Guicciardi, Maria Eugenia

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.

Hepatocyte apoptosis is a hallmark of nonalcoholic steatohepatitis. We have previously observed that the saturated free fatty acids (FFAs) induce hepatocyte apoptosis in part via a death receptor 5 (DR5)-mediated signaling pathway. Cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) proteins are potent inhibitors of death receptor-mediated apoptosis. However, the role of the cIAPs in FFA-mediated hepatocyte apoptosis is unexplored. Our aim was to determine whether cIAPs are dysregulated during hepatocyte lipoapoptosis. cIAP proteins underwent rapid cellular elimination following treatment with the saturated FFAs palmitate (PA) and stearate. In contrast, PA did not decrease cIAP-1 and cIAP-2 mRNA expression in the cells. Degradation of cIAPs was dependent on their E3-ligase activity, suggesting that cIAPs undergo autoubiquitination that leads to proteasomal degradation. Huh-7 cells stably expressing shRNA targeting cIAP-1, but not cIAP-2, displayed enhanced sensitivity to PA-mediated apoptosis. Incubation with the SMAC mimetic JP1584, which induces rapid degradation of cIAPs, also enhanced PA-mediated apoptosis. Hepatocytes isolated from DR5 knockout mice exhibited reduced apoptosis following treatment with PA plus JP1584, implying that degradation of cIAPs sensitizes to DR5-mediated cell death pathways. A decrease of cIAP-1 was also observed in tissue from patients with nonalcoholic steatohepatitis compared with normal obese subjects. Collectively, these results implicate proteasomal degradation of cIAPs by FFA as a mechanism contributing to hepatocyte lipoapoptosis.

Abstract [Background and Aim] TRAIL is a promising therapeutic agent for human malignancies that can activate apoptosis via mitochondria-independent (Type I) or mitochodria-dependent (Type II) death receptor signaling. However, many malignant cells are resistant to TRAIL cytotoxicity and fail to trigger Type I or Type II killing. Using cholangiocarcinoma cells, we identified a role for Hedgehog signaling via GLI transcription factors in the regulation of TRAIL cytotoxicity, and provide new mechanistic insights whereby Hedghog inhibition increases apoptosis. [Materials and Methods] The human cholangiocarcinoma cell lines KMCH, HuCCT-1, and Mz-ChA-1 were employed. Hedgehog signaling was inhibited with cyclopamine or an shRNA construct targeting smootherned (SMO). Type II TRAIL signaling was inhibited with shRNA constructs for Bid or Bim and siRNA for Bax and Bak. Antiapoptotic mediators including Inhibitor of Apoptosis Proteins (IAPs) and Bcl-2 family proteins were examined by immunoblot analysis and/or real time RT-PCR. Apoptosis was quantified morphologically by DAPI and fluorescence microscopy, and biochemically by caspase-3/7 activity. GLI transcription factor binding to the XIAP promoter was examined by EMSA and chromatin immuneprecipitation (ChIP) assay. [Results] Pharmacologic or genetic inhibition of Hedgehog signaling down-regulates both XIAP and cIAP-1 mRNA and protein expression. Knockdown of XIAP, but not cIAP-1, by small-interfering RNA markedly sensitized cells to TRAIL-induced apoptosis, suggesting XIAP is the functional target. Putative GLI-binding sites were identified in the XIAP promoter, and EMSA and ChIP assays demonstrated direct binding by GLI proteins. Sensitization to TRAIL-induced apoptosis was not altered by knockdown of the BH3-only proteins Bim and Bid, or by knockdown of Bax plus Bak, implicating a mitochondria-independent Type I pathway of apoptosis. [Conclusion] These data provide evidence for up-regulation of XIAP by an oncogenic Hedgehog pathway and demonstrate that Hedgehog inhibition sensitizes cells to TRAIL via decreasing XIAP levels. Hedgehog inhibition thereby promotes Type I death receptor signaling and sensitizes cells to apoptosis, which suggests Hedgehog and XIAP inhibition may be a therapeutic approach for TRAIL-resistant neoplasms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4105. doi:10.1158/1538-7445.AM2011-4105

Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH 2 -terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.

