Phosphatase 2 Src Homology-2 Domain-Containing -Induced STAT1 Activation by Regulating Γ Facilitates IFN- Β Glycogen Synthase

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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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 © 2009 American Physical Therapy Association All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 dephosphorylation 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 activation 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, diacylglycerol; 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-␥ treatment (47). After IFN-␥ treatment, neutral SMase- but not acid RAW264.7 and MHS murine macrophages and A549 human lung epithelial SMase-mediated ceramide generation is critical for iNOS/NO bio- cells were obtained from Dr. C. C. Huang (Department of Pediatrics, National synthesis (48, 49). Cytosolic phospholipase A (cPLA2), a phos- Cheng Kung University Hospital, Tainan, Taiwan) and Dr. W. C. Su (Depart- 2 ment of Oncology, National Cheng Kung University Hospital, Tainan, Tai- pholipase that liberates arachidonic acid (AA) from phospholipid wan), respectively. The cells were grown in DMEM (Invitrogen) supple- diacylglycerol (DAG), is essential for activating neutral SMase in mented with 10% heat-inactivated FBS (Invitrogen), 50 U/ml penicillin, and ␮ cytokine signaling (50, 51). Thus, the role of cPLA2/neutral 50 g/ml streptomycin in a humidiﬁed atmosphere with 5% CO2 and 95% air. SMase/ceramide/PPase/GSK-3␤ cascade activation in IFN-␥ sig- For primary splenocyte culture, the mice were i.p. injected with a lethal dose of pentobarbital (200 mg/kg) and their spleens were harvested to prepare a Downloaded from naling requires further investigation. single-cell suspension. Isolated splenocytes were grown in RPMI 1640 (In- 9 Although serine phosphorylation (Ser ) negatively regulates vitrogen) supplemented with 10% heat-inactivated FBS, 50 U/ml penicillin, GSK-3␤, tyrosine phosphorylation (Tyr216) positively regulates and 50 ␮g/ml streptomycin before the experiment. ␤ the catalytic activity of GSK-3 . It is generally regulated either by Cytotoxicity assay tyrosine kinases, such as proline-rich tyrosine kinase 2 (Pyk2), MEK, and Src-like kinase (1–4, 52), or by autophosphorylation We used a colorimetric assay (cytotoxicity detection kit; Roche Diagnos- tics), according to the manufacturer’s instructions, to measure lactate de-

␥ http://www.jimmunol.org/ (53). We have previously (31) shown that IFN- induces phos- ␥ 216 hydrogenase activity, an indicator of cell damage caused by IFN- and all phorylation of GSK-3␤ (Tyr ) via a Pyk2-mediated pathway. inhibitors. Aliquots of the incubation medium were transferred to 96-well Furthermore, Pyk2 is also essential for IFN-␥-induced MAPK ac- microplates and the absorbance was measured using a microplate reader tivation and inflammation (54, 55). Pyk2 is activated by protein (SpectraMax 340PC; Molecular Devices). kinase C (PKC) and Src (56–58) as well as by calcium/calmodu- RT-PCR lin-dependent protein kinase II (59). While Jak2-STAT1 is essential for IFN-␥ signaling, bioactive lipids and their enzymatic gen- We assessed mRNA expression using RT-PCR. Using a reagent (TRIzol; Invitrogen) according to the manufacturer’s instructions, we extracted total erators are also involved (50, 60). Phosphatidylcholine-specific cellular RNA from cells. We quantified RNA concentrations using a spec- phospholipase C (PC-PLC)-mediated DAG generation is required trophotometer (U-2000; Hitachi) at 260 nm. cDNA was prepared using for IFN-␥-induced iNOS/NO biosynthesis (61). Moreover, DAG reverse transcription, and PCR was done using a thermal cycler (GeneAmp by guest on September 28, 2021 directly activates PKC (60, 61) and then activates Src (62). PKC PCR system 2400; PerkinElmer). Based on published sequences (64) and sequences that we designed using Primer3 online software (65), we used regulates neutral SMase by increasing the generation of AA after the following oligonucleotide primers: mouse iNOS, sense, 5Ј-CCCT it has activated cPLA2 (63). We show that activated GSK-3␤ syn- TCCGAAGTTTCTGGCAGCAGCG-3Ј and antisense, 5Ј-GGCTGTCA ergistically facilitates IFN-␥-induced STAT1 activation, which GAGCCTCGTGGCTTTGG-3Ј; TNF-␣, sense, 5Ј-AGCCCACGTCGT then regulates the biosynthesis of iNOS/NO and the production of AGCAAACCACCAA-3Ј and antisense, 5Ј-ACACCCATTCCCTTCA Ј Ј TNF-␣ and RANTES. CAGAGCAAT-3 ; RANTES, sense, 5 -ATATGGCTCGGACACCAC TC-3Ј and antisense, 5Ј-CCCACTTCTTCTCTGGGTTG-3Ј; and ␤-actin, sense, 5Ј-TGGAATCCTGTGGCATCCATGAAAC-3Ј and antisense, Materials and Methods 5Ј-TAAAACGCAGCTCAGTAACAGTCCG-3Ј. Mice The PCR products were analyzed with 1.5% agarose gel electrophoresis, stained with ethidium bromide, and viewed with UV light using a gel Six-week-old progeny of wild-type (WT) C57BL/6 mice and mice defi- camera (UVP). cient in IFN-␥ receptor 1 (B6.129S7-Ifngrtm1Agt/J or IFNGR1Ϫ/Ϫ) (The Jackson Laboratory) were used for study. The mice were fed standard Western