Materials and methods

Materials

8MOP, 1-oleoyl-2-acetylglycerol (OAG), N-2-[p-Bromo-cinnamylamino]ethyl-5-isoquinolinesulfonamide hydrochloride (H89), forskolin, and phorbol 12,13-dibutyrate (PDBu) were purchased from Sigma (St. Louis, MO). 2-[1-(3-(Amidinothio)propyl)-1H-indol-3-yl]-3-1-methylindol-3-ylmaleimide methanesulfonate (Ro31–8220) was purchased from BioMol (Plymouth Meeting, PA). Lactacystin was from Calbiochem (La Jolla, CA). The C5 monoclonal antibody against Mitf was from Lab Vision (Fremont, CA). The PEP7, PEP1, and PEP8 polyclonal antibodies were raised against the carboxyl termini of mouse tyrosinase, Tyrp1, and Dct proteins, respectively (^{Jiménez et al, 1991;Tsukamoto et al, 1992}). Peroxidase-conjugated antimouse and antirabbit antibodies were from Amersham Pharmacia Biotech (Piscataway, NJ).

Cell culture and treatment

Melb-a melanoblasts were a kind gift of Dr. Dorothy C. Bennett (St. George's Hospital, London, U.K.). They were originally derived from C57BL/6 J (black, a/a) mice (^{Sviderskaya et al, 1995}) and were grown in a humidified atmosphere with 10% CO₂ at 37°C. They were routinely passaged in complete RPMI 1640 medium supplemented with 50 nM PDBu, 1 ng basic fibroblast growth factor (bFGF) per ml, 5% fetal bovine serum, and 2 mM L-glutamine, without feeder cells. Cells were harvested by brief treatment with trypsin/ethylenediamine tetraacetic acid (EDTA) (Gibco, Grand Island, NY) and were sedimented and resuspended in complete RPMI 1640 medium. Viable cells, determined by trypan blue exclusion, were counted in a hemocytometer, resuspended in the appropriate volume of complete RPMI 1640 medium, and seeded at 210⁴ cells per cm² in 10 cm cell culture dishes. Stock solutions of 8MOP (100 mM), H89 (10 mM), and Ro31–8220 (10 mM) were prepared weekly in dimethylsulfoxide (DMSO) and were protected from light. Stock solutions were diluted to the appropriate concentrations immediately before use. Melanoblasts were cultured in the presence of the phorbol ester, PDBu, instead of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) because the water-soluble PDBu can be removed by repeated washing. Thus, the influence of residual TPA released from melanoblasts could be avoided (^{Valyi-Nagy et al, 1990}).

To examine the effects of various factors on melanoblasts, PDBu and bFGF were replaced by 100 M 8MOP, 10 M forskolin, or 10 M OAG in the presence of 10% fetal bovine serum. 8MOP was used at 100 M in this study as a previous study by our group showed that this was the maximally effective concentration (^{Lei et al, 2002}); that optimal dose was also confirmed in this study. Ten M forskolin and 10 M OAG are standard concentrations used in the literature to treat melanocytes (^{Hirobe, 1994;Bilodeau et al, 2001}). Equivalent volumes of diluent (DMSO) were added to untreated control dishes. After allowing 1 d for cell attachment, the medium was changed and compounds were added in fresh complete RPMI 1640 medium. Culture media were replaced every 2 d for 6 d (a total of three treatments).

To examine effects of PKA or PKC inhibitors, melanoblasts were pretreated with H89 or Ro31–8220 for 20 min at final concentrations of 1, 5, or 10 M, and then were treated for 6 d in the presence or absence of 100 M 8MOP with H89 or Ro31–8220 at those concentrations, according to previously published protocols (^{Schultz et al, 1997;Rahn Landstrom et al, 2000}).

For evaluating tyrosinase degradation, cells were treated with proteasome inhibitors and/or 8MOP for 1 d or 3 d, as detailed below.

