Nuclear Corepressor Is Required for Inhibition of Phosphoenolpyruvate Carboxykinase Expression by Tumor Necrosis Factor-α

0888-8809/07/$15.00/0 Printed in U.S.A. Molecular Endocrinology 21(7):1630–1641 Copyright © 2007 by The Endocrine Society doi: 10.1210/me.2007-0072 Nuclear Corepressor Is Required for Inhibition of Phosphoenolpyruvate Carboxykinase Expression by Tumor Necrosis Factor-␣ Jinhua Yan, Zhanguo Gao, Gang Yu, Qing He, Jianping Weng, and Jianping Ye Department of Endocrinology (J.Ya., J.W.), The First-Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China; and Pennington Biomedical Research Center (Z.G., G.Y., Q.H., J.Ye), Louisiana State University System, Baton Rouge, Louisiana 70808 Inhibition of phosphoenolpyruvate carboxykinase (PEPCK) by TNF-␣ contributes to the pathogenesis of hypoglycemia in endotoxin shock. In this study, the molecular mechanism underlying the inhibition was investigated in hepatoma cells (rat H4IIE and human HepG2). PEPCK expression was induced by cAMP, and the induction was reduced by TNF-␣ at protein and mRNA levels in H4IIE cells. The inhibition was observed in the PEPCK gene promoter in a PEPCK-luciferase reporter. Activation of nuclear factor ␬B (NF-␬B) pathway was required for the transcriptional inhibition of PEPCK gene. Degradation of NF-␬B inhibitor (I␬B) and p65 nuclear translocation were involved in the inhibition. An interaction of histone deacetylase 3 (HDAC3) and silencing mediator for retinoic acid receptor and thyroid hormone receptor (SMRT) with the PEPCK gene promoter was induced by TNF-␣ and observed in a chromatin immunoprecipitation assay. The TNF-induced inhibition was blocked by HDAC inhibitor or HDAC3 knockdown. The blocking effect was also observed in knockdown of corepressor SMRT. Point mutation suggests that cAMP response element (CRE) is required for TNF-induced inhibition of the PEPCK gene promoter. Phosphorylation of cAMP response element-binding protein at Ser133 and expression of peroxisome proliferator-activated receptor-␥ coactivator 1␣ were not changed by TNF-␣ in H4IIE cells. The transcriptional activity of CRE-binding protein was inhibited by TNF-␣ in a CRE-luciferase reporter. The data suggests that the nuclear corepressor proteins of HDAC3 and SMRT mediate TNF inhibition of PEPCK transcription. The inhibition mechanism is related to activation of NF-␬B and inhibition of CRE-binding protein activity by the corepressor. These data suggest a novel activity of nuclear corepressor in the regulation of PEPCK expression by TNF-␣. (Molecular Endocrinology 21: 1630–1641, 2007) G are mainly controlled by two critical enzymes: phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6 phosphatase (G6Pase). The cytosolic form of phosphoenolpyruvate carboxykinase is encoded by a single copy gene that is highly expressed in the liver, kidney cortex, and adipose tissues (both white and brown) (4). In response to various dietary, hormonal, and environmental stimuli, the activity of the cytosolic form of PEPCK is acutely regulated by gene transcription. Knockout of PEPCK gene in mice led to severe hypoglycemia (5). This is associated with an increase in amino acids in the plasma of PEPCK⫺/⫺ mice. PEPCK is also required for reesterification of free fatty acids into triglycerides (TGs) (6). Expression of PEPCK and G6Pase is inhibited by proinflammation cytokines (1, 2, 7, 8), such as TNF-␣ (7), IL-1 (9), and IL-6 (10). The inhibition occurs at transcriptional level for PEPCK and G6pase (7, 8) and contributes to hypoglycemia in conditions including septic shock (1, 2). PEPCK expression is regulated by many hormones, and the major hormone axis is formed by glucagon and insulin (4, 11). Many transcription factors are involved in the transcriptional regulation of PEPCK by glucagons and insulin (12, 13). These include cAMP response element (CRE)-binding protein (CREB), LUCONEOGENESIS IS IMPORTANT in mammalians for prevention of hypoglycemia in stress conditions, such as starvation and infection (1, 2). A failure in gluconeogenesis leads to hypoglycemic shock that is often seen in septic (or endotoxin) shock. Liver is the most important organ in gluconeogenesis, and it maintains blood glucose level in fasting condition by production of glucose from amino acids (3). The hepatic gluconeogenesis and glucose production First Published Online April 24, 2007 Abbreviations: AP1, Activator protein 1; CBP, CREB-binding protein; ChIP, chromatin immunoprecipitation; CRE, cAMP response element; CREB, cAMP response element-binding protein; G6Pase, glucose 6 phosphatase; HDAC, histone deacetylase; I␬B, inhibitor of NF-␬B; IKK, I␬B kinase; JNK, c-Jun N-terminal protein kinase; N-CoR, nuclear receptor corepressor; NF-␬B, nuclear factor ␬B; PEPCK, phosphoenolpyruvate carboxykinase; PGC-1, peroxisome proliferator-activated receptor-␥ coactivator 1; PMSF, phenylmethylsulfonyl fluoride; Pol II, polymerase II; q, quantitative; RNAi, interference RNA; SMRT, silencing mediator for retinoic and thyroid hormone receptors; SP3, specificity protein 3; ssI␬B␣, super suppressor I␬B␣; TG, triglyceride; TSA, trichostatin A. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community. Yan et al. • Regulation of PEPCK by TNF-␣ CCAAT/enhancer-binding protein ␣ and ␤, glucocorticoid receptor, forkhead transcription factor O1, sterol regulatory element-binding protein, hepatocyte nuclear factor-4, peroxisome proliferator-activated receptor ␥, activating transcription factor 3, and activator protein 1 (AP1) (Fos/Jun heterodimer). Among these nuclear factors, CREB is required for the upregulation of PEPCK by glucagons, which activates CREB through protein kinase A-mediated phosphorylation of Ser133 in the CREB protein. For transcriptional initiation, CREB interacts with several coactivators, such as peroxisome proliferator-activated receptor-␥ coactivator 1␣ (PGC-1) (14), steroid receptor coactivator 1, CREB-binding protein (CBP) (12), and transducer of regulated CREB activity 2 (15–17). In the PEPCK gene promoter, the coactivator PGC-1 also interacts with forkhead transcription factor O1 and hepatocyte nuclear factor-4 for the transcriptional initiation of PEPCK gene (18). Although the coactivators have been well established, the corepressor for CREB remains to be identified and characterized. TNF-␣ induces variety of pathological changes through activation of inhibitor of NF-␬B (I␬B) kinase (IKK)/nuclear factor-␬B (NF-␬B) and c-Jun N-terminal protein kinase (JNK)/AP1 signaling pathways (19, 20). NF-␬B p65 was shown to inhibit PEPCK transcription induced by glucocorticoid or cAMP (21). p65-CBP interaction was proposed to mediate the PEPCK inhibition by TNF-␣. However, it is not clear whether nuclear corepressor is involved in the PEPCK inhibition by TNF-␣. The nuclear corepressor (corepressor in the following text) contains two major subunits. One is histone deacetylase (HDAC) that catalyzes removal of acetyl group from the substrate proteins, s (...truncated)