blot analysis laboratory chow and water ad libitum in the Laboratory Animal Center of We harvested the cells and lysed them with a buffer containing 1% Triton National Cheng Kung University. They were raised and cared for accord- X-100, 50 mM Tris (pH 7.5), 10 mM EDTA, 0.02% NaN3, and a protease ing to the guidelines set by the National Science Council, Taiwan. The inhibitor cocktail (Roche Boehringer Mannheim Diagnostics). Addition- experimental protocol adhered to the rules of the Animal Protection Act of ally, the nuclear lysates were prepared using a compartment ProteoExtract Taiwan and was approved by the Laboratory Animal Care and Use Com- subcellular proteome extraction Kit (Calbiochem) according to the manu- mittee of National Cheng Kung University. facturer’s instructions. After they had been freeze-thawed once, the cell lysates were centrifuged at 9000 ϫ g at 4°C for 20 min. The supernatants Abs and reagents were then collected and boiled in sample buffer for 5 min. Following SDS- Mouse mAb specific for ␤-actin was purchased from Chemicon Interna- PAGE, proteins were transferred to polyvinylidene difluoride membrane tional. Anti-mouse iNOS mAb was from BD Biosciences. Alexa Fluor (Millipore), blocked at 4°C overnight in PBS-T (PBS plus 0.05% Tween 488- and HRP-conjugated goat anti-mouse, goat anti-rabbit, and donkey 20) containing 5% skim milk, and probed with primary Abs at 4°C over- anti-goat IgG were from Invitrogen. Abs against phospho-GSK-3␤ (Ser9), night. After they had been washed with PBS-T, the blots were incubated phospho-GSK-3␤ (Tyr216), GSK-3␣/␤, phospho-GS (Ser641), GS, phos- with a 1/5000 dilution of HRP-conjugated secondary Abs at 4°C for 1 h. pho-Akt (Ser473), Akt, phospho-Pyk2 (Tyr402), Pyk2, phospho-STAT1 The protein bands were visualized using ECL (Pierce Biotechnology). (Tyr701), phospho-STAT1 (Ser727), STAT1, phospho-Jak2 (Tyr1007/1008), Graphical analysis of band density was performed using ImageJ software Jak2, phospho-Src (Tyr416), Src, phospho-ERK1/2 (Thr202/Tyr204), (version 1.41o) from W. Rasband (National Institutes of Health, Bethesda, ERK1/2, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-SHP2 MD) (http://rsb.info.nih.gov/ij/). 542 (Tyr ), SHP2, SOCS1, and SOCS3 were purchased from Cell Signaling Nitrite assay Technology. Polyclonal goat anti-mouse proliferating cell nuclear Ag was from Santa Cruz Biotechnology. Recombinant mouse and human cytokine We assessed NO production by measuring the accumulated levels of nitrite IFN-␥ were from PeproTech. (E)-2-cyano-3-(3,4-dihydrophenyl)-N-(phe- in the supernatant with the Griess reagent as previously described (31). The Journal of Immunology 3

Brieﬂy, 100 ␮l of the culture supernatant was reacted with 100 ␮l of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochlo-

ride, and 2.5% H3PO4) for 10 min at room temperature. The concentration of nitrite was measured using a microplate reader (SpectraMax 340PC; Molecular Devices) at 540 nm, and the nitrite concentration was calculated using a standard curve of sodium nitrite with ELISA software (SoftMax Pro; Molecular Devices). Small interfering RNA (siRNA) and lentiviral-based RNA interference (RNAi) transfection GSK-3␤ expression was silenced using commercial GSK-3␤ siRNA (Santa Cruz Biotechnology; catalog no. sc-35527) in A549 and lentivirus-based RNAi in RAW264.7 cells. siRNA transfection was performed by electro- poration using a microporator (Digital Bio Technology). Before transfection, the A549 cells were washed with serum-free DMEM and mixed with ␤ ␥ siRNA in Opti-MEM medium (Invitrogen) in a volume of 100 ␮l. After FIGURE 1. The effects of inhibiting GSK-3 on IFN- -induced they had been transfected, and before they were stimulated, the cells were iNOS/NO biosynthesis and cytokine expression. A and B, RAW264.7 mu- incubated for 48 h in DMEM supplemented with 10% FBS at 37°C. A rine macrophages were pretreated with SB216763 or BIO for 0.5 h and nonspeciﬁc scramble siRNA was the negative control. GSK-3␤ knock- then treated with IFN-␥ for 24 h. We used RT-PCR and Western blotting down in RAW264.7 cells was performed using lentiviral transduction to to determine the expression of iNOS mRNA and protein, respectively. We stably express short hairpin RNAs (shRNA) that targeted GSK-3␤. determined NO production by using Griess reagent to detect nitrite. C and

