Xiulong Song

Interleukin-6 Alters the Cellular Responsiveness to Clopidogrel, Irinotecan, and Oseltamivir by Suppressing the Expression of Carboxylesterases HCE1 and HCE2

Carboxylesterases constitute a class of enzymes that play important roles in the hydrolytic metabolism of drugs and other xenobiotics. Patients with liver conditions such as cirrhosis show increased secretion of proinflammatory cytokines [e.g., interleukin-6 (IL-6)] and decreased capacity of hydrolysis. In this study, we provide a molecular explanation linking cytokine secretion directly to the decreased capacity of hydrolytic biotransformation. In both primary hepatocytes and HepG2 cells, treatment with IL-6 decreased the expression of human carboxyl-esterases HCE1 and HCE2 by as much as 60%. The decreased expression occurred at both mRNA and protein levels, and it was confirmed by enzymatic assay. In cotransfection experiments, both HCE1 and HCE2 promoters were significantly repressed, and the repression was comparable with the decrease in HCE1 and HCE2 mRNA, suggesting that transrepression is responsible for the suppressed expression. In addition, pretreatment with IL-6 altered the cellular responsiveness in an opposite manner of overexpression of HCE1 and HCE2 toward various ester therapeutic agents (e.g., clopidogrel). Transfection of HCE1, for example, decreased the cytotoxicity induced by antithrombogenic agent clopidogrel, whereas pretreatment with IL-6 increased the cytotoxicity. Such a reversal was observed with other ester drugs, including anticancer agent irinotecan and anti-influenza agent oseltamivir. The altered cellular responsiveness was observed when drugs were assayed at sub- and low-micromolar concentrations, suggesting that suppressed expression of carboxylesterases by IL-6 has profound pharmacological consequences, particularly with those that are hydrolyzed in an isoform-specific manner.

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.

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-4alpha (hepatocyte nuclear receptor-4alpha). 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-4alpha 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-4alpha suggests that the magnitude of the induction is under control through various transcriptional networks.

DNA binding, but not interaction with Bmal1, is responsible for DEC1-mediated transcription regulation of the circadian gene mPer1.

DEC1 (differentially expressed in chondrocytes 1) and DEC2 are E-box-binding transcription factors and exhibit a circadian expression pattern. Recently, both proteins were found to repress the Clock/Bmal1-activated E-box promoters (e.g. mPer1). Yeast two-hybrid assay detected interactions between Bmal1 and DECs. It was hypothesized that DEC-mediated repression on the mPer1 promoter is achieved by binding to E-box elements and interacting with Bmal1. In the present study, we report that E-box binding rather than Bmal1 interaction is responsible for the observed repression. In the absence of Clock/Bmal1, both DEC1 and DEC2 markedly repressed the mPer1 promoter reporter; however, DNA-binding mutants showed no repressive activity. Similarly, DEC1, but not its DNA-binding mutants, repressed the Clock/Bmal1-induced activation. In addition, DEC1(R58P), a DNA-binding mutant with Bmal1 interactivity, repressed neither the mPer1 reporter directly nor the Clock/Bmal1-induced activation, providing direct evidence that DNA binding, rather than Bmal1 interactions, is responsible for the repression on the mPer1 promoter. Furthermore, disruption of the Sp1 site in the proximal promoter of mPer1 increased the repression of DEC1 proteins. Previous studies with mouse DEC2 showed that this factor interacts with Sp1. These findings suggest that DEC proteins regulate the expression of mPer1 through E-box binding and Sp1 interaction. Alterations on circadian systems are increasingly recognized as important risk factors for disease initiation and progression, and the expression of Dec genes is rapidly induced by environmental stimuli and is highly increased in tumour tissues. Therefore de-regulated expression of DEC genes probably alters normal circadian rhythms and contributes significantly to the pathogenesis of many diseases including cancer.

Bile salt export pump is dysregulated with altered farnesoid X receptor isoform expression in patients with hepatocellular carcinoma

