Cross Talk between MyD88 and Kinase Pathways Mirjam B. Zeisel, Vanessa A. Druet, Jean Sibilia, Jean-Paul Klein, Valérie Quesniaux and Dominique Wachsmann This information is current as of September 25, 2021. J Immunol 2005; 174:7393-7397; ; doi: 10.4049/jimmunol.174.11.7393 http://www.jimmunol.org/content/174/11/7393 Downloaded from

References This article cites 24 articles, 7 of which you can access for free at: http://www.jimmunol.org/content/174/11/7393.full#ref-list-1

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Cross Talk between MyD88 and Focal Adhesion Kinase Pathways1

Mirjam B. Zeisel,2* Vanessa A. Druet,2* Jean Sibilia,† Jean-Paul Klein,* Vale´rie Quesniaux,3‡ and Dominique Wachsmann3,4*

Focal adhesion kinase (FAK) is a nonreceptor kinase involved in signaling downstream of , linking bacterial detection, cell entry, and initiation of proinflammatory response through MAPKs and NF-␬B activation. In this study, using protein I/II from Streptococcus mutans as a model activator of FAK, we investigated the potential link between FAK and TLR pathways. Using macrophages from TLR- or MyD88-deficient mice, we report that MyD88 plays a major role in FAK- dependent protein I/II-induced cytokine release. However, response to protein I/II stimulation was independent of TLR4, TLR2, and TLR6. The data suggest that there is a cross talk between FAK and MyD88 signaling pathways. Moreover, MyD88-dependent,

LPS-induced IL-6 secretion by human and murine fibroblasts required the presence of FAK, confirming that MyD88 and FAK Downloaded from pathways are interlinked. The Journal of Immunology, 2005, 174: 7393–7397.

acterial recognition implicates a wide variety of pattern Focal adhesion kinase (FAK), a nonreceptor protein tyrosine recognition receptors specific for pathogen-associated kinase involved in signaling downstream of integrins (4), was B microbial patterns (PAMPs),5 including TLRs, lectins, shown recently to trigger inflammatory responses. Protein I/II, a scavenger receptors, and integrins. Among them, TLRs are critical cell wall component from oral streptococci, binding to ␣ ␤ http://www.jimmunol.org/ for the development of innate immune responses (1). TLR signal- 5 1 induces the production of inflammatory mediators such as ing is mediated by cytoplasmic Toll/IL-1R (TIR) domain homo- or IL-6 and IL-8 by human monocytes, epithelial cells, endothelial heteromeric associations with TIR-containing adaptors (2). cells, and fibroblasts (5, 6). The signaling events leading to this MyD88, an adaptor protein used by all TLRs except TLR3, acti- cytokine release involve FAK, ERK1/2, and JNKs as well as AP- vates MAPKs and NF-␬B, leading to proinflammatory cytokine 1-binding activity and nuclear translocation of NF-␬B (6, 7). FAK expression. MyD88-independent pathways, involving the TIR do- is an interesting candidate to link bacterial detection, initiation of main-containing adaptor inducing IFN-␤ (TRIF) are used by TLR4 proinflammatory responses, and cell entry, because FAK is in- and TLR3, together with TRIF-related adaptor molecule in the case of volved in invasin-mediated bacterial uptake (8). Based on these

