Linking long noncoding RNA to control bile acid signaling and cholestatic liver fibrosis
Authors: John Y.L. Chiang
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. Supported by the National Institutes of Health (DK58379 and DK44442). See Article On Page 1183 Cholestasis is a pathological condition where normal bile flow out of the liver is reduced or disrupted, leading to intrahepatic accumulation of bile acids. High levels of toxic bile acids damage the bile duct epithelium and elevate biliary pressure to rupture the bile duct and expose hepatocytes to high concentrations of bile acids, macrophage infiltration, and inflammation, leading to hepatocyte cell death. Cholestasis can result from genetic defects in canalicular transporters, mechanical obstruction of the bile duct by gallstones or tumors, pregnancy (intrahepatic cholestasis of pregnancy), autoimmune destruction of the bile ducts, or drug‐induced liver toxicity.1 Primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC) are two common types of progressive chronic cholestasis in humans. PSC is small bile duct inflammation and blockage causing accumulation of bile acids in hepatocytes, portal hypertension, and biliary fibrosis and cirrhosis. PBC is a chronic autoimmune liver disease with progressive destruction of the bile ducts. The majority of PSC patients are male, whereas most PBC patients are female. Maintaining bile acid homeostasis is essential for protection against inflammatory liver diseases in the gastrointestinal tract, such as liver fibrosis and cirrhosis, nonalcoholic and alcoholic liver disease, liver cancer, diabetes, and inflammatory bowel diseases. Enterohepatic circulation of bile acids from the liver to the intestine and back to the liver occurs six to eight times a day (Fig. 1). This physiological pathway not only regulates bile acid synthesis and maintains a constant bile acid pool but also controls gut microbial overgrowth and intestinal barrier function. Bile acids are physiological detergents required for nutrient and drug absorption and are signaling molecules that activate farnesoid X receptor (FXR) and the membrane G protein–coupled bile acid receptor‐1 (Gpbar‐1, or Takeda G protein–coupled receptor 5 [TGR5]) in the gastrointestinal tract. Activation of FXR and TGR5 by agonists is known to protect against inflammatory liver diseases. Activation of FXR induces small heterodimer partner (SHP), a negative transcription factor known to inhibit transcription of cytochrome P450 7a1 (Cyp7a1), a key regulatory enzyme in the synthesis of cholic acid and chenodeoxycholic acid, two primary human bile acids (Fig. 1).3Figure 1: Bile acid and lncRNA H19 signaling in hepatic fibrosis. In hepatocytes, Cyp7a1 initiates bile acid synthesis from cholesterol to form cholic acid and chenodeoxycholic acid in human liver. Bile acids activate FXR to induce the repressor SHP, which inhibits H19 expression. In cholestasis, bile acids induce H19, which represses the repressor ZEB1. ZEB1 binds to and represses EpCAM involved in fibrosis. This negative regulatory loop mediates bile acid–induced liver fibrosis. In this study, bile duct ligation induced Cyp7a1 but inhibited Shp mRNA expression. AAV8‐H19 restored Cyp7a1 and Shp involved in bile acid synthesis. The antiapoptotic factor Bcl1 suppresses SHP by promoting SHP decay and reduces H19 expression to protect against liver fibrosis. In hepatocytes and cholangiocytes, taurocholic acid activates the S1PR2/extracellular signal–regulated kinase 1/2 pathway to induce H19. TGR5 is activated by secondary bile acid lithocholic acid and deoxycholic acid and is expressed in cholangiocytes and Kupffer cells but not in hepatocytes. Differential regulation of these bile acid receptors may modulate H19 levels in hepatocytes. H19 may be shuffling between hepatocytes and cholangiocytes through the cholehepatic shunt to cause inflammation and cholestatic liver disease. Hepatic macrophages, Kupffer cells, and stellate cells may also be involved in H19‐mediated fibrosis. In BDL mice and Mdr2 –/– mice, bile acids induced H19 levels. Bile acid activation of TGR5 signaling may protect against cholestasis. The "?" indicates that the underlying mechanism is not clear and needs to be further studied. Abbreviations: cAMP, cyclic adenosine monophosphate; ERK, extracellular signal–regulated kinase; HSC, hepatic stellate cell; KC, Kupffer cell; PKA, protein kinase A; α‐SMA, alpha‐smooth muscle actin; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; T/DCA, TGR5 activated by deoxycholic acid; T/LCA, TGR5 activated by lithocholic acid.Noncoding RNAs including microRNAs and long noncoding RNAs (lncRNAs) play key roles in liver diseases and hepatocellular carcinoma.4 lncRNAs are about 200 nucleotides in length. Recent studies have linked lncRNA H19 to cholestatic liver injury and provide new insight into the mechanism and pathogenesis of cholestasis.5 H19 is imprinted and maternally expressed during embryo development.7 H19 expression is low in adult human liver and highly induced in livers with cholestatic fibrosis and cirrhosis, indicating that H19 may be involved in the pathogenesis of liver fibrosis.5 