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The Inhibitory Effect Of Curcumin Derivative J147 On Melanogenesis And Melanosome Transport By Facilitating ERK-Mediated MITF Degradation Part 2

Apr 07, 2023

J147 Reduced Melanogenesis by Activation MEK/ERK Pathway in Normal Human Epidermal Melanocytes (NHEMs)

We next investigated whether J147 can suppress melanogenesis in normal human epidermal melanocytes (NHEMs). Consistent with the results from B16 melanocytes, J147 also inhibited melanin production in NHEMs (Figure 6A). Western blotting analysis indicated that J147 reduced the protein level of tyrosinase, Myosin Va, Rab27a, and Cdc42 in NHEMs (Figure 6B). In addition, the degradation of MITF protein was also facilitated after J147 treatment in NHEMs (Figure 6C). MEK/ERK activation was also confirmed to be involved in J147-mediated anti-melanogenic in NHEMs (Figures 6D, E).

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J147 Reduced Pigmentation in Zebrafish

Evaluation of candidate compounds based on phenotype provided reliable data for further experiments. The in vivo experiment of J147 was conducted in a zebrafish model, as melanin pigments were on the surface of the fish, which allows simple observation (Choi et al., 2007). PTU is used widely as a positive control in zebrafish research due to its capacity of inhibiting melanogenesis (Kim et al., 2008). As shown in Figure 7, J147 significantly reduced the body pigmentation, likewise the positive control group treated with PTU.

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J147 Reversed UVB-Induced Hyperpigmentation in Guinea Pig Skin

We also investigated the anti-melanogenic effects of J147 in the UVB-induced hyperpigmentation model in brown guinea pigs. J147 (1%) remarkably inhibited pigmentation when compared with the untreated group as shown by representative photographs of guinea pig skin (Figure 8A). To further evaluate the degree of pigmentation, we used a Spectrophotometer to calculate the L value (brightness index). As shown in Figure 8B, the ΔL value of the J147 group was markedly higher after 3 weeks of treatment compared with the vehicle group, indicating that J147 could reverse hyperpigmentation induced by UVB. Consistent with the above observations, a visible decrease in UVB-induced pigmentation in the epidermal basal layer was observed after the J147 treatment by Masson–Fontana ammoniacal silver staining (Figure 8C). In addition, J147 did not affect the melanocyte count as determined by immunohistochemical staining of S100, a melanocyte marker protein (Figures 8D, E). As expected, our findings suggested that J147 has whitening effects on UV-induced hyperpigmentation in guinea pig skin without cytotoxicity.

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DISCUSSION

Global industry analysts (GIA) have predicted that the global whitening market will reach $31.2 billion by 2024 (Lv et al., 2020b). Many research groups are focusing their efforts to elucidate novel and effective whitening compounds. However, few inhibitors underwent studies in vivo and showed good results due to cytotoxicity and weak efficiency (Kim et al., 2008; Pillaiyar et al., 2017). Thus, it is necessary to continue to discover more efficient and safer skin-whitening agents.

The therapeutic use of curcumin and chemically modified curcumin (CMC) for inhibiting tyrosinase activity and melanin formation has been reported recently (Goenka and Simon, 2021). However, the poor bioavailability of curcumin and chemically modified curcumin (CMC) limits its application (Karthikeyan et al., 2020). To solve this problem, J147 is developed as a potent compound of curcumin derivative with greater stability and bioavailability (Li et al., 2020). In this work, we explored the inhibitory influences of J147 on pigment and underlying mechanisms in vitro and in vivo. Our data showed that J147 not only suppressed the basal melanin production but also attenuated α-MSH-induced melanin increase, without influencing cell viability (Figure 1). It is well known that several skin whitening agents exert their hypo melanogenic effects by directly inhibiting tyrosinase activity. In our studies, J147 did not directly inhibit mushroom tyrosinase activity (Figure 2B), which is inconsistent with previous studies suggesting that curcumin directly inhibited mushroom tyrosinase activity (Lee et al., 2010; Tu et al., 2012). The possible reason is that J147 has no phenolic hydroxyl group, which is a critical functional group for tyrosinase activity inhibition. Western blotting analysis demonstrated that J147 inhibited the expression levels of tyrosinase, TRP-1, and TRP-2 (Figure 2C). Our findings indicated that J147 had whitening effects on melanocytes and acted primarily by downregulating the protein levels of three crucial melanogenic enzymes rather than directly suppressing the tyrosinase activity.

