Knockdown of RIPK1 Markedly Exacerbates Murine Immune-Mediated Liver Injury through Massive Apoptosis of Hepatocytes, Independent of Necroptosis and Inhibition of NF-κB.
Receptor-interacting protein kinase (RIPK)1 has an essential role in the signaling pathways triggered by death receptors through activation of NF-κB and regulation of caspase-dependent apoptosis and RIPK3/mixed lineage kinase domain-like protein (MLKL)-mediated necroptosis. We examined the effect of RIPK1 antisense knockdown on immune-mediated liver injury in C57BL/6 mice caused by α-galactosylceramide (αGalCer), a specific activator for invariant NKT cells. We found that knockdown of RIPK1 markedly exacerbated αGalCer-mediated liver injury and induced lethality. This was associated with increased hepatic inflammation and massive apoptotic death of hepatocytes, as indicated by TUNEL staining and caspase-3 activation. Pretreatment with zVAD.fmk, a pan-caspase inhibitor, or neutralizing Abs against TNF, almost completely protected against the exacerbated liver injury and lethality. Primary hepatocytes isolated from RIPK1-knockdown mice were sensitized to TNF-induced cell death that was completely inhibited by adding zVAD.fmk. The exacerbated liver injury was not due to impaired hepatic NF-κB activation in terms of IκBα phosphorylation and degradation in in vivo and in vitro studies. Lack of RIPK1 kinase activity by pretreatment with necrostatin-1, a RIPK1 kinase inhibitor, or in the RIPK1 kinase-dead knock-in (RIPK1D138N) mice did not exacerbate αGalCer-mediated liver injury. Furthermore, RIPK3-knockout and MLKL-knockout mice behaved similarly as wild-type control mice in response to αGalCer, with or without knockdown of RIPK1, excluding a switch to RIPK3/MLKL-mediated necroptosis. Our findings reveal a critical kinase-independent platform role for RIPK1 in protecting against TNF/caspase-dependent apoptosis of hepatocytes in immune-mediated liver injury.
Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033.
Jo
J
Suda
Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033.
Lily
0000-0002-0121-7186
L
Dara
Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033.|Department of Gastroenterology, Bethune First Hospital of Jilin University, Changchuan 130021, China.
Luoluo
0000-0001-8099-9982
L
Yang
Ionis Pharmaceuticals, Carlsbad, CA 92008; and.
Mariam
M
Aghajan
YSL Bioprocess Development Co., Pomona, CA 91767.
Yong
Y
Song
Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033.
Neil
0000-0002-9424-393X
N
Kaplowitz
Research Center for Liver Diseases, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033; zxliu@usc.edu.
Zhang-Xu
0000-0001-7290-3506
ZX
Liu
D000818:Animals; D017209:Apoptosis; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000276:immunology*; D022781:Hepatocytes / Q000502:physiology*; D007093:Imidazoles / Q000008:administration & dosage; D007211:Indoles / Q000008:administration & dosage; D008070:Lipopolysaccharides / Q000276:immunology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D016328:NF-kappa B / Q000378:metabolism; D009336:Necrosis; D016376:Oligonucleotides, Antisense / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D007093:Imidazoles; D007211:Indoles; D008070:Lipopolysaccharides; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; C507699:necrostatin-1; C570694:MLKL protein, mouse; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; C485615:Ripk3 protein, mouse