H19 is induced by c‐Myc oncogene in tumors,8 and antiapoptotic protein Bcl2 activates caspase 8 to degrade SHP, resulting in increased H19 expression and bile acid pool size, and promotes hepatic fibrosis.5 Thus, bile acids may induce SHP to inhibit H19 expression as protection against cholestatic liver injury (Fig. 1). In this issue of hepatology, Song et al.9 further studied the mechanism of H19 in promoting cholestasis and liver fibrosis using bile duct ligation (BDL)–induced cholestasis in mice. They found that adenovirus‐associated virus 8 (AAV8)–mediated overexpression of H19 augmented BDL‐induced liver fibrosis, increased bile acid levels, and induced H19 expression. However, BDL markedly increased liver mRNA levels of Cyp7a1 but reduced Cyp8b1, Cyp7b1, and SHP involved in bile acid synthesis; and AAV8‐H19 restored Cyp7a1 and Shp, but not Cyp8b1 and Cyp7b1, levels. Alteration of bile acid synthesis genes would alter bile acid composition and hepatic metabolic homeostasis. These data contradict their previous report that bile acids induced SHP to repress H19.6 On the other hand, H19 produced in hepatocytes or cholangiocytes may inhibit SHP in hepatocytes (Fig. 1). This BDL+AAV8‐H19 model may complicate the results in this study. Other mouse models, such as the multidrug‐resistant 2 deficient (Mdr−/−) cholestatic mouse model, may be used to verify their findings. It is possible that sphingosine‐1‐phosphate receptor 2 (S1PR2) may induce H19 expression in hepatocytes. Further study is need to confirm whether selective activation of FXRSHP and S1PR2 by primary bile acids in hepatocytes or TGR5 by secondary bile acids in Kupffer cells and cholangiocytes regulates H19 expression (Fig. 1). H19 decreased hepatic zinc finger E box‐binding homeobox 1 (ZEB1) but increased epithelial cell adhesion molecule (EpCAM) expression in BDL mice. Either overexpression of ZEB1 or knockdown of EpCAM ameliorates H19‐induced fibrosis. Hepatic stellate cells are activated to increase α‐smooth muscle actin and fibrogenesis. α‐Smooth muscle actin is increased in H19‐BDL mice and decreased in H19−/−‐BDL mice, suggesting that activation of hepatic stellate cells also contributes to H19‐mediated fibrosis. Further mechanistic study revealed that ZEB1 repressed EpCAM promoter activity and gene transcription by binding to a ZEB1 binding site on the EpCAM gene promoter and that H19 interacted with ZEB1 protein to block its binding to the EpCAM promoter (Fig. 1). Additional in vitro experiments show that bile acids induced H19 in mouse small cholangiocytes but much less so in mouse large cholangiocytes, characteristic of inflammation in small bile ducts in PBC. In addition, they found that H19 expression levels were increased in human PSC and PBC livers. Consistently, EpCAM proteins are significantly induced in PSC and even more so in PBC samples, while ZEB1 proteins were lower in PSC and PBC in livers with high H19. Thus, human patient liver specimens support the role of H19 in the pathogenesis of human cholestatic diseases. Overall, this study demonstrated that activation of hepatic H19 promoted cholestatic liver fibrosis in mice through the ZEB1/EpCAM signaling pathway. The dysregulation of bile acid homeostasis induces H19 in cholangiocytes and hepatocytes and results in induction of EpCAM, leading to biliary hyperplasia. This study provides a mechanistic insight into the role of lncRNA in cholestatic liver diseases. Further study is needed to show how H19 specifically interacts with the zinc finger on the ZEB1 protein and how H19 represses expression of ZEB1 and induces or represses SHP, two repressors involved in the negative regulatory loop in the regulation of H19 expression (Fig. 1). Another study published in hepatology reports that H19 is markedly induced in female Mdr2−/− mice but not in aged‐matched male Mdr2−/− mice at advanced stages of cholestasis, and estrogen might play a role in enhancing H19 expression in intrahepatic cholestasis.6 The taurocholate‐activated S1PR2 may activate extracellular signal–regulated kinase 1/2 to induce H19 by a mechanism that is not yet known. This study did not exam H19 expression in small and large cholangiocytes. The differential expression and induction of H19 by bile acids in different populations of cholangiocytes are not known. It should be noted that the Mdr2−/− mouse is a model of PSC, a male‐dominant cholestatic liver disease. The cause of gender disparity in PSC and PBC is not clear and requires further studies. Currently there is no effective treatment for liver fibrosis, PSC, or PBC. Ursodeoxycholic acid exhibits anti‐inflammatory effects but is not effective for treating PSC. Nor‐ursodeoxycholic acid promotes biliary HCO3− secretion to alleviate liver injury in Mdr2−/− mice. Fibrates, which activate peroxisomal proliferator–activated receptor α, have been used to treat PBC patients who do not respond to ursodeoxycholic acid. Obeticholic acid has been approved recently to treat PBC. H19 lncRNA may be used as a biomarker for diagnosis of cholestasis, and drugs targeted to reduce H19 expression in cholestasis may alleviate chronic cholestatic liver diseases.