TGFb1, TNFa, and insulin signaling crosstalk in regulation of the rat cholesterol 7a-hydroxylase gene expression

The TGFb1/Smad pathway plays a critical role in cholestasis and liver fibrosis. Previous studies show that TGFb1, TNFa, and insulin inhibit cholesterol 7a-hydroxylase (CYP7A1) gene transcription and bile acid synthesis in human hepatocytes. In this study, we investigated insulin, TGFb1, and TNFa regulation of rat Cyp7a1 gene transcription. In contrast to inhibition of human CYP7A1 gene transcription, TGFb1 stimulates rat Cyp7a1 reporter activity. Smad3, FoxO1, and HNF4a synergistically stimulated rat Cyp7a1 gene transcription. Mutations of the Smad3, FoxO1, or HNF4a binding site attenuated the rat Cyp7a1 promoter activity. Furthermore, TNFa and cJun attenuated TGFb1 stimulation of rat Cyp7a1. Insulin or adenovirus-mediated expression of constitutively active AKT1 inhibited FoxO1 and Smad3 synergy. In streptozotocin-induced diabetic rats, Cyp7a1 mRNA expression levels were induced and insulin attenuated CYP7A1 mRNA levels. Chromatin immunoprecipitation assay showed that FoxO1 binding to Cyp7a1 chromatin was increased in diabetic rat livers and insulin reduced FoxO1 binding. These results suggest a mechanistic basis for induction of Cyp7a1 activity and bile acid synthesis in cholestatic rats and in diabetic rats. The crosstalk of insulin, TGFb and TNFa signaling pathways may regulate bile acid synthesis and lipid homeostasis in diabetes, fatty liver disease, and liver fibrosis.—Li, T., H. Ma, and J. Y. L. Chiang. TGFb1, TNFa, and insulin signaling crosstalk in regulation of the rat cholesterol 7a-hydroxylase gene expression. J. Lipid Res. 2008. 49: 1981–1989. Supplementary key words CYP7A1 • bile acid synthesis • liver fibrosis • nuclear receptors • Smad • FoxO1 • TGFb • cholestasis Bile acids are synthesized from cholesterol in the liver, stored in the gallbladder, and secreted into the intestine to facilitate the absorption of dietary lipids and fat-soluble vitamins and disposal of toxic metabolites, drugs, and xenobiotics (1, 2). Bile acids are quantitatively reabsorbed in the intestine and transported back to the liver via portal circulation. Hepatic bile acid synthesis is tightly controlled by virtue of high cytotoxicity of bile acids. During cholestatic liver injury, hepatic nonparenchymal cells including Kupffer cells (hepatic macrophages) and hepatic stellate cells (HSC) release proinflammatory cytokines (IL-1b, TNFa) and growth factors (hepatic growth factor, TGFb), respectively, to hepatocytes. These cytokines have been shown to inhibit bile acid synthesis through transcriptional repression of the gene encoding cholesterol 7a-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis in the liver (3–5). Inhibition of bile acid synthesis and stimulation of bile secretion are adaptive responses to protect hepatocytes from injury. Paradoxically, Cyp7a1 activity and mRNA expression are stimulated in bile duct-ligated (BDL) rat and mouse livers (6–8). TGFb1 is increased in the livers of BDL rats and mice, and in patients with liver fibrosis (7, 9). TGFb1 is a potent profibrogenic factor secreted by activated HSCs to stimulate HSC transdifferentiation and proliferation, extracellular matrix expression, hepatocyte apoptosis, and liver fibrosis (10, 11). Liver fibrosis is associated with nonalcoholic fatty liver disease and diabetes (12, 13). Transcription factors Smad2 and Smad3 are major down stream mediators of TGFb1 signaling (11, 14). TGFb1 binds to and activates cell surface TGFb receptor type I (TbRI) and type II (TbRII) that possess intracellular serine/threonine kinase activity. TGFb1-activated TbRI then recruits and phosphorylates Smad2 and Smad3 at a conserved C-terminal SSXS motif. Phosphorylation of Smads by TbRI increases Smad protein nuclear localization and its transactivating activity. Smad3 binds GTCT and AGAC sequences in its target genes. In contrast, Smad2 does not bind directly to DNA because the amino acids that are responsible for DNA binding are displaced in Smad2. This study is supported by NIH grants DK58379 and DK44442. Manuscript received 14 March 2008 and in revised form 9 May 2008. Published, JLR Papers in Press, May 29, 2008. DOI 10.1194/jlr.M800140-JLR200 Abbreviations: AKT/PKB, protein kinase B; BDL, bile duct ligation; ChIP, chromatin immunoprecipitation; CYP7A1, cholesterol 7ahydroxylase; FoxO1, forkhead transcription factor O1; HNF4a, hepatocyte nuclear factor 4a; HSC, hepatic stellate cells; Smad, mothers against decapentaplegic homolog; STZ, streptozotocin; TGFb1, transforming growth factor b1; TbRI, TGFb1 receptor type I; TbRII, TGFb1 receptor type II; TNFa, tumor necrosis factor a. 1 To whom correspondence should be addressed. e-mail: [email protected] Copyright © 2008 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 49, 2008 1981 by gest, on O cber 9, 2017 w w w .j.org D ow nladed fom Recently, several insulin-regulated genes, FoxO1, FoxO3a and FoxO4, have been identified as interacting partners of Smad3 (15). Smad3 and FoxO1 bind to the cyclindependent kinase inhibitor p21 gene promoter and synergistically stimulate its transcription in neuroepithelial and glioblastoma cell proliferation. A subsequent study has identified many TGFb1 target genes that are synchronously regulated by FoxO-Smad in response to diverse cellular activities including cell cycle control, inflammation, and stress in human epithelial cells (16). FoxO1 plays a critical role in mediating insulin signaling (17). Protein kinase B (AKT/PKB) phosphorylation of FoxO1 results in FoxO1 nuclear exclusion and inhibition of FoxO1-regulated genes. It is thought that FoxO1 converges two antagonistic pathways, PI3K/AKT and TGFb1/Smad (18). In this study, we investigated Smad3 regulation of the rat Cyp7a1 gene, and its possible implications in bile acid homeostasis during liver fibrosis and diabetes. Our results show that TGFb1-activated Smad3 stimulates the rat Cyp7a1 gene. Smad3 directly binds to the rat Cyp7a1 promoter and acts in synergy with FoxO1 and HNF4a to induce rat Cyp7a1 promoter activity. Smad3 and FoxO1 may crosstalk and synchronize insulin inhibition and TGFb1 activation of Cyp7a1 in diabetes and liver fibrosis. On the other hand, TNFa signal antagonizes TGFb1 activation of CYP7A1 gene transcription. These three cell-signaling pathways may crosstalk in response to extracellular signals to regulate bile acid synthesis and maintain lipid homeostasis. MATERIALS AND METHODS