Direct methylation of FXR by Set7/9, a lysine methyltransferase, regulates the expression of FXR target genes.

The farnesoid X receptor (FXR) is a ligand (bile acid)-dependent nuclear receptor that regulates target genes involved in every aspect of bile acid homeostasis. Upon binding of ligand, FXR recruits an array of coactivators and associated proteins, some of which have intrinsic enzymatic activity that modify histones or even components of the transcriptional complex. In this study, we show chromatin occupancy by the Set7/9 methyltransferase at the FXR response element (FXRE) and direct methylation of FXR in vivo and in vitro at lysine 206. siRNA depletion of Set7/9 in the Huh-7 liver cell line decreased endogenous mRNAs of the FXR target genes, the short heterodimer partner (SHP) and bile salt export pump (BSEP). Mutation of the methylation site at K206 of FXR to an arginine prevented methylation by Set7/9. A pan-methyllysine antibody recognized the wild-type FXR but not the K206R mutant form. An electromobility shift assay showed that methylation by Set7/9 enhanced binding of FXR/retinoic X receptor-α to the FXRE. Interaction between hinge domain of FXR (containing K206) and Set7/9 was confirmed by coimmunoprecipitation, GST pull down, and mammalian two-hybrid experiments. Set7/9 overexpression in Huh-7 cells significantly enhanced transactivation of the SHP and BSEP promoters in a ligand-dependent fashion by wild-type FXR but not the K206R mutant FXR. A Set7/9 mutant deficient in methyltransferase activity was also not effective in increasing transactivation of the BSEP promoter. These studies demonstrate that posttranslational methylation of FXR by Set7/9 contributes to the transcriptional activation of FXR-target genes.

The membrane protein ATPase class I type 8B member 1 signals through protein kinase C zeta to activate the farnesoid X receptor.

UNLABELLED: Prior loss-of-function analyses revealed that ATPase class I type 8B member 1 [familial intrahepatic cholestasis 1 (FIC1)] posttranslationally activated the farnesoid X receptor (FXR). Mechanisms underlying this regulation were examined by gain-of-function studies in UPS cells, which lack endogenous FIC1 expression. FXR function was assayed in response to wild-type and mutated FIC1 expression constructs with a human bile salt export pump (BSEP) promoter and a variety of cellular localization techniques. FIC1 overexpression led to enhanced phosphorylation and nuclear localization of FXR that was associated with FXR-dependent activation of the BSEP promoter. The FIC1 effect was lost after mutation of the FXR response element in the BSEP promoter. Despite similar levels of FIC1 protein expression, Byler disease FIC1 mutants did not activate BSEP, whereas benign recurrent intrahepatic cholestasis mutants partially activated BSEP. The FIC1 effect was dependent on the presence of the FXR ligand, chenodeoxycholic acid. The effect of FIC1 on FXR phosphorylation and nuclear localization and its effects on BSEP promoter activity could be blocked with protein kinase C zeta (PKC zeta) inhibitors (pseudosubstrate or small interfering RNA silencing). Recombinant PKC zeta directly phosphorylated immunoprecipitated FXR. The mutation of threonine 442 of FXR to alanine yielded a dominant negative protein, whereas the phosphomimetic conversion to glutamate resulted in FXR with enhanced activity and nuclear localization. Inhibition of PKC zeta in Caco-2 cells resulted in activation of the human apical sodium-dependent bile acid transporter promoter. CONCLUSION: These results demonstrate that FIC1 signals to FXR via PKC zeta. FIC1-related liver disease is likely related to downstream effects of FXR on bile acid homeostasis. Benign recurrent intrahepatic cholestasis emanates from a partially functional FIC1 protein. Phosphorylation of FXR is an important mechanism for regulating its activity.

Role of CYP27A in cholesterol and bile acid metabolism.

