D. Lambertz

Loss of Caspase-8 Protects Mice Against Inflammation-Related Hepatocarcinogenesis but Induces Non-Apoptotic Liver Injury

BACKGROUND & AIMS Disruption of the nuclear factor-κB (NF-κB) essential modulator (NEMO) in hepatocytes of mice (NEMO(Δhepa) mice) results in spontaneous liver apoptosis and chronic liver disease involving inflammation, steatosis, fibrosis, and development of hepatocellular carcinoma. Activation of caspase-8 (Casp8) initiates death receptor-mediated apoptosis. We investigated the pathogenic role of this protease in NEMO(Δhepa) mice or after induction of acute liver injury. METHODS We created mice with conditional deletion of Casp8 in hepatocytes (Casp8(Δhepa)) and Casp8(Δhepa)NEMO(Δhepa) double knockout mice. Acute liver injury was induced by Fas-activating antibodies, lipopolysaccharides, or concanavalin A. Spontaneous hepatocarcinogenesis was monitored by magnetic resonance imaging. RESULTS Hepatocyte-specific deletion of Casp8 protected mice from induction of apoptosis and liver injury by Fas or lipopolysaccharides but increased necrotic damage and reduced survival times of mice given concanavalin A. Casp8(Δhepa)NEMO(Δhepa) mice were protected against steatosis and hepatocarcinogenesis but had a separate, spontaneous phenotype that included massive liver necrosis, cholestasis, and biliary lesions. The common mechanism by which inactivation of Casp8 induces liver necrosis in both injury models involves the formation of protein complexes that included the adaptor protein Fas-associated protein with death domain and the kinases receptor-interacting protein (RIP) 1 and RIP3-these have been shown to be required for programmed necrosis. We demonstrated that hepatic RIP1 was proteolytically cleaved by Casp8, whereas Casp8 inhibition resulted in accumulation of RIP complexes and subsequent liver necrosis. CONCLUSIONS Inhibition of Casp8 protects mice from hepatocarcinogenesis following chronic liver injury mediated by apoptosis of hepatocytes but can activate RIP-mediated necrosis in an inflammatory environment.

Loss of caspase-8 in hepatocytes accelerates the onset of liver regeneration in mice through premature NF-?B activation

The cytokine tumor necrosis factor alpha (TNF‐α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF‐κB) activation, apoptosis signaling by way of caspase‐8 (Casp8), and activation of cJun N‐terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF‐dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor‐interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF‐κB and JNK/cJun related signals. In order to define the role of NF‐κB in this setting we blocked NF‐κB activation in Casp8Δhepa mice by concomitant inactivation of the NF‐κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF‐κB, and JNK activation. While loss of Casp8 triggers NF‐κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

Loss of caspase‐8 in hepatocytes accelerates the onset of liver regeneration in mice through premature nuclear factor kappa B activation

The cytokine tumor necrosis factor alpha (TNF‐α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF‐κB) activation, apoptosis signaling by way of caspase‐8 (Casp8), and activation of cJun N‐terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF‐dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor‐interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF‐κB and JNK/cJun related signals. In order to define the role of NF‐κB in this setting we blocked NF‐κB activation in Casp8Δhepa mice by concomitant inactivation of the NF‐κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF‐κB, and JNK activation. While loss of Casp8 triggers NF‐κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)

Molecular mechanism of Mitomycin C-dependent caspase-8 regulation: implications for apoptosis and synergism with interferon-α signalling

Caspase-8 is frequently mutated or silenced in several tumors including hepatocellular carcinomas (HCC) thereby potentially contributing to chemoresistance. The aim of our present study was to evaluate if chemotherapeutic drugs may mediate their effects through up-regulation of caspase-8 gene transcription. Huh7 hepatoma cells were transfected with a caspase-8 promoter construct fused to a luciferase reporter gene followed by stimulation with a subset of different chemotherapeutic drugs. Several drugs slightly induced caspase-8 promoter activity. However, strong caspase-8 promoter induction was found after Mitomycin C (MMC) treatment and this correlated with an increase in endogenous caspase-8 mRNA expression. Further molecular analysis demonstrated that MMC controls caspase-8 transcription via a c-jun/AP1 site located in the promoter in close proximity to the transcription start site. Inactivation of this c-jun/AP1 site using a dominant-negative c-jun adenovirus or site-directed mutagenesis inhibited MMC-dependent promoter induction. MMC treatment resulted in higher caspase-8 enzymatic activity and apoptosis and could be synergistically enhanced by co-stimulation with interferon-α (IFNα) via independent transcriptional mechanisms. In summary MMC controls caspase-8 expression via a c-jun/AP1 element in its promoter region. MMC-induced up-regulation of caspase-8 triggers apoptosis in target cells which can be further enhanced by IFNα. Therefore these findings also provide a potential new therapeutic approach to treat cancer cells.