shRNA clones were obtained from the National RNAi Core Facility D, ELISA was used to determine the time kinetics of SB216763 on TNF-␣ Downloaded from located at the Institute of Molecular Biology/Genomic Research Center, and RANTES production in IFN-␥-stimulated RAW264.7 cells. E and F, Academia Sinica, Taiwan. The mouse library should be referred to as RAW264.7 cells were pretreated with or without lentiviral-based GSK-3␤ TRC-Mm 1.0. The construct that was most effective in RAW264.7 cells ␣ ␤ (TRCN0000012615 containing the shRNA target sequence 5Ј-CAT RNAi as described in Materials and Methods. GSK-3 and GSK-3 were ␥ GAAAGTTAGCAGAGATAA-3Ј for mouse GSK-3␤) was used to gener- analyzed with Western blotting. Cells were then treated with IFN- for ate recombinant lentiviral particles. Human TE671 cells were cotransfected 24 h. ELISA was used to determine TNF-␣ production. For RT-PCR and with two helper plasmids, pCMVdeltaR8.91 and pMD.G (gift from Dr. Western blot analysis, ␤-actin was the internal control. Data shown are H. K. Sytwu, Graduate Institute of Life Sciences, National Defense Med- representative of three individual experiments. For nitrite detection and http://www.jimmunol.org/ ical Center, Taiwan), plus pLKO.1-puro-shRNA, using GeneJammer trans- ELISA, the data are means Ϯ SD obtained from three individual cultures. .p Ͻ 0.05 compared with the IFN-␥ group ,ء fection reagent (Stratagene). The transfected cells were incubated at 37°C in an atmosphere of 5% CO2 for 24 h, and then the medium was replaced with fresh medium. Cell supernatants containing the viral particles were harvested at 36, 48, 60, and 72 h after transfection. The supernatants were ␮ filtered using a 0.45-␮m low-protein-binding filter and concentrated by Sigma-Aldrich) at a concentration of 5 g/ml at room temperature for 1 h. centrifugation at 20,000 ϫ g at 4°C for 3 h using a JA25.50 (Beckman After another washing with PBS, the cells were covered with mounting Coulter) rotor. The virus pellets were resuspended with fresh medium and fluid and visualized under a confocal laser microscope (Leica TCS SPII). stored at Ϫ80°C. RAW264.7 cells were transduced by lentivirus with ap- Statistical analysis

propriate multiplicity of infection in complete growth medium supple- by guest on September 28, 2021 mented with 8 ␮g/ml polybrene. After transduction for 24 h, protein ex- Student’s t test was used to analyze the data (SigmaPlot 8.0 for Windows; pression was monitored using Western blot analysis. Systat Software). Statistical significance was set at p Ͻ 0.05. ELISA Results The concentrations of TNF-␣ and RANTES in cell-conditioned culture Inhibiting GSK-3␤ reduces IFN-␥-induced iNOS/NO medium were determined using ELISA kits (R&D Systems) according to biosynthesis as well as TNF-␣ and RANTES production the manufacturer’s instructions. RAW264.7 cells treated with 10 ng/ml IFN-␥ showed significantly Sphingomyelinase assay higher NO production as well as iNOS mRNA and protein expression Sphingomyelinase activity was determined from cellular extracts (Amplex via a time-dependent process (data not shown). We also found that the Red sphingomyelinase assay kit; catalog no. A12220, Invitrogen) accord- GSK-3 inhibitors SB216763 and BIO decreased IFN-␥-induced iNOS ing to the manufacturer’s instructions. Briefly, each reaction contained 50 mRNA and protein expression (Fig. 1A) as well as NO production in ␮M Amplex Red reagent, 1 U/ml HRP, 0.1 U/ml choline oxidase, 4 U/ml alkaline phosphatase, and 0.25 mM sphingomyelin in 1ϫ reaction buffer. RAW264.7 (Fig. 1B) and MHS macrophages (data not shown). Co- Reactions were incubated at 37°C for 1 h. Fluorescence was measured treatment with SB216763 significantly reduced IFN-␥-induced using a microplate reader (Fluoroskan Ascent; Thermo Electron) with ex- TNF-␣ (Fig. 1C) and RANTES (Fig. 1D) secretion as well as mRNA citation at 530 nm and emission at 590 nm. expression (data not shown) in RAW264.7 cells. To exclude the in- ␤ PC-PLC assay determinate effects of GSK-3 inhibitors, we silenced GSK-3 expression in RAW264.7 and A549 epithelial cells (supplemental Fig. S1)4 PC-PLC activity was determined from cellular extracts (Amplex Red PC- and then treated the cells with IFN-␥. Western blot analysis showed PLC assay kit; catalog no. A12218, Invitrogen) according to the manufac- ␤ ␤ turer’s instructions. Briefly, each reaction mixture contained 200 ␮M re- down-regulated GSK-3 expression in GSK-3 RNAi (Fig. 1E). agent (10-acetyl-3,7-dihydroxyphenoxazine; Amplex Red), 1 U/ml HRP, 4 ELISA analysis showed that silencing GSK-3␤ significantly reduced U/ml alkaline phosphatase, 0.1 U/ml choline oxidase, 0.5 mM lecithin, and IFN-␥-induced TNF-␣ production (Fig. 1F). These results provide 20–100 mU/ml PC-PLC in 50 mM Tris-HCl (pH 7.4)/140 mM NaCl/10 evidence that IFN-␥ induces iNOS/NO biosynthesis and TNF-␣ and mM dimethylglutarate/2 mM CaCl2. Reactions were incubated at 37°C for ␤ 1 h. Fluorescence was measured using the microplate reader with excitation RANTES production through GSK-3 -regulated mechanisms. at 530 nm and emission at 590 nm. IFN-␥ induces neutral SMase-, PPase-, and Pyk2-regulated Immunostaining of STAT1 nuclear translocation GSK-3␤ activation and inflammation Cells were fixed with 1% formaldehyde in PBS at room temperature for 10 GSK-3␤ is negatively and positively regulated by phosphory- min. After they had been washed twice with PBS, they were stained with lation at Ser9 and Tyr216, respectively (1–4, 32, 33). Western rabbit anti-mouse STAT1 at a final concentration of 1 ␮g/ml at room temperature for 1 h and then incubated with a mixture of Alexa Fluor 488- conjugated goat anti-rabbit IgG plus 4,6-diamidino-2-phenylindole (DAPI; 4 The online version of this article contains supplemental material. 4 GSK-3␤ FACILITATES IFN-␥ SIGNALING

