Shi-Ying Cai

Mrp4−/− mice have an impaired cytoprotective response in obstructive cholestasis†

Mrp4 is a member of the multidrug resistance–associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive upregulation in response to cholestatic injury or bile acid feeding. However, the relative importance of Mrp4 in a protective adaptive response to cholestatic injury is not known. To address this issue, common bile duct ligation (CBDL) was performed in wild‐type and Mrp4−/− mice and animals followed for 7 days. Histological analysis and serum aminotransferase levels revealed more severe liver injury in the absence of Mrp4 expression. Western analyses revealed that Mrp4, but not Mrp3, was significantly increased after CBDL in wild‐type mice. Serum bile acid levels were significantly lower in Mrp4−/− mice than in wild‐type CBDL mice, whereas serum bilirubin levels were the same, suggesting that Mrp4 was required to effectively extrude bile acids from the cholestatic liver. Mrp3 and Ostα‐Ostβ were upregulated in Mrp4−/− mice but were unable to compensate for the loss of Mrp4. High‐performance liquid chromatography analysis on liver extracts revealed that taurine tetrahydroxy bile acid/beta‐muricholic acid ratios were increased twofold in Mrp4−/− mice. In conclusion, hepatic Mrp4 plays a unique and essential protective role in the adaptive response to obstructive cholestatic liver injury. (HEPATOLOGY 2006;43:1013–1021.)

Combination of retinoic acid and ursodeoxycholic acid attenuates liver injury in bile duct-ligated rats and human hepatic cells.

Cholestasis leads to liver cell death, fibrosis, cirrhosis, and eventually liver failure. Despite limited benefits, ursodeoxycholic acid (UDCA) is the only Food and Drug Administration–approved treatment for cholestatic disorders. Retinoic acid (RA) is a ligand for nuclear receptors that modulate bile salt homeostasis. RA also possesses immunomodulatory effects and is used to treat acute promyelocytic leukemia and inflammatory disorders such as psoriasis, acne, and rheumatoid arthritis. To test whether the supplementation of RA with UDCA is superior to UDCA alone for treating cholestasis, male Sprague‐Dawley rats underwent common bile duct ligation (BDL) for 14 days and were treated with phosphate‐buffered saline (PBS), UDCA, all‐trans retinoic acid (atRA), or UDCA and atRA by gavage. Treatment with UDCA and atRA substantially improved animal growth rates, significantly reduced liver fibrosis and bile duct proliferation, and nearly eliminated liver necrosis after BDL. Reductions in the bile salt pool size and liver hydroxyproline content were also seen with treatment with atRA or atRA and UDCA versus PBS and UDCA. Furthermore, atRA and UDCA significantly reduced liver messenger RNA and/or protein expression of transforming growth factor β1 (Tgf‐β1), collagen 1a1 (Col1A1), matrix metalloproteinase 2 (Mmp2), cytokeratin 19, α‐smooth muscle actin (α‐SMA), cytochrome P450 7A1 (Cyp7a1), tumor necrosis factor α, and interleukin‐β1. The molecular mechanisms of this treatment were also assessed in human hepatocytes, hepatic stellate cells, and LX‐2 cells. atRA alone or in combination with UDCA greatly repressed CYP7A1 expression in human hepatocytes and significantly inhibited COL1A1, MMP2, and α‐SMA expression and/or activity in primary human hepatic stellate cells and LX‐2 cells. Furthermore, atRA reduced TGF‐β1–induced Smad2 phosphorylation in LX‐2 cells. Conclusion: Our findings indicate that the addition of RA to UDCA reduces the bile salt pool size and liver fibrosis and might be an effective supplemental therapy with UDCA for cholestatic diseases. (HEPATOLOGY 2011;53:548‐557.)

Aryl hydrocarbon receptor and NF-E2-related factor 2 are key regulators of human MRP4 expression

Multidrug resistance protein 4 (MRP4; ABCC4) is an ATP binding cassette transporter that facilitates the excretion of bile salt conjugates and other conjugated steroids in hepatocytes and renal proximal tubule epithelium. MRP4/Mrp4 undergoes adaptive upregulation in response to oxidative and cholestatic liver injury in human and animal models of cholestasis. However, the molecular mechanism of this regulation remains to be determined. The aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) play important roles in protecting cells from oxidative stress. Here we examine the role of these two nuclear factors in the regulation of the expression of human MRP4. HepG2 cells and human hepatocytes were treated with the AhR and Nrf2 activators, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), or oltipraz and other nuclear receptor agonists. TCDD, 3-MC, and oltipraz significantly increased MRP4 expression at mRNA and protein levels. Computer program analysis revealed three Xenobiotic response element (XRE) and one Maf response element sites within the first 500 bp of the MRP4 proximal promoter. Luciferase reporter assay detected strong promoter activity (53-fold higher than vector control) in this region. TCDD and 3-MC also induced promoter activity in the reporter assays. Mutation of any of these XRE sites significantly decreased MRP4 promoter activity in reporter assays, although XRE2 demonstrated the strongest effects on both basal and TCDD-inducible activity. EMSA and chromatin immunoprecipitation assays further confirmed that both AhR and Nrf2 bind to the proximal promoter of MRP4. Our findings indicate that AhR and Nrf2 play important roles in regulating MRP4 expression and suggest that agents that activate their activity may be of therapeutic benefit for cholestasis.