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Human skin pigmentation is determined by melanin synthesis as well as the distribution of melanosomes. The regulation of melanosome transfer from melanocytes to neighboring keratinocytes is an important mechanism for skin whitening. Recently, Goenka et al. reported that chemically modified curcumin analogs suppressed centricity in melanocytes and inhibited the phagocytosis of FluoSphere beads (melanosome mimics) by HaCaT cells (Goenka and Simon, 2021). The present study expanded upon previous studies and demonstrated that J147 inhibited melanosome transfer to neighboring keratinocytes in the keratinocyte/melanocyte co-culture system (Figure 3A). Mechanisms studies found that the expression levels of Myosin Va, Rab27a, and Cdc42 were significantly decreased by the J147 treatment, while KIF5b was not affected (Figure 3B). These results suggested that J147 suppressed the melanosome transport along actin filaments and inhibited dendrite extension by downregulating the Myosin Va, Rab27a, and Cdc42 expression. However, further investigation of whether J147 affects the melanosome movement along microtubules is needed.

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MITF is a critical regulator in melanocyte development, differentiation, and survival of melanocytes, and it controls the transcription of genes such as tyrosinase, TRP-1, TRP-2, Cdc42, Rab27a and subsequently promotes melanogenesis and melanosome transport (Noguchi et al., 2016). The present studies showed that J147 had no effect on the expression of MITF genes, suggesting that J147 did not affect MITF transcription (Figure 4A). However, western blotting analysis found that J147 remarkedly accelerated MITF protein degradation (Figure 4B). Numerous studies suggested that the MAPK signaling pathway participated in the expression and degradation of MITF (Rodríguez and Setaluri, 2014). Tu et al. reported that curcumin inhibited melanin synthesis through the activation of ERK and p38 signaling pathways (Tu et al., 2012). In the present study, J147 increased the phosphorylation of ERK and p38, whereas no outcome was observed in the phosphorylation of JNK. Furthermore, only PD98059, a specific inhibitor of ERK, significantly attenuated the inhibitory effects of J147 on melanin formation and distribution as well as the expression of tyrosinase, MITF, Rab27a, Myosin Va, and Cdc42 (Figure 5). These data indicated that J147 suppressed melanin formation and distribution mainly through facilitating EKR-mediated MITF degradation. Considering that numerous studies have identified the role of p38 MAPK in melanogenic differentiation, the role of the J147/p38 signaling pathway in melanocytes needs further study.

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Melanogenesis suppressors are very important to the cosmetic industry as skin-lightening agents. Numerous anti-melanogenic agents have been developed, and few agents under in vivo studies, and showed good results (Pillaiyar et al., 2017). J147 is currently in phase I clinical trials for Alzheimer’s disease, which indicates that J147 is a safe and low-toxic agent (Ates et al., 2020). Herein, we investigated the anti-melanogenic effect of J147 in vivo. Zebrafish is an extremely advantageous vertebrate model organism due to its gene sequence and organ system being similar to that of humans (Agalou et al., 2018). And melanin pigments are present on the surface of fish, which is simply easy to observe (Choi et al., 2007). As shown in Figure 7, J147 remarkably reduced the body pigmentation in zebrafish, similar to the positive drug PTU. Furthermore, J147 did not affect the developmental growth or survival of zebrafish. UVB-induced hyperpigmentation in brown guinea pigs is the other kind of experimental model to investigate the effects of J147 on pigmentation. As shown in Figure 8, obvious whitening effects of J147 were found in which hyperpigmentation caused by UVB exposure after the topical application of J147 for 3 weeks to the dorsal skin of a guinea pig. Our results demonstrated that J147 reduced melanin formation in active melanocytes, rather than decreased numbers of melanocytes.

In summary, our study first reported that J147 exerted anti-melanogenic effects in vitro and in vivo, as well as the underlying mechanism. Specifically, J147 significantly inhibited melanin production, in addition to preventing dendrite extension and melanosome distribution. Mechanistically, J147 played these roles mainly by activating the extracellular signal-regulated protein kinase (ERK) pathway. Once activated, it accelerated MITF degradation, subsequently suppressed the protein level of tyrosinase, TRP-1, TRP-2, Myosin Va, Rab27a, and Cdc42, and ultimately suppressed melanin production, dendritic extension, and melanin distribution. Our results indicated that J147 may be applied as a safer skin-whitening agent without cytotoxicity.

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DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

ETHICS STATEMENT

The animal study was reviewed and approved by The animal care and use committee of Changzhou University.

AUTHOR CONTRIBUTIONS

JL, YY, and RG conceived and designed the study, provided critical comments, and edited the manuscripts. YY, BJ, and SL carried out major experiments. XZ performed analysis and interpretation of data on immunoblot analysis assay. JL performed on data collecting. All authors read and approved the final manuscript.

FUNDING

This study was sponsored by the National Natural Science Foundation of China (No. 82103752), the Natural Science Research Project of Colleges and Universities in Jiangsu Province (No. 20KJB310024), Changzhou Science and Technology Program (Grant No. CJ20210125) to JL.

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online

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Conflflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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