Umckalin Promotes Melanogenesis in B16F10 Cells Through the Activation of Wnt/β-Catenin and MAPK Signaling Pathways

Abstract

Melanogenesis is regulated by melanogenic enzymes such as tyrosinase (TYR), TRP-1, and TRP-2, whose expression is controlled by the microphthalmia-associated transcription factor (MITF). Various signaling pathways, including cAMP/PKA, MAPK/ERK, Wnt/β-catenin, and PI3K/Akt, are involved in this process and have been a focal point of research for treating pigmentation disorders. However, developing effective therapies for conditions like vitiligo remains a significant challenge. In this study, the effects of umckalin on melanogenesis and its molecular mechanisms were investigated using B16F10 cells, a mouse melanoma cell line widely used as a model for melanin production studies. B16F10 cells produce melanin via melanosomes and express key melanogenic enzymes such as TYR, TRP-1, and TRP-2, making them a reliable model system. Our findings demonstrate that umckalin promotes melanogenesis in a concentration-dependent manner by upregulating TRP-1 expression and activating the MITF signaling pathway. Additionally, umckalin modulated key signaling pathways, including GSK3β/β-catenin and MAPK, to enhance melanogenesis. In conclusion, umckalin enhances melanogenic enzyme activity by activating critical signaling pathways, thereby promoting melanin synthesis. These findings suggest that umckalin could be a promising candidate for developing therapeutic agents for pigmentation disorders such as vitiligo. Further studies are required to explore its mechanisms and clinical applications in greater detail.

1. Introduction

Melanogenesis is a multifaceted biological and biochemical pathway that governs the synthesis of melanin. Melanin pigment is synthesized in specialized organelles called melanosomes, which are found in melanocytes, dendritic cells derived from the neural crest. These melanocytes are distributed among keratinocytes in the basal layer of the epidermis. By transferring melanin-containing melanosomes to surrounding keratinocytes, melanocytes contribute to melanin deposition in human skin [1,2]. Proper functioning of melanocytes is crucial for maintaining normal pigmentation, while changes in melanocyte numbers or dysregulation of melanin synthesis can lead to pigmentation disorders such as hypopigmentation or hyperpigmentation [3,4].

In mammals, the production of melanin is primarily driven by melanogenic enzymes such as tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). Among these, TYR serves as a key enzyme by catalyzing the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and facilitating the oxidation of DOPA into DOPA quinone. TRP-1 and TRP-2 further regulate subsequent steps in melanin production [5,6]. Microphthalmia-associated transcription factor (MITF) plays a pivotal role in regulating essential melanocyte-specific genes, such as TYR, TRP-1, and TRP-2. It is crucial for melanocyte survival, proliferation, differentiation, and the pigmentation process [7,8].

Melanogenesis is modulated by multiple molecular signaling pathways. Among them, the cAMP/PKA pathway triggers the activation of cyclic adenosine monophosphate (cAMP), which in turn stimulates protein kinase A (PKA), ultimately resulting in the phosphorylation of the cAMP response element-binding protein (CREB). This activation promotes MITF transcription and increases the expression of melanogenic enzymes. The MAPK/ERK pathway maintains balance by regulating cell survival and proliferation while suppressing excessive MITF activity. The Wnt/β-catenin pathway enhances melanin production by promoting MITF transcription, whereas the PI3K/Akt pathway inhibits melanin synthesis by inducing MITF degradation and preventing the overactivation of melanocytes. Reactive oxygen species (ROS) also influence melanogenesis by modulating MITF activity and affecting the expression of melanogenic enzymes. Collectively, these signaling pathways play critical roles in the precise regulation of melanin synthesis [9,10,11].

Despite existing strategies to halt depigmentation and stimulate re-pigmentation, effective therapies for pigmentation disorders such as vitiligo remain limited. Natural plant-based compounds have gained attention as potential treatments due to their minimal long-term side effects. Several studies have identified specific natural compounds with strong melanogenesis-stimulating effects, suggesting their potential as resources for developing anti-vitiligo agents [12,13].

