Scopoletin Stimulates Melanogenesis via cAMP/PKA Pathway and Partially p38 Activation

Abstract

Scopoletin was recently shown to stimulate melanogenesis through cAMP-response element-binding protein (CREB) phosphorylation. In this study, we investigated the molecular events of melanogenesis-induced by scopoletin. After exposure to scopoletin, the protein levels of tyrosinase and tyrosianse related protein-1 (TRP-1) were significantly increased in B16F10 cells. The mRNA levels of tyrosinase and microphthalmia-associated transcription factor (MITF) were also enhanced by scopoletin. cAMP production and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were increased by scopoletin treatment. Scopoletin-mediated increase of intracellular melanin and tyrosinase expression were significantly attenuated by protein kinase A (PKA) inhibitors (H-89 and KT5720), while a protein kinase C (PKC) inhibitor (Ro-32-0432) had no effect and a p38 MAPK inhibitor (SB203580) partially blocked the scopoletin-induced intracellular melanin and tyrosinase expression. Moreover, scopoletin synergistically with cell-permeable cAMP analog (dibutyryl cAMP) significantly induced tyrosinase activity and melanin content in B16F10 cells. The silencing of p38 MAPK by small interfering RNA (siRNA) decreased the scopoletin-induced tyrosinase expression in B16F10 cells. These results suggest that scopoletin could induce melanin synthesis through the cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

Melanin is synthesized in organelles called melanosome and melanosomes are transferred to adjacent keratinocytes. In human, skin hyper-pigmentation is physiologically stimulated by UV radiation.¹⁾ Melanin has a photo-protective effect against harmful UV injury in human skin. Melanin synthesis is mainly regulated by melanogenic enzymes such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2. At transcription level, the expression of melanogenic enzymes is up-regulated by a binding of microphthalmia-associated transcription factor (MITF) and tyrosinase promoter.^2–4) cAMP-responsive element binding protein (CREB) regulates in turn the expression of MITF. Phosphorylation of CREB is regulated by activation of cAMP and protein kinase A (PKA).^5–7)

Abnormal pigmentation conditions can be divided into two types, that is hypermelanosis or hypomelanosis, which involve excessive or insufficient melanin in skin. Many exogenous and endogenous factors are involved in melanin synthesis through intracellular signaling pathways. Especially, cAMP and protein kinase C (PKC) are important factors for melanogenesis pathways.⁷⁾ Downstream signaling of the cAMP pathway during melanogenesis is well-documented. The activation of p38 mitogen-activated protein kinase (MAPK) pathway has been recently shown to induce MITF and tyrosinase expression.⁸⁾ In order to control the abnormal pigmentation conditions, the molecular mechanisms for melanogenesis should be clearly identified.

Scopoletin, 6-methoxy-7-hydroxycoumarin (C₁₀H₈O₄, Fig. 1), is a derivative of coumarin. Some studies have reported that scopoletin has various biological activity including anti-allergy, anti-inflammatory, and antioxidant activity.^9–11) Recently, scopoletin has been demonstrated to activate CREB phosphorylation and tyrosinase expression, which lead to the stimulation of melanin synthesis.¹²⁾ However, the signaling pathway underlying scopoletin-induced melanogenesis are not yet well defined. In this study, we investigated the molecular mechanism of scopoletin-mediated melanogenesis including cAMP, PKC, and p38 MAPK.

Fig. 1. Chemical Structure of Scopoletin (6-Methoxy-7-hydroxycoumarin, C₁₀H₈O₄)

MATERIALS AND METHODS

Cell Cultures and Cell Viability Assay

B16 melanoma (B16F10) cells and human dermal fibroblast neonatal (HDFn) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum at 37°C, 5% CO₂. The viability of cells was determined using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT). The cells (3×10⁴ cells/well) were seeded in 24-well plates and treated with scopoletin (S2500, Sigma, U.S.A.) at various concentrations (0–40 µg/mL) for 24 h. After the exposure period, the medium was changed and incubated with MTT (0.1 mg/mL) for 3 h. The number of viable cells per dish is directly proportional to the production of formazan, which was dissolved in dimethyl sulfoxide (DMSO), and measured spectrophotometrically at 570 nm.