TLR4. TRIF pathway activates IFN regulatory factor 3, leading to the observations, we investigated the possibility of a cross talk be- by guest on September 25, 2021 synthesis of IFN-␣ and -␤. Moreover, receptor interacting protein 1, tween the integrin/FAK and TLR pathways. a component of the TNFR signaling pathway, associates with the Using protein I/II from Streptococcus mutans as a model acti- C-terminal region of TRIF and mediates TLR4 and TLR3-NF-␬B late vator of FAK, together with macrophages from TLR- or MyD88- activation (3). Thus, various PAMP/pattern recognition receptor in- deficient mice, we show that MyD88 and FAK, but neither TLR2, teractions result in MAPK and NF-␬B activation and consequent TLR4, nor TLR6, are involved in the response to protein I/II that expression of proinflammatory cytokines, and there is now strong leads to cytokine release. This cross talk between FAK and evidence that many intracellular signaling molecules are shared MyD88 seems to be a general phenomenon in proinflammatory among the activated signaling pathways. However, the kinases in- cytokine response, because LPS-induced cytokine release also de- volved in these pathways are not yet fully elucidated. pends on the presence of both FAK and MyD88, as evidenced in primary human and murine cells. *Institut National de la Sante´et de la Recherche Me´dicale 392, Faculte´de Pharmacie, Illkirch, France; †Service de Rhumatologie, Hoˆpitaux Universitaires, Strasbourg, Materials and Methods France; and ‡Immunologie et Embryologie Moléculaires 2815, Transgenose Institute, Mice Centre National de la Recherche Scientifique, Orleans, France Six- to 12-wk-old mice deficient for TLR4 (from S. Akira (Department of Received for publication January 5, 2005. Accepted for publication March 25, 2005. Host Defense, Research Institute for Microbial Disease, Osaka University, The costs of publication of this article were defrayed in part by the payment of page Osaka, Japan); Ref. 9), for TLR2 (from C. Kirsching (Institute of Medical charges. This article must therefore be hereby marked advertisement in accordance Microbiology, Immunology and Hygiene, Technical University Munich, with 18 U.S.C. Section 1734 solely to indicate this fact. Munich, Germany); Ref. 10), for TLR6 (11), or for MyD88 (12), and their 1 This work was supported by grants from the French Ministry of Research, Pfizer, control littermates were bred under specific pathogen-free conditions in the Re´gion Alsace, Amgen, Caisse d’Assurance Maladie des Professions Libe´rales de Transgenose Institute animal breeding facility (Orleans, France). Province, Wyeth Lederle, and Centre National de la Recherche Scientifique. 2 M.B.Z. and V.A.D. contributed equally to this work. Cell cultures 3 V.Q. and D.W. contributed equally to this work. Human fibroblast-like synoviocytes (FLSs) were isolated from rheumatoid 4 Address correspondence and reprint requests to Dr. Dominique Wachsmann, Faculte´ arthritis synovial tissues at the time of knee joint arthroscopic synovectomy ϫ 3 de Pharmacie, Universite´Louis Pasteur, 74 route du Rhin, 67400 Illkirch, France. and cultured as previously described (7). FLSs (5 10 cells per well) E-mail address: [email protected] were grown to confluence in 96-well plates and serum-starved for 24 h before activation experiments. FAKϩ/ϩ and FAKϪ/Ϫ primary mouse em- 5 Abbreviations used in this paper: PAMP, pathogen-associated microbial pattern; TIR, Toll/IL-1R; TRIF, TIR domain-containing adaptor inducing IFN-␤; FAK, focal bryo fibroblasts (American Type Culture Collection; CRL-2644 and -2645) adhesion kinase; FLS, fibroblast-like synoviocyte; Pam CSK , S-[2,3-bis(palmitoy- were cultured as previously described (6). Cells were plated in 96-well 3 4 ϫ 4 loxy)-(2-RS)propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH, trihydrochloride; BLP, plates (5 10 cells per well) and serum-starved 24 h before activation bacterial lipoprotein; FRNK, FAK-related nonkinase. experiments.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 7394 CROSS TALK BETWEEN MyD88 AND FAK PATHWAYS

