Rhoa Gtpase Activation by TLR2 and TLR3 Ligands: Connecting Via Src to NF-␬B1

The Journal of Immunology

RhoA GTPase Activation by TLR2 and TLR3 Ligands: Connecting via Src to NF-␬B1

Maria Manukyan,* Perihan Nalbant,† Sylvia Luxen,* Klaus M. Hahn,‡ and Ulla G. Knaus2*

Rho GTPases are essential regulators of signaling networks emanating from many receptors involved in innate or adaptive immunity. The Rho family member RhoA controls cytoskeletal processes as well as the activity of transcription factors such as NF-␬B, C/EBP, and serum response factor. The multifaceted host cell activation triggered by TLRs in response to soluble and particulate microbial structures includes rapid stimulation of RhoA activity. RhoA acts downstream of TLR2 in HEK-TLR2 and monocytic THP-1 cells, but the signaling pathway connecting TLR2 and RhoA is still unknown. It is also not clear if RhoA activation is dependent on a certain TLR adapter. Using lung epithelial cells, we demonstrate TLR2- and TLR3-triggered recruitment and activation of RhoA at receptor-proximal cellular compartments. RhoA activity was dependent on TLR-mediated stimulation of Src family kinases. Both Src family kinases and RhoA were required for NF-␬B activation, whereas RhoA was dispensable for type I IFN generation. These results suggest that RhoA plays a role downstream of MyD88-dependent and -independent TLR signaling and acts as a molecular switch downstream of TLR-Src-initiated pathways. The Journal of Immu- nology, 2009, 182: 3522–3529.

oll-like receptors recognize conserved microbial and viral adapter complexes cause activation of NF-␬B-dependent gene tran- ligands and trigger signaling pathways required for scription and of MAPK/JNK/p38 pathways, although TLR3 mediates T mounting a host immune response against infection by additionally type I IFN generation. Thus, receptor-proximal events these pathogens. In the lung, the pulmonary innate immune system differ between TLR2 and TLR3, connecting TLR-adapter complexes serves as the first line of defense against aerosolized pathogens. A to separate signaling cascades. major component of this lung immune response is the airway ep- Certain signaling molecules have been tentatively connected to ithelium, which provides not only the mechanical barrier and in- MyD88-dependent and -independent TLR signaling. For example, terface between the environment and the host but also is uniquely a PI3K-Akt pathway is triggered by several TLRs, independently equipped for sensing and responding to inhaled bacterial and viral of their adapter usage or cellular localization (8–10). The associ- organisms. Lung epithelial cells respond to many TLR ligands, ation between TLR2 and the p85 regulatory subunit of PI3K re- although the participation of TLR4 under noninflammatory con- quires the tyrosine-phosphorylated intracellular TIR domain of ditions seems to be limited due to the intracellular localization and TLR2 (8). Inducible TLR tyrosine phosphorylation has been linked decreased LPS sensitivity of this receptor in airway epithelial cells to activation of Src family kinases, which seem to be an integral (1–4). In contrast, TLR2 represents an important functional recep- part of the TLR2 and TLR3 signaling complex (11–14). Another tor for bacterial recognition at the lung epithelial cell surface. Key component of TLR complexes is the tyrosine kinase Syk, which bacterial lung pathogens such as Staphylococcus aureus, Strepto- relays signals downstream of integrin engagement and takes part in coccus pneumonia, and Pseudomonas aeruginosa initiate TLR2- ITAM-containing immunoreceptor signal transduction (9, 15, 16). mediated signaling responses (2, 5). Recognition of viral lung Syk activation seems to be the result of receptor cooperation, in pathogens is accomplished by TLR3, located in vesicular endoso- case of TLR2 and TLR4 with the ␤-glucan receptor dectin-1 (17), mal compartments, or by cytosolic receptors such as RIG-I and or with CD36 for TLR2/TLR6 (18, 19). Signaling events down- MDA5 (6, 7). TLR2- and TLR3-initiated signaling differs in its use stream of TLRs have also been connected to Rho family GTPases. of adapter molecules, where TLR2 connects to TIRAP/MyD88, We and others (8, 20, 21) reported a rapid and transient increase in while dsRNA-stimulated TLR3 recruits TIR-domain-containing Rac1 and RhoA activity when TLR2 and TLR4 stimulation by adapter-inducing IFN-␤ (TRIF).3 Further downstream, both TLR- soluble pathogen-associated molecular patterns occurred. Rho GT- Pases are molecular switches that regulate essential cellular processes including actin dynamics, gene transcription and motility. *Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037; †Center for Medical Biotechnology, University of Duisburg- The activity of Rho GTPases is controlled by guanine nucleotide Essen, Essen, Germany; and ‡Department of Pharmacology, University of North exchange factors (GEFs), which are commonly stimulated by ty- Carolina, Chapel Hill, NC 27599 rosine phosphorylation and/or phospholipid binding. In the context Received for publication July 14, 2008. Accepted for publication January 9, 2009. of TLR signaling, the RacGEF Vav-1 is activated via a TLR9-Src The costs of publication of this article were defrayed in part by the payment of page pathway, while the RhoGEF AKAP13 is involved in TLR2 re- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. sponses (22, 23). Previous studies in TLR2-expressing HEK293 cells indicated that RhoA was not an integral part of the TLR2/ 1 This work was supported by National Institutes of Health Grants AI35947 and GM37696 (to U.G.K.) and GM57464 (to K.M.H.). IL-1R-associated kinase/TNFR-associated factor 6 complex but ␨ ␬ 2 Address correspondence and reprint requests to Dr. Ulla G. Knaus, Department of was required for PKC -dependent NF- B transactivation (20). Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, IMM28, La Jolla, CA 92037. E-mail address: uknaus@scripps. GEF, guanine nucleotide exchange factor; MEF, murine embryonic fibroblast; SALE, edu small airway lung epithelial; SI, Src kinase inhibitor I; siRNA, small interfering RNA. 3 Abbreviations used in this paper: TRIF, TIR-domain-containing adapter-inducing IFN-␤; EGF, epidermal growth factor; FRET, fluorescence resonance energy transfer; Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802280 The Journal of Immunology 3523

