Rather Than Autophagy Β Kinase 3 Actions on Akt and Glycogen

Regulation of Inflammatory Response by 3-Methyladenine Involves the Coordinative Actions on Akt and Glycogen Synthase Kinase 3 β Rather than Autophagy This information is current as of September 28, 2021. Yi-Chieh Lin, Hsuan-Cheng Kuo, Jang-Shiun Wang and Wan-Wan Lin J Immunol 2012; 189:4154-4164; Prepublished online 12 September 2012; doi: 10.4049/jimmunol.1102739 Downloaded from http://www.jimmunol.org/content/189/8/4154

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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 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Regulation of Inﬂammatory Response by 3-Methyladenine Involves the Coordinative Actions on Akt and Glycogen Synthase Kinase 3b Rather than Autophagy

Yi-Chieh Lin,* Hsuan-Cheng Kuo,* Jang-Shiun Wang,* and Wan-Wan Lin*,†

3-Methyladenine (3-MA) is one of the most commonly used inhibitors in autophagy research today. However, rather than inhibiting class III PI3K that is involved in autophagy suppression, 3-MA might also interfere with class I PI3K and consequently augment autophagy flux. In this study, we aim to get a thorough understanding on the action mechanisms of 3-MA in TLR4- mediated inflammatory responses in RAW264.7 macrophages and, moreover, to decipher the action of 3-MA in modulation of autophagy. We found that 3-MA could enhance LPS-induced NF-kB activation and production of TNF-a, inducible NO synthase (iNOS), cyclooxygenase-2, IL-1b, and IL-12. In contrast, 3-MA suppressed LPS-induced IFN-b production and STAT signaling. Downloaded from Studies revealed that 3-MA can, through inhibition of Akt as a result of class I PI3K interference, positively regulate p38, JNK, and p65, but negatively regulate TANK-binding kinase 1 and IFN regulatory factor 3 mediated by TLR4. As glycogen synthase kinase 3b (GSK3b) is an important Akt substrate, we further explored its involvement in the actions of 3-MA. 3-MA was found to enhance LPS-induced NF-kB activation, iNOS, and pro–IL-1b expression, and these actions were reversed by either GSK3b inhibitors or small interfering GSK3b. Lastly, we demonstrated that 3-MA acts as an autophagy inducer in RAW264.7 macrophages, but the stimulating effects on NF-kB activation and iNOS and cyclooxygenase-2 expression were not affected in LPS- http://www.jimmunol.org/ stimulated macrophages with small interfering autophagy protein-5 treatment. These results not only shed new light on the action mechanisms of 3-MA to differentially regulate inflammatory outcomes derived from TLR4-mediated MyD88 and Toll/IL-1R domain-containing adapter inducing IFN-b pathways, but also highlight the necessity to check autophagy status upon taking 3- MA as a general autophagy inhibitor. The Journal of Immunology, 2012, 189: 4154–4164.

nnate immunity serves as the ﬁrst line of defense against Recently, there were reports suggesting that activation of PI3K/ invading microorganisms. TLR4 is a pattern recognition re- Akt/glycogen synthase kinase 3b (GSK3b) pathways upon the ceptor that can recognize bacterial LPS and plays a critical early phase of TLR4 stimulation can modulate the MyD88 and

