AP736 induces miR‐125b expression for the efficient whitening and anti‐ageing action in human epidermal cells (2016)

AP736, a recently developed whitening compound, is a benzylbenzamide derivative that strongly inhibits melanin synthesis by inhibiting tyrosinase expression and activity, and suppressing the expression of tyrosinase-related protein (Tyrp)-1 and Tyrp-2, and microphthalmia-associated transcription factor (MiTF).1, 2 MicroRNAs (miRNAs) are known to regulate skin biology, including the pigmentation process in melanocytes.3 For example, miR-340 regulates UVB-induced dendrite formation and melanosome transfer in melanocytes.4 miRNA signatures in vitiligo also regulate melanocyte biology.5 Recently, Kim et al.6 in our research group found that miR-125b is a potent inhibitor of steady-state melanogenesis. In addition, other research groups have reported that miR-125b downregulates the expression of matrix metalloproteinase (MMP)-7 and MMP-13 in cutaneous carcinoma cells, and MMP-2 and MMP-9 in HepG2 cells.7, 8 These studies have suggested that AP736-induced miR-125b expression can be an effective strategy for exerting whitening effects through inhibition of melanogenesis as well as promoting anti-ageing effects through inhibition of MMPs in skin cells. However, intracellular cAMP reduces the expression of miR-125b in melanocytes,6 and ROS also inhibit miR-125b expression by increasing promoter methylation of the miR-125b gene.9 Therefore, decreasing cAMP levels or suppressing ROS production by AP736 can be helpful for increasing miR-125b expression, resulting in whitening and anti-ageing effects, especially in UVB-stimulated epidermal cells. In this study, we examined whether AP736 inhibits melanogenesis in human melanocytes by increasing miR-125b expression, a depigmentation inducer. We also tested whether AP736 suppression of MMP expression and ROS production, which contribute to the anti-ageing efficacy of AP736, involves increased miR-125b expression in human keratinocytes. We used human melanocytes, keratinocytes and the artificial 3D human skin model, Melanoderm (MatTek Corp, Ashland, MA, USA), to investigate the antimelanogenic and anti-ageing effects of AP736 and associated changed in miR-125b expression. Detailed methods can be found in the supplement. In our previous report, we evaluated the whitening effect of AP736 (Figure S1) in melanocytes and an artificial skin model.2 In the present study, we sought to define whether AP736 inhibits melanin synthesis by inducing miR-125b expression. Accordingly, we analysed the expression levels of miR-125b in normal human primary melanocytes following treatment with AP736. As shown in Figure 1A, AP736 significantly increased miR-125b expression approximately threefold. Furthermore, because cAMP decreases miR-125b expression 4 and AP736 inhibits cAMP production,2 we tested whether AP736 restores cAMP-inhibited miR-125b expression in WM266-4 human melanocytes. As expected, the cAMP inducer, forskolin, inhibited miR-125b expression; notably, this inhibitory effect was reversed by AP736, which, like the cAMP inhibitor MDL-12330A (positive control), dramatically increased miR-125b expression in forskolin-treated melanocytes (Figure 1B). These data suggest that AP736 induces miR-125b, possibly through inhibition of cAMP, although other mechanisms are involved in AP736-induced miR-125b expression. Next, we confirmed that AP736 also induces miR-125b expression in an artificial skin model, where it showed whitening efficacy. As shown in Figure 1C,D, AP736 lightened the colour of the skin tissue and significantly changed the ΔL value (the degree of pigmentation), as expected. Haematoxylin and eosin (H&E) and Fontana–Masson (F&M) staining of tissue sections revealed that the normal structure of skin tissue was maintained, melanin was rarely transferred from melanocytes to keratinocytes, the dendritic stretch of melanocytes was inhibited and melanin content were decreased in the presence of AP736 (Figure 1E,F). Expression of the major melanogenic enzymes, tyrosinase, Tyrp-1 and Tyrp-2, and MiTF were significantly suppressed by AP736 treatment (Figure S2A–D). As expected based on results in melanocytes, AP736 dramatically increased miR-125b expression in the skin tissue model (Figure 1G). These data suggest the possibility that AP736 exerts it depigmentation effect by inducing miR-125b expression, and thereby inhibiting melanogenic enzyme expression and melanin synthesis. In addition to its effects in melanocytes, we also found that AP736 increased miR-125b expression in UVB-stimulated keratinocytes (Figure 2A). To define the possible mechanism for miR-125b expression by AP736, we investigated whether AP736 suppresses ROS production because ROS inhibition can cause miR-125b expression.7 As shown in Figure 2B,C, AP736 dramatically scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) in a cell-free system and inhibited ROS production in UVB-stimulated HaCaT keratinocytes. These data suggest that AP736 suppresses ROS production, resulting in increased miR-125b expression in the presence of UVB irradiation. Previous reports have also shown that miR-125b inhibits expression of several MMPs.5, 6 Therefore, we investigated the effect of AP736 on the expression of MMPs in UVB-irradiated keratinocytes using MMP arrays. These analyses showed that UVB dramatically increased secretion of MMP-1 and MMP-3 by keratinocytes, whereas AP736 clearly inhibited both secretion (Figure 2D,E) and mRNA expression (Figure S3A,B) of MMP-1 and MMP-3 induced by UVB irradiation. Inhibition of MMP-1 by AP736 was also confirmed in an artificial skin model (Figure S3C). These results suggest that AP736 exerts antioxidative (ROS inhibition) and anti-ageing effects (MMPs inhibition) in UVB-irradiated keratinocytes. These actions of AP736 could involve an increase in miR-125b expression, although further studies, such as miR-125b knock down, will be required to confirm the detailed mechanism. In conclusion, our current data suggest the possibility that AP736 acts through a novel mechanism involving induction of miR-125b expression to cause the inhibition of melanin synthesis in melanocytes. AP736 also simultaneously suppresses MMP-1 and MMP-3 expression as well as ROS production, at least in part, through a signalling pathway involving miR-125b induction in keratinocytes. Taken together with previous reports, this study provides the first evidence to support a role for increased miR-125b expression in the antimelanogenic and anti-ageing actions of AP736. This work was approved by the Ethical Committee of the AMOREPACIFIC corporation. HS Baek, JA Hwang and IH Bae performed the research. KH Kim and YH Joo contributed essential tools. YJ Kim, HJ Shin, JH Lee and YH Park analysed the data. JW Kim and CS Lee designed the research study and wrote the manuscript. The authors have declared no conflicting interests. Figure S1 The structure of AP736 compound Figure S2 Expression levels of tyrosinase, Tyrp-1, Tyrp-2 and MiTF Figure S3 Expression levels of matrix metalloproteinase Data S1 Materials and Methods Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.