Since RhoA, Src, and Syk play a role in transmitting signals to NF-␬B in several cellular systems, we hypothesized that these proteins could be connected in a TLR-proximal signaling cascade, regulating NF-␬B-dependent gene transcription. Thus, biochemical and spatiotemporal detection of active, GTP-bound RhoA in pathogen-associated molecular pattern-stimulated lung epithelial cells was conducted. RhoA activation was observed in distinct cellular compartments and required Src activity. TLR2 and TLR3 stimulation triggered the Src-RhoA pathway to NF-␬B-dependent gene transcription, whereas RhoA was not involved in TLR3/Src- initiated type I IFN generation.

Materials and Methods Cells and reagents The immortalized primary human lung epithelial cells (small airway lung epithelial (SALE)) were a gift from Dr. W. C. Hahn (Harvard Medical School, Boston, MA) and were described previously (24). SABM medium, SAGM SingleQuots, and trypsin/EDTA used for SALE cells were purchased from Lonza. L929-ISRE cell line (ISRE-luciferase reporter) was provided by Dr. B. Beutler (The Scripps Research Institute, La Jolla, CA). The synthetic lipopeptide MALP-2 was obtained from Alexis, Pam3CSK4 from InvivoGen, and poly(I:C) was purchased from Amersham Bio- sciences and dissolved in pyrogen-free distilled water. dsRNA (a 34-bp sequence from human rhinovirus 16) labeled with Alexa 633 at the 3Ј-end was synthesized by Invitrogen. RhoA mAb (26C4), I␬B␣ polyclonal Ab (C-21), and TLR2 Ab (H-175) were from Santa Cruz Biotechnology; phospho-IRF3 (Ser386) was from IBL; IRF3, phospho-IRF3 (Ser396), phospho- Src (Tyr416), phospho-Syk (Tyr525–526), phospho-p38, p38, Fyn, Hck, Src, Yes, and Lyn Abs were from Cell Signaling Technology; Myc (9E10) mAb was from Covance Research Products; and GM130 mAb was from BD Pharmingen. AKAP-13 Ab was from Bethyl Laboratories. Secondary Ab labeled with Alexa 647 was purchased from Invitrogen. PP2, PP3, Src kinase inhibitor I (SI), and piceatannol were obtained from Calbiochem.

