Bronk, Steven F.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/β inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Toxic bile salts induce hepatocyte apoptosis by a Fas-dependent, Fas ligand-independent mechanism. To account for this observation, we formulated the hypothesis that toxic bile salts induce apoptosis by effecting translocation of cytoplasmic Fas to the cell surface, resulting in transduction of Fas death signals. In McNtcp.24 cells the majority of Fas was cytoplasmic, as assessed by cell fractionation and immunofluorescence studies. However, cell surface Fas increased sixfold after treatment with the toxic bile salt glycochenodeoxycholate (GCDC) in the absence of increased Fas protein expression. Moreover, in cells transfected with Fas-green fluorescence protein, cell surface fluorescence also increased in GCDC-treated cells, directly demonstrating Fas translocation to the plasma membrane. Both brefeldin A, a Golgi-disrupting agent, and nocodazole, a microtubule inhibitor, prevented the GCDC-induced increase in cell surface Fas and apoptosis. In conclusion, toxic bile salts appear to induce apoptosis by promoting cytoplasmic transport of Fas to the cell surface by a Golgi- and microtubule-dependent pathway.

Glycodeoxycholate (GDC) induces apoptosis in hepatocytes by a mechanism associated with DNA cleavage by endonucleases. In many models of apoptosis, proteolysis is required prior to DNA cleavage. Our aims were to determine if enhanced proteolysis is a mechanism causing GDC-mediated apoptosis. In cultured rat hepatocytes exposed to 50 microM GDC for 4 h, nonlysosomal proteolysis increased by 65% compared with controls. The serine protease inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK; 100 microM) reduced cell death from apoptosis by 75% after 4 h of treatment with GDC. TLCK also inhibited DNA fragmentation. There was a twofold increase in nuclear serinelike protease activity during GDC-induced apoptosis accompanied by a 2.5-fold reduction in nonnuclear serine protease activity, suggesting translocation of the protease from the cytosol to the nucleus. Zn2+, an inhibitor of apoptosis, also inhibited nonlysosomal proteolysis and nuclear serinelike protease activity. These novel data suggest that nonlysosomal serinelike protease activity contributes to hepatocyte apoptosis. These data may be important in understanding apoptosis in other cell types and in providing insight into the mechanisms of liver injury during cholestasis.

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) and its cognate receptor(s) are up‐regulated in human and murine nonalcoholic steatohepatitis (NASH); however, the consequence of this enhanced expression on NASH pathogenesis remains unclear. TRAIL may either accentuate liver injury by promoting hepatic steatosis and inflammation or it may mitigate the disease process by improving systemic insulin resistance and averting hepatic fibrosis. Herein, we investigated the role of TRAIL in an obesity‐induced murine model of NASH. C57BL/6 wild‐type mice and Trail –/– mice were placed on a 20‐week standard chow or a high‐fat, high‐fructose, and high‐cholesterol (FFC) diet, which induces obesity, insulin resistance, and NASH. Metabolic phenotype, liver injury, inflammation and fibrosis, and adipose tissue homeostasis were examined. FFC diet‐fed Trail –/– mice displayed no difference in weight gain and metabolic profile when compared to wild‐type mice on the same diet. All FFC‐fed mice developed significant hepatic steatosis, which was attenuated in Trail –/– mice. TRAIL deficiency also significantly decreased FFC diet‐induced liver injury as manifested by reduced serum alanine aminotransferase values, hepatic terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling‐positive cells, and macrophage‐associated inflammation. FFC diet‐associated hepatic stellate cell activation and hepatic collagen deposition were also abrogated in Trail –/– mice. In contrast to the liver, TRAIL deletion did not improve FFC diet‐induced adipose tissue injury and inflammation and actually aggravated insulin resistance. Conclusion: NASH pathogenesis may be dissociated from other features of the metabolic syndrome, and liver‐targeted inhibition of TRAIL signaling may be salutary. (Hepatology Communications 2017;1:648–662)

AbstractThe Hippo pathway effector YAP is implicated in the pathogenesis of cholangiocarcinoma (CCA). The Hippo pathway relies on signaling cross talk for its regulation. Given the importance of platelet derived growth factor receptor (PDGFR) signaling in CCA biology, our aim was to examine potential YAP regulation by PDGFR. We employed human and mouse CCA specimens and cell lines for these studies. Initially, we confirmed upregulation of PDGFRβ and PDGFR ligands in human and mouse CCA specimens and cell lines. YAP, a transcriptional co‐activator, was localized to the nucleus in human CCA specimens and a cell line, as well as patient derived xenografts (PDX). PDGFR pharmacologic inhibition led to a redistribution of YAP from the nucleus to cytosol and downregulation of YAP target genes in a human CCA cell line. siRNA silencing of PDGFR‐β similarly downregulated YAP target genes. YAP activation (nuclear localization and target gene expression) was regulated by Src family kinases (SFKs) downstream of PDGFR. SFK activity resulted in phosphorylation of YAP on tyrosine357 (YAPY357). The importance of YAPY357 phosphorylation in regulating YAP activation was confirmed utilizing the SB‐1 cell line, a mouse cell line expressing YAP S127A precluding canonical serine phosphorylation. PDGFR inhibition decreased cellular abundance of the survival protein Mcl‐1, a known YAP target gene, and accordingly increased cell death in CCA cells in vitro and in vivo. These preclinical data demonstrate that a PDGFR‐SFK cascade regulates YAP activation via tyrosine phosphorylation in CCA. Inhibiting this cascade may provide a viable therapeutic strategy for this human malignancy.