FIGURE 3. IFN-␥ induces PC-PLC-, PKC-, and Src-regulated GSK-3␤ activation and inﬂammation. RAW264.7 cells were pretreated with D609,

Go¨6976, or PP1 for 0.5 h and then treated with IFN-␥. We used Western Downloaded from blotting to determine the phosphorylation of Pyk2 (Tyr402) and GSK-3␤ (Tyr216) 2 h posttreatment and GSK-3␤ (Ser9) 0.5 h posttreatment and iNOS expression 24 h posttreatment (A–C). Griess reagent and ELISA FIGURE 2. IFN-␥ regulates Akt and Pyk2, which then activates GSK-3␤ were used to determine NO (D) 24 h posttreatment and TNF-␣ (E) pro- and inﬂammation. A and C, RAW264.7 cells were treated with IFN-␥ for the duction 6 h posttreatment. For Western blot analysis, ␤-actin was the in- indicated time periods. We used Western blotting to determine phosphoryla- ternal control. Data shown are representative of three individual experi-

9 216 473 http://www.jimmunol.org/ tion of GSK-3␤ (Ser and Tyr ) and Akt (Ser ). B, D, and F, With or ments. The OD of Pyk2 (Tyr402)/total Pyk2 has been clarified using ImageJ without BIO, OA, or Sph-24 pretreatment for 0.5 h, RAW264.7 cells were software. For nitrite detection and ELISA, the data are means Ϯ SD ob- ␥-p Ͻ 0.05 compared with the IFN ,ء .then treated with IFN-␥. Western blotting was used to determine phosphory- tained from three individual cultures 641 473 lation of GS (Ser ) in cells 2 h posttreatment, and Akt (Ser ) and GSK-3␤ group. (Ser9) in cells 0.5 h posttreatment. E, RAW264.7 cells were treated with IFN-␥ for the indicated time periods. We used a kit to determine SMase activity. in IFN-␥-induced inflammation (39, 46–49), we next examined Percentages relative to those of the control are shown. G, Western blotting was ␤ 402 their effects on GSK-3 activation. First, we found that neutral used to determine the time kinetics of phosphorylation of Pyk2 (Tyr )in SMase- (supplemental Fig. S2, A and B) but not acid SMase- (sup- IFN-␥-stimulated RAW264.7 cells. H–J, With or without A9 pretreatment for plemental Fig. S2, C–E) or ceramide synthase-mediated (supple- 0.5 h, RAW264.7 cells were then treated with IFN-␥. Western blotting was ␥ by guest on September 28, 2021 used to determine phosphorylation of GSK-3␤ (Ser9) 0.25 h posttreatment and mental Fig. S2, F–H) ceramide generation was crucial for IFN- - ␥ GSK-3␤ (Tyr216) 2 h posttreatment. Griess reagent was used to determine NO induced iNOS/NO biosynthesis. Second, we found that IFN- production 24 h posttreatment. TNF-␣ production was determined using induced ceramide generation (supplemental Fig. S3A). Both the ELISA 6 h posttreatment. For Western blot analysis, ␤-actin was the internal exogenous administration of ceramide (supplemental Fig. S3, B control. Data shown are representative of three individual experiments. For the and C) and the endogenous accumulation of ceramide (supplemen- activity assay, nitrite detection, and ELISA, the data are means Ϯ SD obtained tal Fig. S3, D and E) increased IFN-␥-induced iNOS/NO biosyn- p Ͻ 0.05 compared with the untreated or the thesis. Third, we found that IFN-␥ up-regulated SMase activity ,ء .from three individual cultures ␥ IFN- group. (Fig. 2E). Neutral SMase inhibitor Sph-24 reduced IFN-␥-induced Akt and GSK-3␤ dephosphorylation (Fig. 2F). Finally, we found blot analysis showed that IFN-␥ rapidly induced GSK-3␤ dephos- that inhibiting PPases (supplemental Fig. S4) and neutral SMase phorylation (Ser9) and then caused time-dependent phosphoryla- (supplemental Fig. S2, A and B) reduced IFN-␥-induced iNOS tion of GSK-3␤ (Tyr216) (Fig. 2A). To further examine the mech- expression as well as NO production. These results indicate that anism of activation of GSK-3␤ by IFN-␥, we analyzed the neutral SMase and OA-sensitive PPases are important for IFN-␥- phosphorylation of glycogen synthase (GS), a GSK-3␤ substrate. induced GSK-3␤ activation as well as for iNOS/NO biosynthesis. Western blotting showed that IFN-␥ time-dependently induced GS Tyrosine phosphorylation of GSK-3␤ is also critical for its ki- phosphorylation (Ser641) (data not shown). Additionally, we con- nase activity (1–4, 52). We previous showed that IFN-␥ induced firmed the dependence of GSK-3␤ on IFN-␥-induced GS phos- GSK-3␤ phosphorylation (Tyr216) through a Pyk2-mediated path- phorylation using GSK-3␤ inhibitor BIO (Fig. 2B). The accumu- way in LPS-activated macrophages (31). Western blot analysis lation of GS expression in BIO-treated cells was also showed as showed that IFN-␥ time-dependently induced Pyk2 phosphoryla- consistent with previous studies (66). These results provide evi- tion (Tyr402) (Fig. 2G). Treating cells with Pyk2 inhibitor A9 de- dence that IFN-␥ causes GSK-3␤ activation. creased IFN-␥-induced GSK-3␤ phosphorylation (Tyr216) (Fig. To further investigate the mechanism of GSK-3␤ activation af- 2H) as well as NO generation (Fig. 2I) and TNF-␣ production (Fig. ter IFN-␥ stimulation, the involvement of Akt (1–4, 32, 33) and 