Cholangiocyte lipoapoptosis: Implications for biliary damage during nonalcoholic fatty liver disease
Authors: Allyson K. Martínez, Shannon S. Glaser
Journal: Hepatology (2014)

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. One third of the population of the United States has NAFLD, 81% of those affected being children.1-3 In a subset of patients, NAFLD progresses to the more severe disease, nonalcoholic steatohepatitis (NASH), which often results in end-stage liver disease and even cancer.4 For these reasons, NAFLD is increasingly becoming a major public health concern. The development of therapeutics for NAFLD requires a basic understanding of the molecular mechanisms involved in the pathogenesis of the disease and the individual target cell types. Free fatty acids (FFAs) are increased in the serum of NAFLD patients.2 FFAs have been shown to induce stress-dependent kinases and cause lipoapoptosis in hepatocytes, which is implicated in the pathogenesis of NAFLD.2, 5 However, the involvement of biliary cell injury and the affect of FFAs on cholangiocytes have only recently been proposed.6, 7 Recently, Friedrich et al. reported that the PNPLA3 rs738409 variant (I148M), which has been associated with an increased risk for development of NAFLD and alcoholic liver disease resulting from hepatocyte lipotoxicity, is a risk factor for reduced survival in male primary sclerosing cholangitis patients with dominant bile duct stenosis, presumably resulting from lipotoxicity at the cholangiocyte level.8-10 In this issue of Hepatology, Natarajan et al.11 provide insights into the novel mechanisms of cholangiocyte lipoapoptosis, which may play an important role in the pathogenesis of biliary damage in a subset of NAFLD patients characterized by cholestasis and biliary cell injury (Fig. 1). In their work, they utilize an in vitro approach with several human cholangiocyte cell lines to demonstrate, for the first time, that cholangiocytes are susceptible to lipoapoptosis. The FFAs, palmitate and stearate, were shown to induce cholangiocyte lipoapoptosis at concentrations of circulating levels of total FFAs found in NAFLD and NASH patients. Working model of the mechanisms regulating cholangiocyte lipoapoptosis. NAFLD is characterized by increased circulating levels of FFAs and hepatic accumulation of lipids, which is commonly associated with metabolic syndrome. Exposure of cholangiocytes to FFAs stimulates activation of phosphorylated (p)-38-MAPK and ERK, with the subsequent nuclear accumulation of FoxO3 resulting in increased expression of the propapoptotic protein, PUMA, and increased caspase-3/7 activity triggering cholangiocyte lipoapoptosis. Natarajan et al. began their analysis of the signaling mechanisms regulating cholangiocyte lipoapoptosis by evaluating major cellular stress signaling kinases, such as c-Jun N-terminal kinase (JNK), p38-MAPK (mitogen-activated protein kinase), and extracellular signal-regulated kinase (ERK) known to play roles in apoptosis. In hepatocytes, lipoapoptosis has been shown to be JNK dependent.2, 12 In contrast to hepatocytes, it was found that inhibition of JNK-dependent signaling did not prevent cholangiocyte lipoapoptosis, which suggests the involvement of other stress-activated kinases. The researchers investigated the roles of ERK and p38-MAPK, also implicated in cholangiocyte lipoapoptosis, using pharmacological inhibitors. However, the specificity of the inhibitors utilized did not allow for the definitive elucidation of the involvement of these two potential candidates. The difference in the signaling mechanisms governing hepatocyte and cholangiocyte lipoapoptosis may be a result of the lack of cellular steatosis observed in cholangiocytes. This lack of cholangiocyte steatosis is consistent with clinical observations that, in NAFLD, there is marked hepatocyte accumulation of lipid without steatosis in cholangiocytes.13, 14 Because cholangiocyte lipoapoptosis was not dependent upon JNK activation, the researchers shifted their focus to FoxO3 and the downstream propapoptotic protein, p53–up-regulated modulator of apoptosis (PUMA). FoxO3 has been shown to activate cell death pathways, which includes up-regulation of PUMA expression and a myriad of other proapoptotic proteins, such as Bim, that has been shown to mediate hepatocyte lipoapoptosis.15, 16 In this study, palmitate stimulated deacetylation and dephosphorylation of nuclear FoxO3 at early time points, but there was increased accumulation of acetylated and phosphorylated nuclear FoxO3 at later time points. It is thought that acetylation and phosphorylation of FoxO3 causes a shift to cell survival by mediating nuclear export of FoxO3. However, the mechanisms regulating nuclear localization of FoxO3 and its role in the mediation of proapoptotic gene expression programs are not well studied in cholangiocytes and other cell types and therefore require further investigation. Other signaling factors, such as sirtuin 1 and protein kinase B, may play a role in fine-tuning the FoxO3-mediated mechanism of lipoapoptosis.17 PUMA has been shown to play an important role in hepatocyte lipoapoptosis as well as a downstream target of FoxO3.12, 15, 16 The role of PUMA in cholangiocyte lipoapoptosis was confirmed by knockdown experiments and evaluation of caspase-3/7 activity. The current findings by Natarajan et al. represent a novel concept that FFAs can induce cholangiocyte lipoapoptosis, which may explain why a subset of NAFLD patients with clinical findings of cholestasis have biliary injury not consistent with that normally observed in NAFLD. Further studies in animal models and human tissue samples are clearly warranted. Obese Zucker rats have been utilized as a model of NAFLD and exhibit impaired bile secretory function and mild cholestasis.18, 19 These studies suggest that defective bile secretory function may be a factor that contributes to greater susceptibility to liver injury and in the case of increased severity of biliary damage and fibrosis in patients with NASH and primary biliary cirrhosis.20 The current findings of FFA-induced cholangiocyte lipoapoptosis and evidence of impaired biliary function should provide a novel direction for the development of innovative therapies for NAFLD alone and in the context of cholestatic liver disease. Allyson K. MartÍnez, Ph.D.3 Shannon S. Glaser, Ph.D.1,2,3 1Central Texas Veterans Health Care System 2Scott & White Digestive Disease Research Center 3Department of Internal Medicine Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, TX Authors names in bold designate shared co-first authorship.