Umckalin, a coumarin-derived compound with the molecular formula C₁₁H₁₀O₅, is primarily present in the roots of Pelargonium sidoides and Pelargonium reniforme. It features a unique structure characterized by a coumarin backbone with hydroxy and methoxy substitutions (7-hydroxy-5,6-dimethoxycoumarin), contributing to its diverse biological activities. Notably, umckalin exhibits antibacterial and antioxidant properties, enhancing its therapeutic potential [14,15,16]. However, no studies to date have explored the relationship between umckalin and melanogenesis.

This study explores the impact of umckalin on melanin synthesis and its associated molecular mechanisms in B16F10 cells. By uncovering these mechanisms, we aim to assess umckalin’s potential as a therapeutic candidate for pigmentation disorders, particularly vitiligo, and to propose novel treatment strategies.

2. Materials and Methods

2.1. Chemicals and Reagents

Umckalin (Cat. No. 50792, purity ≥ 95.0%), α-MSH, sodium hydroxide (NaOH), L-DOPA, sodium phosphate monobasic, and sodium phosphate dibasic were procured from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were sourced from Gibco (Grand Island, NY, USA). The BCA protein assay kit was acquired from Thermo Fisher Scientific (Waltham, MA, USA), while the water-soluble tetrazolium salt (WST) was obtained from DoGenBio (Geumcheon-gu, Seoul, Republic of Korea). Primary antibodies for tyrosinase, TRP-1, TRP-2, and MITF were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies, along with primary antibodies for P-ERK, P-JNK, P-p38, P-AKT, P-GSK-3β, P-β-catenin, P-PKA, P-CREB, T-ERK, T-JNK, T-p38, T-AKT, T-GSK-3β, T-β-catenin, T-PKA, T-CREB, β-actin, and a protease/phosphatase inhibitor cocktail, were obtained from Cell Signaling Technology (Beverly, MA, USA). Bovine serum albumin (BSA) was obtained from Bovogen (Melbourne, Australia). Phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), radioimmunoprecipitation (RIPA) buffer, Tris-buffered saline (TBS), and an enhanced chemiluminescence (ECL) kit were purchased from Biosesang (Sungnam, Republic of Korea). The 4× sample buffer was obtained from Invitrogen (Waltham, MA, USA).

2.2. Cell Culture

B16F10 murine melanoma cells were sourced from ATCC (Manassas, VA, USA) and maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (P/S) at 37 °C in a humidified incubator with 5% CO₂. Cells were subcultured every 3–4 days to ensure optimal growth and viability.

2.3. Measurement of Cell Viability

B16F10 cells were plated in a 24-well plate at a density of 8.0 × 10³ cells per well and incubated for 24 h. The cells were then exposed to various concentrations of umckalin (25, 50, 100, 200, and 400 µM), which was dissolved in DMSO, for 72 h. Following the manufacturer’s instructions, 10 µL of the WST reagent was added to each well, and the plate was incubated for an additional 30 min. Absorbance of the supernatant was then measured at 450 nm using a spectrophotometric microplate reader (Tecan, Salzburg, Austria).

2.4. Melanin Content Assay

The experimental procedure was slightly modified based on the study by Lee et al. [17]. B16F10 cells were plated in 24-well plates at a density of 1.0 × 10⁴ cells per well and incubated for 24 h. The cells were then treated with umckalin (50, 100, and 200 µM) and α-MSH (100 nM) for 72 h. Following treatment, the culture supernatant was discarded, and the cells were washed before being lysed in 1N NaOH containing 10% DMSO at 70 °C for 30 min. Protein concentration was determined using the BCA protein assay kit, and absorbance was measured at 405 nm using a spectrophotometric microplate reader.

2.5. Tyrosinase Activity Assay

The experimental procedure, adapted with slight modifications from Lee et al. [17], followed the same treatment conditions as the melanin content assay. After 72 h, the cells were washed and lysed using RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, 2 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with 1% protease/phosphatase inhibitor cocktail. The lysate was centrifuged at 14,000 rpm for 30 min at 4 °C to collect the supernatant. Protein concentration was determined using the BCA protein assay kit. Subsequently, 20 µL of protein (20 µg/mL) and 80 µL of 2 mg/mL L-DOPA were added to a 96-well plate and incubated at 37 °C for 2 h. Absorbance was then measured at 490 nm using a spectrophotometric microplate reader.

