Nathan W. Werneburg

Free fatty acids promote hepatic lipotoxicity by stimulating TNF-α expression via a lysosomal pathway

Nonalcoholic fatty liver disease (NAFLD) is a serious health problem. Although NAFLD represents a form of lipotoxicity, its pathogenesis remains poorly understood. The aim of this study was to examine the cellular mechanisms involved in free fatty acid (FFA)‐mediated hepatic lipotoxicity. FFA treatment of liver cells resulted in Bax translocation to lysosomes and lysosomal destabilization with release of cathepsin B (ctsb), a lysosomal cysteine protease, into the cytosol. This process was also partially dependent on ctsb. Lysosomal destabilization resulted in nuclear factor κB–dependent tumor necrosis factor α expression. Release of ctsb into the cytoplasm was also observed in humans with NAFLD and correlated with disease severity. In a dietary murine model of NAFLD, either genetic or pharmacological inactivation of ctsb protected against development of hepatic steatosis, liver injury, and insulin resistance with its associated “dysmetabolic syndrome.” In conclusion, these data support a lipotoxic model of FFA‐mediated lysosomal destabilization. Supplemental material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2004;40:185–194.)

Free fatty acids induce JNK dependent hepatocyte lipoapoptosis

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.

Transcriptional suppression of mir-29b-1/mir-29a promoter by c-Myc, hedgehog, and NF-kappaB.

MicroRNAs regulate pathways contributing to oncogenesis, and thus the mechanisms causing dysregulation of microRNA expression in cancer are of significant interest. Mature mir‐29b levels are decreased in malignant cells, and this alteration promotes the malignant phenotype, including apoptosis resistance. However, the mechanism responsible for mir‐29b suppression is unknown. Here, we examined mir‐29 expression from chromosome 7q32 using cholangiocarcinoma cells as a model for mir‐29b downregulation. Using 5′ rapid amplification of cDNA ends, the transcriptional start site was identified for this microRNA locus. Computational analysis revealed the presence of two putative E‐box (Myc‐binding) sites, a Gli‐binding site, and four NF‐κB‐binding sites in the region flanking the transcriptional start site. Promoter activity in cholangiocarcinoma cells was repressed by transfection with c‐Myc, consistent with reports in other cell types. Treatment with the hedgehog inhibitor cyclopamine, which blocks smoothened signaling, increased the activity of the promoter and expression of mature mir‐29b. Mutagenesis analysis and gel shift data are consistent with a direct binding of Gli to the mir‐29 promoter. Finally, activation of NF‐κB signaling, via ligation of Toll‐like receptors, also repressed mir‐29b expression and promoter function. Of note, activation of hedgehog, Toll‐like receptor, and c‐Myc signaling protected cholangiocytes from TRAIL‐induced apoptosis. Thus, in addition to c‐Myc, mir‐29 expression can be suppressed by hedgehog signaling and inflammatory pathways, both commonly activated in the genesis of human malignancies. J. Cell. Biochem. 110: 1155–1164, 2010. Published 2010 Wiley‐Liss, Inc.

Interleukin 6 upregulates myeloid cell leukemia-1 expression through a STAT3 pathway in cholangiocarcinoma cells.

Interleukin 6 (IL‐6) contributes to the pathogenesis of cholangiocarcinoma by upregulating myeloid cell leukemia‐1 (Mcl‐1), a key antiapoptotic Bcl‐2 family member protein. IL‐6 can alter gene transcription via Janus kinases (JAK) and signal transducer and activator of transcription (STAT) signal cascade. We examined this cascade in IL‐6 regulation of Mcl‐1 transcription in human cholangiocarcinoma cell lines. STAT3 was constitutively activated (i.e., tyrosine‐phosphorylated) in cholangiocarcinoma cells but not in nonmalignant cholangiocytes. Treatment with anti–IL‐6 antisera or the JAK inhibitor AG490 or transfection with dominant negative STAT3 diminished Mcl‐1 messenger RNA and protein levels. Likewise, these attempts to interrupt the STAT3 cascade also reduced Mcl‐1 promoter activity. Site‐directed mutagenesis of a putative STAT3 consensus binding sequence decreased Mcl‐1 promoter activity. Chromatin immunoprecipitation analysis demonstrated a direct binding of STAT3 to the putative STAT3 binding sequences in the Mcl‐1 promoter. Downregulation of Mcl‐1 by AG490 sensitized the cells to apoptosis mediated by tumor necrosis factor–related apoptosis‐inducing ligand. In conclusion, we have directly demonstrated a STAT3 regulatory element in the Mcl‐1 promoter. Downregulation of Mcl‐1 transcription by inhibiting this cascade is a potential strategy for the treatment of this cancer.(HEPATOLOGY 2005;42:1329–1338.)