2J). Inhibiting Pyk2 did not affect IFN-␥-induced GSK-3␤ dephos- OA-sensitive PPases (34–40) were tested. Western blot analysis phorylation (Ser9) (Fig. 2H). These results show that IFN-␥-acti- showed that IFN-␥ induced Akt dephosphorylation (Ser473)inthe vated Pyk2 is also essential for GSK-3␤ activation as well as for early stage of stimulation (Fig. 2C). Furthermore, treating cells NO and TNF-␣ production. with OA had the opposite effect (Fig. 2D). These results demon- ␥ strate that IFN-␥-activated OA-sensitive PPases are essential to IFN- induces PC-PLC-, PKC-, and Src-regulated Pyk2 and ␤ inactivate Akt and then activate GSK-3␤. GSK-3 activation and inflammation In general, ceramide activates OA-sensitive PPases (42–46) and PC-PLC-, PKC-, and Src-regulated Pyk2 activation is involved in GSK-3␤ (37, 39, 40). Because ceramide and PPases are involved IFN-␥-induced inflammation (67, 68). We first found that PC-PLC The Journal of Immunology 5 Downloaded from

FIGURE 4. IFN-␥ induces Jak2-regulated activation of PC-PLC, Pyk2, and GSK-3␤ independently of IFNGR1. A, Splenocytes harvested from IFNGR1 wild-type (IFNGR1 WT) and IFNGR1-deﬁcient (IFNGR1Ϫ/Ϫ) FIGURE 5. IFN-␥ activates Jak2-, PC-PLC-, PKC-, and cPLA2-regu- C57BL/6 mice were treated with IFN-␥ for 0.5 h. We used a kit to deter- lated neutral SMase. A and B, RAW264.7 cells were pretreated with AG490, D609, Go¨6976, Sph-24, or BEL for 0.5 h and then treated with mine the activity of PC-PLC. Percentages relative to those of the control http://www.jimmunol.org/ are shown. B, Western blotting was used to determine phosphorylation of IFN-␥ for 0.5 h. We used a kit to determine the activity of SMase. Sph-24 STAT1 (Tyr701) 1 h posttreatment and Pyk2 (Tyr402) and GSK-3␤ (Tyr216) was the positive control. Percentages relative to those of the control are 3 h posttreatment. C, RAW264.7 cells were pretreated with AG490 for shown. C, Western blotting was used to determine the phosphorylation of 9 216 0.5 h and then treated with IFN-␥ for 0.5 h. The activity of PC-PLC was GSK-3␤ (Ser and Tyr ) in IFN-␥-stimulated cells with or without BEL determined using a kit. Percentages relative to those of the control are pretreatment. D and E, Twenty-four hours posttreatment with IFN-␥ with shown. D, Western blotting was used to determine phosphorylation of or without BEL pretreatment, Western blotting and Griess reagent were STAT1 (Tyr701) and Jak2 (Tyr1007/1008) 1 h posttreatment and Pyk2 used to determine the expression of iNOS and the production of NO, re- (Tyr402) and GSK-3␤ (Tyr216) 3 h posttreatment. For the activity assay, the spectively. For the activity assay and nitrite detection, the data are means Ϯ p Ͻ 0.05 compared with the ,ء .p Ͻ 0.05 SD obtained from three individual cultures ,ء .data are means Ϯ SD obtained from three individual cultures compared with the untreated or the IFN-␥ group. For Western blot analysis, IFN-␥ group. For Western blot analysis, ␤-actin was the internal control. by guest on September 28, 2021 ␤-actin was the internal control. Data shown are representative of three Data shown are representative of three individual experiments. individual experiments.

found that AG490 effectively blocked IFN-␥-induced phosphory- inhibitor D609 and PKC inhibitors Cal C and Go¨6976, but not lation of STAT1, Pyk2, and GSK-3␤ (Fig. 4D). These results show PI-PLC inhibitor U73122, significantly reduced IFN-␥-induced that Jak2 acts an upstream regulator role for activating PC-PLC, NO production as well as iNOS expression (supplemental Fig. S5). Pyk2, and GSK-3␤. We also found that PC-PLC and PKC activated Src (supplemental Fig. S6). Western blot analysis showed that treating cells with Signaling of Jak2, PC-PLC, PKC, and cPLA2 are critical for ␥ ␤ D609, Go¨6976, or Src inhibitor PP1 decreased IFN-␥-induced IFN- -induced activation of neutral SMase and GSK-3 as well phosphorylation of Pyk2 (Tyr402) and GSK-3␤ (Tyr216), dephos- as inflammation phorylation of GSK-3␤ (Ser9), and iNOS expression (Fig. 3, A–C). PKC and cPLA2 are neutral SMase-regulating kinases that in- Furthermore, inhibiting PC-PLC, PKC, and Src blocked IFN-␥- crease AA generation from DAG (63). An activity assay showed induced NO (Fig. 3D) and TNF-␣ production (Fig. 3E). These that inhibiting Jak2, PC-PLC, or PKC significantly decreased neu- results indicate that IFN-␥-activated PC-PLC/PKC/Src is also es- tral SMase activity in IFN-␥-stimulated RAW264.7 cells (Fig. 5A). sential for Pyk2 and GSK-3␤ activation and inflammation. Cells treated with neutral SMase inhibitor were the positive control. Further results showed that cPLA2 inhibitor BEL blocked ␥ IFN- induces IFNGR2-associated Jak2-regulated PC-PLC, IFN-␥-induced SMase activation (Fig. 5B), GSK-3␤ dephosphor- ␤ Pyk2, and GSK-3 ylation (Ser9) and phosphorylation (Tyr216) (Fig. 5C), and We next examined the role of IFNGR1 and IFNGR2-associated iNOS/NO biosynthesis (Fig. 5, D and E). All these results show Jak2 in IFN-␥-activated PC-PLC, Pyk2, and GSK-3␤. In spleno- that Jak2/PC-PLC/PKC cross-talk