Convergence of the Mammalian Target of Rapamycin Complex 1- and Glycogen Synthase 3- −β Signaling Pathways Regulates the Innate Inflammatory Response This information is current as of October 1, 2021. Huizhi Wang, Jonathan Brown, Zhen Gu, Carlos A. Garcia, Ruqiang Liang, Pascale Alard, Eléonore Beurel, Richard S. Jope, Terrance Greenway and Michael Martin J Immunol published online 21 March 2011

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published March 21, 2011, doi:10.4049/jimmunol.1002513 The Journal of Immunology

Convergence of the Mammalian Target of Rapamycin Complex 1- and Glycogen Synthase Kinase 3-b–Signaling Pathways Regulates the Innate Inflammatory Response

Huizhi Wang,*,† Jonathan Brown,* Zhen Gu,* Carlos A. Garcia,* Ruqiang Liang,‡ Pascale Alard,* Ele´onore Beurel,x Richard S. Jope,x Terrance Greenway,{ and Michael Martin*,†

The PI3K pathway and its regulation of mammalian target of rapamycin complex 1 (mTORC1) and glycogen synthase kinase 3 (GSK3) play pivotal roles in controlling inflammation. In this article, we show that mTORC1 and GSK3-b converge and that the capacity of mTORC1 to affect the inflammatory response is due to the inactivation of GSK3-b. Inhibition of mTORC1 attenuated GSK3 phosphorylation and increased its kinase activity. Immunoprecipitation and in vitro kinase assays demonstrated that Downloaded from GSK3-b associated with a downstream target of mTORC1, p85S6K, and phosphorylated GSK3-b. Inhibition of S6K1 abrogated the phosphorylation of GSK3-b while increasing and decreasing the levels of IL-12 and IL-10, respectively, in LPS-stimulated monocytes. In contrast, the direct inhibition of GSK3 attenuated the capacity of S6K1 inhibition to influence the levels of IL-10 and IL-12 produced by LPS-stimulated cells. At the transcriptional level, mTORC1 inhibition reduced the DNA binding of CREB and this effect was reversed by GSK3 inhibition. As a result, mTORC1 inhibition increased the levels of NF-kB p65 associated with CREB-binding protein. Inhibition of NF-kB p65 attenuated rapamycin’s ability to influence the levels of pro- or anti- http://www.jimmunol.org/ inflammatory cytokine production in monocytes stimulated with LPS. These studies identify the molecular mechanism by which mTORC1 affects GSK3 and show that mTORC1 inhibition regulates pro- and anti-inflammatory cytokine production via its capacity to inactivate GSK3. The Journal of Immunology, 2011, 186: 000–000.

he ability of the host’s immune system to initially rec- in many inflammatory diseases, an inability to regulate or resolve ognize and respond to microbial components is largely inflammation can lead to detrimental pathological conditions (7). T mediated by the innate immune system via the expression IL-10 has been shown to play a fundamental role in controlling of a family of type I transmembrane receptors, the TLRs (1–3). the immune response to microbial pathogens and is essential for Activation of TLRs by microbial products leads to the engagement maintaining immune homeostasis (8). This function is clearly evi- by guest on October 1, 2021 of a diverse number of intracellular signaling pathways that dictate dent, because IL-10 knockout (KO) mice exhibit hyperinflamma- qualitative and quantitative aspects of the host inflammatory re- tory responses upon bacterial challenge and develop inflammatory sponse (4–6). The host inflammatory response is a critical and bowel disease (8, 9). Owing to the anti-inflammatory impact of necessary event that is required to activate and instruct both the IL-10 on innate and adaptive immune cells, identifying and char- innate and adaptive immune compartments. However, as is evident acterizing the intracellular signaling pathways that regulate IL-10 production are areas of intense investigation. The family of PI3K enzymes is largely responsible for the *Department of Microbiology and Immunology, University of Louisville School phosphorylation of phosphatidylinositol lipids in response to cel- of Medicine, Louisville, KY 40202; †Oral Health and Systemic Disease Research lular stimuli. PI3K is a heterodimeric enzyme that consists of Group, University of Louisville School of Dentistry, Louisville, KY 40202; ‡Gheens Center on Aging, University of Louisville School of Medicine, Louisville, a regulatory (p85) and a catalytic (p110) subunit (10). Initial KY 40202; xDepartment of Psychiatry and Behavioral Neurobiology, University of studies by Arbibe et al. (11) demonstrated that the cytosolic do- { Alabama, Birmingham, AL 35294; and Thad Cochran National Warmwater Aqua- main of TLR2 was involved in the recruitment of PI3K. The culture Center, Mississippi State University, Stoneville, MS 38776 functional relevance of this pathway was further elucidated by Received for publication July 23, 2010. Accepted for publication February 20, 2011. Guha et al. (12), who showed that the inhibition of PI3K resulted This work was supported by National Institute of Mental Health Grant R01MH038752 in an elevated proinflammatory cytokine response and that this (to R.S.J.) and National Institute of Dental Research Grants R01DE017680 and R01DE017921 (to M.M.). effect was associated with increased NF-kB p65 activity. With the The array data presented in this article have been deposited in the Gene Expression generation of PI3K KO mice, the broad impact of the PI3K Omnibus database (www.ncbi.nlm.nih.gov/geo/info/linking.html) under accession pathway on the host inflammatory response was beginning to be number GSE25520. characterized in that dendritic cells (DCs) isolated from mice Address correspondence and reprint requests to Dr. Huizhi Wang, University of deficient in the regulatory subunit of class I PI3K (p85-a) pro- Louisville School of Dentistry, 501 South Preston Street, Room 263C, Louisville, A KY 40202. E-mail address: [email protected] duced elevated levels of IL-12 and exhibited enhanced Th1-like The online version of this article contains supplemental material. immune responses (13, 14). It was subsequently demonstrated that Abbreviations used in this article: BMDC, bone marrow-derived dendritic cell; the activation of the PI3K/Akt pathway exhibited a differential BMDM, bone marrow-derived macrophage; CBP, CREB-binding protein; DC, den- effect on the production of the prototypical anti-inflammatory dritic cell; GSK3, glycogen synthase kinase 3; KO, knockout; MEF, mouse embry- cytokine, IL-10, and the proinflammatory cytokine IL-12 (15). onic fibroblast; mTORC1, mammalian target of rapamycin complex 1. By comparing downstream targets of the PI3K pathway that were Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 affected by either PI3K or Akt inhibition, both of these molecules