DNA constructs FIGURE 1. SALE airway cells contain functional TLR2 and TLR3. A, All plasmids were prepared using endotoxin-free plasmid DNA puriﬁca- I␬B␣ degradation and p38 phosphorylation are induced by MALP-2. tion (Qiagen). RhoA biosensor (RhoA-CFP-RBD-YFP) was described pre- SALE cells were stimulated for indicated time points with MALP-2, and viously (25). An expression plasmid containing the RhoA biosensor, len- lysates were analyzed by immunoblotting. Phospho-p38 and I␬B␣ blots tiviral packaging plasmids, and pVSV-G were provided by Dr. K. Wong were stripped and reprobed for p38 and actin as loading controls. One (University of California, San Francisco, CA (26)). Myc-RhoA wt and representative experiment is shown (n ϭ 3). B,I␬B␣ degradation and p38 Myc-RhoA T19N were described previously (20). The NF-␬B-responsive phosphorylation are induced by poly(I:C). SALE cells were stimulated for luciferase reporter (5ϫ NF-␬B-Luc) was from Promega. c-Src K295M was provided by Dr. D. Schlaepfer (University of California, San Diego, CA). indicated time points with poly(I:C), and lysates were analyzed by immunoblotting. Phospho-p38 and I␬B␣ blots were stripped and reprobed for RhoA biosensor expressing SALE p38 and actin as loading controls. One representative experiment is shown (n ϭ 3). C, IRF3 phosphorylation is induced by poly(I:C). SALE cells were Cotransfection of HEK293T cells with RhoA biosensor, packaging plas- stimulated for indicated time points with poly(I:C), and lysates were ana- mid, and VSV-G was performed by calcium phosphate method. After 2–3 lyzed by immunoblotting. Phospho-IRF3 (S396) and total IRF3 are shown days, virus-containing supernatants were collected and used for transduc- 386 tion of SALE cells. Cells were incubated with viral supernatant for 6–8 h, in upper two panels. Phospho-IRF3 (Ser ) was analyzed by native PAGE, and medium was then replaced with fresh SABM medium with SAGM followed by immunoblotting (panel 3). Blots were stripped and probed for SingleQuots. The procedure was repeated 24 h later. RhoA biosensor-ex- total IRF3 as loading control (panel 4). One representative experiment is pressing SALE cells were positively selected by FACS (98% positive) for shown (n ϭ 2). low to medium expressers.

Transfection and reporter assays Transfection of SALE cells was done using Lipofectamine Plus (Invitro- IFN type I production ϫ 6 gen). SALE cells were plated in 6-well plates at a density of 0.2 10 / L929 cells stably expressing an ISRE luciferase reporter construct were well. Twenty-four hours later, cells were transiently transfected with 100 ␬ provided by Dr. B. Beutler. For measurements of IFN type I production, ng of NF- B luciferase reporter plasmid alone or cotransfected with RhoA L929-ISRE cells were plated in 96-well plate at a density 5 ϫ 104/well. wt (200 ng), RhoA T19N (400 ng), c-Src K295M (400 ng), or empty Twenty-four hours later, medium was removed before adding superna- vector. Eighteen hours later (36 h for cotransfection), cells were starved for tants of SALE cells stimulated with poly(I:C) (100 ␮l/well). After 4 h, 3 h in SABM medium with 0.5% FBS. Stimulation with MALP-2 (10 ␮ L292-ISRE cells were washed with PBS, lysed in luciferase assay buffer ng/ml), Pam3CSK4 (100 ng/ml), or poly(I:C) (10–30 g/ml) was for 4 h. (Promega), and luminescence was measured with a LB 960 Centro Mi- Cells were lysed in luciferase assay buffer (Promega), and luminescence of croplate Luminometer in triplicates (n ϭ 3 independent experiments). lysates was measured in triplicates for each independent assay (n ϭ 3). For small interfering RNA (siRNA) treatment, Stealth Select Syk RNA inter- Rho activation assay ference (HSS110401, HSS110402, and HSS110403) (Invitrogen) was tested using Dharmafect 4 (Dharmacon), and HSS110402 RNA interfer- SALE cells were plated in 10-cm dishes (Fisher Scientiﬁc) at a concen- ence (20 nM) was used for Syk knockdown. Forty-eight hours later, 5ϫ tration of 0.8 ϫ 106/ml in SABM medium with supplements. Forty-eight NF-␬B luc was transfected, followed 24 h later by starvation and cell hours later, cells were starved in SABM/0.5% FBS overnight, followed by stimulation as described earlier. RBD pull-down assay as described previously (20). Stimulation with 3524 RhoA IN TLR SIGNALING

