Tiangang Li

Bile acids and cytokines inhibit the human cholesterol 7α-hydroxylase gene via the JNK/c-jun pathway in human liver cells

Cholesterol 7α‐hydroxylase (CYP7A1) of the bile acid biosynthesis pathway is suppressed by bile acids and inflammatory cytokines. Bile acids are known to induce inflammatory cytokines to activate the mitogen‐activated protein kinase/c‐Jun N‐terminal kinase (JNK) signaling pathway that inhibits CYP7A1 gene transcription. c‐Jun has been postulated to mediate bile acid inhibition of CYP7A1. However, the c‐Jun target involved in the regulation of CYP7A1 is unknown. Human primary hepatocytes and HepG2 cells were used as models to study chenodeoxycholic acid (CDCA) and interleukin‐1β (IL‐1β) regulation of human CYP7A1 gene expression via real‐time polymerase chain reaction, reporter assays, co‐immunoprecipitation and chromatin immunocipitation (ChIP) assays. IL‐1β and CDCA reduced CYP7A1 but induced c‐Jun messenger RNA expression in human primary hepatocytes. IL‐1β inhibited human CYP7A1 reporter activity via the HNF4α binding site. A JNK‐specific inhibitor blocked the inhibitory effect of IL‐1β on HNF4α expression and CYP7A1 reporter activity. c‐Jun inhibited HNF4α and PPARγ coactivator‐1α (PGC‐1α) coactivation of CYP7A1 reporter activity, whereas a dominant negative c‐Jun did not. Co‐immunoprecipitation and ChIP assays revealed that IL‐1β and CDCA reduced HNF4α bound to the CYP7A1 chromatin, and that c‐Jun interacted with HNF4α and blocked HNF4α recruitment of PGC‐1α to the CYP7A1 chromatin. In conclusion, IL‐1β and CDCA inhibit HNF4α but induce c‐Jun, which in turn blocks HNF4α recruitment of PGC‐1α to the CYP7A1 chromatin and results in inhibition of CYP7A1 gene transcription. The JNK/c‐Jun signaling pathway inhibits bile acid synthesis and protects hepatocytes against the toxic effect of inflammatory agents. (HEPATOLOGY 2006;43:1202–1210.)

Bile Acid Signaling in Liver Metabolism and Diseases

Obesity, diabetes, and metabolic syndromes are increasingly recognized as health concerns worldwide. Overnutrition and insulin resistance are the major causes of diabetic hyperglycemia and hyperlipidemia in humans. Studies in the past decade provide evidence that bile acids are not just biological detergents facilitating gut nutrient absorption, but also important metabolic regulators of glucose and lipid homeostasis. Pharmacological alteration of bile acid metabolism or bile acid signaling pathways such as using bile acid receptor agonists or bile acid binding resins may be a promising therapeutic strategy for the treatment of obesity and diabetes. On the other hand, bile acid signaling is complex, and the molecular mechanisms mediating the bile acid effects are still not completely understood. This paper will summarize recent advances in our understanding of bile acid signaling in regulation of glucose and lipid metabolism, and the potentials of developing novel therapeutic strategies that target bile acid metabolism for the treatment of metabolic disorders.

Rifampicin induction of CYP3A4 requires pregnane X receptor cross talk with hepatocyte nuclear factor 4α and coactivators, and suppression of small heterodimer partner gene expression

Bile acids and drugs activate pregnane X receptor (PXR) to induce CYP3A4, which is the predominant cytochrome P450 enzyme expressed in the liver and intestine and plays a critical role in detoxifying bile acids and drugs, and protecting against cholestasis. The aim of this study is to investigate the molecular mechanism of PXR cross talk with other nuclear receptors and coactivators in regulating human CYP3A4 gene transcription. Rifampicin dose dependently induced the CYP3A4 but inhibited small heterodimer partner (SHP) mRNA expression levels in primary human hepatocytes. Rifampicin strongly stimulated PXR and hepatocyte nuclear factor 4α (HNF4α) interaction, and CYP3A4 reporter activity, which was further stimulated by peroxisome proliferators-activated receptorγ co-activator 1α (PGC-1α) and steroid receptor coactivator-1 (SRC-1) but inhibited by SHP. Mutation of the putative HNF4α binding site in the distal xenobiotic responsive element module did not affect CYP3A4 basal promoter activity and synergistic stimulation by PXR and HNF4α. Chromatin immunoprecipitation assays revealed that rifampicin-activated PXR recruited HNF4α and SRC-1 to the CYP3A4 chromatin. On the other hand, SHP reduced PXR recruitment of HNF4α and SRC-1 to the CYP3A4 chromatin. The human SHP promoter was stimulated by HNF4α and PGC-1α. Upon activation by rifampicin, PXR inhibited SHP promoter activity. Results suggest that PXR strongly induces CYP3A4 gene transcription by interacting with HNF4α, SRC-1, and PGC-1α. PXR concomitantly inhibits SHP gene transcription and maximizes the PXR induction of the CYP3A4 gene in human livers. Drugs targeted to PXR may be developed for treating cholestatic liver diseases induced by bile acids and drugs.

