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Isoform- and Cell Type–Specific Roles of Glycogen Synthase Kinase 3 N Isoform- and Cell Type−Specific Roles of Glycogen Synthase Kinase 3 N-Terminal Serine Phosphorylation in Liver Ischemia Reperfusion Injury This information is current as of September 25, 2021. Ming Ni, Haoming Zhou, Jing Zhang, Dan Jin, Tianfei Lu, Ronald W. Busuttil, Jerzy W. Kupiec-Weglinski, Xuehao Wang and Yuan Zhai J Immunol published online 17 July 2020 http://www.jimmunol.org/content/early/2020/07/16/jimmun Downloaded from ol.2000397 Supplementary http://www.jimmunol.org/content/suppl/2020/07/16/jimmunol.200039 http://www.jimmunol.org/ Material 7.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 25, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 17, 2020, doi:10.4049/jimmunol.2000397 The Journal of Immunology Isoform- and Cell Type–Specific Roles of Glycogen Synthase Kinase 3 N-Terminal Serine Phosphorylation in Liver Ischemia Reperfusion Injury Ming Ni,*,†,1 Haoming Zhou,*,†,1 Jing Zhang,* Dan Jin,*,‡ Tianfei Lu,*,x Ronald W. Busuttil,* Jerzy W. Kupiec-Weglinski,* Xuehao Wang,† and Yuan Zhai* Glycogen synthase kinase 3 (Gsk3) a and b are both constitutively active and inhibited upon stimulation by N-terminal serine phosphorylation. Although roles of active Gsk3 in liver ischemia reperfusion injury (IRI) have been well appreciated, whether Gsk3 N-terminal serine phosphorylation has any functional significance in the disease process remains unclear. In a murine liver partial warm ischemia model, we studied Gsk3 N-terminal serine mutant knock-in (KI) mice and showed that liver IRI was decreased in Gsk3aS21A but increased in Gsk3bS9A mutant KI mice. Bone marrow chimeric experiments revealed that the Downloaded from Gsk3a, but not b, mutation in liver parenchyma protected from IRI, and both mutations in bone marrow–derived cells exacer- bated liver injuries. Mechanistically, mutant Gsk3a protected hepatocytes from inflammatory (TNF-a) cell death by the activa- tion of HIV-1 TAT-interactive protein 60 (TIP60)–mediated autophagy pathway. The pharmacological inhibition of TIP60 or autophagy diminished the protection of the Gsk3a mutant hepatocytes from inflammatory cell death in vitro and the Gsk3a mutant KI mice from liver IRI in vivo. Thus, Gsk3 N-terminal serine phosphorylation inhibits liver innate immune activation but suppresses hepatocyte autophagy in response to inflammation. Gsk3 aS21, but not bS9, mutation is sufficient to sustain Gsk4 http://www.jimmunol.org/ activities in hepatocytes and protect livers from IRI via TIP60 activation. The Journal of Immunology, 2020, 205: 000–000. schemia reperfusion (IR) injury (IRI) remains one of the including Kupffer cells (KC)/macrophages, dendritic cells, and primary causes of liver dysfunction and failure after hepatic neutrophils (1, 2). These soluble mediators not only cause direct I tumor resection, liver transplantation, and trauma. Following hepatocellular damage but also recruit/activate more inflammatory the initial ischemic insult, reperfusion-triggered sterile inflam- cells from circulation into the tissue to amplify the immune re- mation is the major driving force of the disease pathogenesis. Host sponse and tissue damage. sentinel pattern recognition receptors are activated by danger- At the cellular level, TLR 4–mediated KC/macrophage activa- associated molecular pattern, such as high mobility group box 1, tion and TNF-a–mediated hepatocyte/endothelium death are by guest on September 25, 2021 derived from necrotic/stressed cells, resulting in the production critical in the pathophysiology of liver IRI (3–8). In the explora- of inflammatory cytokines/chemokines by innate immune cells, tion of regulatory mechanisms of these key processes, glycogen synthase kinase 3 (Gsk3) b has attracted much attention because *Dumont-UCLA Transplant Center, Division of Liver and Pancreas Transplantation, of its ubiquitous expression pattern and indispensable roles in Department of Surgery, David Geffen School of Medicine at University of California, cell metabolism, proliferation, differentiation, and death (9–12). † Los Angeles, Los Angeles, CA 90095; Department of Liver Surgery, First Affili- Gsk3b is a serine/threonine kinase and transduces signals down- ated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210008, China; ‡ a Department of Obstetrics and Gynecology, Shanghai Jiaotong University, Shanghai stream