Yamamoto Takashi, Shimano Hitoshi, Nakagawa Yoshimi, Ide Tomohiro, Yahagi Naoya, Matsuzaka Takashi, Nakakuki Masanori, Takahashi Akimitsu, Suzuki Hiroaki, Sone Hirohito, Toyoshima Hideo, Sato Ryuichiro, Yamada Nobuhiro
Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
J Biol Chem. 2004 Mar 26;279(13):12027-35. doi: 10.1074/jbc.M310333200. Epub 2004 Jan 13.
The hepatocyte nuclear factor-4alpha (HNF-4alpha)/PGC-1 pathway plays a crucial role in the transcriptional regulation of hepatic gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase (PEPCK) and Glc-6-Pase, genes that are activated at fasting and suppressed in a fed state. SREBP-1c dominates the nutritional regulation of lipogenic genes inverse to gluconeogenesis. Here we show the mechanism by which SREBP-1 suppresses expression of gluconeogenic genes. A series of luciferase reporter assays demonstrated that SREBP-1a and -1c effectively inhibited the PEPCK promoter activity that was induced by HNF-4alpha. The HNF-4alpha-binding site in the glucocorticoid-response unit was responsible for the SREBP-1 inhibition, although SREBP-1 did not bind to the PEPCK promoter as demonstrated by electrophoretic mobility shift assays. The inhibitory effect was more potent in the isoform of SREBP-1a than SREBP-1c and was eliminated by deletion of the amino-terminal transactivation domain of SREBP-1. Coimmunoprecipitation experiments demonstrated that these two transcription factors directly interact through the transactivation domain of SREBP-1 and the ligand binding/AF2 domains of HNF-4alpha. Estimation of coactivator recruitment using HNF-4alpha-Gal4DBD fusion assay showed that SREBP-1 competitively inhibited PGC-1 recruitment, a requirement for HNF-4alpha activation. Consistent with these results, hepatic PEPCK and Glc-6-Pase mRNA levels are suppressed by overexpression of SREBP-1a and -1c in the transgenic mice. Our data indicate that SREBP-1 has a novel role as negative regulator of gluconeogenic genes through a cross-talk with HNF-4alpha interference with PGC-1 recruitment.
肝细胞核因子4α(HNF-4α)/过氧化物酶体增殖物激活受体γ共激活因子1α(PGC-1)信号通路在肝脏糖异生酶如磷酸烯醇式丙酮酸羧激酶(PEPCK)和葡萄糖-6-磷酸酶(Glc-6-Pase)的转录调控中起关键作用,这些基因在禁食时被激活,在进食状态下被抑制。固醇调节元件结合蛋白1c(SREBP-1c)主导与糖异生相反的生脂基因的营养调控。在此我们展示了SREBP-1抑制糖异生基因表达的机制。一系列荧光素酶报告基因检测表明,SREBP-1a和-1c有效抑制了由HNF-4α诱导的PEPCK启动子活性。糖皮质激素反应元件中的HNF-4α结合位点是SREBP-1抑制作用的原因,尽管凝胶迁移实验表明SREBP-1不与PEPCK启动子结合。SREBP-1a亚型的抑制作用比SREBP-1c更强,并且通过缺失SREBP-1的氨基末端反式激活结构域可消除这种抑制作用。免疫共沉淀实验表明,这两种转录因子通过SREBP-1的反式激活结构域与HNF-4α的配体结合/AF2结构域直接相互作用。使用HNF-4α-半乳糖苷酶结合结构域(Gal4DBD)融合检测法对共激活因子募集的评估表明,SREBP-1竞争性抑制PGC-1的募集,而PGC-1募集是HNF-4α激活所必需的。与这些结果一致,在转基因小鼠中,SREBP-1a和-1c的过表达会抑制肝脏中PEPCK和Glc-6-Pase的mRNA水平。我们的数据表明,SREBP-1通过与HNF-4α相互作用干扰PGC-1募集,作为糖异生基因的负调节因子发挥新的作用。