| Cholesterol is essential in many biological activities in mammalian cells. Conversion of hepatic cholesterol into bile acids is a major pathway to eliminate cholesterol from the body. However, excess amounts of cholesterol and bile acids are pathogenic. Therefore, the levels of cholesterol and bile acids need to be tightly regulated. Cholesterol 7alpha-hydroxylase (CYP7A1), a liver specific P450 enzyme, is the first and rate-limiting enzyme in this process. Increased levels of bile acids repress transcription of CYP7A1 in a feedback manner. Bile acid-activated FXR increases the transcription of small heterodimer partner (SHP), an orphan nuclear receptor. SHP interacts with hepatic nuclear factor-4 (HNF-4) or liver receptor homologue-1 (LRH-1) on the CYP7A1 promoter, and represses CYP7A1. Recently, an intestinal fibroblast growth factor (FGF) 15/19 has been reported to repress CYP7A1 transcription, which is also depending on SHP expression. In addition to SHP-dependent pathways, xenobiotic nuclear receptors, such as pregnane X receptor (PXR) and constitutive androstane receptor (CAR), have been implicated to play a role in bile acid-mediated repression of CYP7A1. The overall aim of this study is to delineate molecular mechanisms by which the bile acid biosynthesis is regulated in SHP-independent and SHP-dependent pathways.;First, the molecular mechanism of CAR-mediated transcriptional repression of CYP7A1 was examined. It was demonstrated that CAR not only competes with HNF-4 for binding to the CYP7A1, but also competes with HNF4 for common coactivators, such as PGC-1alpha and GRIP-1. These events lead to the dissociation of coactivators from the CYP7A1 promoter, resulting in suppression of the gene. This study provides evidence of a new function for xenobiotic nuclear receptors in regulation of bile acid biosynthesis.;SHP-mediated transcriptional repression of CYP7A1 in a native chromatin context was examined. SHP actively recruits an mSin3A/HDAC-1 deactylase complex and a Swi/Snf-Brm chromatin remodeling complex to the CYP7A1 promoter. Recruitment of these factors results in altered histone modification and chromatin structure, thus repressing the gene. Brm and Brg-1 are two central ATPases of the Swi/Snf complex. Since only Brm, but not Brg-1, was recruited to the CYP7A1 promoter, we speculated that Brm and Brg-1 may function distinctly in bile acid biosynthesis. Therefore, the role of Brm and Brg-1 in FXR/SHP-mediated regulation of CYP7A1 was examined. It was found that Brm, but not Brg-1, is a critical component of the SHP inhibitory complex, and potentiates SHP-mediated suppression of the CYP7A1 promoter, as well as the SHP promoter in an auto-regulatory manner. In contrast, Brg-1, but not Brm, is a coactivator for FXR and activates SHP gene transcription. Thus two Swi/Snf ATPases, Brm and Brg-1, function distinctly in the regulation of bile acid biosynthesis.;Posttranslational modifications profoundly regulate protein stability and activity. Results demonstrated that bile acids function as signaling molecules to increase SHP repressive activity by increasing SHP protein stability and association with corepressors. In addition to bile acid signaling, FGF15/19 was also demonstrated to increase SHP protein stability by activating ERK signaling in cell and mouse in vivo, which provides an explanation of how FGF15/19 inhibits CYP7A1 without inducing SHP transcription.;Interestingly, in this study, SHP stability was found increased in ob/ob mice and mice chronically fed with high fat diet, however SHP gene transcription was not altered. These data strongly suggest that abnormal regulation of SHP protein stability is associated with pathological disease conditions.;SHP is an atypical nuclear receptor, which lacks a DNA binding domain, but contains a putative ligand binding domain. It was found that 3Cl-AHPC potentiates SHP function by increasing SHP stability and interaction with corepressors, including mSin3A/HDAC-1, Brm and G9A. In addition, 3Cl-AHPC promotes the recruitment of SHP and corepressors to the CYPA7A1 promoter, and represses the gene. These data strongly suggest that 3Cl-AHPC is a SHP agonist and potentiates SHP inhibitory function in down regulation of hepatic bile acid biosynthesis.;These combined studies should greatly advance our understanding of how bile acid biosynthesis is regulated in both SHP-dependent and SHP-independent pathways. Importantly, these studies for the first time demonstrate that SHP has a short half-life of 20 to 30 minutes. Bile acids and FGF15/19 signaling pathways, and SHP ligand can dramatically increase SHP protein stability and abnormal regulation of SHP protein stability is associated with pathological disease conditions, indicating that regulation of SHP protein stability is a critical mechanism to regulate SHP activity. Since SHP plays a critical role in diverse cellular pathways, including bile acid/cholesterol and lipid/glucose homeostasis, and cell proliferation, this study to define how SHP activity is modulated by bile acids, FGF15/19 and its ligands, may reveal novel molecular targets for treating disorders in which SHP plays a key regulatory role. (Abstract shortened by UMI.)... |