Measurement of Intracellular Melanin and Tyrosinase Activity

Intracellular melanin content of B16F10 cells were measured according to the slightly modified method.¹³⁾ The colors of cell pellets were visually observed, and pellets were solubilized in 1 M NaOH containing 10% DMSO at 90°C for 1 h. Spectrophotometric analysis of melanin content was performed at 405 nm absorbance. Tyrosinase activity was determined using a modification of the method described.¹⁴⁾ Cells (8×10⁴ cells/well) were cultured at 6-well plates and treated with scopoletin (0–40 µg/mL). After washing, cells were lysed in 200 µL of 0.1 M sodium phosphate buffer (pH 6.8) containing 1% Triton X-100 and 1 M phenylmethylsulfonyl fluoride (PMSF). The supernatant (50 µL), 100 µL of 0.1 M sodium phosphate buffer (pH 6.8) and 50 µL of 0.1% L-3,4-dihydroxyphenylalanine (L-DOPA) were placed into a 96-well plate. Absorbance at 405 nm was read every 30 min for 1 h at 37°C using an enzyme-linked immunosorbent assay (ELISA) plate reader.

L-DOPA Staining

To evaluate L-DOPA reactivity of B16F10 cells, cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 10 min, after treatment with 100% methanol for 10 min. Cells were incubated in L-DOPA (1 mg/mL) for 4 h at 37°C prior to observation using a microscope (Leica, Germany).

Analyses Using Western Blot and RT-PCR

After treatment with scopoletin for 72 h, cells were lysed with RIPA buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.15 NaCl, 0.01 M sodium phosphate (pH 7.2), 2 mM ethylenediaminetetraacetic acid (EDTA), 50 mM sodium fluoride, 0.2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, and 1 µg/mL leupeptin) for 30 min on ice. Protein concentrations of the supernatants were estimated by Bradford assay. Equal amounts of protein from samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. Proteins were probed with tyrosinase, TRP-1, TRP-2, MITF, and phosphor-p38 antibodies (Santa Cruz Biotechnology, U.S.A.). The membranes were probed with a specific horseradish peroxide (HRP)-conjugated secondary antibody and detected using the enhanced chemiluminescent substrate from West zol-plus. The membrane was probed with β-actin primary antibody as a protein loading control.

Total RNA was isolated from B16F10 cells using a Fast Pure RNA kit (TaKaRa Bio, Japan). The first-strand cDNA was synthesized from 1 µg of total RNA with Maxime RT PreMix Oligo dT primer (iNtRON Biotechnology, Korea). The following primers were used for amplification: Tyrosinase, forward 5′-ATT GAT TTT GCC CAT GAA GC-3′ and reverse 5′-GGC AAA TCC TTC CAG TGT GT-3′, TRP-1, forward 5′-GTC ATT GCC ACA AGG AGG TT-3′ and reverse 5′-CCC AGT TGC AAA ATT CCA GT-3′, TRP-2, forward 5′-TGT GCA AGA TTG CCT GTC TC-3′ and reverse 5′-GTT GCT CTG CGG TTA GGA AG-3′, MITF, forward 5′-AGC GTG TAT TTT CCC CAC AG-3′ and reverse 5′-CCT TAG CTC GTT GCT GTT CC-3′, B-actin, foward; 5′-TCA GAA GGA CTC CTA TGT GG-3′ and reverse 5′-TCT CTT TGA TGT CAG CAC G-3′. PCR products were electrophoresed on 2% agarose gels containing ethidium bromide (EtBr).

Radioimmunoassay of cAMP

Intracellular cAMP level was assayed in B16F10 cells with scopoletin or α-melanocyte-stimulating hormone (α-MSH) (10 nM). In order to inhibit phosphodiesterase activity, cells were lysed in 0.1 M of HCl. The supernatants were extracted with two volumes of water-saturated diethylether and concentrated with Speed-vac concentrator (Savant Instrument, U.S.A.). cAMP content was measured by an equilibrated radioimmunoassay as described previously.¹⁵⁾ Standards or samples were introduced in a final volume of 100 µL of 50 mM sodium acetate buffer (pH 4.8), added 100 µL of diluted cAMP antiserum (1 : 1000, Calbiochem-Novabiochem Co., U.S.A.) and iodinated cAMP (10000 cpm/100 µL), and incubated overnight at 4°C. The bound form was separated from the free form by charcoal suspension. Results were discribed as femtomoles of cAMP generated per microgram of protein (fmol/µg of protein).

