Liver receptor homolog 1 transcriptionally regulates human bile salt export pump expression

The metabolic conversion of cholesterol into bile acids in liver is initiated by the rate-limiting cholesterol 7α-hydroxylase (CYP7A1), whereas the bile salt export pump (BSEP) is responsible for the canalicular secretion of bile acids. Liver receptor homolog 1 (LRH-1) is a key transcriptional factor required for the hepatic expression of CYP7A1. We hypothesized that LRH-1 was also involved in the transcriptional regulation of BSEP. In support of our hypothesis, we found that overexpression of LRH-1 induced, whereas knockdown of LRH-1 decreased, BSEP expression. Consistent with its role in transcriptional regulation, LRH-1 dose-dependently transactivated the BSEP promoter. In addition, such transactivation by LRH-1 was required for maximal induction of BSEP expression through the bile acid/farnesoid X receptor (FXR) activation pathway. Bioinformatic and mutational analysis led to the identification of a functional liver receptor homolog 1-responsive element (LRHRE) in the BSEP promoter. Specific binding of LRH-1 to the LRHRE and recruitment of LRH-1 to the BSEP promoter were demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay, respectively. In conclusion, LRH-1 transcriptionally activated the BSEP promoter and functioned as a modulator in bile acid/FXR-mediated BSEP regulation. These results suggest that LRH-1 plays a supporting role to FXR in maintaining hepatic bile acid levels by coordinately regulating CYP7A1 and BSEP for bile acid synthesis and elimination, respectively

DEC1 Negatively Regulates the Expression of DEC2 through Binding to the E-box in the Proximal Promoter

Human DEC (differentially expressed in chondrocytes), mouse STRA (stimulated with retinoic acid), and rat SHARP (split and hairy related protein) proteins constitute a new and structurally distinct class of the basic helix-loop-helix proteins. In each species, two members are identified with a sequence identity of \u3e90% in the basic helix-loop-helix region and ∼40% in the total proteins, respectively. Recently, we have reported that DEC1 is abundantly expressed in colon carcinomas but not in the adjacent normal tissues. The present study was undertaken to extend the expression study of DEC1 and to determine whether DEC1 and DEC2 had similar expression patterns among paired cancer-normal tissues from the colon, lung, and kidney. Without exceptions, DEC1 was markedly higher in the carcinomas, whereas the opposite was true with DEC2. In stable transfectants, tetracycline-induced expression of DEC1 caused proportional decreases in the expression of DEC2. Co-transfection with DEC1 repressed the activity of a DEC2 promoter reporter by as much as 90%. The repression was observed with wild type DEC1 but not its DNA binding-defective mutants. Studies with deletion and site-directed mutants located, in the proximal promoter, an E-box motif that supported the DEC1-mediated repression. Disruption of this E-box markedly abolished the ability of the reporter to respond to DEC1. Our findings assign for DEC1 the first target gene that is regulated through direct DNA binding. DEC/STRA/SHARP proteins are highly identical in the DNA binding domain but much more diverse in other areas. DEC1-mediated repression on the expression of DEC2 provides an important mechanism that these transcription factors regulate the cellular function not only by modulating the expression of their target genes but also the expression of members within the same class

Mechanistic insights into isoform-dependent and species-specific regulation of bile salt export pump by farnesoid X receptor

Expression of bile salt export pump (BSEP) is regulated by the bile acid/farnesoid X receptor (FXR) signaling pathway. Two FXR isoforms, FXRα1 and FXRα2, are predominantly expressed in human liver. We previously showed that human BSEP was isoform-dependently regulated by FXR and diminished with altered expression of FXRα1 and FXRα2 in patients with hepatocellular carcinoma. In this study, we demonstrate that FXRα1 and FXRα2 regulate human BSEP through two distinct FXR responsive elements (FXRE): IR1a and IR1b. As the predominant regulator, FXRα2 potently transactivated human BSEP through IR1a, while FXRα1 weakly transactivated human BSEP through a newly identified IR1b. Relative expression of FXRα1 and FXRα2 affected human BSEP expression in vitro and in vivo. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed the binding and recruitment of FXRα1 and FXRα2 to IR1b and IR1a. Sequence analysis concluded that IR1b was completely conserved among species, whereas IR1a exhibited apparent differences across species. Sequence variations in IR1a were responsible for the observed species difference in BSEP transactivation by FXRα1 and FXRα2. In conclusion, FXR regulates BSEP in an isoform-dependent and species-specific manner through two distinct FXREs, and alteration of relative FXR isoform expression may be a potential mechanism for FXR to precisely regulate human BSEP in response to various physiological and pathological conditions

Pregnane X receptor is required for interleukin-6-mediated down-regulation of cytochrome P450 3A4 in human hepatocytes