As a canalicular bile acid effluxer, the bile salt export pump (BSEP) plays a vital role in maintaining bile acid homeostasis. BSEP deficiency leads to severe cholestasis and hepatocellular carcinoma (HCC) in young children. Regardless of the etiology, chronic inflammation is the common pathological process for HCC development. Clinical studies have shown that bile acid homeostasis is disrupted in HCC patients with elevated serum bile acid level as a proposed marker for HCC. However, the underlying mechanisms remain largely unknown. In this study, we found that BSEP expression was severely diminished in HCC tissues and markedly reduced in adjacent nontumor tissues. In contrast to mice, human BSEP was regulated by farnesoid X receptor (FXR) in an isoform‐dependent manner. FXR‐α2 exhibited a much more potent activity than FXR‐α1 in transactivating human BSEP in vitro and in vivo. The decreased BSEP expression in HCC was associated with altered relative expression of FXR‐α1 and FXR‐α2. FXR‐α1/FXR‐α2 ratios were significantly increased, with undetectable FXR‐α2 expression in one third of the HCC tumor samples. A similar correlation between BSEP and FXR isoform expression was confirmed in hepatoma Huh7 and HepG2 cells. Further studies showed that intrahepatic proinflammatory cytokines, such as interleukin‐6 (IL‐6) and tumor necrosis factor alpha (TNF‐α), were significantly elevated in HCC tissues. Treatment of Huh7 cells with IL‐6 and TNF‐α resulted in a marked increase in FXR‐α1/FXR‐α2 ratio, concurrent with a significant decrease in BSEP expression. Conclusion: BSEP expression is severely diminished in HCC patients associated with alteration of FXR isoform expression induced by inflammation. Restoration of BSEP expression through suppressing inflammation in the liver may reestablish bile acid homeostasis. (HEPATOLOGY 2013)

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.

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. (Hepatology 2014;60:1992–2006)

Simultaneous Substitution of Phenylalanine-305 and Aspartate-318 of Rat Pregnane X Receptor with the Corresponding Human Residues Abolishes the Ability to Transactivate the CYP3A23 Promoter

The pregnane X receptor (PXR) is a key regulator on the expression of genes involved in the elimination of chemicals. As one of the most divergent members in the nuclear receptor family, PXR is activated in a highly species-dependent manner by certain chemicals. Pregnenolone 16α-carbonitrile (PCN), a glucocorticoid antagonist, efficaciously activates rodent but not human PXR. This study was undertaken to investigate the structural basis for PCN-mediated activation of rat PXR. A series of rat-human chimeric PXRs were prepared to gradually replace the ligand-binding domain of human PXR with the corresponding rat sequence at an increasing length of 20 residues. Cotransfection experiments established that region306–326 acted as a transitional conjunction from none to full PCN responsive status. Site-directed mutagenesis study identified two residues (Phe-305 and Asp-318) that were critical in supporting PCN-mediated activation, and simultaneous substitution of both residues abolished the ability of rat PXR to transactivate the CYP3A23 promoter. In addition, substitutions on Phe-305, Asp-318, or both markedly reduced the basal transcriptional activity, and the reduction occurred with the CYP3A4 but not CYP3A23 promoter. Further study with CYP3A4 and CYP3A23 hybrid reporters demonstrated that the region harboring the distal PXR element in the CYP3A4 promoter mediated the repressive activity. PXR has been shown to interact with corepressors in the absence of ligand. The decreased responsiveness toward PCN and reduced basal transcriptional activity suggest that Phe-305 and Asp-318 are involved in both ligand-binding and corepressor interactions.

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.

Estrogen and Estrogen Receptor-α-Mediated Transrepression of Bile Salt Export Pump

Among diseases unique to pregnancy, intrahepatic cholestasis of pregnancy is the most prevalent disorder with elevated serum bile acid levels. We have previously shown that estrogen 17β-estradiol (E2) transrepresses bile salt export pump (BSEP) through an interaction between estrogen receptor (ER)-α and farnesoid X receptor (FXR) and transrepression of BSEP by E2/ERα is an etiological contributing factor to intrahepatic cholestasis of pregnancy. Currently the mechanistic insights into such transrepression are not fully understood. In this study, the dynamics of coregulator recruitment to BSEP promoter after FXR activation and E2 treatment were established with quantitative chromatin immunoprecipitation assays. Coactivator peroxisome proliferator-activated receptor-γ coactivator-1 was predominantly recruited to the BSEP promoter upon FXR activation, and its recruitment was decreased by E2 treatment. Meanwhile, recruitment of nuclear receptor corepressor was markedly increased upon E2 treatment. Functional evaluation of ERα and ERβ chimeras revealed that domains AC of ERα are the determinants for ERα-specific transrepression on BSEP. Further studies with various truncated ERα proteins identified the domains in ERα responsible for ligand-dependent and ligand-independent transrepression. Truncated ERα-AD exhibited potent ligand-independent transrepressive activity, whereas ERα-CF was fully capable of transrepressing BSEP ligand dependently in vitro in Huh 7 cells and in vivo in mice. Both ERα-AD and ERα-CF proteins were associated with FXR in the coimmunoprecipitation assays. In conclusion, E2 repressed BSEP expression through diminishing peroxisome proliferator-activated receptor-γ coactivator-1 recruitment with a concurrent increase in nuclear receptor corepressor recruitment to the BSEP promoter. Domains AD and CF in ERα mediated ligand-independent and ligand-dependent transrepression on BSEP, respectively, through interacting with FXR.