NEDD4L limits cAMP signaling through ubiquitination of CREB-regulated transcription coactivator 3.

The transcription factor cAMP-responsive element-binding protein (CREB) is involved in a variety of physiologic processes. Although its activity appears to be largely correlated with its phosphorylation status, cAMP-mediated dephosphorylation and the subsequent nuclear migration of the CREB-regulated transcription factors (CRTCs) are required to stimulate CREB transcriptional activity. Among the 3 identified mammalian homologs of CRTCs, CRTC3 has been shown to be expressed predominantly in adipose tissues in response to catecholamine signals that regulate lipid metabolism. Here, we show that prolonged cAMP signaling down-regulates CRTC3 in a proteasome-dependent manner and that neural precursor cell-expressed developmentally down-regulated gene 4-like (NEDD4L), a specific ubiquitin ligase for CRTC3, is responsible for this process. By recognizing the PY motif of CRTC3, NEDD4L interacts with CRTC3 and promotes its polyubiquitination. Interaction between NEDD4L and CRTC3 is further boosted by cAMP signaling, and this enhanced interaction appears to be dependent on the cAMP-mediated phosphorylation of NEDD4L at the Ser448 site. Furthermore, we show that food withdrawal stimulates NEDD4L phosphorylation in mice, which then show a decrease of adipose tissue CRTC3 protein levels. Together, these results suggest that NEDD4L plays a key role in the feedback regulation of cAMP signaling by limiting CRTC3 protein levels.-Kim, Y.-H., Yoo, H., Hong, A.-R., Kwon, M., Kang, S.-W., Kim, K., Song, Y. NEDD4L limits cAMP signaling through ubiquitination of CREB-regulated transcription coactivator 3.

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

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.

Therapeutic Potential of Rottlerin for Skin Hyperpigmentary Disorders by Inhibiting the Transcriptional Activity of CREB-Regulated Transcription Coactivators.

Exposure to UVR stimulates the cAMP signaling pathway, which leads to melanin deposits in skin tissues. Although melanogenesis can be beneficial by protecting skin from UVR-induced damage, excessive or uneven deposits of melanin can cause various skin hyperpigmentation disorders. Because cAMP-responsive element binding protein (CREB) and CREB-regulated transcription coactivators (CRTC) play a major role in conveying cAMP signals that induce transcription of microphthalmia-associated transcription factor and melanin production, we screened for a CREB or CRTC inhibitor and identified rottlerin (Rot) as a potent inhibitor of its transcriptional activity. Rot suppressed melanin production in both basal and cAMP-stimulated cultured melanocytes by downregulating melanogenic gene expression. In addition, topical application of Rot on the tails of mice decreased melanin accumulation. Mechanistically, we showed that Rot decreased the mitochondrial membrane potential, which then activated AMPK, leading to the phosphorylation and nuclear exclusion of CRTC3 and suppressing the expression of CREB target genes, including MITF. Our study demonstrates that Rot is an active antimelanogenic agent and suggests that screening for an inhibitor of CREB or CRTC transcriptional activity is a promising strategy by which to discover better drugs to treat skin hyperpigmentary disorders.