2.6. Western Blotting Assay

B16F10 cells (4 × 10⁴ cells/well) were seeded in 6-well plates and incubated for 24 h. The cells were then treated with umckalin (50, 100, and 200 µM) for the designated duration. After treatment, the cells were lysed using RIPA buffer supplemented with 1% protease/phosphatase inhibitor cocktail, and the lysates were centrifuged at 14,000 rpm for 30 min at 4 °C. Protein concentrations were determined using the BCA protein assay kit. The protein samples were mixed with 4× sample buffer and heated at 100 °C for 5 min. Proteins were separated on a 10% SDS-PAGE gel and subsequently transferred to PVDF membranes. Following a blocking step with 5% BSA for 1 h, the membranes were washed four times with TBS containing 0.1% Tween-20 (TBS-T). Primary antibodies (diluted 1:1000 in 5% BSA) were incubated overnight at 4 °C, followed by four washes with TBS-T. Secondary antibodies (diluted 1:1000 in TBS-T) were then applied at room temperature for 1.5 h. After additional washes, protein bands were visualized using an ECL kit.

2.7. Statistical Analysis

All data are expressed as the mean ± standard deviation (SD) from three independent experiments. Statistical significance was determined using one-way ANOVA with IBM SPSS (v. 20, SPSS Inc., Armonk, NY, USA). A p-value < 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Umckalin Promotes Melanin Synthesis in B16F10 Cells

Umckalin, a natural coumarin compound with a 7-hydroxy-5,6-dimethoxy structure (Figure 1a), was evaluated for its effects on melanogenesis. To ensure the observed effects were not influenced by cytotoxicity, we first assessed cell viability using a WST-1 assay. Cytotoxicity can confound results by causing non-specific inhibition or activation of melanogenesis, making this step essential. B16F10 cells, a murine melanoma cell line, were used as the experimental model due to their established reliability in melanogenesis studies. These cells contain melanosomes, express key melanogenic enzymes such as tyrosinase (TYR), TRP-1, and TRP-2, and closely mimic human melanocyte behavior, including their response to UV or α-MSH (melanocyte-stimulating hormone) stimulation [17,18]. To evaluate cytotoxicity, B16F10 cells were treated with varying concentrations of umckalin (25, 50, 100, 200, and 400 μM) for 72 h, and cell viability was measured. No cytotoxic effects were observed at concentrations up to 200 μM, whereas 400 μM caused significant cytotoxicity (Figure 1b). We have recently published a study on the anti-inflammatory effects of umckalin in RAW264.7 macrophages. In this study, RAW264.7 cells were treated with different concentrations of umckalin (72.5, 125, 300, and 500 μM) in the presence of LPS (1 μg/mL) for 24 h. The results demonstrated that umckalin did not exhibit significant cytotoxicity at concentrations up to 300 μM, and even at 500 μM, cell viability remained above 80% [19]. These findings indicate that the cytotoxicity of umckalin does not differ significantly between B16F10 melanoma cells and RAW264.7 macrophages. Therefore, subsequent experiments on melanogenesis and tyrosinase activity were conducted at concentrations ≤ 200 μM. When B16F10 cells were treated with umckalin (50, 100, and 200 μM), intracellular melanin content was significantly increased in a dose-dependent manner, with α-MSH serving as a positive control (Figure 1c). Furthermore, tyrosinase activity, a key enzymatic step in melanogenesis, was analyzed by measuring the conversion of L-DOPA to L-dopachrome via absorbance at 450 nm. Results showed that umckalin dose-dependently enhanced tyrosinase activity (Figure 1d). In summary, umckalin enhances melanogenesis in B16F10 cells by elevating melanin content and stimulating tyrosinase, the key rate-limiting enzyme in the melanogenic pathway. These results indicate that umckalin holds potential as a promising candidate for further research on pigmentation-related therapies.

3.2. Umckalin Promotes Melanin Synthesis by Inducing TRP-1 Expression via the MITF Signaling Pathway

Melanogenesis is governed by key melanogenic enzymes, including tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2). Among these, TYR directly facilitates melanin production by oxidizing the substrate tyrosine. Thus, TYR, TRP-1, and TRP-2 serve as crucial enzymes in melanin synthesis, with their expression levels being directly correlated with melanin production. These enzymes are considered critical indicators for understanding the mechanisms of activation or inhibition of melanogenesis [20].