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002513 2 CONVERGENCE OF mTORC1- AND GSK3-SIGNALING PATHWAYS were found to be involved in the ability of TLRs to mediate ELISA Kit was purchased from Active Motif. The the phosphorylation and inactivation of the constitutively active NF-kB p65 (Ser276) Inhibitory Peptide Set was purchased from IMGE- b b NEX. Cytokine ELISA kits were purchased from eBioscience. Mouse GM- serine/threonine kinase glycogen synthase kinase 3- (GSK3- ; CSF and M-CSF were purchased from Peprotech and R&D Systems, re- serine 9) (12, 15). The small interfering RNA-mediated knock- spectively. down of GSK3-b or pharmacological inhibition of GSK3 using a panel of different GSK3-selective inhibitors suppressed the Preparation of human monocytes, bone marrow-derived production of IL-1b, IL-6, TNF, and IL-12, whereas the pro- macrophages, and bone marrow-derived dendritic cells duction of IL-10 was increased in TLR2-, TLR4-, TLR5-, and PBMCs were obtained from healthy donors per protocols approved by the TLR9-stimulated monocytes (16). The importance of the GSK3-b University of Louisville Institutional Review Board, Human Subjects isoform in controlling the inflammatory response to LPS was Protection Program, study number 503.05. Monocytes were isolated by negative selection using the human Monocyte Isolation Kit II from Miltenyi confirmed in mouse embryonic fibroblasts that were deficient in Biotec. The purity of monocytes was routinely .90%, as determined by GSK3-b. Collectively, these studies identified how the PI3K/ flow cytometry using a FITC-labeled anti-CD14 Ab. GSK3-a/b (S21A/ GSK3-signaling pathway differentially regulated the levels of pro- S9A) knockin mice were previously generated and described (25). IL-10 and anti-inflammatory cytokines in innate immune cells. KO mice were purchased from The Jackson Laboratory. Bone marrow Recent studies have also identified another downstream target from wild-type, GSK3 knockin (S9A), or IL-10 KO mice was isolated by flushing femurs and tibias with HBSS supplemented with 2% FBS, and of the PI3K pathway, mammalian target of rapamycin complex 1 tissue debris was removed by passing cells through a 70-mm cell strainer. (mTORC1), which is involved in differentially regulating the levels Bone marrow-derived macrophages (BMDMs) were generated as pre- of pro- and anti-inflammatory cytokines produced by innate im- viously described (26). For the generation of bone marrow-derived DCs (BMDCs), bone marrow cells were treated with RBC lysis buffer (Sigma), mune cells (17–19). Upon PI3K-Akt activation, Akt can phos- 6 washed in PBS, centrifuged, and resuspended at 2 3 10 cells/ml in RPMI Downloaded from phorylate and inactivate the tuberous sclerosis complex 2–Rheb Complete media containing 10 ng/ml GM-CSF. Cultures were incubated at protein complex that impedes mTORC1 activity. Studies using 37˚C for 10 d in a humidified atmosphere containing 5% CO2. Every 4 d, the mTORC1 inhibitor rapamycin have reported that mTORC1 one-half of the medium was removed and fresh GM-CSF–supplemented activity positively regulates IL-10 production while concurrently culture medium was added. On day 10, nonadherent cells were collected . suppressing the levels of IL-12 and TNF produced by LPS- and analyzed by flow cytometry for CD11c expression; 85% of the cells stained positive for CD11c expression (data not shown). stimulated cells (19). Inhibition of mTORC1 in LPS-stimulated cells has been shown to attenuate the phosphorylation of several Western blot, ELISA, and immunoprecipitation http://www.jimmunol.org/ targets of mTORC1, including p70S6K and 4E-BP1, as well as For experiments using inhibitors for PI3K (LY294002, 25 mM), decrease the levels of phosphorylated STAT3 (17–19). In contrast, mTORC1 (rapamycin, 100 ng/ml), S6K1 (PF4708671, 0.5 mM), or GSK3 mTORC1 inhibition potently increased NF-kB activity, leading to (SB216763, 10 mM), cells were pretreated with inhibitors or with 0.01% enhanced IL-12 production by LPS-stimulated cells (19). Studies DMSO (organic solvent control) for 2 h prior to LPS stimulation. Cell-free by several other groups (18, 20–22) have provided evidence that supernatants were harvested and assayed for cytokine levels by ELISA 20 h after the addition of LPS. Cell lysates were prepared as previously de- inhibiting mTORC1 using rapamycin resulted in the loss of scribed (27). Western blot images were acquired using a Kodak Image GSK3-a/b (S21/S9) phosphorylation, an event required to in- Station 4000MM system (Eastman Kodak, New Haven, CT). Western blot activate GSK3. Turnquist et al. (18) showed that the increased and immunoprecipitation studies were performed as previously described by guest on October 1, 2021 production of IL-12 by mTORC1-inhibited DCs could be sup- (15, 26). The rabbit True Blot Kit (eBioscience) was used for all immu- noprecipitation studies according to the manufacturer’s protocol. pressed using the GSK3 inhibitor lithium. However, it was unclear from these studies whether the ability of mTORC1 inhibition to Phospho-Ab microarray and CREB nuclear binding assay attenuate the phosphorylation and inactivation of GSK3-b is re- The Human mTOR Signaling Phospho-Specific Antibody Microarray was sponsible for the inflammatory properties of rapamycin. More- purchased from and analyzed by Full Moon Biosystem. More than 130 over, it remains to be determined if the negative impact of highly specific and characterized phospho-Abs for the human mTOR- mTORC1 inhibition on GSK3-a/b (S21/9) levels was the result of signaling pathway were immobilized and replicated six times on glass a direct molecular interaction between GSK3-a/b and a kinase slides. The matching nonphosphorylated (total) target Abs were included to allow determination of the relative level of phosphorylation (phosphorylated/ within the mTOR pathway, or an indirect effect of mTORC1 in- total). Ab microarray analysis was carried out following the manufacturer’s hibition due to an alternative cell-signaling pathway involved protocol. The complete data files for Ab array experiments have been de- in the phosphorylation of GSK3-a/b. These studies are further posited in the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/ complicated by the findings of Ohtani et al. (17), who showed that geo/info/linking.html) under the accession number GSE25520. Nuclear rapamycin treatment of bone marrow-derived macrophages did lysates were obtained from human monocytes using a nuclear/cytosolic isolation kit purchased from Active Motif. Nuclear lysates were analyzed not discernibly affect the phosphorylated levels of GSK3 when for DNA-binding levels of phosphorylated CREB (S133), using the CREB stimulated with LPS. Thus, it is yet to be resolved if and how TransAm Kit obtained from Active Motif and performed according to the mTORC1 activity affects GSK3 and if the regulation of GSK3 manufacturer’s protocol. by mTORC1 is responsible for the ability of mTORC1 to regulate In vitro kinase assay the host inflammatory response. Active p70S6K, p85S6K, and inactive GSK3-b (K85A) were purchased from SignalChem. S6K1-mediated phosphorylation of GSK3-b was de- Materials and Methods termined according to the manufacturer’s protocol. Briefly, 10 ml active Media and reagents p70S6K or p85S6K (1 mg/ml or 0.1 mg/ml) was assayed in a total of 25 ml reaction buffer supplemented with 20 mM MgCl2 and 125 mM cold ATP. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 50 b m Then 1 mg/ml GSK3- was added and incubated at 30˚C for 30 min, and M 2-ME, 1 mM sodium pyruvate, 2 mM L-glutamine, 20 mM HEPES, 50 the reaction was stopped by adding 10 ml43 SDS sample buffer and 5 ml U/ml penicillin, and 50 mg/ml streptomycin (RPMI Complete). Ultrapure double distilled H2O. S6K1-mediated phosphorylation of GSK3-b (S9) LPS from E. coli 0111:B4 was purchased from Invivogen. All Abs for was analyzed by Western blot, using a phospho–GSK3-b (S9) Ab and Western blot and immunoprecipitation studies were obtained from Cell reprobed with an Ab for total GSK3-b. Signaling Technology. The GSK3 inhibitor SB216763 and S6K1 inhibitor PF4708671 were purchased from Tocris and have been previously char- Flow cytometry acterized (23, 24). The PI3K inhibitors LY294002 and wortmannin were obtained from LC Laboratories. For mTORC1 inhibition, rapamycin was Purified monocytes were plated at a density of 2.5 3 105 cells per well in used at 100 ng/ml and purchased from Calbiochem. The TransAM CREB a 96-well flat-bottomed plate and pretreated with rapamycin or organic The Journal of Immunology 3 solvent control (DMSO) for 2 h. After 1-h stimulation with LPS, cells were with our microarray data demonstrating that the levels of Akt harvested and transferred to 5-ml polystyrene round-bottomed tubes, (S473) and Akt (T308) were similar and not significantly different washed twice with 2 ml FACS buffer (PBS containing 2% FBS and 0.01% between rapamycin-treated monocytes stimulated with LPS and sodium azide), and then fixed by adding 500 ml formaldehyde to a final concentration of 4% in PBS for 10 min at room temperature. Cells were monocytes stimulated with LPS in the absence of rapamycin washed once in PBS and resuspended in 500 ml 90% methanol and in- (Table I). Taken together, these results demonstrated that rapa- cubated on ice for 10 min. Cells were then washed in PBS containing 2% mycin-induced inhibition of mTORC1 results in reduced phos- FBS and resuspended in PBS containing 2% FBS and an anti-phospho– phorylation of GSK3-b (S9). GSK3-b (R&D Systems) or isotype control Ab. Cells were incubated at room temperature for 30 min, washed twice in PBS containing 2% FBS, To further assess the effects of mTORC1 inhibition on the LPS- and analyzed immediately by flow cytometry. mediated phosphorylation of GSK3, we next monitored the levels of GSK3-b (S9) by Western blot at multiple time points (Fig. 1E, Statistical analysis 1F). As shown in Figs. 1E and 1F, human monocytes pretreated Statistical significance between groups was evaluated by the ANOVA and with rapamycin exhibited reduced levels of GSK3-b (S9) at all the Tukey multiple comparison test, using the InStat program (GraphPad, time points tested. We also used flow cytometry to monitor the San Diego, CA). Differences between groups were considered significant at effects of mTORC1 inhibition on the levels of GSK3-b (S9) at the level of p , 0.05. the single-cell level. Stimulation of human monocytes with LPS resulted in an increase in the mean fluorescent intensity levels of Results phosphorylated GSK3-b, from 3.7 in the nonstimulated control The effect of mTORC1 inhibition on phosphorylated GSK3 group to .9.13 in LPS-stimulated monocytes (Fig. 1G). In con- levels in LPS-stimulated monocytes trast, pretreatment of monocytes with rapamycin reduced the To comprehensively analyze the effects of mTORC1 inhibition mean fluorescent intensity levels of GSK3-b (S9), from 9.13 in Downloaded from on GSK3, we initially used a PI3K/mTOR-specific phospho-Ab LPS-stimulated cells to 4.6 (Fig. 1G). Collectively, these results microarray that analyzed the site-specific phosphorylation of demonstrate that mTORC1 inhibition attenuates the ability of .130 within the PI3K/mTOR pathway. The PI3K/mTOR LPS to mediate the phosphorylation of GSK3-b (S9) without any phospho-Ab microarray identified a list of phosphorylated pro- noticeable effect on the levels of phosphorylated Akt (S473 or teins whose phosphorylation levels decreased in monocytes when T308). Moreover, the data show that the ability of rapamycin to stimulated with LPS in the presence of rapamycin (Table I). A suppress the phosphorylation of GSK3-b (S9) was not due to an http://www.jimmunol.org/ significant (p , 0.01) decrease was observed in the phosphory- effect on Rictor, because similar levels of GSK3-b (S9) were ob- lation of GSK3-b (S9) when LPS-stimulated monocytes were served in both wild-type and Rictor-deficient cells stimulated pretreated with rapamycin, compared with monocytes stimulated with LPS. with LPS alone (Fig. 1A,1B). These results are consistent with Inactivation of the constitutively active kinase GSK3-b occurs those of others (18, 20–22), showing that rapamycin-treated cells via phosphorylation on S9 (29, 30). Because we observed that exhibit suppressed levels of phosphorylated GSK3. mTORC1 inhibition suppressed the capacity of LPS to induce It has been reported that, in certain cell types, prolonged GSK3-b (S9) phosphorylation, we wanted to determine whether treatment with the mTORC1 inhibitor rapamycin can negatively rapamycin was increasing the kinase activity of GSK3 in LPS- by guest on October 1, 2021 affect mTORC2’s capacity to phosphorylate Akt (S473) (28). To stimulated monocytes by assessing the ability of GSK3 to phos- rule out the possibility that the observed effects of rapamycin re- phorylate a GSK3-specific substrate. For these studies, we moni- sulted from mTORC2 inhibition, we also monitored the levels of tored the phosphorylated levels of the GSK3-specific substrate GSK3-b (S9) in LPS-stimulated wild-type and Rictor (essential glycogen synthase (S641) (29, 30). Compared with nonstimulated component of mTORC2)–deficient mouse embryonic fibroblasts cells, LPS induced the phosphorylation of GSK3-b (S9), and this (MEFs). As shown in Fig. 1C and 1D, no discernible difference was accompanied by a reduction in the phosphorylation of the can be seen in the levels of phosphorylated GSK3-b (S9) between GSK3-specific substrate glycogen synthase (S641) (Fig. 1H). How- wild-type and Rictor-deficient cells. These findings are consistent ever, pretreatment of monocytes with rapamycin resulted in at-