Transfections FAKϩ/ϩ cells were transfected with FRNK-YCam (a generous gift from K. Takeda) by the calcium phosphate method as previously described (6). Following transfection, cells were rinsed and then cultured in complete DMEM medium containing geneticin (1.5 mg/ml) for 2 wk. The antibiotic- resistant cells were then pooled and used for further analysis. GFP was used to determine the transfection efficiency. Transient transfection of FLSs was performed using the Nucleofector kit as previously described (6). Cells were then plated in 96-well plates (5 ϫ 104 cells per well) before activation experiments. Protein I/II purification Recombinant protein I/II of S. mutans OMZ 175 was purified from pHBsr- 1-transformed Escherichia coli cell extract by gel filtration and immuno- affinity chromatography as previously described (7). Potential endotoxin in protein I/II preparation was removed using polymyxin B-agarose (Detoxi- Gel), according to the manufacturer’s recommendation (Pierce). Protein I/II had an endotoxin content Ͻ0.01 ng/125 pM protein I/II, as tested by the Limulus chromogenic assay (Charles River Laboratories). Throughout this study, buffers were prepared with apyrogenic water obtained from Braun Medical. Downloaded from Cell activation Murine bone marrow cells were isolated from femurs of TLR- and MyD88- deficient and control mice and cultivated as previously described (13). The bone marrow-derived macrophages were plated in 96-well plates (105 cells per well) before activation experiments. Appropriate stimuli were diluted in serum-free RPMI 1640 or DMEM with antibiotics, except for LPS ac- tivation of fibroblasts, which was performed in medium containing 5% http://www.jimmunol.org/ heat-inactivated FCS. Cells were stimulated with 0.1 ␮g/ml LPS (E. coli, serotype O111:B4; Sigma-Aldrich), 0.5 ␮g/ml synthetic bacterial lipopep- tide S-[2,3-bis(palmitoyloxy)-(2-RS)propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-

Lys4-OH, trihydrochloride (Pam3CSK4; EMC Microcollections), or protein I/II (at the indicated concentrations). After stimulation, the supernatants were harvested and analyzed immediately or stored at Ϫ20°C until further use. Cell number and cell viability were examined by the 3-(4-5-dimethyl- FIGURE 1. Protein I/II stimulation of macrophages induces FAK phos- 2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test. Alternatively, stim- phorylation and requires MyD88 for TNF-␣ and NO secretion. A, Wild- ulated cells were used for Western blotting experiments. type macrophages were either incubated in medium alone or treated with