I by guest on September 28, 2021 role in APCs for host defense and activation of adaptive immune TRIF signaling pathways of TLR4. It was demonstrated that Akt response (1). After activation, TLR4 can induce MyD88- and can negatively regulate MyD88/NF-kB pathways and limit LPS- Toll/IL-1R domain-containing adapter inducing IFN-b (TRIF)– induced production of inflammatory mediators (4–7). Studies dependent upstream signals, leading to production of proinflam- further showed that inhibition of GSK3b following Akt activation matory and anti-inflammatory mediators, respectively (2, 3). To contributes to inhibit NF-kB and is involved in the regulation of achieve these actions, TGF-activated kinase 1 (TAK1), IkB kinase TLR-mediated cytokine production (5, 8–10). Nevertheless, the (IKK), p38 MAPK, JNK, TANK-binding kinase 1 (TBK1), NF-kB, crosstalk between PI3K/Akt and NF-kB pathways remains con- AP-1, and IFN regulatory factor 3 (IRF3) are involved with different troversial. Despite more findings supporting Akt being a negative preferences for target gene transcription. regulator on NF-kB, some studies indicate Akt can positively regulate NF-kB–dependent gene expression via increase of p65 transactivation activity or via activation of IKK and p38 (11–13). The controversy mainly is due to cell-type or stimulation differ- *Department of Pharmacology, College of Medicine, National Taiwan University, ences. In contrast to the major negative role in MyD88-mediated † Taipei 10051, Taiwan; and Graduate Institute of Medical Sciences, Taipei Medical proinflammatory response, TRIF-dependent innate immunity re- University, Taipei 11031, Taiwan sponse seems to be enhanced by Akt itself (14). A very recent Received for publication September 22, 2011. Accepted for publication August 14, 2012. study demonstrated Akt being a downstream molecule of TRIF– This work was supported by grants from the National Science Council (NSC 98- TBK1 and playing an important role in the activation of IRF3 and 2815-C-0020171-B and NSC 99-2815-C-002-009-B) and a grant from the Institute of IFN-b gene expression (15). Besides the NF-kB pathway, PI3K/ Biomedical Science, Academia Sinica (CRC 99-P04). Akt was also reported to regulate MAPKs in a negative manner. Address correspondence and reprint requests to Dr. Wan-Wan Lin, Department of Akt itself can inhibit JNK and p38 MAPK through inhibition of Pharmacology, College of Medicine, National Taiwan University, Taipei 10051, Tai- wan. E-mail address: [email protected] apoptosis signal-regulated kinase 1 (16). Autophagy is a ubiquitous biological process essential for Abbreviations used in this article: Atg5, autophagy protein-5; COX-2, cyclooxygenase-2; GSK3, glycogen synthase kinase 3; IKK, IkB kinase; iNOS, inducible NO maintaining cellular homeostasis among eukaryotes. The cellular synthase; IRF3, IFN regulatory factor 3; 3-MA, 3-methyladenine; mTOR, mamma- events during autophagy follow distinct stages, and the first step is lian target of rapamycin; poly I:C, polyinosinic-polycytidylic acid; p90RSK, p90 ribosomal S6 kinase; p70S6K, p70 ribosomal protein S6 kinase; siRNA, small inter- vesicle nucleation (formation of phagophore), which is differen- fering RNA; TAK1, TGF-activated kinase 1; TBK1, TANK-binding kinase 1; tfLC3, tially regulated by PI3Ks. Activation of class I PI3K and its down- tandem fluorescent-tagged LC3 construct; TRIF, Toll/IL-1R domain-containing stream molecule the mammalian target of rapamycin (mTOR) adapter inducing IFN-b. lead to autophagy suppression. In contrast, activation of class III Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 PI3K (Vps34) positively regulates vesicle nucleation (17). Auto- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102739 The Journal of Immunology 4155 phagy has been shown to intersect with innate immune responses The ECL reagent (Western Lightening Chemiluminescence Reagent Plus) at multiple levels and link to the elimination of intracellular was from PerkinElmer (Wellesley, MA). The FastStart SYBR Green Master pathogens (18–21). It was shown that TLR4 and TLR7 are auto- was from Roche Applied Science (Nutley, NJ). phagy sensors and can induce autophagy by suppressing the in- MTT assay teraction between Beclin 1 and Bcl-2 to control the replication Murine RAW264.7 macrophages (105/ml) plated in 96-well plates were of pathogens (22, 23). Moreover, autophagy was shown to be incubated with indicated agents at 37˚C for 24 h. MTT (5 mg/ml) was essential for TLR7-dependent production of type I IFNs and added for 1 h and then the culture medium was removed. The formazan proinflammatory cytokines, implicating a positive regulatory role granules generated by live cells were dissolved in DMSO and shaken of autophagy in innate immune responses (24). Yet, paradoxically, for 10 min. The OD values at 550 and 630 nm were measured using a microplate reader. The net absorbance (OD550 2 OD630) indicates the there are other studies pointed out that absent or downregulation enzymatic activity of mitochondria and implicates the cell viability. of autophagy results in enhanced production of proinflammatory molecules (25–27). Thus, the distinct roles of autophagy in in- Transfection and luciferase assay flammatory responses implicate that autophagy serves as a dual The iNOS promoter plasmid was kindly provided by Dr. C. K. Glass functional regulator in innate immunity to ensure homeostatic (Department of Medicine, University of California, San Diego, La Jolla, balance of immune responses. CA). The 2.3-kb cDNA for murine iNOS promoter was ligated into the luciferase reporter vector. The NF-kB reporter plasmid containing three 3-Methyladenine (3-MA) is a widely used pharmacological binding sites (pGL2-ELMA-luciferase) was provided by Dr. S. L. Hsieh inhibitor of autophagy today, as it is able to inhibit autophagosome (Yang-Ming University, Taipei, Taiwan). The HA-p65 plasmid was pro- formation by interfering with class III PI3K (28). Nevertheless, vided by Dr. Hiroaki Sakurai (Tanabe Seiyaku, Osaka, Japan). The b some nonspecific effects of 3-MA independently of autophagy -galactosidase expression plasmid (pCR3lacZ; Pharmacia, Stockholm, Downloaded from Sweden) was used for normalization of transfection efficiency. All plas- inhibition have been reported, including inhibition of class I PI3K mids were prepared using an endotoxin-free plasmid preparation kit and mTOR (29), suppression of protein degradation, and cell (Qiagen, Germantown, MD). Following the commercial standard protocol, migration and invasion (30, 31). The preferential action of 3-MA RAW264.7 cells were transfected with 0.25 mg each plasmid by using in distinct PI3Ks was later on shown to be associated with the Lipofectamine 2000 reagent (Invitrogen). After the indicated treatments, cellular setting (32). It was found that prolonged treatment of RAW264.7 cells were lysed in reporter lysis buffer (Promega). Subse- quently, the lysates were reacted with commercial luciferase substrate 3-MA can promote autophagic flux under nutrient-rich conditions, in a luciferase assay system kit (Promega) and then were assayed by http://www.jimmunol.org/ whereas 3-MA is capable of suppressing starvation-induced auto- microplate luminometer (Packard Instrument, Meriden, CT). phagy. Given that autophagy is in close relationship with innate Real-time RT-PCR with SYBR green detection immunity and its involvement or not in the actions of 3-MA in macrophage-mediated inflammatory response remains elusive, The expression of murine iNOS, COX-2, IL-12b,IL-1b,andTNF-a we thus explore the effects and the action mechanisms of 3-MA on mRNA were determined by real-time RT-PCR analysis. After treatment as indicated, cells were homogenized with 500 ml RNA-Bee isolation LPS-induced inflammatory responses in murine macrophages and reagents (Tel-Test), and total RNA was extracted following the commercial also elucidate the involvement or not of autophagy in these events. standard protocol. Reverse transcription was performed using an RT-PCR kit (Promega, Heidelberg, Germany), and 5 mg total RNA was reversely