Gene Silencing by Small Interfering RNA (siRNA) Transfection

The siRNA of p38 MAPK (Ambion, U.S.A.) was transfected into B16F10 cells with Lipofectamine (Invitrogen, CA, U.S.A.) according to the manufacturer’s protocol. Briefly, the cells were grown to 50% confluence in antibiotic-free medium, then the medium was replaced with Opti-MEMTM followed by the transfection with p38 MAPK siRNA or control siRNA using lipofectamine. The cells were replaced with growth medium after transfection for 24 h. After scopoletin treatment, the cells were then subjected to Western blot assay.

Statistical Analysis

The means±standard deviation (S.D.) of the values were calculated; statistical analysis of results was performed using Student’s t-test for independent samples. Values of * p<0.05, and ** p<0.01 were considered significant.

RESULTS

Scopoletin Induced Melanin Synthesis via Up-Regulation of Tyrosinase Activity and MITF

We first confirmed the effects of scopoletin on intracellular melanin content and tyrosinase activity. When B16F10 cells were treated with 20 or 40 µg/mL of scopoletin for 72 h, tyrosinase activity was significantly increased in a dose-dependent manner (Fig. 2A). The color of cell pellets was darker after being treated with scopoletin. Accordingly, intracellular melanin was enhanced by scopoletin when examined by microscopy after DOPA staining (Fig. 2B). α-MSH was used as a positive control.

Fig. 2. Effects of Scopoletin on Tyrosinase, Melanin Content and MITF

Cells were treated with scopoletin at 20 and 40 µg/mL, or 10 nM α-MSH for 72 h. At the end of treatment, tyrosinase activity (A) was measured as Materials and Methods. (B) Intracellular melanin was observed by color of cell pellets and examined by a light microscope after DOPA staining. (C) The expression of tyrosinase, TRP-1, and TRP-2 protein were detected by Western blotting using specific antibodies. (D) The transcript of tyrosinase and MITF were detected by RT-PCR. Results are the mean±S.D. from three independent experiments. ** p<0.01 versus untreated cells.

The effect of scopoletin on the expression of melanogenic enzymes was determined by Western blot analysis. Scopoletin significantly induced tyrosinase and TRP-1 protein levels in a dose-dependent manner (Fig. 2C). mRNA level of tyrosinase was increased after scopoletin treatment, indicating that the induction of this gene expression was occurred at transcription level. As a key transcription factor for melanogenic proteins is MITF, we examined whether MITF is also involved in the scopoletin-induced tyrosinase protein expression. As expected, the mRNA level of MITF was increased by scopoletin (Fig. 2D).

Scopoletin Induced cAMP Production and p38 MAPK Activation

Previous studies have demonstrated that α-MSH activates the cAMP/PKA pathway, which in turn up-regulates MITF transcript to enhance melanin synthesis.¹⁶⁾ To examine the molecular mechanisms involved in the melanogenic effect of scopoletin, we first compared scopoletin-mediated cAMP production with α-MSH-mediated response. After treatment with 40 µg/mL of scopoletin, cAMP production was increased in B16F10 cells (Fig. 3A). Induction of cAMP was observed as early as 15 min after scopoletin treatment and it was reached a peak at 30 min. A similar effect on cAMP production was observed in α-MSH treated cells (Fig. 3B). This result suggests that scopoletin induces melanin synthesis through an elevation of cAMP levels.

Fig. 3. Effect of Scopoletin on Intracellular cAMP Level

Intracellular cAMP levels were determined using a radioimmunoassay. B16F10 cells were treated with scopoletin 40 µg/mL (A) and α-MSH 1 nM (B). Results are the mean±S.D. from triplicate determination. ** p<0.01 versus untreated cells. I.T.: Incubation time.