Cytochrome P450 3A4 (CYP3A4) is the most abundant cytochrome P450 enzyme in human liver and metabolizes more than 60% of prescribed drugs in human body. Patients with liver conditions such as cirrhosis show increased secretion of cytokines (e.g., interleukin-6) and decreased capacity of oxidation of many drugs. In this study, we provided molecular evidence that cytokine secretion directly contributed to the decreased capacity of oxidative biotransformation in human liver. After human hepatocytes were treated with IL-6, the expression of CYP3A4 decreased at both mRNA and protein levels, so did the CYP3A4 enzymatic activity. Meanwhile, the repression of CYP3A4 by IL-6 occurred after the decrease of pregnane X receptor (PXR) in human hepatocytes. The PXR-overexpressed cells (transfected with human PXR) increased the CYP3A4 mRNA level, and the repression of CYP3A4 by IL-6 was greater in the PXR-overexpressed cells than in the control cells. Further, PXR knockdown (transfected with siPXR construct) decreased the CYP3A4 mRNA level with less repression by IL-6 than in the control cells transfected with corresponding vector. Collectively, our study suggests that PXR is necessary for IL-6-mediated repression of the CYP3A4 expression in human hepatocytes

Differential modulation of farnesoid X receptor signaling pathway by the thiazolidinediones

ABSTRACT Thiazolidinediones (TZD), including troglitazone, rosiglitazone, and pioglitazone, are agonists of peroxisome proliferator-activated receptor (PPAR)-␥ and belong to a class of insulin-sensitizing drugs for type 2 diabetes mellitus. However, memberspecific, PPAR␥-independent activities and toxicity have been reported, especially for troglitazone. Currently, the underlying mechanisms are not fully understood. In this study, we demonstrated that troglitazone but not rosiglitazone or pioglitazone modulated expression of farnesoid X receptor (FXR) target genes bile salt export pump (BSEP) and small heterodimer partner (SHP) in Huh-7 cells. More specifically, troglitazone acted as a partial agonist of FXR to weakly increase BSEP and SHP expression but functioned as a potent antagonist to significantly suppress bile acid-induced expression. Consistent with the finding, troglitazone partially induced but markedly antagonized bile acid-mediated BSEP promoter transactivation. However, such modulating effects were not detected with rosiglitazone or pioglitazone. Using the crystal structure of ligand-bound FXR ligand binding domain (LBD), molecular docking predicted that troglitazone, but not rosiglitazone or pioglitazone, could form a stable complex with FXR LBD. The specific ␣-tocopherol side chain of troglitazone significantly contributed to the formation of such a stable complex through extensive interactions with FXR LBD. The docking model was further validated by functional analyses of a series of dockingguided FXR mutants. In summary, the data demonstrated that troglitazone, but not rosiglitazone or pioglitazone, was an FXR modulator and potently antagonized bile acid-induced expression of FXR target genes. Such differential modulation of FXR signaling pathway by TZDs may represent one of the mechanisms for member-specific, PPAR␥-independent activities and toxicity. Thiazolidinediones (TZD), including troglitazone, rosiglitazone, and pioglitazone, are a class of insulin-sensitizing drugs to treat type 2 diabetes mellitus. Such therapeutic effect of TZDs is achieved through activating nuclear receptor peroxisome proliferator-activated receptor (PPAR)-␥, which is directly involved in the regulation of genes controlling glucose homeostasis and lipid metabolism. Studies also show that TZDs exhibit other important activities, such as cardiovascular, hypertension, and anticancer effects in a PPAR␥-dependent or -independent manne

Cellular Origins of EGFR-Driven Lung Cancer Cells Determine Sensitivity to Therapy

Targeting the epidermal growth factor receptor (EGFR) with tyrosine kinase inhibitors (TKIs) is one of the major precision medicine treatment options for lung adenocarcinoma. Due to common development of drug resistance to first- and second-generation TKIs, third-generation inhibitors, including osimertinib and rociletinib, have been developed. A model of EGFR-driven lung cancer and a method to develop tumors of distinct epigenetic states through 3D organotypic cultures are described here. It is discovered that activation of the EGFR T790M/L858R mutation in lung epithelial cells can drive lung cancers with alveolar or bronchiolar features, which can originate from alveolar type 2 (AT2) cells or bronchioalveolar stem cells, but not basal cells or club cells of the trachea. It is also demonstrated that these clones are able to retain their epigenetic differences through passaging orthotopically in mice and crucially that they have distinct drug vulnerabilities. This work serves as a blueprint for exploring how epigenetics can be used to stratify patients for precision medicine decisions

Differential Modulation of Farnesoid X Receptor Signaling Pathway by the Thiazolidinediones