The Bile Acid Glycochenodeoxycholate Induces TRAIL-Receptor 2/DR5 Expression and Apoptosis

Toxic bile salts induce hepatocyte apoptosis by both Fas-dependent and -independent mechanisms. In this study, we examined the cellular mechanisms responsible for Fas-independent, bile acid-mediated apoptosis. HuH-7 cells, which are known to be Fas deficient, were stably transfected with the sodium-dependent bile acid transporting polypeptide. The toxic bile acid glycochenodeoxycholate (GCDC)-induced apoptosis in these cells in a time- and concentration-dependent manner. Apoptosis and mitochondrial cytochrome c release were inhibited by transfection with dominant negative FADD, CrmA transfection, or treatment with the selective caspase 8 inhibitor IETD-CHO. These observations suggested the Fas-independent apoptosis was also death receptor mediated. Reverse transcriptase-polymerase chain reaction demonstrated tumor necrosis factor-R1, tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1/DR4, -R2/DR5, and TRAIL, but not tumor necrosis fator-α expression by these cells. GCDC treatment increased expression of TRAIL-R2/DR5 mRNA and protein 10-fold while expression of TRAIL-R1 was unchanged. Furthermore, aggregation of TRAIL-R2/DR5, but not TRAIL-R1/DR4 was observed following GCDC treatment of the cells. Induction of TRAIL-R2/DR5 expression and apoptosis by bile acids provides new insights into the mechanisms of hepatocyte apoptosis and the regulation of TRAIL-R2/DR5 expression.

Transcriptional Regulation of Bim by FoxO3A Mediates Hepatocyte Lipoapoptosis

Hepatocyte lipoapoptosis, a critical feature of nonalcoholic steatohepatitis, can be replicated in vitro by incubating hepatocytes with saturated free fatty acids (FFA). These toxic FFA induce Bim expression, which is requisite for their cytotoxicity. Because the FoxO3a transcription factor has been implicated in Bim expression, our aim was to determine if FFA induce Bim by a FoxO3a-dependent mechanism. In Huh-7 cells, the saturated FFA, palmitic and stearic acid, increased Bim mRNA 16-fold. Treatment of cells with the saturated FFA induced FoxO3a dephosphorylation (activation) and nuclear translocation and stimulated a FoxO luciferase-based reporter assay; direct binding of FoxO3a to the Bim promoter was also confirmed by a chromatin immunoprecipitation assay. A small interfering RNA-targeted knockdown of FoxO3a abrogated FFA-mediated Bim induction and apoptosis. FoxO3a was activated by a phosphatase 2A-dependent mechanism, since okadaic acid- and small interfering RNA-targeted knockdown of this phosphatase blocked FoxO3a dephosphorylation, Bim expression, and apoptosis. Consistent with these data, phosphatase 2A activity was also stimulated 3-fold by saturated FFA. Immunoprecpitation studies revealed an FFA-dependent association between FoxO3a and protein phosphatase 2A. FFA-mediated FoxO3a activation by protein phosphatase 2A was also observed in HepG2 cells and murine hepatocytes. In conclusion, saturated FFA stimulate protein phosphatase 2A activity, which activates FoxO3a, inducing expression of the intracellular death mediator Bim.

JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis

Free fatty acids (FFA) induce hepatocyte lipoapoptosis by a c-Jun N-terminal kinase (JNK)-dependent mechanism. However, the cellular processes by which JNK engages the core apoptotic machinery during lipotoxicity, especially activation of BH3-only proteins, remain incompletely understood. Thus, our aim was to determine whether JNK mediates induction of BH3-only proteins during hepatocyte lipoapoptosis. The saturated FFA palmitate, but not the monounsaturated FFA oleate, induces an increase in PUMA mRNA and protein levels. Palmitate induction of PUMA was JNK1-dependent in primary murine hepatocytes. Palmitate-mediated PUMA expression was inhibited by a dominant negative c-Jun, and direct binding of a phosphorylated c-Jun containing the activator protein 1 complex to the PUMA promoter was identified by electrophoretic mobility shift assay and a chromatin immunoprecipitation assay. Short hairpin RNA-targeted knockdown of PUMA attenuated Bax activation, caspase 3/7 activity, and cell death. Similarly, the genetic deficiency of Puma rendered murine hepatocytes resistant to lipoapoptosis. PUMA expression was also increased in liver biopsy specimens from patients with non-alcoholic steatohepatitis as compared with patients with simple steatosis or controls. Collectively, the data implicate JNK1-dependent PUMA expression as a mechanism contributing to hepatocyte lipoapoptosis.