Radiometric assay for tyrosinase activity

Tyrosinase activity was measured as previously described (^{Virador et al, 1999}). Briefly, cells were harvested with trypsin/EDTA, 2 ml of fresh complete RPMI 1640 medium was added to inactivate the trypsin, and 100 l aliquots were seeded into flat-bottom 96-well plates for the MTT assay, as described below. The remainder of the cell suspensions were centrifuged for 5 min at 1500g, washed with Dulbecco's phosphate-buffered saline (PBS) (without Ca and Mg), and then solubilized in 240 l extraction buffer [1% Nonidet P-40, 0.01% sodium dodecyl sulfate (SDS), 0.1 M Tris–HCl, pH 7.2, and protease inhibitor cocktail (Roche, Indianapolis, IN)]. Extracts were solubilized at 4°C for 1 h and 30 l of each extract were added to wells of 96-well microtiter plates in quadruplicate. Ten microliters L-[¹⁴C] tyrosine were added to each well along with 10 l 0.25 mM L-Dopa cofactor in 1 M sodium phosphate buffer, pH 7.2, containing 0.01% albumin. Reactions were incubated for 1–2 h at 37°C, after which 100 l 0.1 M HCl with excess unlabeled L-tyrosine was added to each well. The contents of each well were removed with a multichannel pipettor to a dot-blot apparatus (Bio-Rad, Hercules, CA) and acid-insoluble radioactive melanin and melanin precursors were bound to ZetaProbe blotting membranes (Bio-Rad) for 15 min at 23°C. The membranes were then dried under vacuum and washed three times with 250 l 0.1 M HCl; they were removed from the apparatus and washed three times for 20 min each with 100 ml 0.1 M HCl. Membranes were then air-dried and exposed to a Storm phosphor screen; quantitation of radioactive melanin production on the blots was performed using a Storm 860 PhosphorImager and ImageQuant software (Molecular Dynamics, Rockville, MD).

Measurement of cell proliferation and melanin content

An MTT assay kit (Roche) was used to determine cell proliferation, as detailed previously (^{Virador et al, 1999}). After treatment, 100 l aliquots of harvested cells were plated in flat-bottom 96-well microtiter plates, as described above. Cells were allowed to attach and grow over-night at 37°C before performing the MTT assay, according to the manufacturer's instructions. The formazan precipitates were quantitated by absorbance at 562 nm in a SpectraMax 250 ELISA reader (Molecular Devices, Sunnyvale, CA), with a reference wavelength of 690 nm.

Melanin content was determined using a published protocol (^{Curto et al, 1999}) with minor modifications. The cells were lyzed with 200 l 1 N NaOH and pipetted repeatedly to homogenize the extracts. For analysis, the extracts were then transferred into 96-well plates in duplicate. Relative melanin contents were determined by absorbance at 405 nm using a SpectraMax 250 ELISA reader.

Western blot analysis

Cells were treated as indicated in the figure legends. At the end of each treatment period, cells were washed in PBS and were lyzed in extraction buffer containing 1% Nonidet P-40, 0.01% SDS, and the protease inhibitor cocktail. Protein contents were determined with a BCA assay kit (Pierce, Rockford, IL) and equal amounts of each protein extract (5 g per lane) were resolved using 8% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Following transblotting onto Immobilon-P membranes (Millipore, Bedford, MA) and blocking in 5% nonfat milk in saline buffer, the membranes were incubated with PEP7, PEP1, or PEP8 (each at a 1:2000 dilution) or with the C5 monoclonal antibody (at a 1:500 dilution). The membranes were then incubated with horseradish-peroxidase-conjugated antirabbit or mouse IgG at a dilution of 1:2000. Immunoreactive bands were detected by enhanced chemiluminescence using an ECL kit (Amersham, Piscataway, NJ) and were quantitated using ScionImage software (Scion, Frederick, MD).

RNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR)

