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Background and aims Endoplasmic reticulum (ER) stress is an adaptive response to excessive ER demand and contributes to the development of numerous diseases, including nonalcoholic fatty liver disease (NAFLD), which is hallmarked by the accumulation of lipid within hepatocytes. However, the underlying mechanisms remain elusive. MicroRNAs (miRNAs) play an indispensable role in various stress responses, but their implications in ER stress have not yet been systemically investigated. In this study, we identify a negative feedback loop consisting of hepatic ER stress and miR-26a in NAFLD pathogenesis. Approach and results Combining miRNA dot blot array and quantitative PCR, we find that miR-26a is specifically induced by ER stress in liver cells. This induction of miR-26a is critical for cells to cope with ER stress. In human hepatoma cells and murine primary hepatocytes, overexpression of miR-26a markedly alleviates chemical-induced ER stress, as well as palmitate-triggered ER stress and lipid accumulation. Conversely, deficiency of miR-26a exhibits opposite effects. Mechanistically, miR-26a directly targets the eukaryotic initiation factor 2α, a core ER stress effector controlling cellular translation. Intriguingly, miR-26a is reduced in the livers of patients with NAFLD. Hepatocyte-specific restoration of miR-26a in mice significantly mitigates high-fat diet-induced ER stress and hepatic steatosis. In contrast, deficiency of miR-26a in mice exacerbates high-fat diet-induced ER stress, lipid accumulation, inflammation and hepatic steatosis. Conclusions Our findings suggest ER stress-induced miR-26a up-regulation as a regulator for hepatic ER stress resolution, and highlight the ER stress/miR-26a/eukaryotic initiation factor 2α cascade as a promising therapeutic strategy for NAFLD.

Aberrant epigenetic alterations during development may result in long-term epigenetic memory and have a permanent effect on the health of subjects. Constitutive androstane receptor (CAR) is a central regulator of drug/xenobiotic metabolism. Here, we report that transient neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism. CAR activation by neonatal exposure to the CAR-specific ligand 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) led to persistently induced expression of the CAR target genes Cyp2B10 and Cyp2C37 throughout the life of exposed mice. These mice showed a permanent reduction in sensitivity to zoxazolamine treatment as adults. Compared with control groups, the induction of Cyp2B10 and Cyp2C37 in hepatocytes isolated from these mice was more sensitive to low concentrations of the CAR agonist TCPOBOP. Accordingly, neonatal activation of CAR led to a permanent increase of histone 3 lysine 4 mono-, di-, and trimethylation and decrease of H3K9 trimethylation within the Cyp2B10 locus. Transcriptional coactivator activating signal cointegrator-2 and histone demethylase JMJD2d participated in this CAR-dependent epigenetic switch. Conclusion: Neonatal activation of CAR results in epigenetic memory and a permanent change of liver drug metabolism. (HEPATOLOGY 2012)

Gpbar1 (TGR5), a membrane-bound bile acid receptor, is well known for its roles in regulation of energy homeostasis and glucose metabolism. TGR5 also displays strong attenuation of macrophage reactivity in vitro, but the physiological roles of TGR5 in inflammatory response, and its mechanism, is unknown. Here, we demonstrate that TGR5 is a negative modulator of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB)-mediated inflammation. TGR5 activation suppresses the phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), the translocation of p65, NF-κB DNA-binding activity, and its transcription activity. Furthermore, TGR5 activation enhances the interaction of IκBα and β-arrestin2. Suppression of NF-κB transcription activity and its target gene expression by TGR5 agonist are specifically abolished by the expression of anti-β-arrestin2 small interfering RNA. These results show that TGR5 suppresses the NF-κB pathway by mediation of the interaction between IκBα and β-arrestin2. In a lipopolysaccharide (LPS)-induced inflammation model, TGR5−/− mice show more severe liver necroses and inflammation, compared with wild-type (WT) mice. Activation of TGR5 by its agonist ligand inhibits the expression of inflammatory mediators in response to NF-κB activation induced by LPS in WT, but not TGR5−/−, mouse liver. Conclusion: These findings identify TGR5 as a negative mediator of inflammation that may serve as an attractive therapeutic tool for immune and inflammatory liver diseases. (HEPATOLOGY 2011;)

MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by interacting with the 3′ untranslated region (3′-UTR) of multiple mRNAs. Recent studies have linked miRNAs to the development of cancer metastasis. In this study, we show that miR-194 is specifically expressed in the human gastrointestinal tract and kidney. Moreover, miR-194 is highly expressed in hepatic epithelial cells, but not in Kupffer cells or hepatic stellate cells, two types of mesenchymal cells in the liver. miR-194 expression was decreased in hepatocytes cultured in vitro, which had undergone a dedifferentiation process. Furthermore, expression of miR-194 was low in liver mesenchymal-like cancer cell lines. The overexpression of miR-194 in liver mesenchymal-like cancer cells reduced the expression of the mesenchymal cell marker N-cadherin and suppressed invasion and migration of the mesenchymal-like cancer cells both in vitro and in vivo. We further demonstrated that miR-194 targeted the 3′-UTRs of several genes that were involved in epithelial-mesenchymal transition and cancer metastasis. Conclusion: These results support a role of miR-194, which is specifically expressed in liver parenchymal cells, in preventing liver cancer cell metastasis. (HEPATOLOGY 2010;.)