Table I. Effects of rapamycin on the levels of phosphorylated proteins in LPS-stimulated human monocytes

LPS LPS + Rapamycin

Phosphorylation Sites Ratio 95% CIa Ratio 95% CIa Percent Decrease 4E-BP1 (phospho-S65) 2.62 2.39 ∼ 2.85 2.04 1.84 ∼ 2.24 22.13 BAD (phospho-S134) 0.99 0.87 ∼ 1.11 0.81 0.65 ∼ 0.97 18.18 BAD (phospho-S155) 3.99 3.93 ∼ 4.25 2.86 2.68 ∼ 3.04 28.32 BAD (phospho-S136) 3.22 2.98 ∼ 3.46 2.26 2.04 ∼ 2.48 29.81 eIF4G (phospho-S1108) 1.42 1.36 ∼ 1.48 1.07 0.91 ∼ 1.23 24.65 ERK3 (phospho-S189) 5.1 4.82 ∼ 5.38 4.15 3.87 ∼ 4.43 18.62 GSK3-b (phospho-S9) 19.96 18.51 ∼ 18.81 11.18 11.03 ∼ 11.33 43.98 mTOR (phospho-S2448) 1.62 1.52 ∼ 1.82 0.80 0.68 ∼ 0.92 50.61 mTOR (phospho-S2481) 1.87 1.79 ∼ 2.05 1.06 0.96 ∼ 1.16 43.31 P70S6K (phospho-T421) 4.98 4.82 ∼ 5.14 3.01 2.67 ∼ 3.35 39.55 PKC-a (phospho-Y657) 7.78 7.68 ∼ 7.88 6.61 6.54 ∼ 6.68 14.28 PKC a/b II (phospho-T638) 4.75 4.21 ∼ 5.29 3.95 3.89 ∼ 4.01 16.63 AKT(phospho-S473) 1.28 1.26 ∼ 1.30 1.33 1.30 ∼ 1.36 ,5 AKT(phospho-T308) 1.68 1.59 ∼ 1.77 1.73 1.67 ∼ 1.79 ,5 PI3K/mTOR phospho-Ab microarray identified a list of phosphorylated proteins whose phosphorylation states decreased in the presence of rapamycin. The ratio between the phosphorylated protein and the total protein in human monocytes is de- termined in the presence and absence of rapamycin. aA 95% confidence interval (CI) was determined to demonstrate the significance of the signal alternation for each phos- phorylated protein. 4 CONVERGENCE OF mTORC1- AND GSK3-SIGNALING PATHWAYS Downloaded from http://www.jimmunol.org/ by guest on October 1, 2021