transcribed to cDNA following the commercial standard procedures. The by guest on September 28, 2021 Materials and Methods specific primers for iNOS were forward primer 59-CAG CTG GGC TGT Cell culture ACA AAC CTT-39 and reverse primer 59-CAT TGG AAG TGA AGC GTT TCG-39. The specific primers for COX-2 were forward primer 59-GAG Murine RAW264.7 macrophages were obtained from the American Type AGA AGG AAA TGG CTG CAG AA-39 and reverse primer 59-GGC TTC Culture Collection (Manassas, VA), and cultured in DMEM complete CAG TAT TGA GGA GAA CAGA-39. The specific primers for IL-1b were medium containing 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, forward primer 59-TTC AAG GGG ACA TTA GGCAG-39 and reverse and 100 mg/ml streptomycin. Cells were incubated at 37˚C in a humidified primer 59-TGT GCT GGT GCT TCA TTC AT-39. The specific primers for atmosphere of 5% CO in air. Primary murine bone marrow-derived 2 IL-12b were forward primer 59-CAG AAG CTA ACC ATC TCC TGG macrophages were prepared as we previously described (33). TTT G-39 and reverse primer 59-CCG GAG TAA TTT GGT GCT CCA Reagents CAC-39. The specific primers for TNF-a were forward primer 59-ATG AGA AGT TCC CAA ATG GCC-39 and reverse primer 59-TCC ACT TGG LPS, 3-MA, MTT, protease inhibitor cocktails, SB415286, SB216763, and TGG TTT GCT ACG-39. Real-time PCR was performed using the ABI ATP were obtained from Sigma-Aldrich (St. Louis, MO). Bafilomycin A1 Prism 7900 (Applied Biosystems), and PCR fluorescence detection was and Akt inhibitor were obtained from Calbiochem (La Jolla, CA). Murine performed in 96-well plates using FastStart SYBR Green Master (Roche IFN-b was obtained from PBL InterferonSource (Piscataway, NJ). Abs Applied Science) as we previously described (34). directed against IKK, p38, JNK1, IkBa, TAK1, TBK1, JAK1, STAT1, STAT3, GSK3b, cyclooxygenase-2 (COX-2), and HRP-coupled anti-rabbit RNA interference and anti-mouse secondary Abs were purchased from Santa Cruz Bio- The small interfering RNA (siRNA) for Atg5 and GSK3b contained four technology (Santa Cruz, CA). Anti-NLRP3 Ab was purchased from ENZO RNA sequences in a SmartPool selected from the National Center for (Farmingdale, NY). Anti-inducible NO synthase (iNOS) polyclonal Ab Biotechnology Information RefSeq Database using a proprietary algo- and Ab against phosphorylated TBK1 were from BD Biosciences rithm. The control is a pool of four functional nontargeting siRNAs with (Franklin Lakes, NJ). LC3 Ab was purchased from MBL (Woburn, MA). 180 182 183 185 guanine cytosine content comparable to that of the functional siRNA but Abs specific to the phosphorylated p38 (Thr /Tyr ), JNK (Thr /Tyr ), 202 204 176 177 184 187 lacking specificity for the known gene target. We transfected cells with the ERK (Thr /Tyr ), IKKa/b (Ser / ), TAK1 (Thr / ), IRF3 396 536 701 705 100-nM siRNA for 72 h by DharmaFECT 1 (Dharmacon, Chicago, IL) (Ser ), p65 (Ser ), STAT1 (Tyr ), STAT3 (Tyr ), p70 ribosomal 389 followed by drug treatment and then evaluated the gene-silencing effects protein S6 kinase (p70S6K) (Thr ), p90 ribosomal S6 kinase (p90RSK) 380 9 by Western blot analysis. (Ser ), and GSK3b (Ser ), as well as Ab against p70S6K were purchased from Cell Signaling Technology (Beverly, MA). Ab against IRF3 was from Immunoblotting analysis Epitomics (Burlingame, CA). Ab against autophagy protein-5 (Atg5) was purchased from Abcam (Cambridge, U.K.). Ab directed against phos- After indicated treatment, the medium was aspirated. Cells were rinsed with phorylated JAK1 (Tyr1022/1023) was obtained from Invitrogen (Biosource ice-cold PBS and lysed in whole-cell lysis buffer (50 mM Tris-HCl [pH 7.6], International, Nivelles, Belgium). Abs against b-actin and phosphorylated 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% deoxycholate, 2 mM 279 216 GSK3b (Tyr ,Tyr ) were from Upstate Biotechnology (Charlottesville, EDTA, 2 mM NaF, 2 mM Na3VO4, 1 mM PMSF, and protease inhibitor VA). Protein concentration was determined by Bio-Rad protein assay cocktails). After cell harvest, total cell lysates were centrifuged at 12,500 kit (Bio-Rad, Richmond, CA). DMEM, trypsin-EDTA, and antibiotic rpm, 4˚C for 15 min. The protein concentrations were determined using the penicillin/streptomycin were from Invitrogen (Rockville, MD). The RNA- Bio-Rad protein assa (Bio-Rad). Equal amounts of the soluble protein were Bee isolation reagent was purchased from Tel-Test (Friendswood, TX). electrophoresed on 8–10% SDS-PAGE and transferred to Immobilon-P 4156 3-MA REGULATES INFLAMMATION VIA ACTIONS ON Akt AND GSK3b

(Millipore). Nonspecific binding was blocked with 50 mM Tris-HCl [pH affected at early time points (#10 min), both phosphorylations 7.5], 150 mM NaCl, and 0.1% Tween 20 (TBST) containing 5% nonfat seemed to sustain longer under 3-MA treatment compared with milk for 1 h at room temperature. After immunoblotting with the primary those treated with LPS alone (Fig. 2C). Similarly, we found that 3- Abs, membranes were washed three times with TBST and incubated with HRP-conjugated secondary Ab for 1 h. After washing three times with MA also can increase the phosphorylation of p38 and JNK, two TBST, the protein bands were detected with ECL detection reagent (Per- signals downstream of TAK1, at the late stage of LPS activation kinElmer). To ensure equal amounts of sample were applied for electro- (30–120 min) (Fig. 2C). b phoresis and immunoblotting, -actin was used as an internal control. In the aspect of the TRIF-dependent pathway, unlike what have Each immunoblotting trace shown in the figures was representative of at least three reproducible experiments. been observed in the MyD88-dependent pathway, 3-MA did not increase the phosphorylation of TBK1 and IRF3. To be more exact, Fluorescence microscopy analysis 3-MA even slightly reduced the phosphorylation of TBK1 and The tandem fluorescent-tagged LC3 construct (tfLC3), which was gen- IRF3 at the early stage of LPS stimulation (Fig. 2D). It was known erated by Dr. T. Yoshimori’s group (35), was purchased from Addgene that 3-MA can interfere with class I PI3K (29, 32) and that class I (Cambridge, MA). Transfection of the plasmid in RAW264.7 macrophages PI3K is a critical regulator of endocytosis (36). Because LPS- was performed by using Lipofectamine 2000 reagent (Invitrogen). After induced MyD88- and TRIF-dependent pathways are triggered by 24 h of transfection, cells were treated with agents, fixed with 4% para- formaldehyde, and examined by a fluorescent microscope (Zeiss AXIO TLR4 located in the plasma membrane and endosome, respec- Imager A1; Carl Zeiss). tively, we wondered if enhancing TAK1 downstream signals but inhibiting TBK1 downstream signals caused by 3-MA are asso- Statistical evaluation ciated with endocytotic interference. To access this possibility, we