Western blot protein I/II (125 pM) for the indicated times. Cell lysates were analyzed by by guest on September 25, 2021 Western blotting with anti-FAK pY397 or anti-FAK Abs. The results A total of 106 cells was incubated for various times in 100 ␮l of medium shown are representative of two experiments. B and C, Macrophages from with or without protein I/II (125 pM) or LPS (0.1 ␮g/ml). from cell lysates were subjected to SDS-PAGE and transferred electrophoreti- wild-type mice or mice deficient for MyD88 were stimulated with protein ␣ cally to nitrocellulose membranes. FAK was detected using anti-FAK I/II or LPS at the indicated concentrations for 20 h. TNF- levels in culture (pY397; BioSource; Cliniscience) and anti-FAK polyclonal Abs (BD supernatants were quantified by ELISA (B), and nitrite levels were deter- Pharmingen; Cliniscience) as previously described (6). mined using the Griess reaction (C). Results are expressed as mean val- ues Ϯ SD and are representative of three to four experiments with n ϭ 2 Cytokine ELISA mice per genotype. Cell supernatants were assayed for cytokine content using commercially available ELISA reagents for TNF-␣, IL-6, and IL-8 (Duoset; R&D Systems). Nitrite measurements Protein I/II-induced TNF-␣ and NO release is dependent on Nitrite concentrations in cell supernatants were determined using the MyD88 Griess reaction (3% phosphoric acid, 1% p-aminobenzenesulfonamide, 1% In this study, we hypothesized that FAK might be one of the ty- N-(1-napthyl)ethylenediamide) as previously described (14). rosine kinases in cross talk with MyD88. To test this hypothesis, Results we first asked whether MyD88 is involved in the TNF-␣ and NO ␣ ␤ Protein I/II, a ligand of integrin 5 1, induces FAK responses of macrophages activated with protein I/II. Bone-mar- and TNF-␣ and NO production row-derived macrophages from MyD88-deficient mice failed to release significant TNF-␣ and NO concentrations in response to We showed previously that protein I/II, a ligand of integrin ␣ ␤ , 5 1 protein I/II (Fig. 1, B and C). Similar results were obtained with induces cytokine production by human endothelial and epithelial MyD88-deficient bone-marrow-derived dendritic cells (data not cells and FLSs and that this effect is FAK dependent (5, 6). In this shown). Thus, protein I/II-induced activation of cytokine and NO study, we first verified the capacity of protein I/II to stimulate secretion is mediated through MyD88. phosphorylation of FAK in murine bone marrow-derived macro- phages. Cell lysates were analyzed directly by blotting with spe- ␣ cific anti-FAK (pY397) Abs. Stimulation with protein I/II resulted Protein I/II-induced TNF- release is independent of TLR2, -4, -6 in an increasing amount of phosphorylated FAK (Fig. 1A), which It was suggested that TLRs could colocalize with other receptors was detectable within 5 min and remained elevated for at least 30 and that integrins may be part of this complex (15, 16). We next min. In addition, protein I/II induced a strong release of TNF-␣ asked whether TLRs might also play a role in macrophage acti- and NO by activated macrophages, reaching levels similar to those vation by protein I/II. To test this hypothesis, we used TLR2 and obtained after endotoxin stimulation (Fig. 1, B and C, wild-type). TLR4 knockout mice as sources of macrophages. We first focused The Journal of Immunology 7395 on TLR2 and TLR4, because they recognize bacterial ligands ex- pressed by most Gram-positive and Gram-negative bacteria: pep- tidoglycan, lipoteichoic acids, lipoproteins, and endotoxins. We examined TNF-␣ and NO production by TLR2Ϫ/Ϫ, TLR4Ϫ/Ϫ, and wild-type macrophages in response to protein I/II and used syn- thetic bacterial lipopeptide Pam3CSK4 (bacterial lipoprotein FIGURE 3. Protein I/II-induced FAK phosphorylation is independent (BLP)) and LPS as reference TLR2 and TLR4 agonists, respec- of MyD88. Macrophages from mice deficient for MyD88 were either in- tively. As shown in Fig. 2, protein I/II induced a dose-dependent cubated in medium alone or treated with protein I/II (125 pM) for the release of TNF-␣ and N⌷ in wild-type cells, and this release was indicated times. Cell lysates were analyzed by Western blotting with anti- not significantly different in TLR2- or TLR4-deficient cells, FAK pY397 or anti-FAK Abs. The results shown are representative of two whereas these cells did not respond to BLP and LPS, respectively. experiments. These results indicate that neither TLR2 nor TLR4 are essential for protein I/II-induced TNF-␣ and N⌷ release by activated macro- phages. Because TLR6 is known to constitute heterodimers with fore, although MyD88 is involved in the response to protein I/II Ϫ/Ϫ TLR2, we also tested the stimulation of TLR6 macrophages that leads to cytokine release, FAK phosphorylation seems to be with protein I/II and found unimpaired TNF-␣ and N⌷ production largely independent of the presence of MyD88, suggesting that compared with wild-type macrophages (data not shown). phosphorylation of FAK occurs upstream of MyD88 or that both The TLR2 and TLR4 independence of protein I/II activation pathways are parallel. was then confirmed in human FLSs, a cell population known to Downloaded from contribute to joint inflammation in rheumatoid arthritis patients. LPS stimulation induces FAK phosphorylation in macrophages Pretreatment with Abs directed against TLR4 or TLR2 had essen- and FLSs tially no effect on protein I/II-induced IL-8 release by FLSs (data Our data suggesting that MyD88 and FAK are both necessary in not shown). the signaling pathway induced by protein I/II interaction with in- Taken together, these data demonstrate that TLR2, TLR4, and ␣ ␤ tegrin 5 1, we next investigated whether this cross talk between