signaling regulates cPLA2-ac- cytes obtained from IFNGR1 WT or deficient (IFNGR1Ϫ/Ϫ) tivated neutral SMase and then activates GSK-3␤. C57BL/6 mice, a PC-PLC activity assay showed that IFN-␥ in- ␤ ␥ creased PC-PLC activity even in IFNGR1Ϫ/Ϫ cells (Fig. 4A). GSK-3 facilitates IFN- -induced persistent STAT1 activation Western blot analysis showed that IFN-␥ induced phosphorylation by inhibiting SHP2 of STAT1 (Tyr701), but not Pyk2 (Tyr402) or GSK-3␤ (Tyr216), via We next examined the effects of bioactive lipids and their enzy- an IFNGR1-regulated process (Fig. 4B). Notably, inhibiting matic generators—PC-PLC, PKC, Src, Pyk2, cPLA2, neutral IFNGR2-associated Jak2 using specific inhibitor AG490 signifi- SMase, and OA-sensitive PPases—on IFN-␥ signaling. Immuno- cantly blocked IFN-␥-induced PC-PLC activity in splenocytes cytochemical staining and Western blot analysis showed that in- (data not shown) as well as in RAW264.7 cells (Fig. 4C). We hibiting these proteins considerably reduced IFN-␥-induced 6 GSK-3␤ FACILITATES IFN-␥ SIGNALING

FIGURE 6. The effects of inhibiting GSK-3␤ on IFN-␥-induced STAT1 activation. A, RAW264.7 cells were pretreated with BIO for 0.5 h and then treated with IFN-␥ for 3 h. The nuclear translocation of STAT1 was detected with immunocytochemical staining and then fluorescent microscopic analysis. Downloaded from Data shown are representative of three individual experiments. The scale bar is 75 ␮m. B, We used nuclear extraction and then Western blot analysis to determine STAT1 nuclear translocation. PCNA was the nuclear and internal FIGURE 7. GSK-3␤ negatively regulates SHP2. A, Western blotting control. C, We used Western blotting to determine the time kinetic of BIO on was used to determine the time kinetic of BIO on the phosphorylation of 701 727 1007/1008 the phosphorylation of STAT1 (Tyr and Ser ), Jak2 (Tyr ), Src SHP2 (Tyr542) and the expression of SOCS1 and SOCS3 in IFN-␥-stim- 416 202 204 180 182 (Tyr ), ERK1/2 (Thr /Tyr ), and p38 MAPK (Thr /Tyr ) in IFN-␥- ulated RAW264.7 cells. B, RAW264.7 cells were pretreated with or with- http://www.jimmunol.org/ stimulated RAW264.7 cells. D, RAW264.7 cells were pretreated with or without lentiviral-based GSK-3␤ RNAi and then treated with IFN-␥ for3h. out lentiviral-based GSK-3␤ RNAi and then treated with IFN-␥ for 3 h. West- The phosphorylation of SHP2 (Tyr542) was analyzed with Western blot- ern blotting was used to determine the expression of GSK-3␣, GSK-3␤, and ting. C, RAW264.7 cells were pretreated with BIO for 0.5 h and then 701 727 phosphorylation of STAT1 (Tyr and Ser ). ␤-actin was the internal con- treated with IFN-␥ for 3 h. We used a kit to determine the activity of trol. Data shown are representative of three individual experiments. protein phosphatase. Percentages relative to those of the control are shown. D and E, RAW264.7 cells were pretreated with BIO plus NSC-87877 for 0.5 h and then treated with IFN-␥ for 3 h. The nuclear translocation of STAT1 nuclear translocation (supplemental Fig. S7A) and phos- STAT1 was detected with immunocytochemical staining and Western blot- phorylation (supplemental Fig. S7, B–H). These results suggest ting. Data shown are representative of three individual experiments. The that bioactive lipids and their enzymatic generators are indispens- scale bar is 75 ␮m. PCNA was the nuclear and internal control. Western by guest on September 28, 2021 701 able for sustaining IFN-␥ signaling such as STAT1 activation. blotting was used to determine the phosphorylation of STAT1 (Tyr and 727 ␥ We next examined the potential effects of GSK-3␤. Using im- Ser ). F and G, Twenty-four hours posttreatment with IFN- with or munocytochemical staining (Fig. 6A) and Western blot analysis of without BIO plus NSC-87877 pretreatment, Western blotting and Griess reagent were used to determine the expression of iNOS and the production nuclear proteins (Fig. 6B), we found that BIO inhibited STAT1 ␥ of NO, respectively, in RAW264.7 cells. For the activity assay and nitrite nuclear translocation. Co-treatment with BIO inhibited IFN- -in- detection, the data are means Ϯ SD obtained from three individual cultures. 701 727 p Ͻ 0.05 compared with the IFN-␥ or the IFN-␥ plus BIO group. For ,ء duced STAT1 phosphorylation (Tyr and Ser ) in the late stage, but not in the early stage (Fig. 6C). In IFN-␥-stimulated Western blot analysis, ␤-actin was the internal control. Data shown are RAW264.7 and A549 cells (supplemental Fig. S8), silencing representative of three individual experiments. GSK-3␤ (Fig. 6D) reduced STAT1 phosphorylation (Tyr701 and Ser727). Actually, tyrosine kinases Jak2 and Src and MAPKs ERK1/2 and p38 MAPK were critical for IFN-␥-induced NO pro- We next examined the potential role of up-regulated SHP2. Im- duction (data not shown) as well as for STAT1 phosphorylation munocytochemistry and Western blot analysis showed that using (supplemental Fig. S9) (15–20, 69). However, Western blot anal- specific inhibitor NSC-87877 to inhibit SHP2 reversed BIO-in- ysis showed that