PXR induces CYP27A1 and regulates cholesterol metabolism in the intestine

Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes oxidative cleavage of the sterol side chain in the bile acid biosynthetic pathway in the liver and 27-hydroxylation of cholesterol in most tissues. Recent studies suggest that 27-hydroxycholesterol (27-HOC) activates liver orphan receptor α (LXRα) and induces the cholesterol efflux transporters ABCA1 and ABCG1 in macrophages. The steroid- and bile acid-activated pregnane X receptor (PXR) plays critical roles in the detoxification of bile acids, cholesterol metabolites, and xenobiotics. The role of CYP27A1 in the intestine is not known. This study investigated PXR and CYP27A1 regulation of cholesterol metabolism in the human intestinal cell lines Caco2 and Ls174T. A human PXR ligand, rifampicin, induced CYP27A1 mRNA expression in intestine cells but not in liver cells. Rifampicin induced CYP27A1 gene transcription, increased intracellular 27-HOC levels, and induced ABCA1 and ABCG1 mRNA expression only in intestine cells. A functional PXR binding site was identified in the human CYP27A1 gene. Chromatin immunoprecipitation assays revealed that rifampicin induced the PXR recruitment of steroid receptor coactivator 1 to CYP27A1 chromatin. Cholesterol loading markedly increased intracellular 27-HOC levels in intestine cells. Rifampicin, 27-HOC, and a potent LXRα agonist, T0901317, induced ABCA1 and ABCG1 protein expression and stimulated cholesterol efflux from intestine cells to apolipoprotein A-I and HDL. This study suggests an intestine-specific PXR/CYP27A1/LXRα pathway that regulates intestine cholesterol efflux and HDL assembly.

Nuclear receptors in bile acid metabolism

Bile acids are signaling molecules that activate nuclear receptors, such as farnesoid X receptor, pregnane X receptor, constitutive androstane receptor, and vitamin D receptor, and play a critical role in the regulation of lipid, glucose, energy, and drug metabolism. These xenobiotic/endobiotic-sensing nuclear receptors regulate phase I oxidation, phase II conjugation, and phase III transport in bile acid and drug metabolism in the digestive system. Integration of bile acid metabolism with drug metabolism controls absorption, transport, and metabolism of nutrients and drugs to maintain metabolic homeostasis and also protects against liver injury, inflammation, and related metabolic diseases, such as nonalcoholic fatty liver disease, diabetes, and obesity. Bile-acid–based drugs targeting nuclear receptors are in clinical trials for treating cholestatic liver diseases and fatty liver disease.

Regulation of Bile Acid and Cholesterol Metabolism by PPARs

Bile acids are amphipathic molecules synthesized from cholesterol in the liver. Bile acid synthesis is a major pathway for hepatic cholesterol catabolism. Bile acid synthesis generates bile flow which is important for biliary secretion of free cholesterol, endogenous metabolites, and xenobiotics. Bile acids are biological detergents that facilitate intestinal absorption of lipids and fat-soluble vitamins. Recent studies suggest that bile acids are important metabolic regulators of lipid, glucose, and energy homeostasis. Agonists of peroxisome proliferator-activated receptors (PPARα, PPARγ, PPARδ) regulate lipoprotein metabolism, fatty acid oxidation, glucose homeostasis and inflammation, and therefore are used as anti-diabetic drugs for treatment of dyslipidemia and insulin insistence. Recent studies have shown that activation of PPARα alters bile acid synthesis, conjugation, and transport, and also cholesterol synthesis, absorption and reverse cholesterol transport. This review will focus on the roles of PPARs in the regulation of pathways in bile acid and cholesterol homeostasis, and the therapeutic implications of using PPAR agonists for the treatment of metabolic syndrome.