FIGURE 1. Rapamycin inhibits the phosphorylation of GSK3-b (S9) in LPS-stimulated cells. A, Human monocytes were stimulated with LPS (1 mg/ml) in the presence or absence of rapamycin (100 ng/ml) for 60 min, and the levels of phosphorylated GSK3-b (S9) and total GSK3-b were analyzed using a phospho-Ab array. B, The mean ratio of GSK3-b (S9) to total GSK3-b is shown 6 SD of six experiments. C, Wild-type and Rictor-deficient MEFs were treated with LPS (1 mg/ml) over a 2-h time course, and the levels of phosphorylated GSK3-b (S9) were analyzed by Western blot. D, Densitometry scans were performed and the mean ratios of phosphorylated and total GSK3-b are shown 6 SD of three experiments. E, Human monocytes were stimulated with LPS (1 mg/ml) in the presence or absence of rapamycin (100 ng/ml) for up to 24 h, and the levels of phosphorylated GSK3-b (S9) were analyzed by Western blot. F, Densitometry scans were performed, and the mean ratios of phosphorylated and total GSK3-b are shown 6 SD of three experiments. G, The levels of phosphorylated GSK3-b (S9) in LPS-stimulated human monocytes treated with or without rapamycin (100 ng/ml) were monitored by flow cytometry. H, The effects of rapamycin (100 ng/ml) on the levels of phosphorylated GSK3-b (S9) and the GSK3-specific substrate, glycogen synthase (S641), in LPS-stimulated cells were monitored by Western blot. I, Inhibition of mTORC1 using rapamycin (100 ng/ml) and its effects on the levels of phosphorylated mTOR, p70S6K, p85S6K, and 4E-BP1 in LPS-stimulated monocytes. J, Inhibition of GSK3 using SB216763 (10 mM) and its effects on the phosphorylated levels of mTOR, p85S6K, and p70S6K in LPS-stimulated monocytes. Data are representative of three to six separate experiments. **p , 0.01, statistically significant differences between monocytes stimulated with LPS in the presence or absence of rapamycin. The Journal of Immunology 5 tenuated GSK3-b (S9) phosphorylation and enhanced phosphory- lation of glycogen synthase (S641) (Fig. 1H). Therefore, rapamycin inhibits the phospho-inactivation of GSK3 and enhances the ac- tivity of GSK3, as evidenced by the phosphorylation status of glycogen synthase. To assess whether GSK3 inhibition affected the mTORC1- signaling pathway, we next monitored the effects of GSK3 in- hibition on the ability of LPS to phosphorylate mTOR, as well as downstream kinases of mTORC1, including p70S6K, p85S6K, and 4E-BP1. As expected, rapamycin-treated monocytes exhibited reduced phosphorylated levels of mTOR, p70S6K, and p85S6K at all time points tested (Fig. 1I). In contrast, inhibition of GSK3 had no discernible effect on the phosphorylated levels of mTOR, p70S6K, or p85S6K, compared with cells stimulated with LPS alone (Fig. 1J). Thus, although mTORC1 inhibition affected the phosphorylation of GSK3, inhibition of GSK3 did not affect mTOR, p70S6K, or p85S6K phosphorylation.

Regulation of pro- and anti-inflammatory cytokines by mTORC1 inhibition is dependent on GSK3-b Downloaded from Our current data showed that inhibition of mTORC1, using rapa- mycin, attenuated the levels of phosphorylated GSK3 in LPS- stimulated cells. Therefore, we next wanted to determine whether the ability of mTORC1 inhibition to regulate the inflammatory response was due to its capacity to affect GSK3-b (S9). Because the http://www.jimmunol.org/ inhibition of mTORC1 suppressed the phospho-inactivation of FIGURE 2. Ability of rapamycin to regulate the LPS-mediated in- GSK3-b (S9), we predicted that mTORC1 and GSK3 inhibition flammatory response is dependent upon GSK3. The levels of TNF (A), IL- would mediate opposing effects on the levels of pro- and anti- 12 (B), and IL-10 (C) produced by LPS-stimulated monocytes, with or inflammatory cytokines produced by LPS-stimulated monocytes without the mTORC1 inhibitor rapamycin (100 ng/ml), the GSK3 inhibitor m and, if GSK3 was downstream of mTORC1, that the inhibition SB216763 (10 M), or both. For A–C, the GSK3 inhibitor SB216763 of GSK3 would inhibit the inflammatory properties of rapamycin. attenuated the capacity of rapamycin to increase TNF and IL-12 pro- duction and suppress IL-10 levels produced by LPS-stimulated monocytes. Indeed, rapamycin-treated monocytes, compared with cells stim- The levels of IL-12 (D) and IL-10 (E) produced by LPS-stimulated DCs , ulated with LPS alone, produced significantly (p 0.05) higher expressing a constitutively active GSK3 knockin, with or without rapa- levels of TNF and IL-12 (Fig. 2A,2B). In contrast, inhibition of mycin. Data represent the arithmetic mean 6 SD of three separate by guest on October 1, 2021 GSK3 significantly (p , 0.001) reduced the levels of TNF and IL- experiments. *p , 0.05, **p , 0.01, ***p , 0.001, statistically significant 12, compared with levels in LPS-stimulated monocytes (Fig. 2A, differences. 2B). Moreover, analysis of IL-10 levels demonstrated that the inhibition of mTORC1 and GSK3 had opposing effects (Fig. 2C). Specifically, mTORC1 inhibition significantly (p , 0.001) re- strate that the inability to alter GSK3 activity abrogates the ca- pacity of rapamycin to alter pro- and anti-inflammatory cytokine duced IL-10, whereas inactivation of GSK3 significantly (p , production by LPS-stimulated cells. 0.001) increased the levels of IL-10 produced by monocytes stimulated with LPS, compared with cells stimulated with only mTORC1 regulates the phosphorylation of GSK3-b via LPS. To directly determine if the capacity of mTORC1 to regulate p85S6K b the inflammatory response was due to its ability to affect GSK3- To this point, our data have demonstrated that inhibition of (S9) phosphorylation, we next used GSK3 knockin DCs that have mTORC1, using rapamycin, attenuates the ability of LPS to induce b a serine mutated to an alanine at position 9 of GSK3- (S9A) and the phospho-inactivation of GSK3, and that rapamycin-treated a position 21 of GSK3- (S21A) (25). As a result, GSK3 knockin cells exhibited elevated phosphorylation levels of the GSK3-specific DCs express a constitutively active GSK3 that cannot be inacti- substrate glycogen synthase (S641). Moreover, we showed that vated by serine phosphorylation. In wild-type DCs, rapamycin- the direct inhibition of GSK3 abrogates the capacity of rapamycin treated DCs produced significantly (p , 0.01) higher levels of to affect the inflammatory response by LPS-stimulated monocytes. IL-12 than did wild-type DCs stimulated with LPS alone (Fig. In addition, rapamycin was unable to influence the levels of either 2D). In contrast, rapamycin was unable to significantly (p . 0.05) IL-10 or IL-12 produced by LPS-stimulated DCs expressing a affect the levels of IL-12 produced by LPS-stimulated DCs ex- constitutively active knockin of GSK3. These data demonstrate pressing a constitutively active GSK3 (Fig. 2D). Moreover, al- that mTORC1 inhibition affects GSK3 activity and that the ability though rapamycin-treated wild-type DCs produced significantly of rapamycin to influence the phospho-inactivation of GSK3 (p , 0.01) less IL-10 than did wild-type DCs stimulated only with plays a fundamental role in its capacity to regulate the inflamma- LPS, rapamycin did not discernibly alter the levels of IL-10 tory response by LPS-stimulated monocytes. As a result, we next produced by DCs expressing a constitutively active knockin of wanted to determine how mTORC1 inhibition was affecting the GSK3 (Fig. 2E). Similar results were obtained using GSK3 phospho-inactivation of GSK3. We used immunoprecipitation knockin BMDMs (Supplementary Fig. 1A,1B). Furthermore, di- studies to find out whether mTORC1 or an mTORC1-dependent rect inhibition of GSK3 attenuated the ability of rapamycin to kinase was mediating the phosphorylation of GSK3-b (S9). Be- increase proinflammatory cytokines and reduce IL-10 in LPS- cause rapamycin targets and inhibits mTORC1, we immunopreci- stimulated cells (Fig. 2A–C). Taken together, these data demon- pitated GSK3 and probed by Western blot for the association 6 CONVERGENCE OF mTORC1- AND GSK3-SIGNALING PATHWAYS

