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Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury
Sanjoy Roychowdhury1, Megan R McMullen, Sorana G Pisano
1Departments of Pathobiology, Center for Liver Disease Research, Cleveland Clinic, Cleveland, OH, USA. roychos@ccf.org
Unlabelled:
Hepatocyte cell death via apoptosis and necrosis are major hallmarks of ethanol-induced liver injury. However, inhibition of apoptosis is not sufficient to prevent ethanol-induced hepatocyte injury or inflammation. Because receptor-interacting protein kinase (RIP) 3-mediated necroptosis, a nonapoptotic cell death pathway, is implicated in a variety of pathological conditions, we tested the hypothesis that ethanol-induced liver injury is RIP3-dependent and RIP1-independent. Increased expression of RIP3 was detected in livers of mice after chronic ethanol feeding, as well as in liver biopsies from patients with alcoholic liver disease. Chronic ethanol feeding failed to induce RIP3 in the livers of cytochrome P450 2E1 (CYP2E1)-deficient mice, indicating CYP2E1-mediated ethanol metabolism is critical for RIP3 expression in response to ethanol feeding. Mice lacking RIP3 were protected from ethanol-induced steatosis, hepatocyte injury, and expression of proinflammatory cytokines. In contrast, RIP1 expression in mouse liver remained unchanged following ethanol feeding, and inhibition of RIP1 kinase by necrostatin-1 did not attenuate ethanol-induced hepatocyte injury. Ethanol-induced apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive nuclei and accumulation of cytokeratin-18 fragments in the liver, was independent of RIP3.
Conclusion:
CYP2E1-dependent RIP3 expression induces hepatocyte necroptosis during ethanol feeding. Ethanol-induced hepatocyte injury is RIP3-dependent, but independent of RIP1 kinase activity; intervention of this pathway could be targeted as a potential therapeutic strategy.
Insights
Ethanol-induced liver injury involves RIP3-dependent necroptosis, not apoptosis. Cytochrome P450 2E1 (CYP2E1) drives this RIP3 expression, offering a potential therapeutic target for alcoholic liver disease.
Area of Science:
- Hepatology
- Cell Death Pathways
- Molecular Mechanisms of Liver Injury
Background:
- Hepatocyte cell death, including apoptosis and necrosis, is central to alcoholic liver disease.
- Inhibiting apoptosis alone is insufficient to prevent ethanol-induced liver injury and inflammation.
- Receptor-interacting protein kinase 3 (RIP3)-mediated necroptosis is a key non-apoptotic cell death pathway in various pathologies.
Purpose of the Study:
- To investigate if ethanol-induced liver injury is dependent on RIP3 and independent of RIP1.
- To determine the role of cytochrome P450 2E1 (CYP2E1) in ethanol-induced RIP3 expression.
- To explore RIP3-mediated necroptosis as a potential therapeutic target in alcoholic liver disease.
Main Methods:
- Assessed RIP3 and RIP1 expression in mouse livers after chronic ethanol feeding.
- Utilized CYP2E1-deficient mice to evaluate the role of CYP2E1 in RIP3 induction.
- Administered necrostatin-1 (RIP1 inhibitor) to assess its effect on ethanol-induced liver injury.
- Quantified apoptosis markers (TUNEL staining, keratin-18 fragments) to differentiate cell death pathways.
Main Results:
- Increased RIP3 expression was observed in mouse livers and human alcoholic liver disease biopsies.
- Chronic ethanol feeding failed to induce RIP3 in CYP2E1-deficient mice, highlighting CYP2E1's critical role.
- Mice lacking RIP3 showed protection against ethanol-induced steatosis, injury, and inflammation.
- RIP1 expression remained unchanged, and RIP1 inhibition did not prevent ethanol-induced hepatocyte injury.
- Ethanol-induced apoptosis was found to be RIP3-independent.
Conclusions:
- Ethanol metabolism via CYP2E1 drives RIP3 expression, inducing hepatocyte necroptosis.
- Ethanol-induced liver injury is critically dependent on RIP3 but not on RIP1 kinase activity.
- Targeting the RIP3-mediated necroptosis pathway presents a promising therapeutic strategy for alcoholic liver disease.
