Exchange of a nuclear corepressor between NF-kB and CREB mediates inhibition of phosphoenolpyruvate carboxykinase transcription by NF-kB  

作　　者：YAN Jin-hua GAO Zhan-guo YE Jian-ping WENG Jian-ping 

机构地区：[1]Department of Endocrinology Yat-sen University, Guangzhou JH and Weng JP) Third Affiliated Hospital, Sun Guangdong 510630, China [2]Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA 70808, USA

出　　处：《Chinese Medical Journal》2010年第2期221-226,共6页中华医学杂志（英文版）

基　　金：This study was supported by grants from the National Natural Science Foundation of China （No. 30570885）, 973 Program of China （No. 2006CB503902） （to WENG Jian-ping）, the National Institutes of Health Grant （No. DK068036） and American Diabetes Association Research Award （No. 7-04-RA-139） （to YE ,lian-ping）Acknowledgements： We would like to be grateful to Ms. Wei Tseng, Dr. HE Qin and Dr. YIN Jun for their excellent technical assistance.

摘　　要：Background NF-KB p65 was shown to inhibit transcription of phosphoenolpyruvate carboxykinase （PEPCK）, a rate-limiting enzyme in gluconeogenesis in the liver. To understand the mechanism of action of NF-KB p65, we investigated the nuclear receptor corepressor in the regulation of PEPCK transcription. Methods Rat H411E cells, human hepatoma HepG2 cells and human embryo kidney （HEK） 293 cells were used in this study. The transcriptional activity of a rat PEPCK gene promoter （-490/＋100） was analyzed in HepG2 cells, a HepG2 super suppressor IkBa （sslkBa） stable cell line, and HEK 293 cells. The effects of p65 and sslkBa on a rat PEPCK gene promoter were observed using the PEPCK luciferase reporter system. The interaction of the cAMP-response- element-binding （CREB） protein, histone deacetylase 3 （HDAC3） and silencing mediator for retinoic and thyroid hormone receptors （SMRT） with the PEPCK gene promoter were investigated using the chromatin immunoprecipitation （CHIP） assay, p65 cotransfection and RNAi-mediated gene knockdown were used to determine the corepressor involved in the inhibition of PEPCK by NF-KB p65 and the transcriptional regulation of CREB by NF-KB p65. Results NF-KB p65 inhibited PEPCK expression and the inhibition was blocked by sslkBa. The inhibitory effect of p65 was completely blocked in a HepG2 stable cell line in which sslkBa was expressed. HDAC3 or SMRT knockdown led to a significant up-regulation of PEPCK reporter activity in the presence of p65 cotransfection. In the ChIP assay the interaction of HDAC3 and SMRT with the PEPCK gene promoter was induced by p65 activation, but the CREB signal was reduced. Transcriptional activity of CREB was inhibited by NF-kB p65 cotransfection. The inhibitory effect of NF-kB p65 was blocked by HDAC3 RNAi or SMRT RNAi. Conculsions The study showed that the inhibition of PEPCK by NF-kB p65 was dependent on HDAC3 and SMRT, which form a nuclear corepressor complex for transcriptional inhibition. The transcription factors NF-kB p65 aBackground NF-KB p65 was shown to inhibit transcription of phosphoenolpyruvate carboxykinase （PEPCK）, a rate-limiting enzyme in gluconeogenesis in the liver. To understand the mechanism of action of NF-KB p65, we investigated the nuclear receptor corepressor in the regulation of PEPCK transcription. Methods Rat H411E cells, human hepatoma HepG2 cells and human embryo kidney （HEK） 293 cells were used in this study. The transcriptional activity of a rat PEPCK gene promoter （-490/＋100） was analyzed in HepG2 cells, a HepG2 super suppressor IkBa （sslkBa） stable cell line, and HEK 293 cells. The effects of p65 and sslkBa on a rat PEPCK gene promoter were observed using the PEPCK luciferase reporter system. The interaction of the cAMP-response- element-binding （CREB） protein, histone deacetylase 3 （HDAC3） and silencing mediator for retinoic and thyroid hormone receptors （SMRT） with the PEPCK gene promoter were investigated using the chromatin immunoprecipitation （CHIP） assay, p65 cotransfection and RNAi-mediated gene knockdown were used to determine the corepressor involved in the inhibition of PEPCK by NF-KB p65 and the transcriptional regulation of CREB by NF-KB p65. Results NF-KB p65 inhibited PEPCK expression and the inhibition was blocked by sslkBa. The inhibitory effect of p65 was completely blocked in a HepG2 stable cell line in which sslkBa was expressed. HDAC3 or SMRT knockdown led to a significant up-regulation of PEPCK reporter activity in the presence of p65 cotransfection. In the ChIP assay the interaction of HDAC3 and SMRT with the PEPCK gene promoter was induced by p65 activation, but the CREB signal was reduced. Transcriptional activity of CREB was inhibited by NF-kB p65 cotransfection. The inhibitory effect of NF-kB p65 was blocked by HDAC3 RNAi or SMRT RNAi. Conculsions The study showed that the inhibition of PEPCK by NF-kB p65 was dependent on HDAC3 and SMRT, which form a nuclear corepressor complex for transcriptional inhibition. The transcription factors NF-kB p65 a

关 键 词：phosphoenolpyruvate carboxykinase NF-kappa B nuclear receptor corepressor 1 

分 类 号：Q786[生物学—分子生物学] Q513