FIGURE 3. p85S6K associates with and phosphorylates GSK3-b (S9) in LPS-stimulated monocytes. A, Interaction of GSK3-b with p70S6K or p85S6K, as determined by immu- noprecipitation of GSK3-b followed by Western blot for p70S6K or p85S6K in LPS-stimulated monocytes treated with or without rapamycin (100 ng/ml). B, An in vitro kinase assay dem- onstrating that active p85S6K phosphorylates GSK3-b on S9, as determined by Western blot. C, Monocytes were pretreated with the S6K1 inhibitor PF-4708671 (0.5 mM) for 2 h, stimu- lated with LPS, and assessed for phosphorylated GSK3-b and 4E-BP1 levels by Western blot. D and E, Inhibition of S6K1 augmented the pro- duction of IL-12 (D) and decreased the pro- duction of IL-10 (E) by LPS-stimulated mono-

cytes. For D and E, data represent the arithmetic Downloaded from mean 6 SD of three separate experiments. **p , 0.01, ***p , 0.001, statistically signifi- cant differences. http://www.jimmunol.org/ of mTOR. As shown in Fig. 3A, we were unable to detect mTOR mTORC1 inhibition and its effects on STAT3, CREB, and by Western blot when pulling down GSK3. Next, we probed for NF-kB p65 a downstream kinase of mTORC1, S6K1. S6K1 has two isoforms, Weichhart et al. (19) have shown that mTORC1 inhibition in LPS- p70S6K and p85S6K (31). We monitored both of these kinases by stimulated cells resulted in reduced IL-10 production and sup- Western blot to determine if one or both isoforms of S6K1were pressed STAT3 activity. Although it was suggested that reduced associated with GSK3. Probing of blots using a dual-specific Ab STAT3 activity was responsible for the ability of mTORC1 in- that detects both S6K1 isoforms, we ascertained that p85S6K, but hibition to decrease IL-10, it was not shown that the capacity of not p70S6K, was associated with GSK3 (Fig. 3A). Owing to the mTORC1 inhibition to affect STAT3 activity was the result of by guest on October 1, 2021 association of p85S6K with GSK3, we next determined whether reduced IL-10 production by mTORC1-inhibited cells stimulated p85S6K was involved in phosphorylating GSK3. Using inactive with LPS. In fact, previous studies defining the role of STAT3 GSK3-b (K85A) as substrate, we measured the ability of p85S6K activation in LPS-stimulated macrophages demonstrated that to phosphorylate GSK3-b (S9). As shown in Fig. 3B, the in- STAT3 activation was dependent upon an IL-10 feedback mech- cubation of p85S6K with inactive GSK3-b (K85A) resulted in the anism (32). Thus, we next set forth to determine whether the ca- phosphorylation of GSK3-b on S9. Because these data demon- pacity of mTORC1 inhibition to affect STAT3 phosphorylation strate that p85S6K associated with and phosphorylated GSK3-b, was due to its ability to suppress IL-10. Compared with no we then ascertained whether inhibition of S6K1 affected the phosphorylation of GSK3-b and, if so, whether S6K1 inhibition stimulation of cells, LPS stimulation resulted in the phosphory- exhibited a cytokine profile similar to that produced by mTORC1 lation of STAT3 only at greater than 4 h poststimulation with LPS inhibition using rapamycin. Compared with nonstimulated cells, (Fig. 4A). Moreover, pretreatment of LPS-stimulated monocytes S6K1-inhibited monocytes exhibited attenuated levels of phos- with rapamycin suppressed the levels of phosphorylated STAT3, phorylated GSK3-b (S9) (Fig. 3C). Moreover, inhibition of S6K1 in contrast to monocytes stimulated with LPS alone (Fig. 4A). abrogated the capacity of LPS to mediate the phosphorylation of Because IL-10 has been shown to phosphorylate STAT3 and the S6K1 substrate 4E-BP1 (Fig. 3C). Because we showed that mTORC1 inhibition reduced IL-10 levels produced by LPS- rapamycin inhibited the phosphorylation of both GSK3-b and stimulated cells, we next wanted to find out whether IL-10 was S6K1 and that direct inhibition of S6K1 reduced the phosphory- involved in the phosphorylation of STAT3. As shown in Fig. 4B, lated levels of GSK3-b, we next wanted to discover whether S6K1 the addition of an anti–IL-10 Ab potently reduced the phosphor- inhibition would mimic the effects of rapamycin on the levels of ylation of STAT3 in LPS-stimulated monocytes, compared with IL-10 and IL-12 produced by LPS-stimulated monocytes. Indeed, monocytes stimulated with LPS and an isotype control Ab. To S6K1 inhibition in monocytes stimulated with LPS resulted in definitively demonstrate that STAT3 activation was dependent significantly (p . 0.01) increased levels of IL-12, whereas levels of upon IL-10 production, we used IL-10 KO BMDMs and BMDCs IL-10 were significantly (p . 0.001) decreased (Fig. 3D,3E). to examine the phosphorylation of STAT3. As shown in Fig. 4C, Moreover, the ability of S6K1 inhibition to influence the levels of LPS-stimulated IL-10 KO cells, compared with wild-type cells IL-10 and IL-12 produced by LPS-stimulated monocytes was stimulated with LPS, exhibited a complete loss of STAT3 phos- blunted by GSK3-b inhibition (Fig. 3D,3E). Thus, these data in- phorylation. Similar results were obtained using IL-10 KO dicate that S6K1 is involved in the phospho-inactivation of GSK3 BMDCs (Supplementary Fig. 2). These data demonstrate that the and that the direct inhibition of GSK3 abrogates the capacity of phosphorylation of STAT3 by LPS-stimulated cells is dependent S6K1 to alter pro- and anti-inflammatory cytokine production by upon IL-10 feedback. However, these data do not address the LPS-stimulated cells. possibility that mTORC1 inhibition could also be affecting the The Journal of Immunology 7

