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CREB-Regulated Transcriptional Coactivator 2 Proteome Landscape is Modulated by SREBF1.

ABSTRACT: cAMP response element-binding protein (CREB) regulated transcriptional coactivator 2 (CRTC2) is a critical transcription factor that maintains glucose homeostasis by activating CREB. Energy homeostasis is maintained through multiple pathways; therefore, CRTC2 may interact with other transcription factors, particularly under metabolic stress. CRTC2 liver-specific KO mice were created, and the global proteome, phosphoproteome, and acetylome from liver tissue under high-fat diet conditions were analyzed using liquid chromatography-tandem mass spectrometry and bioinformatics analysis. Differentially regulated proteins (DRPs) were enriched in metabolic pathways, which were subsequently corroborated through animal experiments. The consensus DRPs from these datasets were used as seed proteins to generate a protein-protein interaction network using STRING, and GeneMANIA identified fatty acid synthase as a mutually relevant protein. In an additional local-protein-protein interaction analysis of CRTC2 and fatty acid synthase with DRPs, sterol regulatory element binding transcription factor 1 (SREBF1) was the common mediator. CRTC2-CREB and SREBF1 are transcription factors, and DNA-binding motif analysis showed that multiple CRTC2-CREB-regulated genes possess SREBF1-binding motifs. This indicates the possible induction by the CRTC2-SREBF1 complex, which is validated through luciferase assay. Therefore, the CRTC2-SREBF1 complex potentially modulates the transcription of multiple proteins that fine-tune cellular metabolism under metabolic stress.

SUBMITTER: Lim JM

PROVIDER: S-EPMC10522995 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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CREB-Regulated Transcriptional Coactivator 2 Proteome Landscape is Modulated by SREBF1.

Lim Jae Min JM Anwar Muhammad Ayaz MA Han Hye-Sook HS Koo Seung-Hoi SH Kim Kwang Pyo KP

Molecular & cellular proteomics : MCP 20230828 10

cAMP response element-binding protein (CREB) regulated transcriptional coactivator 2 (CRTC2) is a critical transcription factor that maintains glucose homeostasis by activating CREB. Energy homeostasis is maintained through multiple pathways; therefore, CRTC2 may interact with other transcription factors, particularly under metabolic stress. CRTC2 liver-specific KO mice were created, and the global proteome, phosphoproteome, and acetylome from liver tissue under high-fat diet conditions were ana ...[more]

PMID: 37648026

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Project description:The major regulator of the neuroendocrine stress response in the brain is corticotropin releasing factor (CRF), whose transcription is controlled by CREB and its cofactors CRTC2/3 (TORC2/3). Phosphorylated CRTCs are sequestered in the cytoplasm, but rapidly dephosphorylated and translocated into the nucleus following a stressful stimulus. As the stress response is attenuated by oxytocin (OT), we tested whether OT interferes with CRTC translocation and, thereby, Crf expression. OT (1 nmol, i.c.v.) delayed the stress-induced increase of nuclear CRTC3 and Crf hnRNA levels in the paraventricular nucleus of male rats and mice, but did not affect either parameter in the absence of the stressor. The increase in Crf hnRNA levels at later time points was parallel to elevated nuclear CRTC2/3 levels. A direct effect of Thr(4) Gly(7)-OT (TGOT) on CRTC3 translocation and Crf expression was found in rat primary hypothalamic neurons, amygdaloid (Ar-5), hypothalamic (H32), and human neuroblastoma (Be(2)M17) cell lines. CRTC3, but not CRCT2, knockdown using siRNA in Be(2)M17 cells prevented the effect of TGOT on Crf hnRNA levels. Chromatin-immunoprecipitation demonstrated that TGOT reduced CRTC3, but not CRTC2, binding to the Crf promoter after 10 min of forskolin stimulation. Together, the results indicate that OT modulates CRTC3 translocation, the binding of CRTC3 to the Crf promoter and, ultimately, transcription of the Crf gene.Significance statementThe neuropeptide oxytocin has been proposed to reduce hypothalamic-pituitary-adrenal (HPA) axis activation during stress. The underlying mechanisms are, however, elusive. In this study we show that activation of the oxytocin receptor in the paraventricular nucleus delays transcription of the gene encoding corticotropin releasing factor (Crf), the main regulator of the stress response. It does so by sequestering the coactivator of the transcription factor CREB, CRTC3, in the cytosol, resulting in reduced binding of CRTC3 to the Crf gene promoter and subsequent Crf gene expression. This novel oxytocin receptor-mediated intracellular mechanism might provide a basis for the treatment of exaggerated stress responses in the future.

Project description: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.

Dataset Information

CREB-Regulated Transcriptional Coactivator 2 Proteome Landscape is Modulated by SREBF1.

Publications

CREB-Regulated Transcriptional Coactivator 2 Proteome Landscape is Modulated by SREBF1.

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