Western blot analysis, a reliable method for visually assessing changes in protein expression, is widely used to evaluate the effects of compounds on the melanin synthesis pathway and elucidate their mechanisms of action. Based on this approach, we investigated the melanogenesis-inducing mechanism of umckalin by treating B16F10 cells with umckalin at concentrations of 50, 100, and 200 μM for 24 h and analyzing the expression of TYR, TRP-1, TRP-2, and MITF through Western blotting.

As shown in Figure 2, umckalin significantly increased the expression of TRP-1, while it had no notable effect on the expression of TYR and TRP-2 proteins. These results suggest that TRP-1 expression is involved in umckalin-induced melanogenesis. Moreover, the expression of melanogenic enzymes is controlled by MITF, a crucial transcription factor essential for melanogenesis and melanocyte development. MITF itself is regulated by various transcription factors, including activated CREB, which binds to the MITF promoter region in melanocytes and enhances its gene expression [21,22].

To further investigate the mechanism underlying umckalin’s melanogenesis-inducing effects, we analyzed MITF protein expression levels in B16F10 cells. The results showed that umckalin significantly increased MITF protein expression (Figure 3). These findings support that the upregulation of melanogenic enzyme expression by umckalin is attributed to increased MITF expression.

3.3. Umckalin Promotes Melanin Synthesis via GSK-3β/β-Catenin Signaling Pathway

GSK-3β activity is regulated by specific phosphorylation sites, which exert opposing effects on melanogenesis. For instance, phosphorylation at Ser9 inhibits GSK-3β activity, preventing the degradation of β-catenin. This leads to the cytoplasmic accumulation of β-catenin and its eventual translocation into the nucleus. Nuclear β-catenin activates the key transcription factor MITF, promoting the expression of melanogenic genes such as tyrosinase, TRP-1, and TRP-2. Thus, Ser9 phosphorylation acts as a major mechanism to stimulate melanogenesis [23,24]. Conversely, phosphorylation at Tyr216 enhances GSK-3β activity, accelerating the degradation of β-catenin and thereby suppressing melanogenesis. In contrast, dephosphorylation at Tyr216 inhibits GSK-3β activity, stabilizing β-catenin and favoring melanogenesis [25,26].

Based on these mechanisms, this study aimed to investigate whether umckalin promotes Ser9 phosphorylation of GSK-3β, thereby preventing β-catenin degradation, facilitating its nuclear translocation, and inducing melanogenesis. As shown in Figure 4, umckalin increased Ser9 phosphorylation of GSK-3β, inhibiting its enzymatic activity and consequently reducing β-catenin phosphorylation. As a result, β-catenin was not degraded, accumulated in the cytoplasm, and led to the activation of melanogenic gene expression. These findings suggest that umckalin regulates the GSK-3β/β-catenin pathway by stabilizing β-catenin, thereby promoting melanogenesis.

3.4. Umckalin Promotes Melanin Synthesis via MAPK Signaling Pathway

The entire MAP kinase family, including ERK, JNK, and p38 MAPKs, is known to play a critical role in regulating melanogenesis. The activation of the ERK pathway induces the phosphorylation of MITF, promoting ubiquitin-dependent degradation and thereby suppressing melanogenesis [27]. In contrast, the activation of the p38 pathway enhances MITF activation by phosphorylating cAMP response element-binding protein (CREB) and inducing phosphorylation of MITF at Ser301, which stimulates its transcriptional activity. This process promotes melanocyte differentiation and melanin production [28,29]. Additionally, some studies have reported that p38 regulates pigmentation by influencing proteasomal degradation of tyrosinase [30,31].

The JNK/SAPK pathway has been shown to have contrasting roles in melanogenesis. While most studies suggest that JNK activation promotes melanin production, recent research indicates that Ro31-8220 inhibits CREB transcriptional activation and reduces MITF expression by blocking the nuclear translocation of CRTC3 via JNK activation [32]. Similarly, the selective JNK activator anisomycin was found to inhibit melanogenesis by inducing CRTC3 phosphorylation. Conversely, JNK inhibition facilitated CRTC3 dephosphorylation and nuclear translocation, thereby enhancing melanogenesis [33].