FIGURE 2. TLR2- and TLR3-mediated activation of RhoA. A, Time-dependent activation of endogenous RhoA by MALP-2 and poly(I:C) in SALE cells. Upper panel shows RhoA-GTP, and lower panel shows total RhoA in lysates (10% of pull-down lysate). One representative experiment is shown (n ϭ 3). B, RhoA biosensor activation by MALP-2 and poly(I:C). Representative FRET ratio images and RhoA-YFP images of SALE/RhoA cells stimulated with MALP-2 for 5 and 10 min or poly(I:C) for 30 and 60 min (scale bar, 10 ␮m). Quantification of whole-cell emission ratios is shown; number of cells first panel: n (Ϫ) ϭ 59; n (MALP-2, 5 min) ϭ 78; second panel: n (Ϫ) ϭ 70; n (MALP-2, 10 min) ϭ 70; third panel: n (Ϫ) ϭ 29; n (poly(I:C) .(ء) and Յ 0.05 ,(ءء) Յ 0.01 ,(ءءء) min) ϭ 19; fourth panel: n (Ϫ) ϭ 29; n (poly(I:C), 60 min) ϭ 34; error bars represent SEs; values of p Յ 0.001 ,30 C, Colocalization of the RhoA biosensor with dsRNA. Representative FRET ratio images and RhoA-YFP images (upper panel) as well as dsRNA colocalization with FRET and RhoA-YFP (lower panel) in SALE/RhoA cells stimulated with dsRNA/A633 (3 ␮M) for 30 min. Colocalization is in white; the colocalization coefficient for red to green was 0.83 (FRET) and 0.75 (Rho/YFP) analyzed by Image J (version 1.33). FRET ratio images in B and C are scaled so that regions of highest RhoA activity are shown in red. Scale bar, 10 ␮m.

MALP-2 (10 ng/ml) or poly(I:C) (30 ␮g/ml) was performed for the indi- microcystein, 2 ␮g/ml leupeptin, 2 ␮g/ml aprotinin, 1 ␮g/ml pepstatin, and cated times. For some experiments, cells were preincubated with inhibitors 2 mM PMSF). Lysates were cleared by centrifugation and incubated with 30 min before stimulation. Protein samples were analyzed by SDS-PAGE AKAP13, TLR2, or rabbit IgG Abs for 1.5 h at 4°C. Incubation with and immunoblotting with anti-RhoA Ab. Sepharose G beads was performed for 45 min at 4°C. After four washes, samples were resuspended in sample buffer, boiled for 5 min, and analyzed Immunoblotting by SDS-PAGE and immunoblotting. For the detection of phospho-IRF3 (Ser386), the procedure was as described previously (27). All other immunoblots were performed as described else- where (28). Densitometry of immunoblots was performed by using the Sample preparation for confocal microscopy GS-800 Calibrated Densitometer (BioRad) and Quantity One software For fluorescence resonance energy transfer (FRET) experiments SALE/ (version 4.5.2). Average density of the protein bands was normalized to the RhoA cells (0.2 ϫ 105) were seeded on glass coverslips in 35-mm tissue corresponding bands of the loading controls. culture dishes (Fisher Scientific). Stimulation with MALP-2 (10 ng/ml) for ␮ Immunoprecipitation 0–10 min or poly(I:C) (30 g/ml) for 0–30 min in SABM with 0.5% FBS was performed after overnight starvation in the same media. When indi- SALE cells stably expressing Myc-RhoA were plated in 10-cm dishes cated, SI (1 ␮M) was added to cells 30 min before stimulation. Cells were (Fisher Scientific) at a concentration of 1 ϫ 106/ml in SABM medium with fixed with 4% paraformaldehyde for 15 min at room temperature and supplements. Forty-eight hours later, cells were starved in SABM/0.5% mounted in Pro Long Gold mounting medium (Invitrogen). For colocal- FBS overnight. After stimulation with MALP-2 (10 ng/ml) for 10 min, ization experiments, cells were incubated with dsRNA-Alexa 633 (3 ␮M) cells were washed with PBS and lysed (10 mM Tris-Cl, 100 mM NaCl, 1 for 30 min and then fixed with 4% paraformaldehyde for 15 min at room

mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na2P2O7, 1% Triton X-100, temperature and mounted in Pro Long Gold mounting medium (Invitro- 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 2 mM orthovanadate, 1 ␮M gen). An average of 50 cells was analyzed per condition at each time point. The Journal of Immunology 3525