␣ http://www.jimmunol.org/ TLR6 are not involved in protein I/II-induced TNF- and NO FAK and MyD88 was a general phenomenon by assessing the role release by activated murine macrophages and in protein I/II-in- of FAK in signaling pathways known to involve MyD88. Cytokine duced IL-6 and IL-8 release by activated human FLSs. response to LPS is largely MyD88 dependent, and we examined the capacity of LPS to induce FAK phosphorylation in murine Protein I/II-induced FAK phosphorylation is independent of macrophages. After stimulation, cell lysates were analyzed directly MyD88 by blotting with specific anti-FAK (pY397) Abs as above. Stim- We then tested the role of MyD88 in FAK phosphorylation. Stim- ulation by LPS resulted in an increasing amount of phosphorylated Ϫ/Ϫ ulation of MyD88 macrophages with protein I/II resulted in an FAK (Fig. 4A), which was detectable within 5 min and remained increasing amount of phosphorylated FAK (Fig. 3), which was elevated for at least 30 min. Similar FAK phosphorylation was by guest on September 25, 2021 detectable within 5 min and maximal after 15 min of stimulation, obtained after LPS stimulation of human FLSs (Fig. 4B). Thus, comparable to what was shown in wild-type cells (Fig. 1A). There- LPS induced FAK phosphorylation in murine and human primary cells.

LPS-induced IL-6 release occurs via a FAK-dependent pathway We next asked whether FAK could contribute to the signaling events leading to cytokine release from LPS-activated cells. First, using FAKϩ/ϩ and FAKϪ/Ϫ primary mouse embryo fibroblasts, we showed that LPS induced IL-6 release by FAKϩ/ϩ fibroblasts, but there was essentially no IL-6 production by FAKϪ/Ϫ fibro- blasts ( p Ͻ 0.01). This suggested that FAK is involved in LPS- induced release of IL-6. To rule out the possibility that the lack of

FIGURE 2. Protein I/II-induced TNF-␣ and NO secretion is indepen- dent of TLR2 and TLR4. Macrophages from wild-type mice or mice de- FIGURE 4. LPS induces FAK phosphorylation in murine macrophages ficient for TLR2 or TLR4 were stimulated with protein I/II, BLP, or LPS and human synoviocytes. Murine macrophages (A) or human FLSs (B) at the indicated concentrations for 20 h. TNF-␣ levels in culture superna- were either incubated in medium alone or stimulated with protein I/II (125 tants were quantified by ELISA (A), and nitrite levels were determined pM) for the indicated times. Cell lysates were analyzed by Western blotting using the Griess reaction (B). Results are expressed as mean values Ϯ SD with anti-FAK pY397 or anti-FAK Abs. The results shown are represen- and are representative of three experiments with n ϭ 2 mice per genotype. tative of two experiments. 7396 CROSS TALK BETWEEN MyD88 AND FAK PATHWAYS