To assess the effects of 8MOP or forskolin on mRNA expression, semiquantitative RT-PCR was performed. Total RNA was extracted from cultured cells using an RNeasy total RNA isolation kit (QIAGEN, Valencia, CA) following the supplied protocol. Five hundred nanograms of each RNA sample was treated with RNase-free DNase I (Gibco) containing 1 U RNase inhibitor (Gibco) to remove contaminating DNA. The DNase-treated RNA samples were then reverse transcribed at 50°C for 32 min using the SuperScript One-Step RT-PCR kit (Gibco). The cDNA was amplified in a Perkin-Elmer Cetus thermal cycler using Taq DNA polymerase. PCR was run for Mitf (26 cycles at 96°C for 0.5 min, 66°C for 1.5 min), tyrosinase (30 cycles at 94°C for 0.5 min, 55°C for 1 min, 72°C for 1 min), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (26 cycles at 94°C for 0.5 min, 64°C for 1.5 min) with a final extension at 72°C for 5 min. PCR products were separated on a 1.8% agarose gel containing ethidium bromide (500 ng per ml) and sizes were estimated with 100 bp DNA ladder (InVitrogen, Carlsbad, CA). Oligonucleotide primers used for PCR were based on published mRNA sequences and were as follows: mouse tyrosinase (^{Chakraborty et al, 1999}) (forward) 5'-TTC AAA GGG GTG GAT GAC-3' and (reverse) 5'-GAC ACA TAG TAA TGC ATC-3'; mouse Mitf (^{Powers and Davidson, 1996}) (forward) AGT CAC TAC CAG GTG CAG AC-3' and (reverse) 5'-CTT GCT TCA GAC TCT GTGGG-3'; and mouse GAPDH (^{Ogawa et al, 2000}). With these primer sets, the PCR products for tyrosinase, Mitf, and GAPDH were 341 bp, 569 bp, and 729 bp, respectively. Blots were quantitated using ScionImage software.

Results

8MOP and/or forskolin stimulates melanoblast pigmentation

In this study, we observed that immortalized melb-a melanoblasts could be grown and subcultured in RPMI 1640 medium containing PDBu and bFGF, but in the absence of feeder cells. The addition of PDBu and bFGF sustained the proliferation and phenotype of these murine melanoblasts, and inhibited their pigmentation as previously reported (^{Sviderskaya et al, 1995}). Once they were deprived of those factors, they rarely but occasionally began to spontaneously synthesize melanin. The characteristic appearance of melanoblasts is mainly bipolar, tripolar, polypolar, or dendritic and with small dark nuclei (^{Bennett et al, 1989;Hirobe, 1992}), as seen by phase optics in Figure 1. When melanoblasts were exposed to 8MOP for several days or longer, more of them became pigmented and dendritic compared with untreated controls (the pigmentation is easiest to see in the bright field panels). Forskolin, a cAMP-elevating agent, had a similar stimulating effect on melanoblasts and, when used in conjunction with 8MOP, stimulated their pigmentation more obviously, whereas OAG, a PKC activator, had no such effect. These results show that 8MOP can directly stimulate melanoblast pigmentation in vitro.

Figure 1.

8MOP and/or forskolin stimulates melanoblast pigmentation. Melb-a melanoblasts were cultured for 6 d in RPMI 1640 medium (without PDBu or bFGF) containing 8MOP, forskolin, and/or OAG as noted. Identical fields are shown by phase-contrast (top panels) and by bright-field optics (bottom panels); bar=100 m (5.8 mm). Photographic parameters, such as light levels, were matched for comparability. Control, DMSO vehicle only; 8MOP, 100 M 8MOP; FOR, 10 M forskolin; OAG, 10 M OAG.

Full figure and legend (209K)

8MOP and/or forskolin stimulates the pigmentation but not the proliferation of melanoblasts

We then determined the tyrosinase activity, melanin content, and proliferation rates of melanoblasts exposed to 8MOP, forskolin, and/or OAG. When melanoblasts were treated with 8MOP for 2, 3, or 6 d, significant increases in tyrosinase activity and in melanin content occurred in a time-dependent manner (data not shown). Maximal stimulation of melanogenesis was obtained following treatment with 100 M 8MOP for 6 d (Figure 2). Compared with controls, tyrosinase activity was increased 7-fold and melanin content was increased 3-fold after 6 d of treatment with 8MOP. Forskolin stimulated tyrosinase activity and melanin content 12-fold and 3-fold, respectively, and both parameters of melanogenesis were significantly higher when exposed concomitantly to forskolin and 8MOP for 6 d. In contrast, but consistent with the results shown in Figure 1, OAG did not stimulate tyrosinase activity or melanin content of melanoblasts. In general, none of these agents significantly affected rates of melanoblast growth except forskolin, which stimulated it slightly. Taken together, these results suggest that melanoblast pigmentation is stimulated by 8MOP through activation of the PKA pathway rather than the PKC pathway.