Elevated hepatic multidrug resistance-associated protein 3/ATP-binding cassette subfamily C 3 expression in human obstructive cholestasis is mediated through tumor necrosis factor alpha and c-Jun NH2-terminal kinase/stress-activated protein kinase–signaling pathway †‡

Multidrug resistance‐associated protein 3 (MRP3, ABC subfamily C [ABCC]3) plays an important role in protecting hepatocytes and other tissues by excreting an array of toxic organic anion conjugates, including bile salts. MRP3/ABCC3 expression is increased in the liver of some cholestatic patients, but the molecular mechanism of this up‐regulation remains elusive. In this report, we assessed liver MRP3/ABCC3 expression in patients (n = 22) with obstructive cholestasis caused by gallstone blockage of bile ducts and noncholestatic patient controls (n = 22). MRP3/ABCC3 messenger RNA (mRNA) and protein expression were significantly increased by 3.4‐ and 4.6‐fold, respectively, in these cholestatic patients where elevated plasma tumor necrosis factor alpha (TNFα) (4.7‐fold; P < 0.01) and hepatic specificity protein 1 transcription factor (SP1) and liver receptor homolog 1 expression (3.1‐ and 2.1‐fold at mRNA level, 3.5‐ and 2.5‐fold at protein level, respectively) were also observed. The induction of hepatic MRP3/ABCC3 mRNA expression is significantly positively correlated with the level of plasma TNFα in these patients. In HepG2 cells, TNFα treatment induced SP1 and MRP3/ABCC3 expression in a dose‐ and time‐dependent manner, where increased phosphorylation of c‐Jun NH2‐terminal kinase/stress‐activated protein kinase (JNK/SAPK) was also detected. These inductions were significantly reduced in the presence of the JNK inhibitor, SP600125. TNFα treatment enhanced HepG2 cell nuclear extract‐binding activity to the MRP3/ABCC3 promoter, but was abolished by SP600125, as demonstrated by electrophoretic mobility shift assay (EMSA). An increase in nuclear protein‐binding activity to the MRP3/ABCC3 promoter, consisting primarily of SP1, was also observed in liver samples from cholestatic patients, as assessed by supershift EMSA assays. Conclusions: Our findings indicate that up‐regulation of hepatic MRP3/ABCC3 expression in human obstructive cholestasis is likely triggered by TNFα, mediated by activation of JNK/SAPK and SP1. (HEPATOLOGY 2012)

Nuclear factor erythroid 2–related factor 2 is a positive regulator of human bile salt export pump expression†‡

The bile salt export pump (BSEP) is the major determinant of bile salt–dependent bile secretion, and its deficiency leads to cholestatic liver injury. BSEP/Bsep gene expression is regulated by the nuclear farnesoid X receptor. However, BSEP expression, though reduced, is retained in the livers of Fxr−/− mice, indicating that additional transcriptional factors may regulate its expression. Nuclear factor erythroid 2–related factor 2 (Nrf2) plays a major role in response to oxidative stress by binding to antioxidant‐responsive elements that regulate many hepatic phase I and II enzymes as well as hepatic efflux transporters. Computer software analysis of human BSEP reveals two musculo‐aponeurotic fibrosacroma (Maf) recognition elements (MAREs) from the sequence in the proximal promoter region where Nrf2 may bind. In this study, we assessed whether Nrf2 plays a role in human BSEP expression and if this might be mediated by MAREs. Oltipraz, a potent activator of Nrf2, increased BSEP messenger RNA expression by approximately seven‐fold in HepG2 cells and protein by approximately 70% in human hepatocytes. Small interfering RNAs lowered NRF2 expression in HepG2 cells and prevented the up‐regulation of BSEP by oltipraz. Human BSEP promoter activity was stimulated by Nrf2 in a dose‐dependent manner in luciferase reporter assays. Mutations of the predicted MARE1, but not MARE2, abolished this Nrf2 transcriptional activation. Chromatin immunoprecipitation assays also demonstrated that Nrf2 specifically bound to MARE1, but not MARE2 regions in the BSEP promoter in HepG2 cells. Electrophoretic mobility shift assays further demonstrated direct binding of MARE1 in the BSEP promoter. Conclusion: Nrf2 is a positive transcriptional regulator of human BSEP expression. Pharmacological activation of Nrf2 may be beneficial for cholestatic liver injury. (HEPATOLOGY 2009.)