inhibiting GSK-3␤ did not decrease the activation duced inhibition of STAT1 nuclear translocation (Fig. 7D) as well of Src, ERK1/2, and p38 MAPK but blocked Jak2 phosphorylation as phosphorylation (Tyr701 and Ser727) (Fig. 7E). Notably, co- (Tyr1007/1008) in the late stage (3 h posttreatment) of IFN-␥ stim- treatment with NSC blocked the BIO-induced inhibition of iNOS ulation (Fig. 6C). These results strongly suggest that GSK-3␤ is expression (Fig. 7F) and NO production (Fig. 7G) in IFN-␥-stim- critical for IFN-␥-activated Jak2 and STAT1 but has no effect on ulated RAW264.7 cells. All of these results indicate that inhibiting Src, ERK1/2, or p38 MAPK. GSK-3␤ reduces IFN-␥-induced iNOS/NO biosynthesis and per- Feedback from both SHP2 and SOCS protein down-regulate sistent STAT1 activation by up-regulating SHP2. IFN-␥-induced STAT1 activation (21–23). Interestingly, Western blot analysis showed that using BIO in IFN-␥-stimulated Discussion RAW264.7 cells (Fig. 7A) or RNAi in IFN-␥-stimulated GSK-3␤ regulates IFN-␥ signaling and is involved in IFN-␥-in- RAW264.7 (Fig. 7B) and A549 cells (supplemental Fig. S10) to duced inflammation (13, 14, 31, 67). IFN-␥ activates the proin- inhibit GSK-3␤ caused higher phosphorylation of SHP2 (Tyr542). flammatory expression of TNF-␣, RANTES, and iNOS (15, 24– However, inhibiting GSK-3␤ had no effect on SOCS1 or SOCS3. 27). We provide the first evidence that RANTES and iNOS, as Furthermore, a protein phosphatase activity assay showed that in- well as TNF-␣ (31), expression is GSK-3␤-dependent. Addition- hibiting GSK-3␤ increased IFN-␥-induced protein phosphatase ac- ally, IFN-␥-inducible protein such as caspase-1 is also induced via tivation (Fig. 7C). These results suggest that GSK-3␤ negatively a GSK-3␤-regulated manner (data not shown). The specific targets regulates SHP2; however, the mechanism remains unclear. for GSK-3␤ in IFN-␥ signaling need further investigation. However, The Journal of Immunology 7

tion directly or indirectly by dephosphorylating Akt or other GSK- 3␤-phosphorylating kinases (1–4, 32–40). Consistent with previous studies (37, 39, 40) on IFN-␥ signaling, we showed the involvement of neutral SMase- and PPase-mediated Akt inactivation followed by GSK-3␤ activation. cPLA2, which is activated by PKC, is required for DAG to generate AA (63). Furthermore, cPLA2 is important for ceramide generation (50, 51). Others (60–62) have reported that both PC- PLC- and PI-PLC-mediated DAG generation are involved in IFN- ␥-induced PKC activation. Our results, however, show an independent role for PI-PLC in IFN-␥-induced iNOS/NO biosynthesis. Consistent with previous studies (50, 51, 61, 63), we showed the essential roles of PC-PLC, PKC, and cPLA2 for neutral SMase FIGURE 8. Schematic model for GSK-3␤-facilitated IFN-␥-induced activation and GSK-3␤ dephosphorylation (Ser9) and inflamma- persistent STAT1 activation and inflammation. In this study, we provide tion. Based on our findings, we hypothesize that PC-PLC and cer- ␤ ␥ evidence of the essential role of GSK-3 in IFN- -induced signal trans- amide synergistically activate GSK-3␤ through IFN-␥ signaling. duction and inflammatory responses, including iNOS/NO biosynthesis and Pyk2 acts upstream of IFN-␥-induced STAT1 activation (54). cytokine production. After IFN-␥ stimulation, bioactive lipids and their enzymatic generators, including PC-PLC, cPLA2, and ceramide, synergis- However, the mechanism of Pyk2 activation and its effects remain tically induce GSK-3␤ activation. Physiologically, activated GSK-3␤ neg- unclear. We provide evidence that PKC is also essential for IFN- Downloaded from atively regulates SHP2, which facilitates the persistent activation of ␥-activated Pyk2 via a Src-mediated process. This is not consistent STAT1 signaling. However, the mechanism remains unknown. Based on with current studies suggesting that type I IFN-␣ activates Pyk2 our findings, inhibiting GSK-3␤ blocks IFN-␥ signaling and inflammation via calcium/calmodulin-dependent protein kinase II (56). After by up-regulating SHP2. The relationships between GSK-3␤, SHP2, and IFN-␣ stimulation, Pyk2 activation positively regulates Jak-STAT STAT1 in IFN-␥-induced signal activation and feedback regulation need signaling. Pyk2 activates GSK-3␤ by phosphorylating its tyrosine further investigation. residue (1–4, 52). Consistent with our previous study (31), we http://www.jimmunol.org/ further show that IFN-␥ activates GSK-3␤ via PC-PLC-, PKC-, Src-, and Pyk2-regulated signaling. Thus, we hypothesize that the mechanism of GSK-3␤ activation and its effects on IFN-␥ IFN-␥ regulates GSK-3␤ activation either by PPase-mediated de- signaling remain unknown. Our findings lead us to hypothesize, phosphorylation (Ser9) or by Pyk2-mediated phosphorylation however, that, after IFN-␥ stimulation, neutral SMase- and PPase- (Tyr216). Furthermore, we showed that IFNGR2-associated Jak2, mediated dephosphorylation at Ser9 and Pyk2-mediated phosphor- but not IFNGR1, is important for activating PC-PLC, Pyk2, and ylation at