Values were expressed as the mean 6 SEM of at least three independent pretreated RAW264.7 cells with 3-MA and then stimulated with Downloaded from experiments, which were performed in duplicate. ANOVA was used to a direct activator of the TRIF pathway, polyinosinic-polycytidylic , assess the statistical significance of the differences, and a p value 0.05 acid (poly I:C; 25 mg/ml), which can bind to TLR3 on the was considered statistically significant. endosomal membrane and activate the TRIF pathway independent of the process of endocytosis. As shown in Fig. 2E, 3-MA (1 mM) Results could still inhibit poly I:C-induced IRF3 phosphorylation. Thus, 3-MA enhances LPS-induced inflammatory responses in we suggest that 3-MA (1 mM) may negatively regulate LPS- http://www.jimmunol.org/ RAW264.7 macrophages induced IRF3 activation through a mechanism independent of To investigate the effects of 3-MA on innate inflammatory re- TLR4 endocytosis. Together, these findings indicate that 3-MA sponse, LPS-stimulated RAW264.7 macrophages were used in can differentially regulate MyD88- and TRIF-dependent signaling this study. Cell viability was first examined after 3-MA and LPS pathways downstream of TLR4 activation. treatment. Low concentration of 3-MA (1 mM), either alone or in b company with LPS, did not cause significant effect on cell viability, 3-MA inhibits PI3K–Akt–GSK3 signaling pathway whereas higher concentration of 3-MA (3 mM) decreased cell Previous studies showed that 3-MA inhibits not only class III PI3K, viability (Fig. 1A). Notably, the cytotoxicity of 3-MA (3 mM) can but also interferes with class I PI3K. Next, we determined the be diminished by LPS (1 mg/ml) cotreatment. effects of 3-MA on PI3K/Akt/GSK3b pathways. Akt was phos- by guest on September 28, 2021 First of all, we determined the effects of 3-MA on LPS-induced phorylated under LPS stimulation, and 3-MA exhibited an in- inflammatory response. Results revealed that LPS-induced iNOS hibitory effect in this event (Fig. 3A). Given that the PI3K/Akt protein expression (Fig. 1B) and iNOS promoter activation (Fig. pathway could activate mTOR, which in turn activates p70S6K, 1C) were enhanced by 3-MA in a concentration-dependent man- we next examined p70S6K phosphorylation in LPS-activated ner. To exclude the interference of cell viability on the inflam- macrophages. As expected, 3-MA inhibited p70S6K phosphory- matory responses, 3-MA (1 mM) was adopted as the optimal lation under LPS stimulation, in accordance with its inhibitory experimental condition in the following experiments. Likewise, action on Akt (Fig. 3A). These results indicate that 3-MA indeed the transcription of several proinflammatory genes, including is a negative regulator of PI3K/Akt/mTOR pathway. iNOS, pro–IL-1b, IL-12b, TNF-a, and COX-2, was also enhanced PI3K-dependent activation of Akt leads to phosphorylation of by 3-MA after LPS stimulation (Fig. 1D). Consistently, COX-2 GSK3b, which is active in resting cells but inactivated by phos- and pro–IL-1b protein expression induced by LPS was increased phorylation (9). Because 3-MA has been demonstrated to inhibit by 3-MA addition (Fig. 1E). Altogether, these data suggest that PI3K/Akt in LPS-stimulated RAW264.7 macrophages, it inspires 3-MA could enhance the inflammatory responses in LPS-activated us to determine if 3-MA also activates GSK3b following its effect RAW264.7 macrophages. on Akt inhibition. As shown in Fig. 3B, 3-MA alone decreased the inactivating phosphorylation of GSK3b at Ser9, indicating that 3- Effects of 3-MA on LPS-induced upstream signaling pathways MA could activate GSK3b. LPS (1 mg/ml) stimulation was shown k and NF- B activation to increase the inactivating GSK3b phosphorylation, meaning that Because NF-kB was reported to be one of the main transcription LPS itself exerts an inhibitory effect on GSK3b activity. The factors mediating inflammatory response downstream of TLR4, GSK3b-activating effect of 3-MA also existed under LPS stimu- we evaluated the effect of 3-MA on NF-kB activation. As shown lation, as 3-MA significantly decreased LPS-induced Ser9-inacti- in the luciferase reporter assay, 3-MA could concentration- vating phosphorylation. GSK3 is also known to be phosphorylated dependently augment NF-kB activation under LPS stimulation at a tyrosine residue (Tyr279 in GSK3a and Tyr216 in GSK3b), and (Fig. 2A). However, 3-MA exerted no effect on NF-kB activity in several studies pointed out that this Tyr phosphorylation also p65-overexpressing RAW264.7 macrophages (Fig. 2B). We next contributes to GSK3 activity (37). However, in our study, we did analyzed the signaling cascade downstream of TLR4 activation. In not detect any effects of 3-MA and LPS on this phosphorylation the aspect of MyD88-dependent pathway, which provides stronger (Fig. 3B), which is consistent with previous findings in LPS- signals than the TRIF pathway for IKK and MAPK activation, 3- stimulated macrophages (38). MA did not exert significant influence on the phosphorylation of Notably, the ATP-competitive GSK3 inhibitor SB216763, which IKK or IkBa degradation. In contrast, although the intensities of is supposed to inhibit GSK3b activity, appeared to decrease the LPS-induced phosphorylation of TAK1 and p65 were not much inactivation phosphorylation at Ser9 (Fig. 3B). This phenomenon The Journal of Immunology 4157 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. 3-MA augmented LPS-stimulated inflammatory responses in RAW264.7 macrophages. RAW264.7 macrophages were pretreated with 3-MA at the concentrations indicated for 30 min, followed by the stimulation with LPS (1 mg/ml). (A) After LPS treatment for 24 h, the cell viability was assessed by MTT assay. (B) Total cell lysates were subjected to SDS-PAGE and immunoblotting for iNOS. The increased iNOS expression was normalized with b-actin level and expressed as fold of control response of LPS without 3-MA treatment. (C) RAW264.7 macrophages were transfected with iNOS promoter construct followed by treatment with LPS (1 mg/ml) and/or 3-MA at the concentrations indicated. After 24 h of incubation, the luciferase activity was determined, normalized with b-galactosidase activity, and expressed relative to the control group. (D) RAW264.7 macrophages were pretreated with 3-MA (1 mM) for 30 min prior to stimulation with LPS (1 mg/ml). After incubation for different periods, total RNA was extracted and reversely transcribed for quantitative PCR analyses of iNOS, COX-2, IL-12, IL-1b, and TNF-a mRNA. Values were normalized to b-actin gene expression and expressed relative to the control group. Data were the mean 6 SEM from three independent experiments. (E) RAW264.7 macrophages were treated with LPS (1 mg/ml) and/or 3-MA (1 mM) as indicated, followed by immunoblotting to determine protein expression. *p , 0.05, indicating significant effects of 3-MA as compared with control (A) or LPS response alone (B–D). has also been observed in previous studies (39) and was ascribed we explored p90RSK phosphorylation. As shown in Fig. 3C, 3-MA to the nonspecific effect of GSK3 inhibitors on p90RSK, another did not affect p90RSK phosphorylation induced by LPS. Collec- upstream kinase of GSK3b (40). To understand if 3-MA could tively, these results demonstrate that 3-MA could inhibit PI3K/Akt decrease GSK3b Ser9 phosphorylation through inhibiting p90RSK, signaling and in turn activate GSK3b. 4158 3-MA REGULATES INFLAMMATION VIA ACTIONS ON Akt AND GSK3b