Figure 2.

8MOP and/or forskolin stimulates the pigmentation but not the growth of melanoblasts. Tyrosinase activity, melanin content, and cell proliferation of melanoblasts treated with DMSO (control), 100 M 8MOP, 10 M forskolin (FOR), and/or 10 M OAG for 6 d are shown. Data are reported as n-fold compared with the controls and represent the means (SEM) of three separate experiments. Significance of differences is assessed by the two-tailed and paired Student's test. ^*p< 0.05 from control, ^**p< 0.01 from control, ^***p< 0.001 from 8MOP or FOR.

Full figure and legend (19K)

Tyrosinase expression is increased in melanoblasts exposed to 8MOP and/or forskolin

Melanin synthesis takes place in melanocytes after their differentiation from melanoblasts (^{Bertolotto et al, 1998}). Three melanocyte-specific enzymes, tyrosinase (^{Hearing, 1987}), tyrosinase-related protein 1 (Tyrp1) (^{Jiménez-Cervantes et al, 1994;Kobayashi et al, 1994}), and tyrosinase-related protein 2 (Dct) (^{Jackson et al, 1992}), are involved in catalytic processes that convert tyrosine to melanin. To further characterize those enzymes during 8MOP-induced pigmentation, we examined their expression using Western blot analysis (Figure 3). Tyrosinase in untreated melanoblasts was just barely detectable, but treatment with 8MOP induced significant (6- to 9-fold) increases in tyrosinase within 6 d, which was consistent with the enzyme assays reported above. Furthermore, treatment with forskolin alone or in the presence of 8MOP dramatically enhanced (> 25-fold) levels of tyrosinase, but again, OAG had no effect in the presence or absence of 8MOP. In contrast, the two other melanogenic enzymes, Tyrp1 and Dct, were readily detectable in the untreated controls, and 8MOP (or forskolin or OAG) had little or no effect on those levels. Therefore, these results demonstrate that the de novo synthesis of tyrosinase accounts for the 8MOP stimulation of melanoblast pigmentation, and further suggest that the PKA pathway (and not the PKC pathway) is crucial for that process.

Figure 3.

Tyrosinase, but not Tyrp1 or Dct, expression is stimulated by 8MOP and/or forskolin. Cells were treated for 6 d with 8MOP, forskolin, and/or OAG as noted. Ten micrograms total protein from extracts of untreated cells (control) or from cells treated with 100 M 8MOP, 10 M forskolin (FOR), and/or 10 M OAG were separated on 8% SDS-PAGE gels and transferred to PVDF membranes. Specific detection of tyrosinase, Tyrp1, and Dct was performed with the antibodies PEP7, PEP1, and PEP8, respectively. Molecular masses, indicated on the left, are expressed in kDa. The blots shown are representative of three different experiments that had similar results. Arrows point to the bands of interest, and the number at the bottom of each gel represents densitometric scans of the bands reported as n-fold compared with the controls.

Full figure and legend (49K)

Inhibitors of PKA abrogate the stimulation of melanoblast pigmentation elicited by 8MOP

To further characterize the signaling mechanisms involved in the stimulation of melanoblast pigmentation by 8MOP, we examined the effects of inhibiting the PKA or PKC pathways using potent inhibitors of PKA (H89) or PKC (Ro31–8220) (^{Carsberg et al, 1994;Yoshida et al, 2000}). As expected from the results presented above, 100 M 8MOP significantly stimulated the morphologic changes (Figure 4a), and tyrosinase activity and melanin content (Figure 4b), but had no effect on proliferation (Figure 4b). Treatment with H89, however, suppressed the stimulatory effects of 8MOP on the morphology, tyrosinase activity, and melanin content of melb-a melanoblasts in a dose-dependent manner (Figure 4a, b). In contrast, Ro31–8220 had no significant effect on the stimulation of melanoblasts by 8MOP. Both H89 and Ro31–8220 were toxic at the higher concentrations tested, but at concentrations that were not toxic (1 or 5 M H89 and 1 M Ro31–8220), Western blot analysis showed that H89 significantly suppressed the stimulation of tyrosinase expression by 8MOP, whereas Ro31–8220 had no effect (Figure 4c). Thus, these results support the conclusion that 8MOP stimulates the pigmentation of melanoblasts via the PKA, but not the PKC, pathway.

