Differential Feedback Regulation of Δ\u3csup\u3e4\u3c/sup\u3e-3- Oxosteroid 5β-Reductase Expression by Bile Acids

Δ4-3-oxosteroid 5β-reductase is member D1 of the aldo-keto reductase family 1 (AKR1D1), which catalyzes 5β-reduction of molecules with a 3-oxo-4-ene structure. Bile acid intermediates and most of the steroid hormones carry the 3-oxo-4-ene structure. Therefore, AKR1D1 plays critical roles in both bile acid synthesis and steroid hormone metabolism. Currently our understanding on transcriptional regulation of AKR1D1 under physiological and pathological conditions is very limited. In this study, we investigated the regulatory effects of primary bile acids, chenodeoxycholic acid (CDCA) and cholic acid (CA), on AKR1D1 expression. The expression levels of AKR1D1 mRNA and protein in vitro and in vivo following bile acid treatments were determined by real-time PCR and Western blotting. We found that CDCA markedly repressed AKR1D1 expression in vitro in human hepatoma HepG2 cells and in vivo in mice. On the contrary, CA significantly upregulated AKR1D1 expression in HepG2 cells and in mice. Further mechanistic investigations revealed that the farnesoid x receptor (FXR) signaling pathway was not involved in regulating AKR1D1 by bile acids. Instead, CDCA and CA regulated AKR1D1 through the mitogen-activated protein kinases/c-Jun N-terminal kinases (MAPK/JNK) signaling pathway. Inhibition of the MAPK/JNK pathway effectively abolished CDCA and CA-mediated regulation of AKR1D1. It was thus determined that AKR1D1 expression was regulated by CDCA and CA through modulating the MAPK/JNK signaling pathway. In conclusion, AKR1D1 expression was differentially regulated by primary bile acids through negative and positive feedback mechanisms. The findings indicated that both bile acid concentrations and compositions play important roles in regulating AKR1D1 expression, and consequently bile acid synthesis and steroid hormone metabolism

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

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

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

Activation of CDK4 Triggers Development of Non-alcoholic Fatty Liver Disease

The development of non-alcoholic fatty liver disease (NAFLD) is a multiple step process. Here, we show that activation of cdk4 triggers the development of NAFLD. We found that cdk4 protein levels are elevated in mouse models of NAFLD and in patients with fatty livers. This increase leads to C/EBPα phosphorylation on Ser193 and formation of C/EBPα-p300 complexes, resulting in hepatic steatosis, fibrosis, and hepatocellular carcinoma (HCC). The disruption of this pathway in cdk4-resistant C/EBPα-S193A mice dramatically reduces development of high-fat diet (HFD)-mediated NAFLD. In addition, inhibition of cdk4 by flavopiridol or PD-0332991 significantly reduces development of hepatic steatosis, the first step of NAFLD. Thus, this study reveals that activation of cdk4 triggers NAFLD and that inhibitors of cdk4 may be used for the prevention/treatment of NAFLD

Inhibition of MAPK/JNK signaling pathway abolished CA-mediated regulation of AKR1D1.

<p>(A) HepG2 cells were treated with CA (50μM) in the absence or presence of MAPK/JNK inhibitor SP600125 (1μM), MAPK/ERK1/2 inhibitor PD98059 (5μM) or vehicle for 30 hrs, followed by detection of AKR1D1 mRNA by real-time PCR and (B) AKR1D1 protein by Western blot. (C) quantification of AKR1D1 protein levels in (B). One-way ANOVA was applied to analyze the data, followed by Tukey post-hoc test for multiple comparisons. * p<0.05.</p

FXR signaling was not involved in regulating AKR1D1 by bile acids <i>in vivo</i> in mice.

<p>(A) two groups of mice (n = 6/group) were treated with GW4064 (5mg/kg) or vehicle propanediol through intraperitoneal injection twice a day for 3 days. The expression levels of Akr1d1 and Bsep were quantified by real-time PCR and (B) Western blot. (C) quantification of Akr1d1 and (D) Bsep protein levels in (B). (E) the expression levels of Akr1d1 and Bsep in wt and FXR-knockout (FXR-/-) mice were determined by real-time PCR and (F) Western blot. (G) quantification of Akr1d1 and (H) Bsep protein levels in (F). The data are presented as mean ± SD of the groups of mice. The Student’s t-test was applied to pair-wise comparison. ** p<0.01.</p

Inhibition of MAPK/JNK signaling pathway abolished CDCA-mediated regulation of AKR1D1.

<p>(A) HepG2 cells were treated with CDCA (25μM) in the absence or presence of MAPK/JNK inhibitor SP600125 (1μM), MAPK/ERK1/2 inhibitor PD98059 (5μM) or vehicle for 30 hrs, followed by detection of AKR1D1 mRNA by real-time PCR and (B) AKR1D1 protein by Western blot. (C) quantification of AKR1D1 protein levels in (B). One-way ANOVA was applied to analyze the data, followed by Tukey post-hoc test for multiple comparisons. * p<0.05 and ** p<0.01.</p