Tyr216 regulate the activation of GSK-3␤. These pro- GSK-3␤. In contrast, Chang et al. (62) showed that IFN-␥-medi- cesses are universal with various stimuli (1–4, 31, 34–37, 40). ated activation of Jak1/Jak2 was essential for PI-PLC-mediated Thus, neutral SMase and PPases act synergistically with Pyk2 to PKC and Src activation and ICAM-1 expression. Because our by guest on September 28, 2021 regulate GSK-3␤ activation. We also showed that a PC-PLC/PKC/ results and previous studies (61) showed that PC-PLC, but not Src regulated process-activated Pyk2, that an IFNGR2-associated PI-PLC, is required for IFN-␥-induced iNOS/NO biosynthesis, Jak2-mediated process potentially regulated PC-PLC/Pyk2/ the regulation of PC-PLC activation by Jak2 requires further GSK-3␤ activation, and that Jak2/PC-PLC/PKC/cPLA2 signaling investigation. activated neutral SMase. An unknown mechanism may inhibit Proinflammatory IFN-␥, a Th1 cytokine, down-regulates the SHP2 when GSK-3␤ is activated. We hypothesize that bioactive signaling of antiinflammatory Th2 IL-10 (15, 24, 25). IFN-␥ lipids and their enzymatic generators regulate GSK-3␤ activation, facilitates TLR-mediated inflammation through a mechanism in- and that GSK-3␤ in turn inhibits SHP2, which facilitates IFN-␥- volving GSK-3-mediated CREB inactivation and then IL-10 induced persistent Jak2-STAT1 activation and inflammation. down-regulation (13, 31, 67). In contrast, IL-10 also inhibits IFN- Based on these findings, we have created a schematic summary for ␥-induced Jak-STAT signaling by inducing SOCS proteins (21, GSK-3␤ activation and its effects on IFN-␥ signaling and inflam- 22). In general, IFN-␥ automatically induces feedback regulation matory activation (Fig. 8). that diminishes Jak2-STAT1 activation through a mechanism in- Bioactive lipids and their enzymatic generators (ceramide, volving the activation of SOCS1, SOCS3, and SHP2 (21–23, 68– cPLA2, and PC-PLC) are generally involved in cytokine (TNF-␣, 70). However, the mechanism that activates feedback regulation IL-1, and IFN-␥)-mediated signaling (45–47), and their down- remains unclear. We showed that, in the early stage of IFN-␥ stim- stream targets and effects are abundant. The generation of cer- ulation, STAT1 activation is fully regulated by IFNGR1 as well as amide through neutral SMase-mediated SM hydrolysis is essential IFNGR2-associated Jak2, but that it is independent of GSK-3␤. for IFN-␥ synergized with LPS to induce iNOS/NO biosynthesis This is consistent with current studies (14) suggesting that GSK-3 and cytokine production (47–49). We have provided evidence that dependence is selective for activation of STAT3 and STAT5, IFN-␥ alone causes neutral SMase-regulated signaling and inflam- whereas STAT1 and STAT6 activation are GSK-3-independent. mation. Furthermore, ceramide increases IFN-␥-induced inflam- Although our results and those of Beurel and Jope (14) showed mation. Ceramide also activates PPases such as PP1 and PP2A that inhibiting GSK-3 does not inhibit Jak2 phosphorylation (42–46), which are required for IFN-␥-synergized with LPS to (Tyr1007/1008) and STAT1 phosphorylation (Tyr701) in the early induce iNOS/NO biosynthesis (41). We showed the potency of stage of IFN-␥ stimulation (within 0.5–1 h posttreatment), we pro- OA-sensitive PPases in IFN-␥-induced STAT1 activation and vide further evidence showing that, after the early stage of IFN-␥ inflammation. stimulation, activated GSK-3␤ is critical for extending Jak2- The targets of OA-sensitive PPases are diverse. Notably, cer- STAT1 activation. Indeed, we showed that inhibiting GSK-3␤ did amide induces GSK-3␤ activation (37, 39, 40). For GSK-3␤ acti- not reduce IFN-␥-activated IRF-1 (data not shown), an early tran- vation, PP1- or PP2A-mediated serine residue dephosphorylation scriptional regulator of IFN-␥ signaling, but blocked iNOS, is essential (34–40). PPases may cause GSK-3␤ dephosphoryla- TNF-␣, and RANTES expression as well as caspase-1 (data not 8 GSK-3␤ FACILITATES IFN-␥ SIGNALING shown). Thus, we hypothesize a mechanism by which GSK-3␤ ticancer, antimicrobe, and immunomodulation. This hypothesis facilitates persistent Jak2-STAT1 activation after the early stage of needs further investigation. IFN-␥ stimulation. Moreover, our results exclude the possible ef- ␤ ␥ 416 fects of inhibiting GSK-3 on IFN- -activated Src (Tyr ), Disclosures ERK1/2 (Thr202/Tyr204), and p38 MAPK (Thr180/Tyr182), even The authors have no financial conflicts of interest. though they are required for IFN-␥-induced STAT1 activation. Since NF-␬B is activated by IFN-␥-activated Jaks, PKR, and IKK␣/␤ (71, 72), we currently demonstrate that inhibiting References GSK-3␤ decreases NF-␬B activation through SHP2-regulated 1. Frame, S., and P. Cohen. 2001. GSK3 takes centre stage more than 20 years after its discovery. Biochem. J. 359: 1–16. pathway (data not shown). In addition to Jak2-STAT1, the role for 2. Cohen, P., and S. Frame. 2001. The renaissance of GSK3. Nat. Rev. Mol. 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