Confocal microscopy and image processing Imaging was performed with a confocal microscope (2100 Radiance; Bio- Rad) using a ϫ60 oil immersion objective. Intramolecular FRET, as previously described (29), was measured by exciting CFP with a 405-nm laser line and using the sequential scan acquisition mode for the CFP and FRET (YFP) channels; additionally, a YFP image was acquired by excitation with a 488-nm laser line. Three images were acquired in the same order: CFP, FRET, and YFP in all experiments. Ratiometric image analysis of FRET was performed using Image Pro 3DS software (version 6.0; Media Cyber- netics) and LSM examiner software. The YFP image with high signal-to- noise ratio served to create a binary mask with a value of zero outside and value of one inside the cell using a threshold-based procedure. Subse- quently, CFP and FRET images were multiplied by the mask. Dividing a FRET-masked image by a CFP-masked image and multiplying this value by a factor of 1000, a ratio image was obtained. Colocalization analysis was performed using NIH image analysis software packages Image J (version 1.33) and LSM examiner software (version 6; BioRad-Zeiss Laser- Sharp 2000). Statistics Indicated experimental data were analyzed for statistical significance using the Student t test for paired samples. Significance is indicated by asterisks and described in figure legends. Results Responsiveness of SALE cells to TLR ligands Human airway epithelial (SALE) cells express a variety of TLRs, although they are not very responsive to LPS stimulation (30). Cell surface expression of TLR2 and CD14 as well as intracellular expression of TLR3 was confirmed by flow cytometry (data not shown). TLR2 and TLR3 reside in different cellular compartments and use distinct adapters such as TIRAP/MyD88 and TRIF. SALE cells were analyzed for their responsiveness to TLR2 and TLR3 FIGURE 3. Inhibition of RhoA blocks NF-␬B transactivation by TLR2 ligands by using diacylated lipopeptide (MALP-2), Pam3CSK4, and TLR3 stimulation. SALE cells were transiently transfected with NF- and dsRNA (poly(I:C)). The specificity of TLR2 and TLR3 ligands ␬B-luc and plasmids as indicated before stimulation with MALP-2 (A)or was confirmed using TLR2 blocking Ab or chloroquine pretreat- poly(I:C) (B and C) for 4 h. Lysates of A and B were analyzed in chemi- ment, followed by NF-␬B-luciferase reporter assays (data not luminescence and by immunoblot for protein expression (data not shown), shown). For initial screening of the NF-␬B pathway and of MAPK and supernatants (C) were collected and used to determine type I IFN -to stim (ءءء) production. Error bars represent SDs; p values are Յ0.001 activation, I␬B␣ degradation and p38 phosphorylation by these ulated vector control. Data of one representative experiment are shown ligands were assessed. Airway cells did not respond well to the (n ϭ 3). TLR2 ligand Pam3CSK4 (see NF-␬B activation; supplemental Fig. 1A).4 In contrast, MALP-2-induced degradation of I␬B␣ was detected after 15 min, and I␬B␣ was completely resynthesized in visualization of signaling molecule recruitment. To visualize local 90 min (Fig. 1A). Phosphorylation of p38 by MALP-2 occurred at RhoA activity changes caused by TLR-mediated signals, a single- 30 min. A similar pattern was observed with poly(I:C) (Fig. 1B), chain FRET sensor was introduced into SALE cells. A RhoA bi- although the overall kinetic was delayed due to internalization of osensor, which permits detection of RhoA activation in real time, the TLR ligand and the TLR3 response from the endosomal com- was lentivirally transduced into SALE cells. These cells, termed partment. It has been previously shown that TLR3 stimulation gen- SALE/RhoA, were characterized for their RhoA content and erates IRF3-dependent type I IFN. Accordingly, poly(I:C) stimu- screened for their TLR ligand responsiveness. SALE/RhoA cells lation of SALE cells induced IRF3 phosphorylation at Ser396 and expressed similar levels of endogenous RhoA and the RhoA bio- at Ser386. Dimer formation of phospho-IRF3 (S386), a prerequisite sensor (supplemental Fig. 1B). Expression of the RhoA biosensor for IRF3 transcriptional activity, was observed after 60 min (Fig. did not disturb TLR signaling, because SALE/RhoA cells re- 1C). Thus, major TLR2 and TLR3 signaling pathways are func- sponded equally well to TLR ligands as nontransduced SALE cells tional in SALE cells. (supplemental Fig. 1, C and D). To study the spatiotemporal dy- TLR2 and TLR3 signaling leads to RhoA activity at distinct namics of TLR-triggered RhoA activation, SALE/RhoA cells were cellular compartments either left unstimulated or stimulated with MALP-2 or poly(I:C) for various time periods. Cells were fixed and analyzed by confo- TLR2 stimulation triggers activation of the Rho GTPase RhoA in cal microscopy for increased FRET. RhoA activation is presented TLR2-expressing model cell lines and monocytic cells (20). Thus, as CFP/FRET emission ratio and is visualized by color-coding the RhoA activation was determined in SALE cells stimulated either images with scaling from low (blue) to high (red) FRET, whereas with MALP-2 or for comparison with poly(I:C). Pull-down assays biosensor localization is shown in RhoA-YFP images. Short stim- showed a rapid increase in RhoA activity after 5 min, whereas ulation of SALE/RhoA cells with MALP-2 (5 min) increased the poly(I:C)-induced RhoA activation was delayed, as expected for emission ratios with areas of the highest intensity close to cell signaling by intracellular TLR3 (Fig. 2A). SALE cells are charac- edges. At the same time, enrichment of RhoA was detected at terized by large, spreading cell bodies, which is advantageous for membrane ruffles (Fig. 2B, panel 1). Active RhoA moved from the cell edges to the cytoplasm at later time points (10 min shown). 4 The online version of this article contains supplemental material. Performance of the RhoA biosensor was validated by coexpression 3526 RhoA IN TLR SIGNALING