with LPS in the presence or absence of rapamycin. In the absence of LPS stimulation, rapamycin and the GSK3 inhibitor SB216763 did not noticeably affect the levels of CREB (S133), compared with the unstimulated control group (Fig. 5A). Rapamycin-treated monocytes stimulated with LPS exhibited a significant reduction (p , 0.001) in the nuclear levels of CREB (S133), compared with levels in cells stimulated with LPS alone (Fig. 5A). In sharp contrast, inhibition of GSK3 significantly (p , 0.05) increased the nuclear levels of CREB (S133) over those in monocytes stimu- lated with LPS alone (Fig. 5A). These data reveal that inhibition of mTORC1 and GSK3 differentially affect the nuclear DNA- binding levels of CREB (S133). Optimal transcriptional activity of NF-kB p65 (S276) occurs when associated with CBP (33, 34). The nuclear amounts of CBP are limiting, and phosphorylated CREB (S133) and NF-kB p65 (S276) compete for CBP binding. Past studies have demonstrated that an increased association of CREB and CBP suppresses NF- Downloaded from FIGURE 4. STAT3 activation and the ability of rapamycin to affect STAT3 phosphorylation in LPS-stimulated cells are dependent upon IL-10 feedback. A, Monocytes were treated with rapamycin (100 ng/ml) and stimulated with LPS (1 mg/ml) for up to 4 h, and cell lysates were probed by Western blot for phosphorylated STAT3. B, Monocytes were treated with IC or neutralizing anti–IL-10 Ab, stimulated with LPS (1 mg/ml) for up to 4 h, and cell lysates were probed by Western blot for phosphorylated http://www.jimmunol.org/ STAT3. C, Wild-type and IL-10 KO macrophages were stimulated with LPS (1 mg/ml) for up to 12 h, and cell lysates were probed by Western blot for phosphorylated STAT3. D, Monocytes were stimulated with IL-10 (10 ng/ml) in the presence or absence of rapamycin (100 ng/ml) for up to 4 h, and cell lysates were probed by Western blot for phosphorylated STAT3. Data are representative of three to five separate experiments. IC, isotype control; WT, wild-type. by guest on October 1, 2021 capacity of the IL-10 receptor to mediate the activation of STAT3. As shown in Fig. 4D, cells stimulated with exogenous IL-10 in the presence or absence of rapamycin did not exhibit any discerni- ble differences in the levels of STAT3 phosphorylation. Thus, mTORC1 inhibition does not affect the IL-10/IL-10 receptor– mediated activation of STAT3. Taken together, these data show that phosphorylation of STAT3 in LPS-stimulated cells treated with or without rapamycin is mediated by an IL-10 feedback mechanism. Previous studies have indicated that the ability of GSK3 to differentially regulate pro- and anti-inflammatory cytokines de- pended upon the transcription factors CREB and NF-kBp65 (16). Specifically, GSK3 inhibition increased the association of CREB with the coactivator of transcription CREB-binding protein (CBP) while concurrently decreasing the levels of NF-kB p65 associated with CBP. The previous findings of the current study were as follows: 1) mTORC1 inhibition abrogated the phosphor- FIGURE 5. GSK3 and mTORC1 inhibition differentially affect the ylation of GSK3; 2) mTORC1 inhibition was unable to affect the nuclear DNA-binding levels of CREB (S133). A, Transcription factor inflammatory response in DCs expressing a constitutively active binding assay measuring the levels of CREB (S133) in 10 mg of nuclear knockin of GSK3; 3) inhibition of GSK3 or mTORC1 induced op- extract from LPS-stimulated monocytes treated with SB216763 (10 mM) posing effects on the inflammatory response; 4) direct inhibition or rapamycin (100 ng/ml). B, Rapamycin (100 ng/ml) increases and of GSK3 abrogated the capacity of mTORC1 inhibition to in- decreases the association of NF-kB p65 (S276) and CREB (S133) with the crease proinflammatory cytokine production and decrease IL-10; coactivator of transcription CBP, respectively, as determined by immuno- k and 5) the downstream target of mTORC1, p85 S6K, associated precipitation of CBP followed by Western blot for NF- B p65 (S276) or CREB (S133). C and D, The functional effect of rapamycin (100 ng/ml) with and phosphorylated GSK3. On the basis of these findings, we increasing the levels of NF-kB p65 (S276) activity in LPS-stimulated determined whether mTORC1 inhibition affected the levels of monocytes was assessed by pretreatment of cells with a control peptide or k CREB and NF- B p65. Because GSK3 inhibition has been shown NF-kB p65 (S276) inhibitory peptide (100 mM), stimulated with LPS for to increase the nuclear DNA-binding levels of CREB (S133) (16), 20 h; the levels of IL-12 (C) or IL-10 (D) were determined by ELISA. For we set forth to assess the nuclear levels of CREB capable of A, C, and D, data represent the arithmetic mean 6 SD of three separate binding its DNA consensus sequence in monocytes stimulated experiments. *p , 0.05, ***p , 0.001, statistically significant differences. 8 CONVERGENCE OF mTORC1- AND GSK3-SIGNALING PATHWAYS kB p65 activity (33–36). On the basis of our observation that monocytes, compared with control monocytes stimulated with rapamycin negatively regulated the activation and DNA-binding LPS. These data demonstrate that the ability of rapamycin to in- activity of CREB (S133), we investigated whether mTORC1 in- crease the association of NF-kB p65 with CBP is responsible for hibition, using rapamycin, affected the levels of CREB (S133) and its capacity to affect pro- and anti-inflammatory cytokine pro- NF-kB p65 (S276) associated with CBP in LPS-stimulated cells. duction. Moreover, taken collectively, findings from the current LPS-stimulated monocytes, compared with nonstimulated ones, study have shown that the mTORC1 pathway regulates the levels exhibited increased levels of both CREB (S133) and NF-kB p65 of pro- and anti-inflammatory cytokine production by LPS- (S276) associated with CBP (Fig. 5B). However, monocytes stimulated cells via its ability to affect GSK3-b activity and the stimulated with LPS in the presence of rapamycin showed a con- subsequent association of NF-kB p65 or CREB with the coac- siderable increase in the association of NF-kB p65 (S276) with tivator of transcription CBP (Fig. 6). CBP, whereas the levels of CREB (S133) associated with CBP were reduced (Fig. 5B). These data indicate that inhibition of Discussion GSK3 and mTORC1 differentially affect the nuclear levels and The present findings demonstrated that mTORC1 inhibition re- DNA-binding activity of CREB (S133) and that mTORC1 in- sulted in the loss of GSK3-a/b (S21/9) phosphorylation and that hibition mediates opposing effects on CREB (S133) and NF-kB this effect was responsible for the ability of rapamycin to influence p65 (S276) associated with CBP. the levels of pro- and anti-inflammatory cytokine production in Our group previously showed that inhibition of GSK3 increased LPS-stimulated cells. Upon identifying that rapamycin affected the association of CREB (S133) with CBP, whereas the levels of phospho–GSK3-b levels, we showed that the ability of mTORC1 NF-kB p65 (S276) were reduced (16). The present findings have to influence GSK3-b (S9) was due to p85S6K. Specifically, we demonstrated that mTORC1 inhibited the phospho-inactivation of showed that the immunoprecipitation of GSK3-b resulted in Downloaded from GSK3, increased the association of NF-kB p65 (S276) with CBP, pulling down p85S6K, an effect that could be blunted by rapa- and decreased the association of CREB (S133) with CBP. Because mycin. Using an in vitro kinase assay, we demonstrated that GSK3 inhibition increases IL-10 and suppresses proinflammatory p85S6K directly phosphorylated GSK3-b (S9). In addition, we cytokine production by LPS-stimulated cells via its capacity to were able to show the functional role of S6K1 in that inhibition increase CREB (S133) bound to CBP, and mTORC1 inhibition of S6K1 abrogated the phosphorylation of GSK3-b in LPS- attenuates GSK3 inactivation, we predicted that the ability of stimulated cells. An assessment of GSK3 activity in rapamycin- http://www.jimmunol.org/ mTORC1 inhibition to increase the association of NF-kB p65 with treated monocytes revealed increased phosphorylation of the CBP was responsible for the capacity of mTORC1 inhibition to GSK3-specific substrate, glycogen synthase. The pro- and anti- increase proinflammatory cytokine production and decrease IL-10 inflammatory effects of mTORC1 inhibition were also shown to levels by LPS-stimulated monocytes. As shown in Fig. 5C and 5D, be due to its regulation of GSK3, because an inability of the inhibition of NF-kB p65 prevented the ability of rapamycin to mTORC1 pathway to inactivate GSK3-b in DCs expressing a increase IL-12 and decrease IL-10 production by LPS-stimulated constitutively active GSK3 resulted in a loss of rapamycin to aug- by guest on October 1, 2021