Bile acid signaling in lipid metabolism: Metabolomic and lipidomic analysis of lipid and bile acid markers linked to anti-obesity and anti-diabetes in mice

Bile acid synthesis is the major pathway for catabolism of cholesterol. Cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme in the bile acid biosynthetic pathway in the liver and plays an important role in regulating lipid, glucose and energy metabolism. Transgenic mice overexpressing CYP7A1 (CYP7A1-tg mice) were resistant to high-fat diet (HFD)-induced obesity, fatty liver, and diabetes. However the mechanism of resistance to HFD-induced obesity of CYP7A1-tg mice has not been determined. In this study, metabolomic and lipidomic profiles of CYP7A1-tg mice were analyzed to explore the metabolic alterations in CYP7A1-tg mice that govern the protection against obesity and insulin resistance by using ultra-performance liquid chromatography-coupled with electrospray ionization quadrupole time-of-flight mass spectrometry combined with multivariate analyses. Lipidomics analysis identified seven lipid markers including lysophosphatidylcholines, phosphatidylcholines, sphingomyelins and ceramides that were significantly decreased in serum of HFD-fed CYP7A1-tg mice. Metabolomics analysis identified 13 metabolites in bile acid synthesis including taurochenodeoxycholic acid, taurodeoxycholic acid, tauroursodeoxycholic acid, taurocholic acid, and tauro-β-muricholic acid (T-β-MCA) that differed between CYP7A1-tg and wild-type mice. Notably, T-β-MCA, an antagonist of the farnesoid X receptor (FXR) was significantly increased in intestine of CYP7A1-tg mice. This study suggests that reducing 12α-hydroxylated bile acids and increasing intestinal T-β-MCA may reduce high fat diet-induced increase of phospholipids, sphingomyelins and ceramides, and ameliorate diabetes and obesity. This article is part of a Special Issue entitled Linking transcription to physiology in lipodomics.

Cannabinoid Receptor Type 1 (CB1R) Signaling Regulates Hepatic Gluconeogenesis via Induction of Endoplasmic Reticulum-bound Transcription Factor cAMP-responsive Element-binding Protein H (CREBH) in Primary Hepatocytes

Activated cannabinoid 1 receptor (CB1R) signaling has been implicated in the development of phenotypes associated with fatty liver, insulin resistance, and impaired suppression of hepatic glucose output. Endoplasmic reticulum stress-associated liver-specific transcription factor CREBH is emerging as a critical player in various hepatic metabolic pathways and regulates hepatic gluconeogenesis in diet-induced obese settings. In this study, we elucidated the critical role of CREBH in mediating CB1R signaling to regulate glucose homeostasis in primary rat and human hepatocytes. mRNA and protein levels and glucose production were analyzed in primary rat and human hepatocytes. ChIP assays were performed together with various transcriptional analyses using standard techniques. CB1R activation by 2-arachidonoylglycerol (2-AG) specifically induced CREBH gene expression via phosphorylation of the JNK signaling pathway and c-Jun binding to the AP-1 binding site in the CREBH gene promoter. 2-AG treatment significantly induced hepatic gluconeogenic gene expression and glucose production in primary hepatocytes, and we demonstrated that the CREBH binding site mutant significantly attenuated 2-AG-mediated activation of the gluconeogenic gene promoter. Endogenous knockdown of CREBH led to ablation of 2-AG-induced gluconeogenic gene expression and glucose production, and the CB1R antagonist AM251 or insulin exhibited repression of CREBH gene induction and subsequently inhibited gluconeogenesis in both rat and human primary hepatocytes. These results demonstrate a novel mechanism of action of activated CB1R signaling to induce hepatic gluconeogenesis via direct activation of CREBH, thereby contributing to a better understanding of the endocannabinoid signaling mechanism involved in regulating the hepatic glucose metabolism.