ABSTRACTSaturated fatty acids (SFA) and their toxic metabolites contribute to hepatocyte lipotoxicity in nonalcoholic steatohepatitis (NASH). We previously reported that hepatocytes, under lipotoxic stress, express the potent macrophage chemotactic ligand C‐X‐C motif chemokine 10 (CXCL10), and release CXCL10‐enriched extracellular vesicles (EV) by a mixed lineage kinase (MLK) 3‐dependent mechanism. In the current study, we sought to examine the signaling pathway responsible for CXCL10 induction during hepatocyte lipotoxicity. Here, we demonstrate a role for signal transducer and activator of transcription (STAT) 1 in regulating CXCL10 expression. Huh7 and HepG2 cells were treated with lysophosphatidylcholine (LPC), the toxic metabolite of the SFA palmitate. In LPC‐treated hepatocytes, CXCL10 induction is mediated by a mitogen activated protein kinase (MAPK) signaling cascade consisting of a relay kinase module of MLK3, MKK3/6, and p38. P38 in turn induces STAT1 Ser727 phosphorylation and CXCL10 upregulation in hepatocytes, which is reduced by genetic or pharmacological inhibition of this MAPK signaling cascade. The binding and activity of STAT1 at the CXCL10 gene promoter were identified by chromatin immunoprecipitation and luciferase gene expression assays. Promoter activation was attenuated by MLK3/STAT1 inhibition or by deletion of the consensus STAT1 binding sites within the CXCL10 promoter. In lipotoxic hepatocytes, MLK3 activates a MAPK signaling cascade, resulting in the activating phosphorylation of STAT1, and CXCL10 transcriptional upregulation. Hence, this kinase relay module and/or STAT1 inhibition may serve as a therapeutic target to reduce CXCL10 release, thereby attenuating NASH pathogenesis. J. Cell. Biochem. 118: 3249–3259, 2017. © 2017 Wiley Periodicals, Inc.

Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage‐associated inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage‐driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C‐X‐C motif) ligand 10 (CXCL10)‐laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine, CXCL10, in the EVs, which was markedly enriched in EVs isolated from LPC‐treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)‐tagged CXCL10 was present in vesicular structures and colocalized with the red fluorescent protein (RFP)‐tagged EV marker, CD63, after LPC treatment of cotransfected Huh‐7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone‐marrow–derived macrophages with lipotoxic hepatocyte‐derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10‐neutralizing antisera. MLK3‐deficient mice fed a NASH‐inducing diet had reduced concentrations of total plasma EVs and CXCL10 containing EVs compared to wild‐type mice. Conclusions: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10‐bearing vesicles from hepatocytes, which are chemotactic for macrophages. (Hepatology 2016;63:731–744)

Cholangiocarcinoma (CCA) is a lethal hepatobiliary neoplasm originating from the biliary apparatus. In humans, CCA risk factors include hepatobiliary inflammation and fibrosis. The recently identified interleukin (IL)−1 family member, IL‐33, has been shown to be a biliary mitogen which also promotes liver inflammation and fibrosis. Our aim was to generate a mouse model of CCA mimicking the human disease. Ectopic oncogene expression in the biliary tract was accomplished by the Sleeping Beauty transposon transfection system with transduction of constitutively active AKT (myr‐AKT) and Yes‐associated protein. Intrabiliary instillation of the transposon–transposase complex was coupled with lobar bile duct ligation in C57BL/6 mice, followed by administration of IL‐33 for 3 consecutive days. Tumors developed in 72% of the male mice receiving both oncogenes plus IL‐33 by 10 weeks but in only 20% of the male mice transduced with the oncogenes alone. Tumors expressed SOX9 and pancytokeratin (features of CCA) but were negative for HepPar1 (a marker of hepatocellular carcinoma). Substantive overlap with human CCA specimens was revealed by RNA profiling. Not only did IL‐33 induce IL‐6 expression by human cholangiocytes but it likely facilitated tumor development in vivo by an IL‐6–sensitive process as tumor development was significantly attenuated in Il‐6–/– male animals. Furthermore, tumor formation occurred at a similar rate when IL‐6 was substituted for IL‐33 in this model. Conclusion: The transposase‐mediated transduction of constitutively active AKT and Yes‐associated protein in the biliary epithelium coupled with lobar obstruction and IL‐33 administration results in the development of CCA with morphological and biochemical features of the human disease; this model highlights the role of inflammatory cytokines in CCA oncogenesis. (Hepatology 2015;61:1627–1642)