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The aim of this study was to explore why in rabbits activation of farnesoid X receptor (FXR) is dominant over activated liver X receptor-[alpha] (LXR[alpha]) in the regulation of CYP7A1. We cloned the rabbit CYP7A1 promoter and found a fetoprotein transcription factor (FTF) binding element embedded within the LXR[alpha] binding site (LXRE). Gel shift assays demonstrated that FTF competes with LXR[alpha] for binding to LXRE. Short heterodimer partner (SHP) enhances the competitive ability of FTF. Studies in HepG2 cells showed that SHP combined with FTF had more powerful effect to offset the stimulation of CYP7A1 by LXR[alpha]. Gel shift and chromatin immunoprecipitation assays demonstrated that SHP with FTF diminished LXR[alpha] binding to the CYP7A1 promoter. In vivo studies in rabbits fed cholesterol for 10 days showed that hepatic expression of SHP but not FTF rose and LXR[alpha]-bound LXRE decreased. We propose that the SHP/FTF heterodimer occupies LXRE via the embedded FTF binding element and blocks LXR[alpha] from recruiting to LXRE. Therefore, activation of FXR, which upregulates SHP expression, will eliminate the stimulatory effect of LXR[alpha] on the CYP7A1 promoter because increased levels of SHP combined with FTF diminish the recruitment of LXR[alpha] to CYP7A1 promoter. cholesterol 7[alpha]-hydroxylase; regulation; LXR binding site; short heterodimer partner; fetoprotein transcription factor; farnesoid X receptor; liver X receptor-[alpha]

Sterol 12[alpha]-hydroxylase (CYP8B 1) catalyzes cholic acid synthesis in the liver and is feedback inhibited by bile acids. In addition to activating farnesoid X receptor (nuclear receptor subfamily 1H4), bile acids also induce inflammatory cytokines in hepatocytes. The objective of this study was to investigate the mechanism by which inflammatory cytokines inhibit human CYP8BI gene transcription. Real-time PCR assays revealed that both chenodeoxycholic acid (CDCA) and interleukin- 1 [beta] (IL-I[beta]) markedly reduced CYP8B1, cholesterol 7[alpha]-hydroxylase CYP7A1 and hepatic nuclear factor 4[alpha] (HNF4[alpha]) mRNA expression levels in human primary hepatocytes. However, CDCA induced, but IL-1[beta] reduced, small heterodimer partner (SHP) mRNA expression. IL-1[beta] inhibited human CYP8B1 reporter activity only in liver cells, and a c-Jun [NH.sub.2]-terminal kinase (JNK)-specific inhibitor-blocked IL-1[beta] inhibition. Activated JNK1 or c-Jun inhibited, whereas their dominant negative forms blocked, IL-1[beta] inhibition of CYP8B1 transcription. Mutagenesis analyses mapped an IL-1[beta] response element to a previously identified bile acid response element, which contains an HNF4[alpha] binding site. A dominant negative HNF4[alpha] inhibited CYP8B1 gene transcription and ectopically expressed HNF4c[alpha] blocked IL-1[beta] inhibition. Furthermore, IL-1[beta] inhibited HNF4[alpha] gene transcription, protein expression, and binding to the CYP8B1 gene. JNK1 phosphorylated HNF4[alpha] and a JNK-specific inhibitor blocked the IL-1[beta] inhibition of HNF4[alpha] expression. These results suggest that IL-1[beta] inhibits CYP8B1 gene transcription via a mitogen-activated protein kinase/ JNK pathway that inhibits HNF4[alpha] gene expression and its DNA-binding ability. This mechanism may play an important role in the adaptive response to inflammatory cytokines and in the protection of the liver during cholestasis. bile acid synthesis; nuclear receptor; interleukin-1[beta]; hepatic nuclear factor 4[alpha]; cholestasis