of both TLR4 and TNF- . Distinctive from MAPKs, 200025, China; and xLiver Surgery, Renji Hospital, Shanghai Jiaotong University, Gsk3b differentially regulates pro- and anti-inflammatory cyto- Shanghai 200025, China kine gene transcriptions (10). Gsk3b also determines the sensi- 1 M.N. and H.Z. contributed equally to this work. tivity of hepatocyte response to TNF-a that global Gsk3b ORCIDs: 0000-0003-1064-374X (M.N.); 0000-0001-8157-1531 (R.W.B.). knockout (KO) is lethal because of severe TNF-a toxicity in livers Received for publication April 10, 2020. Accepted for publication June 21, 2020. (13, 14). Additionally, Gsk3b has been found to regulate non– This work was supported by National Institutes of Health National Institute of receptor-mediated intrinsic cell death via the mitochondrial per- Allergy and Infectious Diseases Grant P01Al120944 (to Y.Z. and J.W.K.-W.), the meability transition pore (9, 15), which is critically involved in Dumont Research Foundation, and National Nature Science Foundation of China Grants 81600450, 81870448, and 3193002 (to H.Z. and X.W.). ischemia-induced cell death mechanism. a b Address correspondence and reprint requests to Dr. Xuehao Wang or Dr. Yuan Zhai, Gsk3 has two isoforms, and , which share extensive ho- Department of Liver Surgery, First Affiliated Hospital of Nanjing Medical University, mology in the kinase domain but are distinctive in their N- and 300 Guanzhou Road, Nanjing, People’s Republic of China (X.W.) or Dumont-UCLA C-terminals. The majority of Gsk3 research has been focused on Transplant Center, The David Geffen School of Medicine at University of California, b Los Angeles, 77-120 CHS, 10833 Le Conte Avenue, Los Angeles, CA 90095 (Y.Z.). the isoform using small molecule inhibitors. However, these E-mail addresses: [email protected] (X.W.) or [email protected] (Y.Z.) inhibitors actually do not discriminate between kinase domains of The online version of this article contains supplemental material. Gsk3 isoforms (16). Although Gsk3 isoforms have overlapping Abbreviations used in this article: EM, electron microscopy; ER, endoplasmic functions, isoform-specific properties have begun to be revealed in reticulum; Gsk3, glycogen synthase kinase 3; HIF, hypoxia-inducible factor; IR, recent studies. Whereas global Gsk3b KO mice are embryonic ischemia reperfusion; IRI, IR injury; KC, Kupffer cell; KI, knock-in; KI-WT, WT a mice that received mutant KI; KO, knockout; LDH, lactate dehydrogenase; 3-MA, lethal (13), Gsk3 KO mice are viable and “normal” (17). It has 3-methyladenine; NPC, nonparenchymal cell; sALT, serum alanine aminotransfer- been shown in myocardial disease models that Gsk3 a and b ase; Tg, thapsigargin; TIP60, HIV-1 TAT-interactive protein 60; WT, wild type; played distinctive or even opposite functions (18–22). WT-KI, mutant KI mice that received WT; WT-WT, WT mice that received WT. Gsk3 is a unique intracellular signaling kinase that is consti- Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 tutively active in resting cells and inhibited by phosphorylation in www.jimmunol.org/cgi/doi/10.4049/jimmunol.2000397 2 ACTIVE Gsk3a PROMOTES HEPATOCYTE AUTOPHAGY response to various stimuli. The N-terminal phosphorylation of isoflurane and placed in a designed warm container (HTP-1500 Heat serine residues of Gsk3 a at Ser21 and b at Ser9 are the major Therapy Pump; Adroit Medical Systems, Loudon, TN) to keep the tem- mechanism of Gsk3 inactivation, which are induced in hearts and perature at 29˚C. After 90 min of partial hepatic warm ischemia, the clip was removed, initiating liver reperfusion. Mice were sacrificed after 1, 6, livers by ischemia reperfusion (IR) (23, 24). Multiple intracellular or 24 h postreperfusion. Sham controls underwent the same procedure but signaling pathways can act on the serine site of Gsk3s. Among without vascular occlusion. HIV-1 TAT-interactive protein 60 (TIP60) in- them, the PI3K–Akt pathway triggered by growth factors/insulin hibitor TH1834 (50 mg/kg; Axon Medchem, Reston, VA) or autophagy and inflammation and the AMPK–mTOR–S6K pathway induced inhibitor 3-methyladenine (3-MA, 30 mg/kg; Sigma, St. Louis, MO) or PBS (vehicle control) was administered i.p. 1 h prior to the onset of liver by nutrient/energy alterations are highly relevant to organ IRI. We ischemia. Serum alanine aminotransferase (sALT) levels were measured have shown in our previous
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