FIGURE 4. Src and Syk mediate TLR2- and TLR3-induced NF-␬B-dependent gene transcription. A, SALE cells were stimulated with MALP-2 for 10 min with or without preincubation with PP2. Lysates were analyzed for Src activation (phospho-Src Tyr416). Actin served as control. B, SALE cells were stimulated with MALP-2 or EGF (1 ng/ml), and lysates were analyzed for Syk activation using phospho-Syk Tyr525/526 Ab. To- tal Syk served as control. C and F, SALE cells were transiently transfected with NF-␬B-luc and preincubated with SI (10 ␮M), PP2 (10 ␮M), PP3 (10 ␮M), or piceatannol (25 ␮M) before incubation with MALP-2 or poly(I:C) for 4 h. D and E, SALE cells were either cotransfected with c-Src K295M (24 h) and 5ϫ NF-␬B luc (D) or treated with Syk siRNA (no. 2, 20 nM, 48 h) before 5ϫ NF-␬B luc transfection (E). Lysates of C–F were analyzed for chemiluminescence. Super- natants (G) from F were collected to determine type I IFN production. Er- ror bars represent SDs; p values are -to stimu (ء) and Յ0.05 (ءء) Յ0.01 lated control. Data of one representative experiment are shown (n ϭ 3).

of dominant-negative RhoA, which reduced the emission ratio of harboring a NF-␬B responsive, luciferase-coupled promoter in- MALP-2-stimulated, Myc-RhoA DN-positive cells (supplemental hibited TLR2 NF-␬B activation. Overexpression of RhoA wt Fig. 1E). An increase in FRET was also observed after 15–30 min (ϳ3- to 6-fold) in these cells augmented NF-␬B activity. RhoA of poly(I:C) stimulation (Fig. 2B, panel 3), although highest RhoA signaling was also required for TLR3-mediated NF-␬B activa- activity was in this case concentrated in perinuclear, vesicular tion (Fig. 3B), although production and release of IFN-␣␤ was structures. After 60 min of poly(I:C) stimulation, RhoA activity independent of RhoA (Fig. 3C). was still maintained. Thus, the imaging time course of RhoA activity correlated well with the biochemical assay. To confirm co- Src family kinase activity is required for TLR2 and TLR3 ␬ localization of active RhoA with dsRNA-containing vesicles, signaling to NF- B which are TLR3 signaling compartments (11), SALE/RhoA cells Recent reports (11, 14) placed Src family kinases as receptor-prox- were incubated with labeled dsRNA. After 30 min of stimulation, imal regulators of TLR signaling. Similarly, the tyrosine kinase the labeled TLR3 ligand colocalized with areas of high FRET sig- Syk was implicated in TLR pathways (9, 15, 16). When analyzing nal (active RhoA) (Fig. 2C, upper panel) as well as with RhoA- MALP-2-induced Src kinase activation, several immunoreactive YFP (Fig. 2C, lower panel). bands were detected using pan-phospho-Src Tyr416 Ab (Fig. 4A). Tyrosine phosphorylation of these proteins was blocked by the Src ␬ RhoA is required for NF- B activation, but dispensable for type kinase inhibitor PP2. Immunoblotting revealed that SALE cells I IFN generation express multiple Src family kinases. The tyrosine-phosphorylated Previous studies in HEK293-TLR2 cells demonstrated