FIGURE 6. Convergence of mTORC1 and GSK3-b in LPS-stimulated cells. LPS stimulation induces PI3K/Akt activity, which, in turn, mediates activation of the mTORC1 pathway. Activation of mTORC1 results in the phosphorylation and activation of p85S6K. Activated p85S6K binds with and phos- phorylates GSK3-b, resulting in the suppression of GSK3-b activity. Suppressed GSK3-b activity en- hances the nuclear binding levels of CREB that promote IL-10 and decrease IL-12 production. The phosphorylation of STAT3 in LPS-stimulated cells depends upon IL-10. The Journal of Immunology 9 ment the inflammatory response and suppress the levels of IL-10. stimulated with LPS exhibited an increased association of NF-kB In contrast, the effects of rapamycin on the levels of pro- and anti- p65 with CBP and that inhibiting the enhanced NF-kB p65 ac- inflammatory cytokines could be rescued by directly inhibiting tivity in rapamycin-treated monocytes attenuated the capacity of GSK3. These findings indicate how mTORC1 inhibition influen- rapamycin to affect the levels of IL-10 and IL-12 production by ces GSK3-b (S9) and highlight how mTORC1 differentially LPS-stimulated cells. controls the levels of pro- and anti-inflammatory cytokine pro- In summary, we have identified how mTOR regulates the activity duction by LPS-stimulated cells. of GSK3-b and characterized the molecular interaction by which Mapping of the PI3K pathway by several groups revealed that this occurs. The ability of mTOR to increase the inflammatory the ability of the PI3K pathway to dictate regulation of pro- and response via its regulation of GSK3-b highlights how targeting anti-inflammatory cytokines largely depended upon the inactiva- GSK3 in the presence of mTORC1 inhibition abrogated the in- tion of the serine/threonine kinase GSK3-b (16, 37, 38). Small flammatory properties of rapamycin. These findings may be of interfering RNA knockdown of GSK3-b or the inhibition of GSK3 major consequence, considering that inflammation-associated side using a variety of pharmacological inhibitors resulted in sup- effects have been reported in patients using rapamycin (40–42). pressing the production of inflammatory cytokines while in- However, because of the ability of GSK3 to also positively affect creasing IL-10 levels in TLR-stimulated cells. These findings IL-2 production and cell cycle progression of naive CD4+ T cells, were definitively confirmed using GSK3-b KO MEFs and studies future studies will be required to assess whether GSK3 inhibition by Hu et al. (37) using chimeric mice reconstituted with GSK3-b– has a bearing on the immunosuppressive effects of rapamycin on deficient fetal liver cells. These initial studies have been verified T cells (43). and extended by many others that show the anti-inflammatory Downloaded from properties of GSK3 in other cell types and in response to differ- Disclosures ent stimuli (39). Recently, it has been reported that the PI3K The authors have no financial conflicts of interest. pathway also differentially regulated the inflammatory response of LPS-stimulated cells by influencing mTORC1 activity (17–19). Of interest, the reported effects of mTORC1 inhibition were similar References to those reported for PI3K inhibition, but inverse to those of GSK3 1. Medzhitov, R., P. Preston-Hurlburt, and C. A. Janeway, Jr. 1997. A human ho- mologue of the Drosophila Toll protein signals activation of adaptive immunity. http://www.jimmunol.org/ inhibition (17–19). These data are in agreement with the known Nature 388: 394–397. effects of PI3K on both mTORC1 and GSK3 activity. However, it 2. Medzhitov, R., P. Preston-Hurlburt, E. Kopp, A. Stadlen, C. Chen, S. Ghosh, and C. A. Janeway, Jr. 1998. MyD88 is an adaptor protein in the hToll/IL-1 receptor was unclear if these pathways were distinct or if there was po- family signaling pathways. Mol. Cell 2: 253–258. tential convergence of mTORC1 and GSK3. In this regard, earlier 3. Yang, R. B., M. R. Mark, A. Gray, A. Huang, M. H. Xie, M. Zhang, A. Goddard, studies performed by Peyrollier et al. (20) found that amino acid- W. I. Wood, A. L. Gurney, and P. J. Godowski. 1998. Toll-like receptor-2 b mediates lipopolysaccharide-induced cellular signalling. Nature 395: 284–288. induced phosphorylation of GSK3- (S9) was inhibited by rapa- 4. Kawai, T., and S. Akira. 2007. TLR signaling. Semin. Immunol. 19: 24–32. mycin. Several studies since then have further elucidated that the 5. Kumar, H., T. Kawai, and S. Akira. 2009. Toll-like receptors and innate im- inhibitory effects of mTORC1 inhibition on GSK3-b (S9) were munity. Biochem. Biophys. Res. Commun. 388: 621–625. 6. Kumar, H., T. Kawai, and S. Akira. 2009. Pathogen recognition in the innate due to a loss in S6K1 activity (22). More recently, studies by immune response. Biochem. J. 420: 1–16. by guest on October 1, 2021 Turnquist et al. (18) have made a similar observation in LPS- 7. Dinarello, C. A. 2000. Proinflammatory cytokines. Chest 118: 503–508. 8. Ku¨hn, R., J. Lo¨hler, D. Rennick, K. Rajewsky, and W. Mu¨ller. 1993. Interleukin- stimulated DCs and reported that rapamycin treatment resulted 10-deficient mice develop chronic enterocolitis. Cell 75: 263–274. in a reduction in the levels of GSK3-a/b (S21/9). Using a PI3K- 9. Sellon, R. K., S. Tonkonogy, M. Schultz, L. A. Dieleman, W. Grenther, specific kinase array, we confirmed the findings reported by E. Balish, D. M. Rennick, and R. B. Sartor. 1998. Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation Turnquist and others (20, 22) and further identified that the in interleukin-10-deficient mice. Infect. Immun. 66: 5224–5231. phosphorylation levels of several GSK3-specific substrates were 10. Cantley, L. C. 2002. The phosphoinositide 3-kinase pathway. Science 296: 1655– reduced. The current findings also showed that the effects of 1657. 11. Arbibe, L., J. P. Mira, N. Teusch, L. Kline, M. Guha, N. Mackman, mTORC1 on GSK3 activity were the result of P85S6K interacting P. J. Godowski, R. J. Ulevitch, and U. G. Knaus. 2000. Toll-like receptor 2- with and influencing the phosphorylation of GSK3-b (S9). Taken mediated NF-kappa B activation requires a Rac1-dependent pathway. Nat. Immunol. 1: 533–540. together, these studies define the molecular interaction by which 12. Guha, M., and N. Mackman. 2002. The phosphatidylinositol 3-kinase-Akt mTORC1 inhibition affects GSK3-b (S9). pathway limits lipopolysaccharide activation of signaling pathways and ex- Studies by Weichhart et al. (19) showed that mTOR inhibition pression of inflammatory mediators in human monocytic cells. J. Biol. Chem. 277: 32124–32132. resulted in suppressing the production of IL-10 in LPS-stimulated 13. Fukao, T., M. Tanabe, Y. Terauchi, T. Ota, S. Matsuda, T. Asano, T. Kadowaki, monocytes and that the ability of rapamycin to negatively in- T. Takeuchi, and S. Koyasu. 2002. PI3K-mediated negative feedback regulation fluence STAT3 was responsible for this effect. Our present study of IL-12 production in DCs. Nat. Immunol. 3: 875–881. 14. Fukao, T., T. Yamada, M. Tanabe, Y. Terauchi, T. Ota, T. Takayama, T. Asano, demonstrated that the effects of rapamycin on STAT3 were an T. Takeuchi, T. Kadowaki, J. Hata Ji, and S. Koyasu. 2002. Selective loss of indirect result of rapamycin’s impact on IL-10 levels whereby gastrointestinal mast cells and impaired immunity in PI3K-deficient mice. Nat. Immunol. 3: 295–304. STAT3 levels were reduced. In fact, IL-10 KO mice exhibited no 15. Martin, M., R. E. Schifferle, N. Cuesta, S. N. Vogel, J. Katz, and S. M. Michalek. detectable levels of STAT3 phosphorylation upon LPS stimula- 2003. Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of tion. Our findings that STAT3 activation in LPS-stimulated cells is IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide. J. Immunol. 171: 717–725. dependent upon IL-10 are consistent with those of Chang et al. 16. Martin, M., K. Rehani, R. S. Jope, and S. M. Michalek. 2005. Toll-like receptor- (32), who showed that the loss of IL-10 inhibits STAT3 activation mediated cytokine production is differentially regulated by glycogen synthase in LPS-stimulated macrophages. Although these data demonstrate kinase 3. Nat. Immunol. 6: 777–784. 17. Ohtani, M., S. Nagai, S. Kondo, S. Mizuno, K. Nakamura, M. Tanabe, that the negative effects of rapamycin on STAT3 activity were T. Takeuchi, S. Matsuda, and S. Koyasu. 2008. Mammalian target of rapamycin likely the result of reduced IL-10 levels, they do not address the and glycogen synthase kinase 3 differentially regulate lipopolysaccharide- induced interleukin-12 production in dendritic cells. Blood 112: 635–643. possibility that rapamycin could affect the IL-10 receptor–medi- 18. Turnquist, H. R., J. Cardinal, C. Macedo, B. R. Rosborough, T. L. Sumpter, ated activation of STAT3. However, we also showed that, in the D. A. Geller, D. Metes, and A. W. Thomson. 2010. mTOR and GSK-3 shape the presence of exogenous IL-10, mTORC1 inhibition did not affect CD4+ T-cell stimulatory and differentiation capacity of myeloid DCs after ex- posure to LPS. Blood 115: 4758–4769. the phosphorylation of STAT3. In contrast, the data provided 19. Weichhart, T., G. Costantino, M. Poglitsch, M. Rosner, M. Zeyda, in the current study revealed that rapamycin-treated monocytes K. M. Stuhlmeier, T. Kolbe, T. M. Stulnig, W. H. Ho¨rl, M. Hengstschla¨ger, et al. 10 CONVERGENCE OF mTORC1- AND GSK3-SIGNALING PATHWAYS