FIGURE 2. Effects of 3-MA on TLR4-mediated signaling pathway. (A) RAW264.7 macrophages transfected with kB reporter construct were pretreated with 3-MA at the concentrations indicated for 30 min prior to LPS (1 mg/ml) stimulation. After LPS incubation for 24 h, the luciferase activity was determined, normalized with b-galactosidase activity, and expressed relative to the control group. (B) RAW264.7 macrophages were transfected with 2 mg of control plasmid CMV2 or HA-p65 plasmid. Transfection also included 1 mg of NF-kB reporter construct. At 24 h after transfection, cells were treated with 3-MA for another 24 h. Downloaded from The luciferase activity was determined, normalized with b-galactosidase activity, and expressed relative to the control group. RAW264.7 macrophages were pretreated with 3-MA (1 mM) for 30 min prior to the http://www.jimmunol.org/ stimulation with LPS (1 mg/ml) (C, D) or poly I:C (25 mg/ml) (E). After incubation for different periods as indicated, the total cell lysates were subjected to SDS-PAGE followed by immunoblotting for p-TAK1, p-IKKa/ b, p-p65, p-p38, p-JNK, p-TBK1, IkBa, and p-IRF3. The total TAK1, IKKa/b, p65, p38, JNK1, TBK1, and

IRF3 levels were considered as internal by guest on September 28, 2021 controls. *p , 0.05, indicating sig- niﬁcant enhancement of LPS-induced NF-kB activation by 3-MA.

Akt but not GSK3b is involved in differentially regulating by 3-MA, unlike Akt, is not involved in regulating TLR4 signaling LPS-induced p38, JNK, p65, and IRF3 activation of p38 and JNK. Akt signaling has been shown to negatively regulate MyD88- 3-MA augments LPS-induced proinflammatory responses dependent pathway, whereas it positively regulates the TRIF- through GSK3b-dependent NF-kB activation dependent pathway upon TLR4 activation (4–7, 14, 15). Because Next, we demonstrated if 3-MA–enhanced inflammatory responses 3-MA could significantly attenuate LPS-induced Akt phosphory- and NF-kB activation were associated with GSK3b activation. lation, we wonder if the differentially regulatory effects of 3-MA To this end, pharmacological GSK3b inhibitors (SB216763 and on LPS signaling as shown previously in Fig. 2C–E were mediated SB415286) were used. We found that NF-kB activity triggered by by Akt. As shown in Fig. 4A, pharmacological inhibition of Akt LPS itself was inhibited by SB216763 and SB415286, suggesting by Akt inhibitor (10 mM) enhanced the phosphorylation of p38, that GSK3b plays a positive regulatory role in LPS-induced NF- JNK, and p65 in response to LPS stimulation, whereas it dimin- kB activation. Moreover, 3-MA–induced NF-kB enhancement ished the phosphorylation of IRF3. Such phenomena appear to under LPS stimulation was also significantly blunted by both correspond with those affected by 3-MA upon LPS stimulation GSK3b inhibitors (Fig. 5A). Confirming results using a kinase as shown above. inhibitor, LPS-elicited NF-kB activity either in the presence or Recently, GSK3b was proposed as a key downstream target of absence of 3-MA was reduced in cells pretreated with si-GSK3b Akt to regulate TLR-mediated cytokine production (9). Thus, to (Fig.5A).ToaddressifGSK3b-mediated NF-kB results from understand if 3-MA regulation of LPS-induced signaling is as- p65 phosphorylation, we determined this via immunoblotting. As a sociated with GSK3b activation, we tested a GSK3b inhibitor result, SB216763 did not change LPS-induced p65 phosphorylation, and found that 3-MA–induced enhancement of JNK and p38 unlike the effect of p38 inhibitor (SB203580) (Fig. 5B). phosphorylation were not affected by SB216763 (10 mM). LPS- In agreement with the response of NF-kB, SB216763 can induced IKK phosphorylation was not affected by 3-MA or suppress the enhancing effect of 3-MA on the LPS-induced iNOS SB216763 (Fig. 4B). These results suggest that GSK3b activation and pro–IL-1b protein expression (Fig. 5C). In parallel, GSK3 The Journal of Immunology 4159

expression induced by LPS was markedly reduced in cells treated with GSK3b siRNA (Fig. 5E). To understand if current findings for GSK3b-mediated proinflammatory response display the cell-type specificity, we tested the effects of 3-MA and SB216763 in primary bone marrow- derived macrophages. As expected, protein levels of proinflammatory mediators like NLRP3, iNOS, COX-2, and pro–IL-1b were induced by LPS, and all of these effects were diminished in the presence of GSK3b inhibitor. Moreover, as in the response shown in RAW264.7 macrophages (Fig. 5C), 3-MA further enhanced pro–IL-1b expression under LPS stimulation, and this effect of LPS plus 3-MA is still sensitive to GSK3b inhibitor (Fig. 5F). In summary, we suggest that 3-MA–augmented LPS-induced proinflammatory response, at least in part, is ascribed to the positive regulation of the GSK3b-dependent NF-kB pathway. 3-MA negatively regulates the JAK/STAT1 pathway