Targeting CCl4-induced liver fibrosis by RNA interference - mediated inhibition of Cyclin E1 in mice

Initiation and progression of liver fibrosis requires proliferation and activation of resting hepatic stellate cells (HSCs). Cyclin E1 (CcnE1) is the regulatory subunit of the cyclin‐dependent kinase 2 (Cdk2) and controls cell cycle re‐entry. We have recently shown that genetic inactivation of CcnE1 prevents activation, proliferation, and survival of HSCs and protects from liver fibrogenesis. The aim of the present study was to translate these findings into preclinical applications using an RNA interference (RNAi)‐based approach. CcnE1‐siRNA (small interfering RNA) efficiently inhibited CcnE1 gene expression in murine and human HSC cell lines and in primary HSCs, resulting in diminished proliferation and increased cell death. In C57BL/6 wild‐type (WT) mice, delivery of stabilized siRNA using a liposome‐based carrier targeted approximately 95% of HSCs, 70% of hepatocytes, and 40% of CD45+ cells after single injection. Acute CCl4‐mediated liver injury in WT mice induced endogenous CcnE1 expression and proliferation of surviving hepatocytes and nonparenchymal cells, including CD45+ leukocytes. Pretreatment with CcnE1‐siRNA reverted CcnE1 induction to baseline levels of healthy mice, which was associated with reduced liver injury, diminished proliferation of hepatocytes and leukocytes, and attenuated overall inflammatory response. For induction of liver fibrosis, WT mice were challenged with CCl4 for 4‐6 weeks. Co‐treatment with CcnE1‐siRNA once a week was sufficient to continuously block CcnE1 expression and cell‐cycle activity of hepatocytes and nonparenchymal cells, resulting in significantly ameliorated liver fibrosis and inflammation. Importantly, CcnE1‐siRNA also prevented progression of liver fibrosis if applied after onset of chronic liver injury. Conclusion: Therapeutic targeting of CcnE1 in vivo using RNAi is feasible and has high antifibrotic activity. (Hepatology 2017;66:1242‐1257).

Inhibition of Caspase-8 does not protect from alcohol-induced liver apoptosis but alleviates alcoholic hepatic steatosis in mice

Hepatic apoptosis is involved in the progression of alcoholic liver disease (ALD). Caspase-8, the apical initiator in death receptor-mediated apoptosis, has been implicated in acute liver injury and in non-alcoholic steatohepatitis. However, the relevance of Caspase-8 in the pathogenesis of ALD remains unclear. In the present study, we investigated the impact of Caspase-8 in human and murine alcohol-induced apoptosis and in ALD. We investigated human samples from ALD patients, primary mouse hepatocytes, and hepatocyte-specific Caspase-8 knockout (Casp8Δhepa) mice in acute and chronic models of ethanol (EtOH) administration. Caspase-8 activation was detected in liver biopsies from ALD patients, as well as in livers of wild-type (WT) mice after chronic ethanol feeding for 8 weeks using the Lieber-DeCarli model. Lack of Caspase-8 expression in Casp8Δhepa animals failed to prevent alcohol-induced liver damage and apoptosis. Instead, inhibition of Caspase-8 shifted the ethanol-induced death signals towards pronounced activation of the intrinsic, mitochondria-dependent apoptosis pathway in Casp8Δhepa livers involving enhanced release of cytochrome c, stronger Caspase-9 activation and specific morphological changes of mitochondria. In vitro and in vivo intervention using a pan-caspase inhibitor markedly attenuated alcohol-induced hepatocyte damage in a Caspase-8-independent manner. Surprisingly, EtOH-fed Casp8Δhepa mice displayed significantly attenuated steatosis and reduced hepatic triglyceride and free fatty acids content. Caspase-8 is dispensable for alcohol-induced apoptosis, but plays an unexpected role for alcohol-dependent fat metabolism. We provide evidence that simultaneous inhibition of extrinsic and intrinsic apoptosis signaling using pan-caspase inhibitors in vivo might be an optimal approach to treat alcohol-induced liver injury.