2008. The TSC-mTOR signaling pathway regulates the innate inflammatory 32. Chang, E. Y., B. Guo, S. E. Doyle, and G. Cheng. 2007. Cutting edge: in- response. Immunity 29: 565–577. volvement of the type I IFN production and signaling pathway in lipo- 20. Peyrollier, K., E. Hajduch, A. S. Blair, R. Hyde, and H. S. Hundal. 2000. L- polysaccharide-induced IL-10 production. J. Immunol. 178: 6705–6709. leucine availability regulates phosphatidylinositol 3-kinase, p70 S6 kinase and 33. Sheppard, K. A., D. W. Rose, Z. K. Haque, R. Kurokawa, E. McInerney, glycogen synthase kinase-3 activity in L6 muscle cells: evidence for the in- S. Westin, D. Thanos, M. G. Rosenfeld, C. K. Glass, and T. Collins. 1999. volvement of the mammalian target of rapamycin (mTOR) pathway in the L- Transcriptional activation by NF-kappaB requires multiple coactivators. Mol. leucine-induced up-regulation of system A amino acid transport. Biochem. J. Cell. Biol. 19: 6367–6378. 350: 361–368. 34. Zhong, H., R. E. Voll, and S. Ghosh. 1998. Phosphorylation of NF-kappa B p65 21. Gulati, P., L. D. Gaspers, S. G. Dann, M. Joaquin, T. Nobukuni, F. Natt, by PKA stimulates transcriptional activity by promoting a novel bivalent in- S. C. Kozma, A. P. Thomas, and G. Thomas. 2008. Amino acids activate mTOR teraction with the coactivator CBP/p300. Mol. Cell 1: 661–671. complex 1 via Ca2+/CaM signaling to hVps34. Cell Metab. 7: 456–465. 35. Parry, G. C., and N. Mackman. 1997. Role of cyclic AMP response element- 22. Zhang, H. H., A. I. Lipovsky, C. C. Dibble, M. Sahin, and B. D. Manning. 2006. binding protein in cyclic AMP inhibition of NF-kappaB-mediated transcription. S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition J. Immunol. 159: 5450–5456. of Akt. Mol. Cell 24: 185–197. 36. Parker, D., K. Ferreri, T. Nakajima, V. J. LaMorte, R. Evans, S. C. Koerber, 23. Cross, D. A., D. R. Alessi, P. Cohen, M. Andjelkovich, and B. A. Hemmings. C. Hoeger, and M. R. Montminy. 1996. Phosphorylation of CREB at Ser-133 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein induces complex formation with CREB-binding protein via a direct mechanism. Mol. Cell. Biol. kinase B. Nature 378: 785–789. 16: 694–703. 24. Pearce, L. R., G. R. Alton, D. T. Richter, J. C. Kath, L. Lingardo, J. Chapman, 37. Hu, X., P. K. Paik, J. Chen, A. Yarilina, L. Kockeritz, T. T. Lu, J. R. Woodgett, and L. B. Ivashkiv. 2006. IFN-gamma suppresses IL-10 production and syner- C. Hwang, and D. R. Alessi. 2010. Characterization of PF-4708671, a novel and gizes with TLR2 by regulating GSK3 and CREB/AP-1 proteins. Immunity 24: highly specific inhibitor of p70 ribosomal S6 kinase (S6K1). Biochem. J. 431: 563–574. 245–255. 38. Rodionova, E., M. Conzelmann, E. Maraskovsky, M. Hess, M. Kirsch, T. Giese, 25. McManus, E. J., K. Sakamoto, L. J. Armit, L. Ronaldson, N. Shpiro, A. D. Ho, M. Zo¨ller, P. Dreger, and T. Luft. 2007. GSK-3 mediates differenti- R. Marquez, and D. R. Alessi. 2005. Role that phosphorylation of GSK3 plays in ation and activation of proinflammatory dendritic cells. Blood 109: 1584–1592. insulin and Wnt signalling defined by knockin analysis. EMBO J. 24: 1571– 39. Beurel, E., S. M. Michalek, and R. S. Jope. 2010. Innate and adaptive immune 1583. responses regulated by glycogen synthase kinase-3 (GSK3). Trends Immunol. 31: 26. Wang, H., C. A. Garcia, K. Rehani, C. Cekic, P. Alard, D. F. Kinane, T. Mitchell, 24–31. Downloaded from and M. Martin. 2008. IFN-beta production by TLR4-stimulated innate immune 40. Dittrich, E., S. Schmaldienst, A. Soleiman, W. H. Ho¨rl, and E. Pohanka. 2004. cells is negatively regulated by GSK3-beta. J. Immunol. 181: 6797–6802. Rapamycin-associated post-transplantation glomerulonephritis and its remission 27. Rehani, K., H. Wang, C. A. Garcia, D. F. Kinane, and M. Martin. 2009. Toll-like after reintroduction of -inhibitor therapy. Transpl. Int. 17: 215–220. receptor-mediated production of IL-1Ra is negatively regulated by GSK3 via the 41. Haidinger, M., M. Hecking, T. Weichhart, M. Poglitsch, W. Enkner, K. Vonbank, MAPK ERK1/2. J. Immunol. 182: 547–553. D. Prayer, A. Geusau, R. Oberbauer, G. J. Zlabinger, et al. 2010. Sirolimus in 28. Sarbassov, D. D., S. M. Ali, S. Sengupta, J. H. Sheen, P. P. Hsu, A. F. Bagley, renal transplant recipients with tuberous sclerosis complex: clinical effectiveness A. L. Markhard, and D. M. Sabatini. 2006. Prolonged rapamycin treatment and implications for innate immunity. Transpl. Int. 23: 777–785.

inhibits mTORC2 assembly and Akt/PKB. Mol. Cell 22: 159–168. 42. Thaunat, O., C. Beaumont, L. Chatenoud, S. Lechaton, M. F. Mamzer-Bruneel, http://www.jimmunol.org/ 29. Jope, R. S., and G. V. Johnson. 2004. The glamour and gloom of glycogen B. Varet, H. Kreis, and E. Morelon. 2005. Anemia after late introduction of synthase kinase-3. Trends Biochem. Sci. 29: 95–102. sirolimus may correlate with biochemical evidence of a chronic inflammatory 30. Doble, B. W., and J. R. Woodgett. 2003. GSK-3: tricks of the trade for a multi- state. Transplantation 80: 1212–1219. tasking kinase. J. Cell Sci. 116: 1175–1186. 43. Garcia, C. A., M. R. Benakanakere, P. Alard, M. M. Kosiewicz, D. F. Kinane, 31. Shima, H., M. Pende, Y. Chen, S. Fumagalli, G. Thomas, and S. C. Kozma. 1998. and M. Martin. 2008. Antigenic experience dictates functional role of glycogen Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and synthase kinase-3 in human CD4+ T cell responses. J. Immunol. 181: 8363– a new functional S6 kinase. EMBO J. 17: 6649–6659. 8371. by guest on October 1, 2021