The farnesoid X receptor (FXR) is a nuclear receptor that plays key roles in hepatoprotection by maintaining the homeostasis of liver metabolism. FXR null mice display strong hepatic inflammation and develop spontaneous liver tumors. In this report, we demonstrate that FXR is a negative modulator of nuclear factor kappaB (NF-kappaB)-mediated hepatic inflammation. Activation of FXR by its agonist ligands inhibited the expression of inflammatory mediators in response to NF-kappaB activation in both HepG2 cells and primary hepatocytes cultured in vitro. In vivo, compared with wild-type controls, FXR(-/-) mice displayed elevated messenger RNA (mRNA) levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interferon-inducible protein 10, and interferon-gamma in response to lipopolysaccharide (LPS). Examination of FXR(-/-) livers showed massive necroses and inflammation after treatment with LPS at a dose that does not induce significant liver damage or inflammation in wild-type mice. Moreover, transfection of a constitutively active FXR expression construct repressed the iNOS, COX-2, interferon-inducible protein 10 and interferon-gamma mRNA levels induced by LPS administration. FXR activation had no negative effects on NF-kappaB-activated antiapoptotic genes, suggesting that FXR selectively inhibits the NF-kappaB-mediated hepatic inflammatory response but maintains or even enhances the cell survival response. On the other hand, NF-kappaB activation suppressed FXR-mediated gene expression both in vitro and in vivo, indicating a negative crosstalk between the FXR and NF-kappaB signaling pathways. Our findings reveal that FXR is a negative mediator of hepatic inflammation, which may contribute to the critical roles of FXR in hepatoprotection and suppression of hepatocarcinogenesis.

The constitutive androstane receptor (CAR) modulates xeno- and endobiotic hepatotoxicity by regulating detoxification pathways. Whether activation of CAR may also protect against liver injury by directly blocking apoptosis is unknown. To address this question, CAR wild-type (CAR+/+) and CAR knockout (CAR-/-) mice were treated with the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and then with the Fas agonist Jo2 or with concanavalin A (ConA). Following the administration of Jo2, hepatocyte apoptosis, liver injury, and animal fatalities were abated in TCPOBOP-treated CAR+/+ but not in CAR-/- mice. Likewise, acute and chronic ConA-mediated liver injury and fibrosis were also reduced in wild-type versus CAR(-/-) TCPOBOP-treated mice. The proapoptotic proteins Bak (Bcl-2 antagonistic killer) and Bax (Bcl-2-associated X protein) were depleted in livers from TCPOBOP-treated CAR+/+ mice. In contrast, mRNA expression of the antiapoptotic effector myeloid cell leukemia factor-1 (Mcl-1) was increased fourfold. Mcl-1 promoter activity was increased by transfection with CAR and administration of TCPOBOP in hepatoma cells, consistent with a direct CAR effect on Mcl-1 transcription. Indeed, site-directed mutagenesis of a putative CAR consensus binding sequence on the Mcl-1 promoter decreased Mcl-1 promoter activity. Mcl-1 transgenic animals demonstrated little to no acute liver injury after administration of Jo2, signifying Mcl-1 cytoprotection. In conclusion, these observations support a prominent role for CAR cytoprotection against Fas-mediated hepatocyte injury via a mechanism involving upregulation of Mcl-1 and, likely, downregulation of Bax and Bak.

Elucidating the mechanism of liver regeneration could lead to life-saving therapy for a large number of patients, especially elderly patients, after segmental liver transplantation or resection of liver tumors. The forkhead box m1b (Foxm1b) transcription factor is required for normal liver regeneration. Here we report that Foxm1b is the first direct farnesoid X receptor (FXR) target gene known to be involved in cell cycle regulation and that aging regenerating livers have delayed activation of FXR, which results in defective induction of Foxm1b and thereby contributes to defective liver regeneration. An inverted repeat 0 (IR-0) FXR response element, acting as an enhancer in intron 3 of the Foxm1b gene, was identified by a combination of transcriptional reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays. Diminished FXR binding to the IR-0 element was found in aging regenerating livers. FXR activation by a novel ligand in aging livers induced Foxm1b expression and elevated hepatocyte DNA replication to about 70% of the levels found in young regenerating livers, which were specifically suppressed by hepatic expression of anti-Foxm1b short hairpin RNA. Conclusion: Our results have revealed Foxm1b as the first known direct FXR target gene involved in cell cycle regulation and have demonstrated that defective activation of FXR could be an intrinsic defect in aging regenerating livers. Activation of FXR alone is largely able to alleviate age-related liver regeneration defects. These findings highlight FXR as a potential target of drug design for promoting liver regeneration in older subjects. (HEPATOLOGY 2010.)