The regulation of the rabbit apical sodium-dependent bile acid transporter (ASBT) was studied both in vivo and in vitro. New Zealand White rabbits were fed 0.5% deoxycholic acid (DCA) or SC-435, a competitive ASBT inhibitor, for 1 wk. In DCA-fed rabbits, ASBT expression was repressed, associated with actiwtted FXR, and evidenced by increased ileal short heterodimer partner (SHP) mRNA. Feeding SC-435 to the rabbits blocked bile acid absorption, decreased SHP mRNA, and increased ASBT expression. A 1.9-kb rabbit ASBT 5'-flanking region (promoter) was cloned, and a cis-acting element for [alpha]-fetoprotein transcription factor (FTF) was identified (-1166/-1158). The effects of transcriptional factors and different bile acids on the rabbit ASBT promoter were studied in Caco-2 cells. FTF stimulated the rabbit ASBT promoter activity fourfold but not after the FTF binding site was deleted from the promoter. Increasing the SHP protein notably inhibited FTF-dependent trans-activation of rabbit ASBT. Adding hydrophobic bile acids deoxycholic acid, chenodeoxycholic acid, and cholic acid, activating ligands for FXR, inhibited rabbit ASBT promoter activity in Caco-2 cells, but this inhibitory effect was abolished after the FTF binding site was deleted. Ursodeoxycholic acid and ursocholic acid, nonactivating ligands for FXR, did not repress ASBT promoter activity. Thus the rabbit ASBT promoter is negative-feedback regulated by bile acids via a functional FTF binding site. Only FXR-activating ligands can downregulate rabbit ASBT expression through the regulatory cascade FXR-SHP-FTF.

Bile acids, steroids, and drugs activate steroid and xenobiotic receptor pregnane X receptor (PXR: NRI12), which induces human cytochrome P4503A4 (CYP3A4) in drug metabolism and cholesterol 7[alpha]-hydroxytase (CYP7AI) in bile acid synthesis in the liver. Rifampicin, a human PXR agonist, inhibits bile acid synthesis and has been used to treat cholestatic diseases. The objective of this study is to elucidate the mechanism by which PXR inhibits CYP7A1 gene transcription. The mRNA expression levels of CYP7A1 and several nuclear receptors known to regulate the CYP7A1 gene were assayed in human primary hepatocytes by quantitative real-time PCR (Q-PCR). Rifampicin reduced CYP7A1 and small heterodimer partner (SHP: NRO2B) mRNA expression suggesting that SHP was not involved in PXR inhibition of CYP7A1. Rifampicin inhibited CYP7A1 reporter activity and a PXR binding site was localized to the bile acid response element-1. Mammalian two-hybrid assays revealed that PXR interacted with hepatic nuclear factor 4[alpha] (HNF4[alpha], NR2A1) and rifampicin was required. Coimmunoprecipitation assay confirmed PXR interaction with HNF4[alpha] PXR also interacted with peroxisome proliferator-activated receptor [gamma] coactivator (PGC-1[alpha]), which interacted with HNF4[alpha] and induced CYP7A1 gene transcription. Rifampicin enhanced PXR interaction with HNF4[alpha] and reduced PGC-1[alpha] interaction with HNF4[alpha]. Chromatin immunoprecipitation assay showed that PXR, HNF4[alpha], and PGC-1[alpha] bound to CYP7A1 chromatin, and rifampicin dissociated PGC-1[alpha] from chromatin. These results suggest that activation of PXR by rifampicin promotes PXR interaction with HNF4[alpha] and blocks PGC-1[alpha] activation with HNF4[alpha] and results in inhibition of CYP7A1 gene transcription. Rifampicin inhibition of bile acid synthesis may be a protectixe mechanism against drug and bile acid-induced cholestasis. bile acid synthesis; gene regulation: nuclear receptors; peroxisome proliferator-activated receptor [gamma] coactivator

Bile acids are physiological detergents that facilitate excretion, absorption, and transport of fats and sterols in the intestine and liver. Recent studies reveal that bile acids, also are signaling molecules that activate several nuclear receptors and regulate many physiological pathways and processes to maintain bile acid and cholesterol homeostasis. Mutations of the principal regulatory genes in bile acid biosynthetic pathways have recently been identified in human patients with hepatobiliary and cardiovascular diseases. Genetic manipulation of key regulatory genes and bile acid receptor genes in mice have been obtained. These advances have greatly improved our understanding of the molecular mechanisms underlying complex liver physiology but also raise many questions and controversies to be resolved. These developments will lead to early diagnosis and discovery of drugs for treatment of liver and cardiovascular diseases. liver orphan receptor; farnesoid X receptor; cholesterol 7[alpha]-hydroxylase cytochrome P-450; cholesterol homeostasis; gene regulation