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Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

Andrea Iorga¹, Katherine Donovan², Layla Shojaie²

¹Division of Gastrointestinal and Liver Diseases, Department of Medicine, Keck School of Medicine of University of Southern California, Los Angeles, California, USA; USC Research Center for Liver Disease, Keck School of Medicine of University of Southern California, Los Angeles, California, USA.

The Journal of Biological Chemistry

|January 18, 2021

Abstract:

Acetaminophen (APAP)-induced liver necrosis is a form of regulated cell death (RCD) in which APAP activates the mitogen-activated protein kinases (MAPKs) and specifically the c-Jun-N-terminal kinase (JNK) pathway, leading to necrotic cell death. Previously, we have shown that receptor interacting protein kinase-1 (RIPK1) knockdown is also protective against APAP RCD upstream of JNK. However, whether the kinase or platform function of RIPK1 is involved in APAP RCD is not known. To answer this question, we used genetic mouse models of targeted hepatocyte RIPK1 knockout (RIPK1^HepCKO) or kinase dead knock-in (RIPK1^D138N) and adult hepatocyte specific knockout of the cytoprotective protein A20 (A20^HepCKO), known to interact with RIPK1, to study its potential involvement in MAPK signaling. We observed no difference in injury between WT and RIPK^1D138N mice post APAP. However, RIPK1^HepCKO was protective. We found that RIPK1^HepCKO mice had attenuated pJNK activation, while A20 was simultaneously upregulated. Conversely, A20^HepCKO markedly worsened liver injury from APAP. Mechanistically, we observed a significant upregulation of apoptosis signal-regulating kinase 1 (ASK1) and increased JNK activation in A20^HepCKO mice compared with littermate controls. We also demonstrated that A20 coimmunoprecipitated (co-IP) with both RIPK1 and ASK1, and that in the presence of RIPK1, there was less A20-ASK1 association than in its absence. We conclude that the kinase-independent platform function of RIPK1 is involved in APAP toxicity. Adult RIPK1^HepCKO mice are protected against APAP by upregulating A20 and attenuating JNK signaling through ASK1, conversely, A20^HepCKO worsens injury from APAP.

Insights

Receptor interacting protein kinase-1 (RIPK1) acts as a platform, not a kinase, in acetaminophen-induced liver injury. Its absence protects against liver damage by upregulating A20 and reducing JNK signaling.

Area of Science:

Hepatology
Molecular Toxicology
Cell Death Research

Background:

Acetaminophen (APAP) overdose causes liver necrosis via regulated cell death (RCD) involving mitogen-activated protein kinases (MAPKs), particularly JNK.
Receptor interacting protein kinase-1 (RIPK1) knockdown protects against APAP-induced RCD upstream of JNK, but its specific function (kinase vs. platform) remains unclear.

Purpose of the Study:

To elucidate the role of RIPK1's kinase or platform function in APAP-induced liver injury.
To investigate the interplay between RIPK1, A20, and the JNK/ASK1 signaling axis in APAP toxicity.

Main Methods:

Utilized genetic mouse models: RIPK1 hepatocyte-specific knockout (RIPK1^HepCKO), RIPK1 kinase-dead knock-in (RIPK1^D138N), and A20 hepatocyte-specific knockout (A20^HepCKO).
Administered APAP to induce liver injury and assessed outcomes including pJNK activation, A20 and ASK1 levels, and liver damage.
Performed co-immunoprecipitation (co-IP) to analyze protein interactions (A20, RIPK1, ASK1).

Main Results:

RIPK1^D138N mice showed no difference in APAP-induced injury compared to wild-type (WT).
RIPK1^HepCKO mice were protected from APAP, exhibiting attenuated pJNK activation and increased A20 levels.
A20^HepCKO mice showed exacerbated liver injury, with upregulated apoptosis signal-regulating kinase 1 (ASK1) and increased JNK activation.
A20 co-immunoprecipitated with RIPK1 and ASK1, with RIPK1 presence reducing A20-ASK1 association.

Conclusions:

The kinase-independent platform function of RIPK1 is critical in APAP-induced liver toxicity.
RIPK1 mediates APAP toxicity by facilitating ASK1/JNK signaling, while A20 acts protectively.
Targeting RIPK1 or modulating A20 levels may offer therapeutic strategies for APAP overdose.

Read full analysis

Keywords:

RIPK1 RIPK3 TNFAIP3 cell death drug action drug induced liver injury hepatotoxicity necroptosis necrosis receptor interacting protein

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