Figure 4.

Inhibitors of PKA abrogate the stimulation of melanoblast pigmentation by 8MOP. (a) Phenotypic changes of melb-a melanoblasts were observed by phase-contrast (left column) and by bright-field optics (right column) after treatment for 6 d with DMSO only (control) or with 8MOP, with or without H89 or Ro31–8220; bar=100 m (6.6 mm). (b) Tyrosinase activity, melanin content, and cell proliferation of melanoblasts treated with various concentrations of H89 or Ro31–8220 in the absence (open bars) or presence (closed bars) of 100 M 8MOP for 6 d. Data are reported as n-fold compared with the control and represent the means (SEM) of three separate experiments. (c) Western blot analysis of tyrosinase protein in melanoblasts treated for 6 d with agents as noted. Ten micrograms total protein from extracts of cells treated with DMSO only (control) or from cells treated with 100 M 8MOP (8MOP) in the presence or absence of 5 M H89 or 1 M Ro31–8220 (Ro) were separated on 8% SDS-PAGE gels and transferred to PVDF membranes. Specific detection of tyrosinase was performed with the antibody PEP7. The arrow points to the band of interest, and the number at the bottom of each gel represents densitometric scans of the bands reported as n-fold compared with the control. The results are representative of two experiments.

Full figure and legend (95K)

8MOP and/or forskolin rapidly stimulate Mitf mRNA and protein expression compared with tyrosinase

Earlier studies showed that the Mitf locus encodes a helix–loop–helix (HLH) transcription factor that plays a pivotal role in determining melanocyte survival, development, and differentiation (^{Hodgkinson et al, 1993;Busca and Ballotti, 2000;Tachibana, 2000}). Thus, we examined whether Mitf expression is involved in the stimulation of melanoblast pigmentation by 8MOP and/or forskolin. mRNA and protein levels for Mitf and tyrosinase were assessed using semiquantitative RT-PCR and Western blot analysis, respectively. Incubation with 8MOP and/or forskolin elicited increases in Mitf mRNA (Figure 5) within 3 h, which were maximized at 6 h. Levels of Mitf protein were similarly increased in response to 8MOP and/or forskolin (Figure 6) within 3–6 h. Mitf protein levels had returned to baseline levels by 6 d, as had Mitf mRNA levels. In a similar but delayed fashion, 8MOP and/or forskolin had a detectable effect on tyrosinase expression at the mRNA and protein levels, but only at 1 d and thereafter. Thus, our observations show that treatment of melanoblasts with 8MOP and/or forskolin leads to a rapid, transient increase in Mitf mRNA and protein. Similarly, 8MOP and/or forskolin elicited dramatic increases in tyrosinase mRNA and protein that were detectable after 1 d of treatment.

Figure 5.

Mitf mRNA expression is rapidly increased by 8MOP and/or forskolin whereas induction of tyrosinase mRNA expression is slower, as assessed by semiquantitative RT-PCR. Specific primers, as described in Materials and Methods, were used to amplify cDNA obtained from melanoblasts following treatment with DMSO solvent only, 100 M 8MOP, and/or 10 M forskolin (FOR) for 3 h, 6 h, 1 d, 3 d, or 6 d, as noted. Amplification products of tyrosinase (341 bp), Mitf (569 bp), and GAPDH (729 bp) were separated on 1.8% agarose gels; a 100 bp DNA ladder was used as markers. Results are representative of three independent experiments that had similar results. Bands of interest are identified by arrows and the numbers at the bottom of the blots represent densitometric scans of the bands calculated as n-fold against the relevant control.

Full figure and legend (113K)

Figure 6.