Upon activation of TLR4, the transcription of type I IFN can be Downloaded from induced via the TRIF-dependent pathway, and it in turn via an autocrine action leads to the activation of the type I IFN receptor- mediated JAK/STAT pathway. Next, we were interested to understand whether 3-MA also inﬂuences the LPS-induced JAK/ STAT signaling pathway, type I IFN production, and their rele- vance to GSK3b activation. We found that 3-MA appeared to http://www.jimmunol.org/ diminish LPS-triggered JAK1, STAT1, and STAT3 phosphorylation (Fig. 6A). In parallel, 3-MA could attenuate LPS-induced IFN-b protein expression, and this effect was not altered by SB216763 (Fig. 6B), suggesting the noninvolvement of GSK3b in regulating molecular events underlying the inhibitory actions of 3-MA for IFN-b gene transcription. In addition to inhibit IFN-b production, we examined if 3-MA might also affect JAK/STAT signaling at the receptor level. Thus,

b by guest on September 28, 2021 FIGURE 3. 3-MA inhibited PI3K/Akt/mTOR, but activated GSK3b we stimulated RAW264.7 macrophages with exogenous IFN- signaling pathways. RAW264.7 macrophages were pretreated with 3-MA and determined the effect of 3-MA on STAT phosphorylation. (1 mM) and/or SB216763 (10 mM) for 30 min prior to stimulation with Results showed that 3-MA failed to affect STAT1 phosphorylation LPS (1 mg/ml). After incubation for different periods as indicated, the total upon IFN-b treatment, but can inhibit IFN-b–induced STAT3 cell lysates were subjected to SDS-PAGE followed by immunoblotting for phosphorylation (Fig. 6C). Both effects of 3-MA were still present Akt, p70S6K (A), GSK3b (B), and p90RSK (C). when treating SB216763, which itself has no signiﬁcant effects in IFN-b–induced STAT1 and STAT3 phosphorylation. These results inhibition signiﬁcantly reduced the level of LPS-induced IL-1b suggest the ability of 3-MA to interrupt LPS-induced IFN-b sig- mRNA expression. 3-MA–augmented IL-1b mRNA expression naling through both indirect (i.e., inhibition of IFN-b production) in response to LPS stimulation was also inhibited by GSK3b and direct (i.e., inhibition of IFN-b–mediated STAT3 phosphor- inhibitors (Fig. 5D). Consistently, iNOS and pro–IL-1b protein ylation) mechanisms.

FIGURE 4. Akt inhibitor, but not GSK3b inhibitors, regulates the upstream signaling pathways of LPS. RAW264.7 macrophages were pretreated with Akt inhibitor (AktI) (10 mM) (A), SB216763 (10 mM), and/or 3-MA (1 mM) (B) for 30 min prior to the stimulation with LPS (1 mg/ml). After incubation for different periods, the total cell lysates were subjected to SDS-PAGE followed by immunoblotting for p38, JNK, p65, IRF3, and IKKa/b. 4160 3-MA REGULATES INFLAMMATION VIA ACTIONS ON Akt AND GSK3b Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. 3-MA augmented LPS-triggered NF-kB activation and proinflammatory response via GSK3b activation. (A) RAW264.7 macrophages transfected with NF-kB reporter construct were pretreated with SB216763, SB415286, and/or 3-MA (1 mM) for 30 min prior to LPS (1 mg/ml) stimulation. In some experiments, cells were pretreated with siRNA of GSK3b prior to addition of 3-MA and/or LPS. After incubation for 24 h, the luciferase activity was determined, normalized with b-galactosidase activity, and expressed relative to the control group. (B) RAW264.7 macrophages were treated with LPS, SB216763, and/or SB203580 for different periods, and p65 phosphorylation was determined by immunoblotting. (C) RAW264.7 macrophages were pretreated with 3-MA (1 mM) prior to LPS (1 mg/ml) stimulation, either in the absence or presence of SB216763 (10 mM). After LPS incubation for 8, 12, 18 (bottom panel), or 24 h (top panel), equivalent protein amounts of total cell lysates were subjected to SDS-PAGE, followed by immunoblotting. The b-actin level was considered as an internal control. (D) After treatment with indicated agents (1 mM 3-MA, 10 mM SB216763, 10 mM SB415286) and LPS for 3 h, total RNA was extracted and reversely transcribed for quantitative PCR analysis of IL-1b mRNA. Values were normalized to b-actin gene expression and expressed relative to the control group. (E) RAW264.7 macrophages pretreated with GSK3b siRNA were stimulated with LPS (1 mg/ml). (F) Bone marrow- derived macrophages were treated with LPS (1 mg/ml), 3-MA (1 mM), and/or SB216763 (10 mM) as indicated for different periods. Protein expression was determined in cell lysates by immunoblotting. *p , 0.05, indicating significant enhancing effects of 3-MA or inhibitory effects of GSK3b inhibitors or GSK3b siRNA on LPS response, #p , 0.05, indicating the significant reversal effects of GSK3b inhibitors or GSK3b siRNA on the action of 3-MA.

Autophagy activation by 3-MA is not involved in NF-kB conjugation of LC3 leading to the conversion of the soluble form activation of LC3 (LC3-I) to the lipidated form (LC3-II) and resulting in Although 3-MA is a commonly used autophagy inhibitor today, formation of the autophagosome is a widely used marker of recent evidence has suggested that this pharmacological agent had autophagy, we determined LC3-II level and intracellular LC3 other off-target effects, including the augmentation of autophagic staining. As shown in Fig. 7A, 3-MA enhanced the conversion ﬂux (32, 41). Thus, we aimed to understand the role of autophagy from LC3-I to LC3-II regardless of the presence or not of LPS, in the action of 3-MA in activated macrophages. Because lipid indicating its ability to induce autophagy. We also used tfLC3 The Journal of Immunology 4161 Downloaded from