miR-25 targets TNF-related apoptosis inducing ligand (TRAIL) death receptor-4 and promotes apoptosis resistance in cholangiocarcinoma

It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. Although targets and functional roles for a number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR‐25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR‐25 expression, suggesting Hedgehog signaling stimulates miR‐25 production. Functionally, miR‐25 was shown to protect cells against TNF‐related apoptosis‐inducing ligand (TRAIL)‐induced apoptosis. Correspondingly, antagonism of miR‐25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor‐4 (DR4) as a potential novel miR‐25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays. Conclusion: These data implicate elevated miR‐25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR‐25 target DR4 provides a mechanism by which miR‐25 contributes to evasion of TRAIL‐induced cholangiocarcinoma apoptosis. (HEPATOLOGY 2012)

Tumor Necrosis Factor-related Apoptosis-inducing Ligand Activates a Lysosomal Pathway of Apoptosis That Is Regulated by Bcl-2 Proteins

The present studies were performed to determine whether lysosomal permeabilization contributes to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity and to reconcile a role for lysosomes with prior observations that Bcl-2 family members regulate TRAIL-induced apoptosis. In KMCH cholangiocarcinoma cells stably expressing Mcl-1 small interference RNA (siRNA), treatment with TRAIL induced a redistribution of the cathepsin B from lysosomes to the cytosol. Pharmacological and small hairpin RNA-targeted inhibition of cathepsin B attenuated TRAIL-mediated apoptosis as assessed by morphological, biochemical, and clonogenic assays. Neither Bid siRNA nor Bak siRNA prevented cathepsin B release. In contrast, treatment of the cells with Bim siRNA or the JNK inhibitor SP600125 attenuated lysosomal permeabilization and cell death. Moreover, Bim and active Bax co-localized to lysosomes in TRAIL-treated cells in a JNK-dependent manner, and Bax siRNA reduced TRAIL-induced lysosomal permeabilization and cell death. Finally, BH3 domain peptides permeabilized isolated lysosomes in the presence of Bax. Collectively, these data suggest that TRAIL can trigger an apoptotic pathway that involves JNK-dependent activation of Bim, which in turn induces Bax-mediated permeabilization of lysosomes.

Proteasome inhibition induces hepatic stellate cell apoptosis

Induction of hepatic stellate cell (HSC) apoptosis attenuates hepatic fibrosis, and, therefore, mechanisms to induce HSC cell death are of therapeutic interest. Proteasome inhibitors induce apoptosis in transformed cells, especially those cells dependent upon nuclear factor kappa B (NF‐κB) activation. Because stimulated HSCs also trigger NF‐κB activation, the aim of this study was to determine if proteasome inhibitors induce HSC apoptosis. The immortalized human HSC line, LX‐2, and primary rat HSCs were treated with the proteasome inhibitors bortezomib and MG132. Both proteasome inhibitors induced HSC apoptosis. Proteasome inhibition blocked NF‐κB activation and, more importantly, NF‐κB inhibition by Bay11‐7082–triggered HSC apoptosis. Activated HSC survival is dependent upon the NF‐κB target gene A1, an anti‐apoptotic Bcl‐2 family member, as siRNA targeted knockdown of A1‐induced HSC apoptosis. In contrast, proteasome inhibition–induced alterations in TRAIL, death receptor 5, and Bim could not be implicated in the apoptotic response. The relevance of these findings was confirmed in the bile‐duct–ligated mouse where bortezomib reduced hepatic markers of stellate cell activation and fibrosis. In conclusion, proteasome inhibition is a potential therapeutic strategy for inducing HSC apoptosis and inhibiting liver fibrogenesis. (HEPATOLOGY 2006;43:335–344.)