Retinoic acid represses CYP7A1 expression in human hepatocytes and HepG2 cells by FXR/RXR-dependent and independent mechanisms

Cholesterol 7α-hydroxylase (CYP7A1) plays a key role in maintaining lipid and bile salt homeostasis as it is the rate-limiting enzyme converting cholesterol to bile acids. Deficiency of CYP7A1 leads to hyperlipidemia in man and mouse. Hyperlipidemia is often seen in patients when treated with high-dose retinoic acid (RA), but the molecular mechanisms remain elusive. Our present study revealed that CYP7A1 mRNA expression is greatly repressed by RA in both human hepatocytes and HepG2 cells where increased fibroblast growth factor 19 (FGF19) and small heterodimer partner (SHP) expressions were also observed, suggesting farnesoid X receptor (FXR) and retinoid X receptor (RXR) were activated. Promoter reporter assays demonstrate that all-trans RA (atRA) specifically activated FXR/RXR. However, detailed molecular analyses indicate that this activation is through RXR, whose ligand is 9-cis RA. Knocking down of FXR or RXRα by small interference RNA (siRNA) in human hepatocytes increased CYP7A1 basal expression, but the repressive effect of atRA persisted, suggesting there are also FXR/RXR-independent mechanisms mediating atRA repression of CYP7A1 expression. Chromatin immunoprecipitation (ChIP) assay and cell transfection results indicate that PGC-1α plays a role in the FXR/RXR-independent mechanism. Our findings may provide a potential explanation for hyperlipidemic side effects observed in some patients treated with high-dose RA.

FXR: a target for cholestatic syndromes?

The nuclear farnesoid X receptor (FXR) plays a pivotal role in maintaining bile acid homeostasis by regulating key genes involved in bile acid synthesis, metabolism and transport, including CYP7A1, UGT2B4, BSEP, MDR3, MRP2, ASBT, I-BABP, NTCP and OSTα-OSTβ in humans. Altered expression or malfunction of these genes has been described in patients with cholestatic liver diseases. This review examines the rationale for the use of FXR ligand therapy in various cholestatic liver disorders and includes potential concerns.

A Primitive ATP Receptor from the Little Skate Raja erinacea

P2Y ATP receptors are widely expressed in mammalian tissues and regulate a broad range of activities. Multiple subtypes of P2Y receptors have been identified and are distinguished both on a molecular basis and by pharmacologic substrate preference. Functional evidence suggests that hepatocytes from the little skateRaja erinacea express a primitive P2Y ATP receptor lacking pharmacologic selectivity, so we cloned and characterized this receptor. Skate hepatocyte cDNA was amplified with degenerate oligonucleotide probes designed to identify known P2Y subtypes. A single polymerase chain reaction product was found and used to screen a skate liver cDNA library. A 2314-base pair cDNA clone was generated that contained a 1074-base pair open reading frame encoding a 357-amino acid gene product with 61–64% similarity to P2Y1 receptors and 21–37% similarity to other P2Y receptor subtypes. Pharmacology of the putative P2Y receptor was examined using the Xenopus oocyte expression system and revealed activation by a range of nucleotides. The receptor was expressed widely in skate tissue and was expressed to a similar extent in other primitive organisms. Phylogenetic analysis suggested that this receptor is closely related to a common ancestor of the P2Y subtypes found in mammals, avians, and amphibians. Thus, the skate liver P2Y receptor functions as a primitive P2Y ATP receptor with broad pharmacologic selectivity and is related to the evolutionary forerunner of P2Y1 receptors of higher organisms. This novel receptor should provide an effective comparative model for P2Y receptor pharmacology and may improve our understanding of nucleotide specificity among the family of P2Y ATP receptors.

Adult sea lamprey tolerates biliary atresia by altering bile salt composition and renal excretion.

The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia, as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the postmetamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic, with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (∼10 μM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free‐swimming adults revealed that ∼35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72‐hour period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts, and BSP, were predominantly excreted by way of the kidney, with minor amounts also detected in feces. Gene expression analysis detected marked up‐regulation of orthologs of known organic anion and bile salt transporters in the kidney, with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis. (HEPATOLOGY 2013;57:2418–2426)

Evolution of substrate specificity for the bile salt transporter ASBT (SLC10A2)

The apical Na+-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbts indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution.