NF-κB 和 CREB 之间的核共抑制因子交换介导 NF-κB 对磷酸烯醇式丙酮酸羧激酶转录的抑制。
Exchange of a nuclear corepressor between NF-kappaB and CREB mediates inhibition of phosphoenolpyruvate carboxykinase transcription by NF-kappaB.
机构信息
Department of Endocrinology, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China.
出版信息
Chin Med J (Engl). 2010 Jan 20;123(2):221-6.
BACKGROUND
NF-kappaB 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-kappaB p65, we investigated the nuclear receptor corepressor in the regulation of PEPCK transcription.
METHODS
Rat H4IIE 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 IkBalpha (ssIkBalpha) stable cell line, and HEK 293 cells. The effects of p65 and ssIkBalpha 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-kappaB p65 and the transcriptional regulation of CREB by NF-kappaB p65.
RESULTS
NF-kappaB p65 inhibited PEPCK expression and the inhibition was blocked by ssIkBalpha. The inhibitory effect of p65 was completely blocked in a HepG2 stable cell line in which ssIkBalpha 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-kappaB p65 cotransfection. The inhibitory effect of NF-kappaB p65 was blocked by HDAC3 RNAi or SMRT RNAi.
CONCLUSIONS
The study showed that the inhibition of PEPCK by NF-kappaB p65 was dependent on HDAC3 and SMRT, which form a nuclear corepressor complex for transcriptional inhibition. The transcription factors NF-kappaB p65 and CREB share the same corepressor HDAC3-SMRT, and the corepressor exchange leads to inhibition of PEPCK gene transcription by NF-kappaB p65.
背景
NF-κB p65 被证明能抑制磷酸烯醇丙酮酸羧激酶（PEPCK）的转录，PEPCK 是肝脏糖异生过程中的限速酶。为了了解 NF-κB p65 的作用机制，我们研究了核受体共抑制子在 PEPCK 转录调节中的作用。
方法
本研究使用大鼠 H4IIE 细胞、人肝癌 HepG2 细胞和人胚肾（HEK）293 细胞。在 HepG2 细胞、HepG2 超抑制型 IkBα（ssIkBα）稳定细胞系和 HEK 293 细胞中分析大鼠 PEPCK 基因启动子（-490/+100）的转录活性。使用 PEPCK 荧光素酶报告系统观察 p65 和 ssIkBα 对大鼠 PEPCK 基因启动子的影响。采用染色质免疫沉淀（ChIP）实验研究 cAMP 反应元件结合（CREB）蛋白、组蛋白去乙酰化酶 3（HDAC3）和视黄酸和甲状腺激素受体沉默调节剂（SMRT）与 PEPCK 基因启动子的相互作用。p65 共转染和 RNAi 介导的基因敲低用于确定 NF-κB p65 抑制 PEPCK 表达所涉及的共抑制子以及 NF-κB p65 对 CREB 的转录调节。
结果
NF-κB p65 抑制 PEPCK 表达，ssIkBα 可阻断该抑制作用。在表达 ssIkBα 的 HepG2 稳定细胞系中，p65 的抑制作用完全被阻断。p65 共转染时，HDAC3 或 SMRT 敲低可显著上调 PEPCK 报告基因活性。ChIP 实验表明，p65 激活诱导 HDAC3 和 SMRT 与 PEPCK 基因启动子相互作用，但 CREB 信号减少。NF-κB p65 共转染抑制 CREB 的转录活性。NF-κB p65 的抑制作用可被 HDAC3 RNAi 或 SMRT RNAi 阻断。
结论
本研究表明，NF-κB p65 对 PEPCK 的抑制作用依赖于 HDAC3 和 SMRT，它们形成转录抑制的核共抑制子复合物。转录因子 NF-κB p65 和 CREB 共享相同的共抑制子 HDAC3-SMRT，共抑制子的交换导致 NF-κB p65 抑制 PEPCK 基因转录。
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