Glycogen Synthase Kinase-3β Facilitates IFN- γ-Induced STAT1 Activation by Regulating Src Homology-2 Domain-Containing Phosphatase 2 This information is current as of September 28, 2021. Cheng-Chieh Tsai, Jui-In Kai, Wei-Ching Huang, Chi-Yun Wang, Yi Wang, Chia-Ling Chen, Yi-Ting Fang, Yee-Shin Lin, Robert Anderson, Shun-Hua Chen, Chiung-Wen Tsao and Chiou-Feng Lin J Immunol published online 19 June 2009 Downloaded from http://www.jimmunol.org/content/early/2009/06/19/jimmuno l.0804033 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2009/06/19/jimmunol.080403 Material 3.DC1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published June 19, 2009, doi:10.4049/jimmunol.0804033 The Journal of Immunology Glycogen Synthase Kinase-3␤ Facilitates IFN-␥-Induced STAT1 Activation by Regulating Src Homology-2 Domain-Containing Phosphatase 21 Cheng-Chieh Tsai,*†‡ Jui-In Kai,†§ Wei-Ching Huang,*†§ Chi-Yun Wang,*† Yi Wang,†§ Chia-Ling Chen,§ Yi-Ting Fang,*§ Yee-Shin Lin,*§¶ Robert Anderson,ʈ Shun-Hua Chen,*§ Chiung-Wen Tsao,‡ and Chiou-Feng Lin2*†‡§ Glycogen synthase kinase-3␤ (GSK-3␤)-modulated IFN-␥-induced inflammation has been reported; however, the mechanism that activates GSK-3␤ and the effects of activation remain unclear. Inhibiting GSK-3␤ decreased IFN-␥-induced inflammation. IFN-␥ treatment rapidly activated GSK-3␤ via neutral sphingomyelinase- and okadaic acid-sensitive phosphatase-regulated dephos- phorylation at Ser9, and proline-rich tyrosine kinase 2 (Pyk2)-regulated phosphorylation at Tyr216. Pyk2 was activated through Downloaded from phosphatidylcholine-specific phospholipase C (PC-PLC)-, protein kinase C (PKC)-, and Src-regulated pathways. The activation of PC-PLC, Pyk2, and GSK-3␤ was potentially regulated by IFN-␥ receptor 2-associated Jak2, but it was independent of IFN-␥ receptor 1. Furthermore, Jak2/PC-PLC/PKC/cytosolic phospholipase A2 positively regulated neutral sphingomyelinase. Inhibiting GSK-3␤ activated Src homology-2 domain-containing phosphatase 2 (SHP2), thereby preventing STAT1 activation in the late stage of IFN-␥ stimulation. All these results showed that activated GSK-3␤ synergistically affected IFN-␥-induced STAT1 acti- vation by inhibiting SHP2. The Journal of Immunology, 2009, 183: 0000–0000. http://www.jimmunol.org/ lycogen synthase kinase-3␤ (GSK-3␤),3 a serine/threo- and presentation, microbial killing, and proinflammatory cytokine nine kinase, regulates cellular inflammation (1–4). In- production (15, 16). IFN-␥ activates Jak2-STAT1, which then reg- G hibiting GSK-3␤ protects cells from inflammatory stimuli, ulates IFN-␥-inducible gene expression (15, 17, 18). The IFN-␥ including TNF-␣ (5), endotoxemia (6), experimental colitis, type II receptor (IFNGR) consists of IFNGR1 and IFNGR2, which inter- collagen-induced arthritis (7), OVA-induced asthma (8), experimental act with Jak1 and Jak2, respectively (15, 17, 18). IFN-␥ binding autoimmune encephalomyelitis (9), and bacterial infection (10). No- induces Jak2 autophosphorylation and activation, which leads to tably, recent studies have reported that GSK-3␤ is a potent regulator Jak1 transphosphorylation. Activated Jak1 then phosphorylates by guest on September 28, 2021 of TLR- (11, 12) and IFN-␥-mediated inflammation (13, 14). How- IFNGR1, creating a docking site for STAT1, which, mediated by ever, the molecular targets of GSK-3␤ and the mechanism of GSK-3␤ Jak2, then phosphorylates tyrosine residue 701 (Tyr701) (15, 17, activation after IFN-␥ stimulation remain unclear. 18). Subsequently, IFN-␥-activated MAPKs, which include IFN-␥, an immune IFN produced by T cells and NK cells, is a ERK1/2 and p38 MAPK, cause serine phosphorylation of STAT1 potent macrophage-activating factor that promotes Ag processing (Ser727). STAT1 phosphorylation (Tyr701 and Ser727) is essential for its dimer formation, nuclear translocation, and DNA binding stability (19, 20). It is also essential for STAT1 to attain maximal *Institute of Basic Medical Sciences and †Institute of Clinical Medicine, National ␥ Cheng Kung University Medical College, Tainan, Taiwan; ‡Department of Nursing, capacity and initiate or suppress the transcription of IFN- -induc- Chung Hwa University of Medical Technology, Tainan, Taiwan; §Department of ible genes (15, 19, 20). For feedback regulation, it is now known Microbiology and Immunology, National Cheng Kung