Department of Biomedical Sciences, University of Ulsan College of Medicine, Seoul 05505, Korea.
Bio-Medical Institute of Technology (BMIT), University of Ulsan College of Medicine, Seoul 05505, Korea.
Theranostics. 2020 Mar 4;10(9):4017-4029. doi: 10.7150/thno.41502. eCollection 2020.
Melanogenesis is a critical self-defense mechanism against ultraviolet radiation (UVR)-induced skin damage and carcinogenesis; however, dysregulation of melanin production and distribution causes skin-disfiguring pigmentary disorders. Melanogenesis is initiated by UVR-induced cAMP generation and ensuing activation of transcription factor CREB, which induces expression of the master melanogenic regulator MITF. Recent studies have demonstrated that recruitment of CRTCs to the CREB transcription complex is also required for UVR-stimulated melanogenesis. Therefore, modulation of cAMP-CRTC/CREB-MITF signaling may be a useful therapeutic strategy for UVR-associated skin pigmentary disorders. : We identified the small-molecule Ro31-8220 from CREB/CRTC activity screening and examined its melanogenic activity in cultured mouse and human melanocytes as well as in human skin. Molecular mechanisms were deciphered by immunoblotting, RT-PCR, promoter assays, tyrosinase activity assays, immunofluorescent examination of CRTC3 subcellular localization, and shRNA-based knockdown. : Ro31-8220 suppressed basal and cAMP-stimulated melanin production in melanocytes and human melanocyte co-culture as well as UVR-stimulated melanin accumulation in human skin through downregulation of MITF and tyrosinase expression. Mechanistically, down regulation of MITF expression by Ro31-8220 was due to inhibition of transcriptional activity of CREB, which was resulted from phosphorylation-dependent blockade of nuclear translocation of CRTC3 via JNK activation. The selective JNK activator anisomycin also inhibited melanin production through phosphoinhibition of CRTC3, while JNK inhibition enhanced melanogenesis by stimulating CRTC3 dephosphorylation and nuclear migration. : Melanogenesis can be enhanced or suppressed via pharmacological modulation of a previously unidentified JNK-CRTC/CREB-MITF signaling axis. As Ro31-8220 potently inhibits UVR-stimulated melanin accumulation in human skin, suggesting that small-molecule JNK-CRTC signaling modulators may provide therapeutic benefit for pigmentation disorders.
黑色素生成是对抗紫外线 (UVR) 诱导的皮肤损伤和致癌作用的关键自我防御机制；然而，黑色素生成和分布的失调会导致皮肤色素紊乱。黑色素生成是由 UVR 诱导的 cAMP 生成和随后激活转录因子 CREB 引发的，CREB 诱导主黑色素生成调节剂 MITF 的表达。最近的研究表明，CRTCs 向 CREB 转录复合物的募集对于 UVR 刺激的黑色素生成也是必需的。因此，调节 cAMP-CRTC/CREB-MITF 信号可能是一种有用的治疗 UVR 相关皮肤色素紊乱的策略。
我们从 CREB/CRTC 活性筛选中鉴定出小分子 Ro31-8220，并在培养的小鼠和人类黑素细胞以及人类皮肤中检查了其黑色素生成活性。通过免疫印迹、RT-PCR、启动子测定、酪氨酸酶活性测定、CRTC3 亚细胞定位的免疫荧光检查以及基于 shRNA 的敲低来解析分子机制。
Ro31-8220 通过下调 MITF 和酪氨酸酶的表达，抑制黑素细胞和人黑素细胞共培养中的基础和 cAMP 刺激的黑色素生成以及 UVR 刺激的人皮肤中黑色素的积累。从机制上讲，Ro31-8220 下调 MITF 表达是由于 CREB 的转录活性受到抑制，这是由于 JNK 激活导致 CRTC3 的核易位磷酸化依赖性阻断所致。选择性 JNK 激活剂 anisomycin 也通过对 CRTC3 的磷酸抑制来抑制黑色素生成，而 JNK 抑制通过刺激 CRTC3 去磷酸化和核迁移来增强黑色素生成。
通过对先前未识别的 JNK-CRTC/CREB-MITF 信号轴的药理学调节，可以增强或抑制黑色素生成。由于 Ro31-8220 可有效抑制人皮肤中 UVR 刺激的黑色素积累，因此小分子 JNK-CRTC 信号调节剂可能为色素沉着紊乱提供治疗益处。