FIGURE 6. 3-MA negatively regulated JAK/STAT signaling pathway. RAW264.7 macrophages were pretreated with 3-MA (1 mM) and/or SB216763 (10 mM) for 30 min as indicated prior to the stimulation with LPS (1 mg/ml) or IFN-b (10 ng/ml). After incubation for different periods as indicated, the http://www.jimmunol.org/ total cell lysates were subjected to SDS-PAGE followed by immunoblotting for STAT1, JAK1, and STAT3 (A, C). In some experiments, secreted IFN-b in the culture medium was determined by ELISA (B). *p , 0.05 indicates significant suppression of IFN-b protein expression by 3-MA. construct to confirm 3-MA–induced autophagic flux. This double- of 3-MA and much scientific research in the field of autophagy tagged LC3 construct can monitor the maturation processes of commonly using 3-MA for autophagy inhibition, it is indeed autophagosome and autolysosome with different fluorescent sig- worthwhile taking notice of the actual action mechanisms of 3- nals (42). As the greenish fluorescent signal would be quenched MA. Thus, in this study, we determined the action of 3-MA in under low pH condition, yellow puncta represents autophagosome, macrophages with respect to the regulation of both inflammatory by guest on September 28, 2021 whereas red puncta represents autolysosome. As shown in Fig. 7B, response and autophagy. Moreover, the link from Akt, GSK3b, RAW264.7 macrophages under starvation with treatment of HBSS and autophagy to inflammatory signals (e.g., NF-kB, MAPKs, and exhibited increased red puncta formation compared with the STATs) was elucidated in this study to provide a new insight on control group. In contrast, bafilomycin A1, which inhibits acidi- the action of 3-MA. fication inside the lysosome and thus impairs autolysosomal mat- To investigate how 3-MA affects inflammatory responses, we uration, only increased yellow puncta. Under 3-MA and/or LPS used LPS-stimulated murine RAW264.7 macrophages as a cellular treatment, obvious red puncta were detected. These data suggest model. According to our finding, it showed that 3-MA inhibited that 3-MA is a positive regulator of autophagy in RAW264.7 LPS-induced phosphorylation of Akt as well as one of the macrophages. downstream substrates of mTOR, p70S6K (Fig. 3A). These find- To further understand if the enhancing effect of 3-MA on LPS- ings indicate that 3-MA may exert inhibitory effects on class I induced inflammatory response was associated with autophagy, PI3K-mediated signaling. Before investigating the role of 3-MA in we adopted the siRNA approach to abrogate autophagy. Depri- LPS-induced inflammatory responses, the concentration-gradient vation of Atg5 by siRNA significantly increased LPS-triggered response of 3-MA in cell viability was assessed (Fig. 1A). NF-kB activation, and under this condition, the enhancing effect Moreover, to exclude the interference of cell viability on 3-MA– of 3-MA on LPS-induced NF-kB activation was still observed affected inflammatory responses, 3-MA (1 mM) was chosen as the (Fig.7C).InconsistencyofNF-kB activation, 3-MA–induced optimal experimental condition. increasesofiNOSandCOX-2proteinexpression(Fig.7D)and To date, there is a large body of evidence suggesting that PI3K/ COX-2mRNAexpression(Fig.7E)causedbyLPSwerealso Akt pathway plays a negative regulatory role in TLR-triggered present in autophagy-compromised cells. These findings suggest inflammation, particularly in MyD88-dependent pathways like that the proinflammatory actions of 3-MA in LPS-stimulated p38, JNK, p65, and NF-kB (5, 16, 44, 45). Consistently in this macrophages are unrelated to autophagy induction. Taken together, study, we also observed that 3-MA enhanced the production of 3-MA actions in macrophages shown in this study are summarized various LPS-induced proinflammatory responses, including the in Fig. 8. transcriptional expression of iNOS, COX-2, TNF-a, IL-12, and IL-1b (Figs. 1, 5F), as well as the activation of p65, p38, JNK, and Discussion NF-kB (Fig. 2). We further conducted an experiment using Akt 3-MA is a widely used autophagy inhibitor that was known to take inhibitor to confirm a negative regulation loop of Akt on MAPKs effect via interference with class III PI3K, hVps34 (43). Never- and 65 (Fig. 4A). Because p65 phosphorylation can be inhibited theless, induction of autophagy and other cellular actions via its by SB203580 (an inhibitor of p38) (Fig. 5B) as previously re- inhibition on class I PI3K and independent of autophagy inhibition ported (12), we suggest two possible routes contribute to the p65 were also demonstrated (29–32). Based on these conflict actions phosphorylation under 3-MA treatment. One is mediated by the 4162 3-MA REGULATES INFLAMMATION VIA ACTIONS ON Akt AND GSK3b Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 7. Autophagy induced by 3-MA does not contribute to regulate NF-kB and inflammation response. (A) RAW264.7 macrophages were treated with 3-MA (1 mM) and/or LPS (1 mg/ml) for different periods as indicated. Total cell lysates were subjected to immunoblotting for LC3. (B) RAW264.7 macrophages were transfected with tfLC3 plasmid and treated with 3-MA (1 mM), LPS (1 mg/ml), bafilomycin A1 (BA; 50 nM), or HBSS for 8 h. Cells were prepared and subjected to fluorescence microscopy. (C) RAW264.7 macrophages were transfected with either small interfering Atg5 (si-Atg5) or control siRNA and were also transfected with NF-kB reporter construct followed by pretreatment with 3-MA (1 mM) for 30 min prior to LPS (1 mg/ml) stimulation. After LPS incubation for 24 h, the luciferase activity was determined, normalized with b-galactosidase activity, and expressed relative to the control group. (D and E) RAW264.7 macrophages were transfected with si-Atg5 or control siRNA. Then cells were treated with 3-MA (1 mM) and LPS (1 mg/ml) for 24 (D)or3h(E). Expression levels of iNOS, COX-2, and Atg5 proteins (D) as well as COX-2 mRNA (E) were determined. *p , 0.05, indicating significant increasing actions of 3-MA on the LPS response in control siRNA cells, #p , 0.05, indicating significant increasing actions of 3-MA alone and on the LPS response in si-Atg5 cells. inhibition of Akt, which is a negative regulator of p38, and the p38 and JNK activation than for IKK activation (2, 46). Phos- other is mediated by the increase of TAK1 activation (see below). phorylation of TAK1 is dispensable for IKK activation upon TLR4 TAK1 is recognized as an upstream signaling molecule of activation in certain cellular contexts, indicating that the role of MAPKs and NF-kB signaling in response to LPS stimulation. TAK1 as an adaptor protein rather than its kinase activity accounts However, it has been suggested that TAK1 is more important for for activation of IKK (47). In our case, TAK1 phosphorylation The Journal of Immunology 4163 after 60 min was enhanced by 3-MA, whereas IKK phosphory- STAT3pathwaycanalsobeinducedbyIL-6andIL-10(54). lation occurring as fast as 10 min and lasting to 120 min was Currently, we cannot rule out the effect of 3-MA on both cytokine unaffected (Fig. 2C). It is possible that similar to previous find- production and signaling transduction. Taken together, our current ings, increased activity of TAK1 in 3-MA–treated cells has data suggest that both the decreased cytokine production of IFN-b a greater influence on MAPKs than on IKK. We speculate TAK1 as well as the dampened mTOR activity contribute to the reduc- might be involved to mediate the effect of 3-MA in enhancing the tion of phosphorylated STATs by 3-MA. late-phase response (.60 min) of p38, JNK, and p65. To date, many studies have suggested an intimate interplay In contrast to what has been observed in the MyD88-dependent between autophagy and TLR-mediated inflammatory responses. pathway, 3-MA appeared to negatively regulate the TRIF-dependent TLRs can mediate autophagy (23, 55, 56), whereas conversely, the pathway, because phosphorylation of TBK1 and IRF3 as well as autophagy process or autophagy proteins themselves can also in- IFN-b production were all dampened upon 3-MA treatment (Figs. fluence inflammatory responses (57–59). In this study, we found 2D, 2E, 6B). Recent studies pointed out that Akt plays a dual role that actions of 3-MA stand at the convergence of TLR-induced in regulating TLR4 signaling, in which Akt serves as a negative inflammation and autophagy. Consistent with the abilities of 3- regulator of MyD88-dependent signaling, whereas it positively MA to inhibit Akt and subsequent mTOR activation, 3-MA indeed regulates the TRIF-dependent pathway (48). With respect to the promoted autophagic flux in our study, as indexed by the increased TRIF-dependent pathway, Akt was shown to act downstream of LC3-II expression (Fig. 7A) and autolysosome formation (Fig. TBK1 but upstream of IRF3, in which Akt can positively regulate 7B). It has been well established that autophagy played a negative activation of IRF3 (15). Consistent with this notion, our results regulatory role in innate immune responses (57). However, in our showed the ability of the Akt inhibitor to diminish IRF3 phos- study, silencing of Atg5 failed to affect the effects of 3-MA on Downloaded from phorylation upon LPS stimulation (Fig. 4A). Thus, the action of NF-kB activation (Fig. 7C), iNOS and COX-2 protein expression 3-MA in TRIF-mediated responses is primarily ascribed to inhibition (Fig. 7D), or COX2 gene expression (Fig. 7E). We thus suggest of Akt. that the 3-MA–upregulated autophagy flux is not involved in its Although many have suggested the negative regulation of Akt effect on LPS-induced inflammatory response. on NF-kB activation, the detailed mechanism underlying this In conclusion, our results demonstrate that 3-MA is capable of