Thiazolidinediones (TZD), including troglitazone, rosiglitazone, and pioglitazone, are agonists of peroxisome proliferator-activated receptor (PPAR)-γ and belong to a class of insulin-sensitizing drugs for type 2 diabetes mellitus. However, member-specific, PPARγ-independent activities and toxicity have been reported, especially for troglitazone. Currently, the underlying mechanisms are not fully understood. In this study, we demonstrated that troglitazone but not rosiglitazone or pioglitazone modulated expression of farnesoid X receptor (FXR) target genes bile salt export pump (BSEP) and small heterodimer partner (SHP) in Huh-7 cells. More specifically, troglitazone acted as a partial agonist of FXR to weakly increase BSEP and SHP expression but functioned as a potent antagonist to significantly suppress bile acid-induced expression. Consistent with the finding, troglitazone partially induced but markedly antagonized bile acid-mediated BSEP promoter transactivation. However, such modulating effects were not detected with rosiglitazone or pioglitazone. Using the crystal structure of ligand-bound FXR ligand binding domain (LBD), molecular docking predicted that troglitazone, but not rosiglitazone or pioglitazone, could form a stable complex with FXR LBD. The specific α-tocopherol side chain of troglitazone significantly contributed to the formation of such a stable complex through extensive interactions with FXR LBD. The docking model was further validated by functional analyses of a series of docking-guided FXR mutants. In summary, the data demonstrated that troglitazone, but not rosiglitazone or pioglitazone, was an FXR modulator and potently antagonized bile acid-induced expression of FXR target genes. Such differential modulation of FXR signaling pathway by TZDs may represent one of the mechanisms for member-specific, PPARγ-independent activities and toxicity

The far and distal enhancers in the CYP3A4 gene co-ordinate the proximal promoter in responding similarly to the pregnane X receptor but differentially to hepatocyte nuclear factor-4α

CYP3A4 (cytochrome P450 3A4) is involved in the metabolism of more than 50 % of drugs and other xenobiotics. The expression of CYP3A4 is induced by many structurally dissimilar compounds. The PXR (pregnane X receptor) is recognized as a key regulator for the induction, and the PXR-directed transactivation of the CYP3A4 gene is achieved through a co-ordinated mechanism of the distal module with the proximal promoter. Recently, a far module was found to support constitutive expression of CYP3A4. The far module, like the distal module, is structurally clustered by a PXR response element (F-ER6) and elements recognized by HNF-4α (hepatocyte nuclear receptor-4α). We hypothesized that the far module supports PXR transactivation of the CYP3A4 gene. Consistent with the hypothesis, fusion of the far module to the proximal promoter of CYP3A4 markedly increased rifampicin-induced reporter activity. The increase was synergistically enhanced when both the far and distal modules were fused to the proximal promoter. The increase, however, was significantly reduced when the F-ER6 was disrupted. Chromatin immunoprecipitation detected the presence of PXR in the far module. Interestingly, HNF-4α increased the activity of the distal-proximal fused promoter, but decreased the activity of the far-proximal fused promoter. Given the fact that induction of CYP3A4 represents an important detoxification mechanism, the functional redundancy and synergistic interaction in supporting PXR transactivation suggest that the far and distal modules ensure the induction of CYP3A4 during chemical insults. The difference in responding to HNF-4α suggests that the magnitude of the induction is under control through various transcriptional networks. © The Authors

Transcriptional dynamics of bile salt export pump during pregnancy: Mechanisms and implications in intrahepatic cholestasis of pregnancy

Bile salt export pump (BSEP) is responsible for biliary secretion of bile acids, a rate-limiting step in the enterohepatic circulation of bile acids and transactivated by nuclear receptor farnesoid X receptor (FXR). Intrahepatic cholestasis of pregnancy (ICP) is the most prevalent disorder among diseases unique to pregnancy and primarily occurs in the third trimester of pregnancy, with a hallmark of elevated serum bile acids. Currently, the transcriptional regulation of BSEP during pregnancy and its underlying mechanisms and involvement in ICP are not fully understood. In this study the dynamics of BSEP transcription in vivo in the same group of pregnant mice before, during, and after gestation were established with an in vivo imaging system (IVIS). BSEP transcription was markedly repressed in the later stages of pregnancy and immediately recovered after parturition, resembling the clinical course of ICP in human. The transcriptional dynamics of BSEP was inversely correlated with serum 17β-estradiol (E2) levels before, during, and after gestation. Further studies showed that E2 repressed BSEP expression in human primary hepatocytes, Huh 7 cells, and in vivo in mice. Such transrepression of BSEP by E2 in vitro and in vivo required estrogen receptor α (ERα). Mechanistic studies with chromatin immunoprecipitation (ChIP), protein coimmunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays demonstrated that ERα directly interacted with FXR in living cells and in vivo in mice. Conclusion: BSEP expression was repressed by E2 in the late stages of pregnancy through a nonclassical E2/ERα transrepressive pathway, directly interacting with FXR. E2-mediated repression of BSEP expression represents an etiological contributing factor to ICP and therapies targeting the ERα/FXR interaction may be developed for prevention and treatment of ICP