All-trans-retinoic acid ameliorates hepatic steatosis in mice by a novel transcriptional cascade

Mice deficient in small heterodimer partner (SHP) are protected from diet‐induced hepatic steatosis resulting from increased fatty acid oxidation and decreased lipogenesis. The decreased lipogenesis appears to be a direct consequence of very low expression of peroxisome proliferator‐activated receptor gamma 2 (PPAR‐γ2), a potent lipogenic transcription factor, in the SHP−/− liver. The current study focused on the identification of a SHP‐dependent regulatory cascade that controls PPAR‐γ2 gene expression, thereby regulating hepatic fat accumulation. Illumina BeadChip array (Illumina, Inc., San Diego, CA) and real‐time polymerase chain reaction were used to identify genes responsible for the linkage between SHP and PPAR‐γ2 using hepatic RNAs isolated from SHP−/− and SHP‐overexpressing mice. The initial efforts identify that hairy and enhancer of split 6 (Hes6), a novel transcriptional repressor, is an important mediator of the regulation of PPAR‐γ2 transcription by SHP. The Hes6 promoter is specifically activated by the retinoic acid receptor (RAR) in response to its natural agonist ligand, all‐trans retinoic acid (atRA), and is repressed by SHP. Hes6 subsequently represses hepatocyte nuclear factor 4 alpha (HNF‐4α)‐activated PPAR‐γ2 gene expression by direct inhibition of HNF‐4α transcriptional activity. Furthermore, we provide evidences that atRA treatment or adenovirus‐mediated RAR‐α overexpression significantly reduced hepatic fat accumulation in obese mouse models, as observed in earlier studies, and the beneficial effect is achieved by the proposed transcriptional cascade. Conclusions: Our study describes a novel transcriptional regulatory cascade controlling hepatic lipid metabolism that identifies retinoic acid signaling as a new therapeutic approach to nonalcoholic fatty liver diseases. (Hepatology 2014;59:1750–1760)

Curcumin Differentially Regulates Endoplasmic Reticulum Stress through Transcriptional Corepressor SMILE (Small Heterodimer Partner-interacting Leucine Zipper Protein)-mediated Inhibition of CREBH (cAMP Responsive Element-binding Protein H)

Background: Curcumin has been reported to play an important role in ER stress. Results: Curcumin blocks the CREBH-mediated transactivation of target gene, whereas it has no such effect on ATF6-mediated transactivation. Conclusion: Curcumin differentially regulates ER stress-induced genes. Significance: Curcumin may provide a way to ameliorate ER stress. Curcumin (diferuloylmethane), a major active component of turmeric (Curcuma longa), is a natural polyphenolic compound. Herein the effect of curcumin on endoplasmic reticulum (ER) stress responsive gene expression was investigated. We report that curcumin induces transcriptional corepressor small heterodimer partner-interacting leucine zipper protein (SMILE) gene expression through liver kinase B1 (LKB1)/adenosine monophosphate-activated kinase (AMPK) signaling pathway and represses ER stress-responsive gene transcription in an ER-bound transcription factor specific manner. cAMP responsive element-binding protein H (CREBH) and activating transcription factor 6 (ATF6) are both ER-bound bZIP family transcription factors that are activated upon ER stress. Of interest, we observed that both curcumin treatment and SMILE overexpression only represses CREBH-mediated transactivation of the target gene but not ATF6-mediated transactivation. Knockdown of endogenous SMILE significantly releases the inhibitory effect of curcumin on CREBH transactivation. Intrinsic repressive activity of SMILE is observed in the Gal4 fusion system, and the intrinsic repressive domain is mapped to the C terminus of SMILE spanning amino acid residues 203–269, corresponding to the basic region leucine zipper (bZIP) domain. In vivo interaction assay revealed that through its bZIP domain, SMILE interacts with CREBH and inhibits its transcriptional activity. Interestingly, we observed that SMILE does not interact with ATF6. Furthermore, competition between SMILE and the coactivator peroxisome proliferator-activated receptor α (PGC-1α) on CREBH transactivation has been demonstrated in vitro and in vivo. Finally, chromatin immunoprecipitation assays revealed that curcumin decreases the binding of PGC-1α and CREBH on target gene promoter in a SMILE-dependent manner. Overall, for the first time we suggest a novel phenomenon that the curcumin/LKB1/AMPK/SMILE/PGC1α pathway differentially regulates ER stress-mediated gene transcription.