RIP3 and MLKL regulate ER stress in alcohol-associated liver disease and pharmacological ER stress models: Insights beyond necroptosis.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is an important contributor to liver disease progression, including alcohol-associated liver disease. Although receptor-interacting protein kinase-3 (RIP3) and mixed lineage kinase domain-like pseudokinase (MLKL) are known for their roles in necroptosis, emerging evidence highlights their non-canonical functions in metabolic regulation and cellular-stress responses. However, their specific role in regulating hepatic ER stress remains unclear. This study investigates how RIP3 and MLKL regulate ER stress pathways during chronic ethanol exposure and pharmacological ER stress induction.

METHODS: Rip3 -/- , Rip3 K51A/K51A , and Mlkl -/- mice alongside wild-type (WT) and pharmacological necroptosis inhibitors were used to study the role of RIP3 and MLKL in modulating ER stress. Chronic ethanol feeding and pharmacological agents were utilized to induce ER stress in vivo and in isolated primary hepatocytes. ER stress markers were assessed by qPCR and Western blot, ER expansion by confocal microscopy, and viability by MTS assay. (n = 8-11 for ethanol; n = 5-8 for tunicamycin; n = 3-12 for hepatocyte experiments).

RESULTS: Chronic ethanol increased expression of ER stress markers in WT mice; this response was attenuated in Rip3 -/- mice (p <0.05). Tunicamycin exposure increased hepatic ER stress markers in WT mice; this response was diminished in Rip3 -/- , Rip3 K51A/K51A, and Mlkl -/- mice (p <0.05). In primary hepatocytes, genetic and pharmacological inhibition of RIP3 and MLKL also reduced thapsigargin-induced ER stress responses (p <0.05). Hepatocytes lacking Rip3, RIP3 kinase activity, or Mlkl showed enhanced viability (p <0.05) and greater ER expansion and sheet formation compared to WT hepatocytes (p <0.05), suggesting improved adaptive remodeling.

CONCLUSIONS: This study highlights a novel function of RIP3 and MLKL in regulating hepatic ER stress responses, expanding their known roles beyond programmed necrosis.

IMPACT AND IMPLICATIONS: This study provides new mechanistic insight into how RIP3 and MLKL regulate hepatic ER stress responses, extending their roles beyond necroptosis. By demonstrating that genetic or pharmacological inhibition of Rip3, RIP3 kinase activity and Mlkl attenuates ER stress signaling, reduces cell death, and promotes adaptive ER remodeling, our findings identify these proteins as key modulators of hepatocyte survival under stress. These results are important for researchers and clinicians focused on alcohol-associated liver disease and other ER stress-driven liver disorders, as they highlight novel therapeutic targets. In practical terms, modulation of the RIP3-MLKL axis could inform the development of interventions aimed at enhancing ER stress resilience, with potential applications in drug development for ER stress-associated liver injury.