Next, we examined whether scopoletin could induce the phosphorylation of p38 MAPK. Scopoletin significantly elevated the phosphorylation of p38 MAPK in B16F10 cells (Fig. 4). This result indicates that p38 MAPK activation contributes to the melanogenic effect of scopoletin.

Fig. 4. Effect of Scopoletin on p38 Phosphorylation

After incubation of B16F10 cells with 40 µg/mL scopoletin for the indicated time periods, whole-cell lysates were subjected Western blot analysis using specific antibody against phosphor-p38. Equal protein loadings were confirmed using β-actin antibodies.

Scopoletin Stimulated Melanin Synthesis through cAMP/PKA Pathway and Partially Activation of p38 MAPK

Using selective inhibitors, including H-89 and KT5720 (PKA inhibitors), Ro-32-0432 (PKC inhibitor), and SB203580 (p38 MAPK inhibitor), we further investigated the molecular mechanism by which scopoletin enhances melanogenesis. Melanin content increased by scopoletin was completely decreased by H-89 and KT5720, while Ro-32-0432 had no effect and SB203580 partially blocked the scopoletin-induced melanin synthesis (Figs. 5A, B). Also, the level of tyrosinase protein was completely suppressed by H-89 and KT5720, but its expression level was not altered by Ro-32-0432 and partially blocked by SB203580 (Figs. 6A, B). Furthermore, we investigated whether cell-permeable cAMP analog, dibutyryl cAMP (dbcAMP), could mimic the effect of scopoletin on melanogenesis. dbcAMP increased tyrosinase activity and melanin content in B16F10 cells. Scopoletin synergistically with dbcAMP significantly induced tyrosinase activity and melanin content (Fig. 7). The contribution of p38 MAPK to melanogenic effect of scopoletin was tested by introducing p38 MAPK targeting siRNA into B16F10 cells. The silencing of p38 MAPK by siRNA decreased the scopoletin-induced tyrosinase expression in B16F10 cells (Fig. 8). These results suggest that scopoletin stimulated melanin synthesis through cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

Fig. 5. Effect of Various Kinase Inhibitors on Scopoletin-Enhanced Melanin Synthesis

Cells were incubated with or without scopoletin (40 µg/mL) for 72 h either in the presence and absence of H-89, KT5720, Ro-32-0432 and SB203580. Melanin content was determined as described in Materials and Methods (A). Intracellular melanin was observed by DOPA staining (B). Results are the mean±S.D. from triplicate determination. * p<0.01 versus untreated cells, ^# p<0.01 versus scopoletin treated cells.

Fig. 6. Effect of Various Kinase Inhibitors on Scopoletin-Enhanced Tyrosinase Expression

Cells were incubated with or without scopoletin (40 µg/mL) for 72 h either in the presence and absence of H-89, KT5720, Ro-32-0432 and SB203580. Tyrosinase expression was detected by Western blotting using specific antibodies (A). Fold increases over the control were determined by densitometric analysis (B). Results are the mean±S.D. from triplicate determination. * p<0.01 versus untreated cells, ^# p<0.05, ^## p<0.01 versus scopoletin treated cells.

Fig. 7. Effect of cAMP Analog and Scopoletin on Melanin Synthesis

Cells were treated with scopoletin (40 µg/mL), or dbcAMP (500 µM) for 48 h. At the end of treatment, tyrosinase activity (A) and melanin content (B) were measured as Materials and Methods. Results are the mean±S.D. from three independent experiments. * p<0.05, ** p<0.01 versus untreated cells.

Fig. 8. Effect of p38 MAPK siRNA on Scopoletin-Enhanced Tyrosinase Expression

Cells were transfected with a specific siRNA of p38 MAPK or a non-silencing control. Following transfection for 24 h, the cells were incubated with or without scopoletin (40 µg/mL) for 72 h. The knockdown was evaluated by Western blotting. Results are the mean±S.D. from triplicate determination. ** p<0.01 versus untreated cells, ^## p<0.01 versus scopoletin treated cells.