University Medical College, that suppressor of cytokine signaling (SOCS) proteins SOCS1 and Tainan, Taiwan; ¶Center for Gene Regulation and Signal Transduction Research, National Cheng Kung University, Tainan, Taiwan; and ʈDepartment of Microbiology SOCS3 interact with Jak2 and inhibit its catalytic activity to sup- and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada press IFN-␥ signaling (21, 22). In addition to SOCS, dual-phos- Received for publication December 3, 2008. Accepted for publication May 8, 2009. phatase Src homology-2 domain-containing phosphatase 2 (SHP2) The costs of publication of this article were defrayed in part by the payment of page deactivates STAT1 (23). However, the mechanisms for feedback charges. This article must therefore be hereby marked advertisement in accordance regulation are not well understood. with 18 U.S.C. Section 1734 solely to indicate this fact. IFN-␥ causes the production of TNF-␣, IFN-inducible protein 1 This work was supported by Grant NSC 96-2320-B-006-018-MY3 from the Na- tional Science Council, Taiwan, and the Landmark Project C020 of National Cheng 10, MCP-1, and RANTES and the expression of adhesion mole- Kung University, Taiwan. cule ICAM-1, but it decreases the production of IL-10 (15, 24, 25). 2 Address correspondence and reprint requests to Dr. Chiou-Feng Lin, Institute of IFN-␥ also induces inducible NO synthase (iNOS) expression and Clinical Medicine, College of Medicine, National Cheng Kung University, 1 Univer- then NO generation (26, 27). These proinflammatory responses by sity Road, Tainan 701, Taiwan. E-mail address: cfl[email protected] IFN-␥ are activated through a mechanism involving the activation 3 Abbreviations used in this paper: GSK-3␤, glycogen synthase kinase-3␤; IFNGR, IFN-␥ receptor; SOCS, suppressor of cytokine signaling; SHP2, Src homology-2 of transcription factors: STAT1 (28), IFN regulatory factor 1 domain-containing phosphatase 2; iNOS, inducible NO synthase; IRF-1, IFN regu- (IRF-1) (29), and NF-␬B (30). The recent finding that GSK-3␤ is latory factor 1; OA, okadaic acid; PPase, protein phosphatase; SMase, sphingomy- involved in the IFN-␥-induced production of TNF-␣ and inhibition elinase; cPLA2, cytosolic phospholipase A2; AA, arachidonic acid; DAG, diacylglyc- erol; Pyk2, proline-rich tyrosine kinase 2; PKC, protein kinase C; PC-PLC, of IL-10 (13, 31) suggests a novel role for GSK-3␤ in IFN-␥ sig- phosphatidylcholine-specific phospholipase C; WT, wild type; BIO, 6-bromo-indiru- naling. GSK-3␤ is also involved in IFN-␥-activated STAT3 and bin-3Ј-oxime; siRNA, short interference RNA; RNAi, RNA interference. STAT5 (14). In general, serine phosphorylation (Ser9) negatively Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 regulates GSK-3␤ primarily through PI3K-Akt pathways (1–4, 32, www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804033 2 GSK-3␤ FACILITATES IFN-␥ SIGNALING 33). Okadaic acid (OA)-sensitive serine/threonine protein phos- nylmethyl)-2-propenamide (AG490), PP1, and 8-hydroxy-7-(6-sulfonaph- phatases (PPases) such as PP1 and PP2A may concomitantly de- thalen-2-yl)diazenyl-quinoline-5-sulfonic acid (NSC-87877) were pur- phosphorylate and activate GSK-3␤ directly or indirectly by de- chased from Tocris Bioscience. Sphingolactone-24 (Sph-24) was from ␥ Alexis Biochemicals. U73122, Go¨6976, and calphostin C (Cal C) were phosphorylating Akt (34–40). IFN- -induced iNOS expression purchased from Calbiochem. 3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-in- requires the activity of PP1 and PP2A (41). Ceramide activates dol-3-yl)-1H-pyrrole-2,5-dione (SB216763), 6-bromo-indirubin-3Ј-oxime PP1 and PP2A (42–46) and is also involved in activating GSK-3␤ (BIO), OA, 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (tyrphos- (37, 39, 40). Ceramide is a bioactive lipid generated in response to tin A9), and tricyclodecan-9-yl-xanthogenate (D609) were from Sigma- Aldrich. All drug treatments in cells were assessed for their cytotoxic ef- various stresses, such as apoptotic and inflammatory stimuli, by de fects using cytotoxicity and viability assays. Doses determined to be novo synthesis or sphingomyelinase (SMase)-mediated hydrolysis harmless were used. of sphingomyelin (39, 46). Intracellular levels of ceramide and Cell culture iNOS expression correlatively increase after LPS and IFN-␥ treat- ment