IL-6 release could be due to a nonspecific inhibition of other cel- of LPS to phosphorylate FAK in MyD88Ϫ/Ϫ cells. FAK phosphor- lular functions, FAKϩ/ϩ fibroblasts were transfected with FAK- ylation was not inhibited in MyD88Ϫ/Ϫ macrophages stimulated related nonkinase (FRNK), the C-terminal region of FAK, which is with LPS (Fig. 6), indicating that phosphorylation of FAK oc- known to block FAK activation when overexpressed. FAKϩ/ϩ fi- curred independently of MyD88. broblasts transfected with a GFP-expressing vector were used as a Taken together, our results show that MyD88 is involved in the ␣ ␤ control. As shown in Fig. 5A, overexpression of FRNK strongly response to protein I/II-integrin 5 1 interaction that leads to cy- inhibited IL-6 release from LPS-activated FAKϩ/ϩ cells ( p Ͻ tokine release through FAK phosphorylation, and this cross talk 0.01), confirming the involvement of FAK in LPS-induced IL-6 between the MyD88 and FAK pathways seems to be a general secretion. phenomenon in proinflammatory cytokine response, because Second, to further demonstrate the requirement of FAK in LPS- MyD88-dependent, LPS-induced IL-6 secretion also depends on induced cytokine release, we analyzed the cytokine response of the presence of FAK. human FLSs transiently transfected with FRNK. FLSs transfected with a GFP-expressing vector were used as a control. Fig. 5, B and Discussion C, shows that overexpression of FRNK significantly inhibited IL-6 In the present study, we first demonstrated that absence of MyD88 and IL-8 release from LPS-activated FLSs. Thus, FAK is an es- abrogates or severely impairs TNF-␣ and NO production by pro- sential component of the LPS-induced signaling pathways leading tein I/II-activated macrophages. These data suggest that MyD88, a to cytokine stimulation, both in murine and human fibroblastic key downstream adaptor of the TLR pathway, plays a major role cells. in protein I/II-integrin-induced cytokine release. We showed pre- Ϫ Ϫ viously, using FAK / and FRNK-transfected cells, that FAK is Downloaded from LPS-dependent FAK phosphorylation is independent of MyD88 essential in IL-6 and IL-8 release by protein I/II-activated FLSs To further characterize whether FAK phosphorylation was down- (6). Thus, we examined next how FAK and MyD88 pathways may stream of LPS-induced MyD88 recruitment, we tested the ability interact. Two hypotheses were formulated, namely, protein I/II may activate MyD88 by interacting with a member of the TLR family, or alternatively, protein I/II might trigger a signaling path-

way involving an intracellular cross talk between FAK and http://www.jimmunol.org/ MyD88. To address these questions, we first used macrophages isolated from TLR2Ϫ/Ϫ and TLR6Ϫ/Ϫ mice insofar as these TLRs recognize various components from Gram-positive bacteria. TLR4, the receptor of LPS from Gram-negative bacteria that also ␣ ␤ interacts with fibronectin, a ligand of integrin 5 1, was also tested. Our results clearly show that TLR2, TLR4, and TLR6 are not essential for the stimulation of macrophages by protein I/II. Also, Abs to TLR4 and TLR2 failed to inhibit protein I/II-induced IL-8 release by human FLSs (data not shown). These data are in by guest on September 25, 2021 agreement with the observation that macrophage activation by group B streptococci requires MyD88 but is independent of TLR1, -2, -4, and -6 (17). Thus, we excluded TLR2, TLR4, and TLR6 as potential receptors for protein I/II. We next showed that FAK plays an important role downstream of TLRs, because cytokine response to LPS is totally abrogated in FAKϪ/Ϫ cells. Furthermore, FAK-expressing cells transfected with the FAK inhibitor FRNK produced significantly lower amounts of IL-6 upon LPS challenge. This is in line with previous results showing that LPS induced FAK phosphorylation in a mu- rine monocytic cell line (18). We further demonstrated that other TLR ligands may also induce FAK phosphorylation at Tyr397 and require FAK for cytokine release because comparable results have

been obtained with the TLR2 ligand Pam3CSK4 (data not shown). Also, our findings extend to FAK the observation that TLR2 and TLR4 agonists induced tyrosine phosphorylation of the - rich 2 (Pyk2), which in turn increases tyrosine phosphorylation of paxillin, an adaptor protein involved in integrin

FIGURE 5. FAK is necessary for LPS-induced cytokines release. FAKϪ/Ϫ, FAKϩ/ϩ primary mouse embryo fibroblasts (A) and human FLSs FIGURE 6. LPS-induced FAK phosphorylation is independent of (B and C) transfected or not with FRNK were stimulated with LPS (0.1 MyD88. Macrophages from MyD88-deficient mice were stimulated with ␮g/ml) for 20 h. IL-6 and IL-8 levels in culture supernatants were deter- LPS (0.1 ␮g/ml) for the indicated times. Cell lysates were analyzed by mined by ELISA. Results are expressed as mean values Ϯ SD for triplicate Western blotting with anti-FAK pY397 or anti-FAK Abs. The results determinations and are representative of three experiments. shown are representative of two experiments. The Journal of Immunology 7397

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