase compared to AdGFP-infected cells (Fig. 3).

Taken together, these results showed that Wnt3a inhibited melan-a cell proliferation and this was associated with decreased population of cells in the S phase and an increase in the G₁ phase.

We analyzed the effect of Wnt3a on the melanogenesis of melan-a cells by assessing melanin production and tyrosinase activity. Although almost all the cells contained melanin pigment, AdWnt3a-infected cells showed obvious higher level of melanin accumulation compared to AdGFP-infected cells (Fig. 4A and B).

To analyze whether melanin synthesis is activated via a direct effect on tyrosinase, tyrosinase activity assay was adopted to analyze tyrosinase activity in melan-a cells. As shown in Fig. 4C, a greatly significant increase of tyrosinase activity was detected in AdWnt3a-infected cells compared with AdGFP-infected cells.

To investigate how Wnt3a promotes melanin synthesis, we studied the expression of microphthalmia-associated transcription factor (MITF), the transcriptional master regulator of melanocytes, and its downstream target genes, including tyrosinase, TRP1, and TRP2 (25–27). Western blot analyses showed that Wnt3a significantly increased the expression levels of MITF, tyrosinase, and TRP1 in melan-a cells, but did not affect the expression of TRP2 (Fig. 5). Subsequently, we infected melan-a cells with AdSimMITF to knockdown the endogenous MITF. According to western blot analysis, the expression of MITF, and its downstream target genes, tyrosinase and TRP1, were dramatically decreased in the cells infected with AdSimMITF (Fig. 6). Co-infection of melan-a cells with AdSimMITF and AdWnt3a demonstrated that Wnt3a rescued the expression of MITF, tyrosinase and TRP1 (Fig. 6).

These results suggested that Wnt3a contributed to increase melanin synthesis through upregulation of the expression of MITF and its downstream genes, tyrosinase and TRP1.