The kinetics of Mitf and tyrosinase protein expression are similar to mRNA expression following treatment with 8MOP and/or forskolin. Melb-a melanoblasts were harvested at 3 h, 6 h, 1 d, 3 d, or 6 d following treatment with solvent only (control), 100 M 8MOP, and/or 10 M forskolin (FOR). Ten micrograms of total protein were electrophoresed in 8% SDS-PAGE gels and transferred to PVDF membranes. Specific detection of Mitf and tyrosinase was then performed using the C5 and PEP7 antibodies, respectively, as detailed in Materials and Methods. The C5 monoclonal antibody recognizes two isoforms of the Mitf protein in melb-a cell lysates, the 52 kDa and the 56 kDa isoforms, which represent the inactive (unphosphorylated) and the active (phosphorylated) isoforms, respectively (noted with arrows). The doublet band seen at 69–70 kDa probably represents a nonspecific reactivity. The PEP7 antibody also recognizes two forms of tyrosinase, the lower molecular weight form (65 kDa) being the de novo synthesized protein and the higher molecular weight form (68 kDa) representing the glycosylated and processed forms (noted with arrows). Molecular masses, indicated on the left, are expressed in kDa. The blots shown are representative of three different experiments with comparable results. The numbers at the bottom of each gel represent densitometric scans of the bands of interest calculated as n-fold compared with the relevant control.

Full figure and legend (114K)

Inhibiting proteasome activity mimics the effect of 8MOP on tyrosinase

Degradation of tyrosinase by proteasomes has been recently reported to regulate the level of melanin production at the post-transcriptional level (^{Halaban et al, 1997;Ando et al, 1999;Toyofuku et al, 2001}). To assess whether the inhibition of tyrosinase degradation by proteasomes might contribute to the increased pigmentation of melanoblasts elicited by 8MOP, we used Western blot analysis to examine the effects of the known proteasome inhibitor, lactacystin, on tyrosinase degradation in the presence or absence of 8MOP. Treatment of melanoblasts with lactacystin alone for 1 d elicited an increase in tyrosinase protein levels compared with the control (Figure 7). Treatment with lactacystin and 8MOP led to even greater levels of tyrosinase, which was more than the increase elicited by 8MOP alone. More dramatic effects were seen at 3 d, and it should be noted that the melb-a melanoblasts tolerated these reagents quite well, remaining viable and proliferative throughout the time course of these experiments.

Figure 7.

Inhibiting proteasome activity mimics the effect of 8MOP on tyrosinase protein. Melanoblasts were treated for 1 d or 3 d with agents as noted. Ten micrograms total protein from extracts of cells treated with DMSO only (control) or from cells treated with 100 M 8MOP (8MOP) and/or 30 M lactacystin (LC) were separated on 8% SDS-PAGE gels and transferred to PVDF membranes. Tyrosinase protein was detected with the PEP7 antibody, as detailed in Materials and Methods. Molecular masses, indicated on the left, are expressed in kDa. The blot shown is representative of two different experiments. The numbers at the bottom of each gel represent densitometric scans of the bands calculated as n-fold compared with the relevant control.

Full figure and legend (30K)

Discussion

These data clearly demonstrate that 8MOP can stimulate melanoblast pigmentation by increasing the expression of tyrosinase, which is accompanied by upregulation of tyrosinase catalytic function and melanin production. Melb-a melanoblasts are immortalized cells derived from neonatal black mouse skin, which lack a functional tyrosinase catalytic activity. We show in this study that when unpigmented melanoblasts are exposed to 8MOP, many of them become pigmented, but with no effect on rates of cell growth. Tyrosinase catalytic function increased about 7-fold within 6 d and melanin content increased about 3-fold compared with untreated controls. Interestingly, these cells express other components of the melanogenic machinery, such as Tyrp1 and Dct, and levels of those proteins are relatively unaffected by treatment with 8MOP. Because tyrosinase is the rate-limiting enzyme in melanin production, its function is really the critical factor regulating pigmentation. Melanocyte stimulating hormone and UV light are well-known stimulants of melanocyte function, and interestingly, both of them also have their primary effects on expression of tyrosinase, whereas Tyrp1 and Dct are relatively unaffected (^{Jiménez et al, 1988;Abdel-Malek et al, 1995;Mengeaud and Ortonne, 1996}).