In this study, we investigated the effects of umckalin on the activation of p38, JNK, and ERK MAPKs to understand the molecular mechanisms by which umckalin influences melanogenesis. As shown in Figure 5, umckalin (50–200 μM) reduced the phosphorylation of ERK and JNK while having no significant effect on p38 phosphorylation. These findings suggest that umckalin contributes to melanogenesis by suppressing ERK and JNK phosphorylation, which in turn enhances MITF activation and the expression of melanogenic genes. Although this study elucidates the molecular mechanisms underlying umckalin’s melanogenesis-promoting activity, additional inhibitor-based experiments are needed to further clarify the roles of ERK and JNK. Future research addressing these limitations will provide a deeper understanding of umckalin’s mechanism of action and its potential therapeutic applications.

3.5. Umckalin Promotes Melanin Synthesis Independently of the PI3K/AKT Signaling Pathway

The PI3K/AKT signaling pathway exhibits a dual and intricate role in melanogenesis, with its effects influenced by cellular context, activation timing, and interactions with other signaling pathways [34]. Typically, AKT directly phosphorylates MITF at Ser510, targeting it for proteasomal degradation. This process downregulates the expression of melanogenic enzymes, including tyrosinase, TRP-1, and TRP-2, leading to the suppression of melanin production [35]. Additionally, the PI3K/AKT signaling pathway regulates melanogenesis through its interaction with GSK3β. The activation of PI3K/AKT leads to the phosphorylation of GSK3β at Ser9, inhibiting its activity. While this prevents β-catenin degradation, it simultaneously limits MITF activation, ultimately reducing melanogenesis [36]. In summary, the PI3K/AKT signaling pathway collaborates with GSK3β to negatively regulate MITF activity, thereby suppressing melanin synthesis.

In this study, we investigated the effects of umckalin on the activation of the PI3K/AKT signaling pathway to further understand the molecular mechanisms underlying umckalin’s melanogenesis-promoting effects. However, as shown in Figure 6, contrary to expectations, umckalin (50–200 μM) tended to increase the phosphorylation of both PI3K and AKT. This finding suggests that umckalin’s melanogenesis-promoting effects may be independent of the PI3K/AKT signaling pathway. The increase in melanin production despite PI3K/AKT pathway activation cannot be solely explained by a simple correlation; instead, it is likely the result of interactions among multiple signaling pathways. The temporal activation pattern of the PI3K/AKT pathway may play a crucial role. Initially, the activation of PI3K/AKT may induce MITF degradation and suppress melanogenesis. However, the subsequent activation of other signaling pathways could restore MITF expression and promote melanin production. These findings reflect the complex interplay between the PI3K/AKT pathway and other pathways, including ERK and GSK3β.

In conclusion, umckalin’s melanogenesis-promoting effects appear to be largely independent of the PI3K/AKT signaling pathway and are likely influenced by interactions with multiple signaling pathways, the duration of activation, and external stimuli. Further studies are required to elucidate these mechanisms, which could provide valuable insights into umckalin’s role in regulating melanogenesis and its potential as a therapeutic agent for pigmentation disorders.

4. Conclusions

This study explored the melanogenesis-promoting effects of umckalin and its underlying molecular mechanisms in B16F10 cells, a well-established murine melanoma model. The findings demonstrated that umckalin significantly enhances melanin production in a dose-dependent manner by upregulating melanogenic enzymes, particularly TRP-1, and activating the MITF signaling pathway. Furthermore, umckalin modulated several key signaling pathways, including GSK-3β/β-catenin, MAPK, and PI3K/AKT, to regulate melanin synthesis. Interestingly, umckalin was shown to activate GSK-3β/β-catenin and inhibit ERK and JNK phosphorylation while promoting MITF expression. Despite its apparent activation of the PI3K/AKT pathway, the results suggest that umckalin’s melanogenesis-promoting effects are largely independent of this pathway and involve complex interactions with other signaling cascades. These data position umckalin as a promising candidate for developing therapeutic agents aimed at treating pigmentation disorders such as vitiligo. However, further research, including inhibitor-based studies and in vivo evaluations, is necessary to comprehensively understand its mechanisms and potential clinical applications. This work provides a foundation for future investigations into the therapeutic potential of umckalin in pigmentation-related conditions.