JNK suppresses melanogenesis by interfering with CREB-regulated transcription coactivator 3-dependent MITF expression

Abstract

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. Methods: 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. Results: 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. Conclusions: 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.

Keywords: CREB; CREB regulated transcription coactivator 3 (CRTC3); JNK; Ro31-8220; melanogenesis.

Figure 1

Suppression of cAMP-stimulated CREB activity and melanin production by Ro31-8220. (A) Ro31-8220 (0.1-2 µM) suppressed forskolin (FSK)-stimulated CREB activity as measured by hEVX1 promoter activity. (B, C) Melanin content of Mel-Ab cells was dose-dependently reduced by Ro31-8220 treatment for 96 h. Bars = 2000 µm. (D) Mel-Ab cell viability was not substantially reduced following treatment with 0.1-2 µM Ro31-8220 for 96 h according to MTT assay. CTRL: vehicle-treated controls, FSK: forskolin, Ro31: Ro31-8220, number in (C) indicates concentration (µM) of Ro31-8220.

Figure 2

Ro31-8220 downregulates cAMP-stimulated melanin accumulation and melanogenic gene expression in melanocytes. Ro31-8220 suppressed FSK-induced induction of melanin and melanogenic genes. (A, B) Basal and FSK-stimulated melanin content of Mel-Ab cells after 96 h of vehicle or Ro31-8220 treatment. Bars = 2000 µm. (C, D) Expression levels of melanogenic genes at the protein level (C) and mRNA level (D) in Mel-Ab cells treated with vehicle (CTRL), FSK, or Ro31-8220 plus FSK (Rot + FSK) as revealed by western blotting and qRT-PCR, respectively. Ro31-8220 suppressed FSK-induced upregulation of tyrosinase activity. (E) Tyrosinase activity of Mel-Ab cells after treatment with vehicle (CTRL), Ro31-8220 (Ro31), FSK, or Ro31-8220 + FSK for 96 h (expressed as percent change from vehicle-treated control).

Figure 3

Ro31-8220 suppresses MITF expression through phosphorylation-dependent inhibition of CRTC3 nuclear translocation. (A) Short-term effects of FSK, and Ro31-8220 (Ro31) + FSK treatment (1-6 h) on expression levels of melanogenic gene mRNAs. (B) Short-term effects Ro31, FSK, and Ro31 + FSK treatment (4 h) on expression of melanogenic proteins. (C) The effect of Ro31-8220 (Ro31) on MITF and tyrosinase transcription levels using MITF and Tyr promoter-luc-based promoter reporter assays, respectively. (D) Effect of Ro31 on FSK- and CRTC3-stimulated MITF promoter activity assessed using a MITF-luc promoter reporter assay. (E, F) Expression levels and phosphorylation status of CRTC2/3, CREB, and AMPK signaling pathway proteins (AMPK, Raptor, ACC) analyzed by immunoblotting in Mel-Ab cells (E) and mouse embryonic fibroblasts (MEFs) (F) following 1 h of FSK treatment with or without 1 h of Ro31-8220 pretreatment. (G) Effects of Ro31-8220, FSK, and both on the subcellular localization of CRTC3 in B16F10 cells transfected with CRTC3-EGFP. Bar = 100 µm.

Figure 4

JNK mediates Ro31-8220-induced suppression of melanogenesis through inhibitory phosphorylation of CRTC. (A) Mel-Ab cells were treated with FSK or Ro31-8220 for 1 h and the expression and phosphorylation levels of CRTC2/3, CREB, AMPK, and JNK were examined by western blotting. (B) Mel-Ab cells were treated with anisomycin, FSK, or anisomycin for 1 h followed by FSK for 1 h, and the phosphorylation and expression level of CRTC2/3, CREB, AMPK, and JNK were analyzed by western blotting. (C) B16F10 cells transfected with CRTC3-EGFP were treated with anisomycin (ANS), FSK, anisomycin plus FSK, SP600125 (SP), or SP600125 + Ro31-8220, then subcellular localization of CRTC3 was examined. Bar = 100µm (D) Effect of JNK activation by anisomycin (ANS) on CREB/CRTC3 transcriptional activity. (E) Protein expression of melanogenic genes in Mel-Ab cells after 4 h of FSK or anisomycin + FSK treatment examined by western blotting. (F) Effect of JNK inhibition by SP600125 on phosphorylation and expression levels of CRTC2/3, CREB, AMPK, and JNK analyzed by western blotting. (G) CRTC3 phosphorylation in HEK-293T cells transfected with CRTC3 alone and CRTC3 with Flag tagged JNK1, JNK2, or JNK3 was compared by western blotting.

Figure 5

JNK suppresses cAMP-stimulated melanogenesis in melanocytes. (A-C) Mel-Ab cells were treated with anisomycin (ANS), FSK, or anisomycin + FSK for 96 h and examined for melanin content (A, B) and expression of melanogenic genes (C). Bars = 2000 µm in A and B. (D) Melanin content of Mel-Ab cells treated with SP600125, Ro31-8220, or Ro31-8220 + Sp600125 for 96 h. (E, F) Control (shUsi) or CRTC3 knockdown (shCRTC3i) Mel-Ab cells were treated with (E) Ro31-8220 (Ro31), FSK, or Ro31-8220 + FSK or (F) anisomycin (ANS), FSK, or anisomycin + FSK for 96 h and melanin content measured.

Figure 6

Ro31-8220 suppresses UVR-induced melanin accumulation in melanocyte-keratinocytes cocultures and ex vivo human skin. (A) Human primary melanocytes and keratinocytes in co-culture were treated with FSK or pretreated with Ro31-8220 or anisomycin (ANS) followed by FSK treatment. After 96 h, melanin content was examined. (B) Human primary melanocytes and keratinocytes co-culture were treated with ET-1/SCF or pretreated with Ro31-8220 or anisomycin (ANS) followed by ET-1/SCF. After 96 h of treatment, melanin content was examined. Ex vivo human skin tissues were exposed to UVB (150 mJ/cm²) or co-treated with Ro31-8220 plus UVB. After 96 h, skin tissues were harvested. (C) Quantification of melanin accumulation (melanin index). (D) Representative images of Fontana-Masson-stained paraffin-embedded sections treated with vehicle (CTRL), UVB + vehicle, or UVB + Ro31-8220. Bars = 100 µm. (E) Microscopic images of melan-A immunohistochemistry for melanocyte staining. Arrow heads indicate positive melan-A staining (Red color) Bar = 100 µm. (F) Protein expression of melanogenic genes in human skin tissue exposed to vehicle only (CTRL), UVR, or UVR+Ro31-8220 as assessed by western blotting.

Figure 7

Schematic model of melanogenic modulation by JNK-CRTC3/CREB-MITF signaling. In response to UVR exposure, αMSH secreted from keratinocytes activates melanocortin receptor 1 (MC1R) on melanocytes, which in turn triggers cAMP generation, PKA activation, CREB phosphorylation, and nuclear entry of CRTC3, which promotes MITF expression and induction of downstream melanogenic genes. Activation of JNK by RO31-8220 or anisomycin directly phosphorylates CRTC3, thereby inhibiting nuclear translocation and suppressing UV-induced MITF expression and melanogenesis.