The PKA and PKC signal pathways have been shown to play important roles in regulating melanogenesis and the proliferation of melanocytes in vitro (^{Hirobe, 1992;Mengeaud and Ortonne, 1994}). Therefore, we addressed the question of whether the PKA and/or the PKC pathways were involved in regulating the stimulation of melanoblast pigmentation by 8MOP. In this study, we found that tyrosinase activity and melanin production of melanoblasts exposed concomitantly to forskolin (a cAMP-elevating agent) and 8MOP was higher than in cells treated with 8MOP alone or with forskolin alone. In contrast, OAG (an activator of the PKC pathway) did not stimulate the pigmentation of melanoblasts, suggesting that the PKC pathway is not critically important in that response. Similar results were obtained from Western blot analysis; 8MOP significantly increased tyrosinase protein levels in melanoblasts following exposure to 8MOP after 1 d. This striking stimulation by 8MOP was further enhanced by forskolin, but not by OAG. Inhibitors of the PKA or PKC pathway substantiated those effects. Collectively, our results show that the PKA-dependent signal pathway is involved in the stimulation of melanoblast pigmentation by 8MOP, and that the PKC signal pathway is not involved in this process.

In recent years, there has been increasing evidence that Mitf plays a pivotal role as a master gene that regulates melanocyte development, survival, and differentiation (^{Busca and Ballotti, 2000;Tachibana, 2000}). Mitf is a basic HLH transcription factor containing a leucine-zipper domain. The basic region is responsible for the DNA binding, whereas the HLH motif and the leucine-zipper domain are involved in protein–protein interactions. Studies on the cell-specific expression of tyrosinase have shown that the M-box in the upstream promoter region of the tyrosinase gene is required for its efficient expression in melanocytes (^{Busca and Ballotti, 2000;Tachibana, 2000}). This motif contains a canonical CANNTG motif that is recognized and bound by Mitf. To gain more insight into what roles might be played by Mitf in the melanoblast response to 8MOP, we used Western blot analysis and semiquantitative RT-PCR to assess the protein and mRNA expression levels of Mitf in melanoblasts treated with 8MOP for varying times. Treatment with 8MOP rapidly increased Mitf mRNA and protein levels within 3–6 h. In contrast, tyrosinase mRNA and protein had delayed responses to 8MOP, and took 1 d or longer to show increased levels. A reasonable interpretation of these results is that 8MOP increases Mitf expression, which in turn activates tyrosinase gene expression, and this occurs via activation of the PKA signaling pathway. The involvement of proteasomes in regulating tyrosinase levels has been recently shown (^{Halaban et al, 1997;Ando et al, 1999;Toyofuku, 2001}) and our data further supports that. Proteasomes have also been shown to be important to the regulation of Mitf function (^{Busca and Ballotti, 2000;Wu et al, 2000;Xu et al, 2000}), and the effects noted in this study may well be regulated at the level of effects on Mitf. As is shown in this study, even untreated melanoblasts have ample levels of Mitf mRNA but levels of Mitf protein are negligible in those cells. We hypothesize that the increase in Mitf protein (which would lead to increased tyrosinase expression) that is elicited by 8MOP may well depend on its effects on proteasome function, and future study will naturally be directed toward further elucidating that possibility. The sum of these results suggests that proteasomes may be intimately involved in regulating the pigmentation of melanoblasts in response to 8MOP at several levels.

Previous studies have demonstrated that there are specific binding sites for psoralens (psoralen receptors) located at the mammalian cell membrane (^{Laskin et al, 1986}) and that psoralens can elevate intracellular cAMP levels (^{Mengeaud and Ortonne, 1994}). It was therefore suspected that 8MOP might modulate Mitf expression through binding to a psoralen receptor and activating the PKA signal pathway to elicit melanoblast pigmentation in vitro. These results therefore support a potential mechanism whereby 8MOP activates epidermal melanoblasts, which may be involved in the response of vitiligo skin to PUVA therapy.

In conclusion, this study demonstrates that 8MOP can stimulate melanoblast pigmentation by increasing the expression of Mitf and by subsequently increasing the stability and enzymatic function of tyrosinase. The PKA signal pathway appears to be involved in the stimulation of melanoblast pigmentation by 8MOP, whereas the PKC signal pathway seems to be relatively unimportant to this process. New advances in understanding the molecular mechanisms of repigmentation induced by psoralens should be useful in the future to develop new treatments or drugs for vitiligo. This should lead to an effective approach for treating vitiligo, via activation of precursor melanocytes in the ORS of hair follicles.

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Acknowledgments

We would like to thank Dr. Yuji Yamaguchi for quantitating the gel bands in this study.