The CYP27A gene encodes a mitochondrial cytochrome P450 enzyme, sterol 27-hydroxylase, that is expressed in many different tissues and plays an important role in cholesterol and bile acid metabolism. In humans, CYP27A deficiency leads to cerebrotendinous xanthomatosis. To gain insight into the roles of CYP27A in the regulation of cholesterol and bile acid metabolism, cyp27A gene knockout heterozygous, homozygous, and wild-type littermate mice were studied. In contrast to homozygotes, heterozygotes had increased body weight and were mildly hypercholesterolemic, with increased numbers of lipoprotein particles in the low density lipoprotein size range. Cyp7A expression was not increased in heterozygotes but was in homozygotes, suggesting that parts of the homozygous phenotype are secondary to increased cyp7A expression and activity. Homozygotes exhibited pronounced hepatomegaly and dysregulation in hepatic cholesterol, bile acid, and fatty acid metabolism. Hepatic cholesterol synthesis and synthesis of bile acid intermediates were increased; however, side chain cleavage was impaired, leading to decreased bile salt concentrations in gallbladder bile. Expression of Na-taurocholate cotransporting polypeptide, the major sinusoidal bile salt transporter, was increased, and that of bile salt export pump, the major canalicular bile salt transporter, was decreased. Gender played a modifying role in the homozygous response to cyp27A deficiency, with females being generally more severely affected. Thus, both cyp27A genotype and gender affected the regulation of hepatic bile acid, cholesterol, and fatty acid metabolism.

Transporter-mediated bile acid uptake causes Ca2+-dependent cell death in rat pancreatic acinar cells.

BACKGROUND & AIMS: The mechanism by which cholelithiasis increases the risk of acute pancreatitis remains obscure. Because bile acids can enter the pancreas either by luminal diffusion or by interstitial leakage during gallstone impaction and pancreatitis is associated with impaired Ca(2+) signaling, we examined the effect of bile acids on pancreatic acinar cell signaling and the associated intracellular events. METHODS: Rat pancreatic acinar cells were isolated by collagenase digestion and the effects of bile acids on [Ca(2+)](i) signaling, cell survival, inflammatory signals, and the molecular and functional expressions of bile uptake transporters were analyzed. RESULTS: Bile acids specifically inhibited the sarco/endoplasmic reticulum Ca(2+) ATPase pump to chronically deplete part of the Ca(2+) stored in the endoplasmic reticulum. This in turn led to the activation of capacitative Ca(2+) entry and a chronic [Ca(2+)](i) load. The increase in [Ca(2+)](i) and Ca(2+) load activated the inflammation-associated signals of c-Jun amino-terminal kinases and NF-kappaB and led to cell death, which was inhibited by buffering [Ca(2+)](i) with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid. A comprehensive molecular analysis of bile acid transporters revealed that pancreatic acinar cells express the bile uptake transporters Na(+)-taurocholate co-transporting polypeptide and organic anion transporting polypeptide in the luminal and basolateral membranes, respectively. Bile acid uptake into acinar cells was in part Na(+)-dependent and in part Na(+)-independent, suggesting that both transporters contribute to bile acid influx into acinar cells. CONCLUSIONS: These results suggest that bile acids can be transported into pancreatic acinar cells through specific membrane transporters and induce cell death by impairing cellular Ca(2+) signaling.

Hypercholesterolemia and changes in lipid and bile acid metabolism in male and female cyp7A1-deficient mice.

Cholesterol 7alpha-hydroxylase, a rate-limiting enzyme for bile acid synthesis, has been implicated in genetic susceptibility to atherosclerosis. The gene, CYP7A1, encoding a protein with this activity, is expressed normally only in hepatocytes and is highly regulated. Our cyp7A1 gene knockout mouse colony, as young adults on a chow diet, is hypercholesterolemic. These mice were characterized extensively to understand how cyp7A1 affects lipid and bile acid homeostasis in different tissue compartments and whether gender plays a modifying role. Both male and female cyp7A1-deficient mice had decreased hepatic LDL receptors, unchanged hepatic cholesterol synthesis, increased intestinal cholesterol synthesis and bile acid transporters, and decreased fecal bile acids but increased fecal sterols. In females, cyp7A1 deficiency also caused changes in hepatic fatty acid metabolism, decreased hepatic canalicular bile acid transporter, Bsep, and gallbladder bile composition altered to a lithogenic profile. Taken together, the data suggest that cyp7A1 deficiency results in a proatherogenic phenotype in both genders and leads to a prolithogenic phenotype in females.