phenomenon still remains poorly understood. Recently, GSK3b regulating various LPS-induced inflammatory responses through http://www.jimmunol.org/ has become the most characterized candidate downstream of Akt Akt- and GSK3b-mediated signaling pathways in murine macro- that participates in its linkage to NF-kB. It was shown that Akt can phages (Fig. 8). They involve the interference with class I PI3K, negatively regulate GSK3b activity, while GSK3b can activate inhibition of Akt, and activation of GSK3b, which all orchestrate NF-kB (49, 50). In this respect, as reported by Martin et al. (49), cooperatively in modulating the signaling downstream of activated GSK3b inhibition led to increased binding of CREB to CBP, thus TLR4. In contrast, by suppressing the PI3K/Akt pathway, 3-MA decreasing the association of NF-kB p65 to CBP. However, nei- can promote autophagy flux, but does not contribute to balance ther the phosphorylation of p65 (Ser276 or Ser536) nor the DNA NF-kB activation or other inflammatory responses enhanced by 3- binding of NF-kB p65 and p50 was affected by GSK3b activity MA. These results not only shed new light on the action mecha- (49). As demonstrated in Fig. 3B, 3-MA, either alone or in the nisms of 3-MA to differentially regulate inflammatory outcomes by guest on September 28, 2021 presence of LPS, decreased inactivating Ser9 phosphorylation of derived from TLR4-mediated MyD88 and TRIF pathways, but GSK3b, indicating the role of 3-MA in promoting GSK3b acti- also highlight the necessity to check autophagy status upon taking vation as a consequence of Akt inhibition. In agreement with 3-MA as a general autophagy inhibitor. previous findings that GSK3b is involved to augment production of various proinflammatory cytokines (49), inhibition of GSK3b activity via pharmacological inhibitor of GSK3 or genetic silencing could reverse 3-MA–enhanced proinflammatory signaling, including NF-kB activation (Fig. 5A), iNOS, and pro–IL-1b protein expression (Fig. 5C, 5E) and pro–IL-1b mRNA expression (Fig. 5D). It has been established that type I IFN possesses anti- inflammatory potential (51). Upon TLR4 activation, several different mechanisms orchestrate in the regulation of type I IFN production. Activated TRIF-dependent pathways can mediate transcription of IFN-b via IRF3. As mentioned above, activated Akt contributes to IRF3 activation; therefore, it is reasonable that inhibition Akt by 3-MA can diminish transcription of IFN-b (Fig. 6B) and in turn reduce the signaling evoked by type I IFN (i.e., activation of JAK1, STAT1, and STAT3) (Fig. 6A). In ad- FIGURE 8. Summary of 3-MA in regulating LPS-induced inflammatory dition, unlike a previous report showing that GSK3 can negatively responses. 3-MA exerts dual effects on the MyD88- and TRIF-mediated regulate IFN-b by promoting c-Jun degradation (52), our study signaling and inflammatory responses evoked by LPS. Upon LPS stimu- ruled out this possibility because GSK3b inhibitor failed to re- lation and Akt activation, 3-MA can inhibit Akt as a result of class I PI3K verse the inhibitory action of 3-MA on IFN-b production (Fig. interference, which results in the inhibition of mTOR as well as activation b 6B). Furthermore, we dissected the direct effect of 3-MA on of GSK3 . 3-MA can enhance proinflammatory response mediated by MyD88 by removing the negative role played by Akt on p38 and JNK and IFN-b and found that STAT3 rather than STAT1 activation was inducing GSK3b- and p38-dependent NF-kB activation. In contrast, 3-MA inhibited by 3-MA (Fig. 6C). The possible mechanism for 3-MA– inhibits TRIF-mediated IFN-b production primarily through inhibition suppressed STAT3 activation is that mTOR can positively regulate of Akt-dependent IRF3 activation. In resting and LPS-stimulated macro- STAT3 (53), and 3-MA could inhibit mTOR. It is reasonable that phages, unlike commonly recognizing as an autophagy inhibitor, 3-MA is the decrease in STAT3 activation by 3-MA may be partly mean autophagy inducer, and this action is associated with suppression of diated by mTOR inhibition. Besides IFN-b, the engagement of class I PI3K/Akt/mTOR. 4164 3-MA REGULATES INFLAMMATION VIA ACTIONS ON Akt AND GSK3b

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