The hepatocyte is especially vulnerable to injury due to its central role in xenobiotic metabolism including drugs and alcohol, participation in lipid and fatty acid metabolism, its unique role in the enterohepatic circulation of bile acids, the widespread prevalence of hepatotropic viruses, and its existence within a milieu of innate immune responding cells. Apoptosis and necrosis are the most widely recognized forms of hepatocyte cell death. The hepatocyte displays many unique features regarding cell death by apoptosis. It is quite susceptible to death receptor-mediated injury, and its death receptor signaling pathways involve the mitochondrial pathway for efficient cell killing. Also, death receptors can trigger lysosomal disruption in hepatocytes which further promote cell and tissue injury. Interestingly, hepatocytes are protected from cell death by only two anti-apoptotic proteins, Bcl-x L and Mcl-1, which have nonredundant functions. Endoplasmic reticulum stress or the unfolded protein response contributes to hepatocyte cell death during alterations of lipid and fatty acid metabolism. Finally, the current information implicating RIP kinases in necrosis provides an approach to more fully address this mode of cell death in hepatocyte injury. All of these processes contributing to hepatocyte injury are discussed in the context of potential therapeutic strategies.

ABSTRACT Death receptors are members of the tumor necrosis factor receptor superfamily characterized by a cytoplasmic region known as the “death domain” that enables the receptors to initiate cytotoxic signals when engaged by cognate ligands. Binding to the ligand results in receptor aggregation and recruitment of adaptor proteins, which, in turn, initiates a proteolytic cascade by recruiting and activating initiator caspases 8 and 10. Death receptors were once thought to primarily induce cytotoxic signaling cascades. However, recent data indicate that they initiate multiple signaling pathways, unveiling a number of nonapoptosis‐related functions, including regulation of cell proliferation and differentiation, chemokine production, inflammatory responses, and tumor‐promoting activities. These noncytotoxic cascades are not simply a manifestation of inhibiting proapoptotic pathways but are intrinsically regulated by adaptor protein and receptor internalization processes. Insights into these various death receptor signaling pathways provide new therapeutic strategies targeting these receptors in pathophysiological processes.—Guicciardi, M.E., Gores, G.J. Life and death by death receptors. FASEB J. 23, 1625–1637 (2009)

Although the ultimate outcome of prolonged exposure of cells to stress is often death, the early response appears to be the activation of survival pathways that are likely to give the cell an opportunity to repair low-level damage. How these stress-initiated survival pathways influence B cell lymphoma/leukemia 2 (Bcl-2) proteins, the core cell death machinery, has remained unclear; however, two papers now provide insight into stress-mediated survival mechanisms. The liver is unusually resistant to p53-mediated apoptosis. It appears that p53-mediated induction of the gene that encodes insulin-like growth factor–binding protein-1 (IGFBP1) attenuates the cell death response in hepatocytes by preventing the formation of a complex between p53 and the proapoptotic protein BAK. This is especially interesting as IGFBP1 is not a member of the Bcl-2 family, yet it inhibited BAK. In three unrelated cell lines, another regulatory interaction that influences cell survival occurs at the mitochondria. In this case, protein phosphatase 1γ (PP1γ) regulated the phosphorylation status of the Bcl-2/Bcl-X L –associated death promoter (BAD). The prefoldin family member URI is normally phosphorylated by S6 kinase 1, which liberates PP1γ from a URI-PP1γ complex. However, the withdrawal of growth factors or nutrients stabilizes this complex, which renders PP1γ inactive. The net response of this stress stimulus is an increased abundance of phosphorylated BAD, which raises the threshold required to trigger cell death. These two studies have identified new players and mechanisms that integrate stress responses and cell death.