DISCUSSION

We investigated the molecular events of scopoletin on melanogenesis pathway including cAMP, PKC, and p38 MAPK. First, we confirmed that scopoletin induced melanin biosynthesis and tyrosinase activity in B16F10 cells. Here, we found that scopoletin-induced melanogenesis occurs via the cAMP/PKA signaling pathway and partially p38 MAPK activation.

Since melanin is synthesized by an enzymatic cascade of melanogenic enzymes, we ascetained the effect of scopoletin on the expression of these enzymes. Scopoletin augmented the protein levels of tyrosinase and TRP-1. Especially the mRNA level of tyrosinase was increased after scopoletin treatment, indicating that the induction of this gene expression was occurred at transcription level. MITF is a key transcription factor of melanogenic proteins,^2–4) scopoletin significantly increased the expression of MITF mRNA. These data suggest that scopoletin up-regulated tyrosinase and MITF at the transcription level.

cAMP is a key factor in the sequential processes required for melanin synthesis, the activations of PKA and CREB, followed by the expression of MITF.^4,17) However, it has been recently reported that transforming growth factor β (TGF-β) activates PKA via a cAMP-independent pathway, which is mediated by the smad3/4 complex.^18,19) Thus, we investigated whether scopoletin-induced melanin synthesis is regulated by cAMP-dependent pathway. In our study, the cAMP levels increased when cells were treated with 40 µg/mL of scopoletin. dbcAMP increased tyrosinase activity and melanin content, and scopoletin synergistically with dbcAMP significantly induced melanin synthesis. This result shows that scopoletin stimulate melanin synthesis through an increase in cAMP levels. Moreover, scopoletin-induced intracellular melanin and tyrosinase expression were significantly reduced by specific inhibitors of PKA treatment, suggesting that the induction of melanin synthesis by scopoletin was markedly inhibited when cAMP/PKA pathway was blocked by inhibition of PKA.

Although the biological effects of cAMP inducing agents are mostly regulated by cAMP-dependent PKA activation, some studies have reported that there are cross-talk between the PKA and PKC signaling pathway.^20,21) For example, bee venom has augmented melanin biosynthesis via phospholipase A₂ (sPLA₂) activation and cAMP production, suggesting that the effect of bee venom is mediated by PKC as well as PKA.^22,23) In our study, the inhibition of PKC by Ro-32-0432 had no effect on melanin synthesis and tyrosinase protein expression. This result suggests that the induction of melanin synthesis by scopoletin is not mediated to the activation of PKC.

The MAPK family proteins, including p38 MAPK, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), are known to play important roles in melanin synthesis. The ERK and/or JNK/stress activated protein kinase (SAPK) pathways cause downregulation of melanin synthesis.²⁴⁾ On the other hand, the phosphorylation of p38 MAPK activates MITF and eventually stimulates melannogenesis.²⁵⁾ In our experiment, the p38 MAPK phosphorylation was elevated by scopoletin. Treatment of cells with SB203580, a p38 MAPK inhibitor, partially blocked the scopoletin-stimulated melanin content and tyrosinase protein expression. Although SB203580 is widely used as a specific inhibitor of p38 MAPK, there are many reports that SB203580 activates other signaling molecules including JNK/SAPK and Raf-1. In this study, we showed that siRNA-mediated knockdown of p38 MAPK significantly eliminated the scopoletin-induced tyrosinase expression in B16F10 cells. These results mean that the induction of melanin synthesis by scopoletin is partially mediated to the p38 MAPK activation.

In conclusion, we confirmed that the melanogenic activity of scopoletin was mediated by the cAMP production and p38 MAPK activation. PKA inhibitors completely blocked scopoletin-mediated increase of melanin synthesis and tyrosinase expression, while partially blocked by SB203580 and p38 MAPK siRNA. Collectively, these results suggest that scopoletin can stimulate melanogenesis by the cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

Acknowledgments

This work was supported by the National Research Foundation of Korea [NRF] Grant funded by the Korea government [MSIP] [No. 2008-0062484] and the NRF Grant funded by the MSIP [No. 2015M3A9E3051054].

Conflict of Interest

The authors declare no conflict of interest.

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