a regulatory band at 68 kDa may represent c-Src or Yes kinases; the 130-kDa role for RhoA in TLR2-initiated NF-␬B transactivation. These data band remains unidentified (supplemental Fig. 2). In addition, rapid were verified in SALE cells, which present a more physiologically activation of Syk by the TLR ligand MALP-2 was detected, using relevant cell type. As shown in Fig. 3, expression of dominant- epidermal growth factor (EGF) stimulation of SALE cells as pos- negative RhoA or Clostridium botulinum C3 toxin in SALE cells itive control (Fig. 4B). The Journal of Immunology 3527

FIGURE 5. TLR2- and TLR3-mediated RhoA activation requires Src. A, MALP-2 and poly(I:C)-stimulated RhoA activity is inhibited by Src kinase inhibitors. SALE cells were stimulated with MALP-2 for 10 min with or without preincubation with SI or PP2. Lysates were analyzed for RhoA activity by pull-down assay (RhoA-GTP, total RhoA 10% of lysate) and quantitated by densitometry (see Materials and Methods). B, RhoA activity was analyzed by FRET using RhoA biosensor (representative FRET ratio images and RhoA-YFP images of SALE/RhoA cells are shown; scale bar is 10 ␮m). FRET ratio images are scaled so that regions of highest RhoA activity are shown in red. Quantiﬁcation of whole-cell emission ratios are shown; number of cells ,C .(ءء) and Յ 0.01 (ءءء) analyzed was n (Ϫ) ϭ 32; n (MALP-2) ϭ 52; n (SI/MALP-2) ϭ 45; error bars represent SEs; values of p Յ 0,001 MALP-2-stimulated RhoA activity is inhibited by Syk kinase inhibitor. SALE cells were stimulated with MALP-2 for 10 min with or without piceatannol preincubation. Lysates were used for RBD pull-down assay and total RhoA immunoblotting. Data in A–C are representative of three independent experiments.

Next, the involvement of Src family kinases and Syk in TLR2- inhibition (Fig. 5B). Overall, these data indicate that Src signaling and TLR3-initiated pathways was probed. Inhibition of Src kinases is essential for transmitting TLR2 and TLR3 signals to RhoA. with PP2 or SI, and of Syk with piceatannol, significantly reduced MALP-2-stimulated NF-␬B activation (Fig. 4C). These data were Syk kinase is upstream of TLR2-induced RhoA activation confirmed using transfection of dominant-negative c-Src or by Syk seems to be involved in transmitting TLR2 and TLR3 signals siRNA knockdown of Syk (Fig. 4, D and E). Similarly, poly(I:C)- to NF-␬B in SALE cells. Analysis of MALP-2-stimulated SALE stimulated NF-␬B activation was abolished by Src kinase inhibi- cells showed that Syk kinase activity was necessary for RhoA tion (Fig. 4F), whereas PP3, an inactive analog of PP2, did not activation (Fig. 5C). Although piceatannol treatment of SALE alter NF-␬B-dependent gene transcription. Piceatannol treatment cells increased RhoA activity in unstimulated cells, MALP-2-trig- of SALE cells had a modest effect on TLR3 ligand-stimulated gered RhoA activity was decreased. Interestingly, poly(I:C)-medi- NF-␬B activation. Both, Src kinase and Syk inhibitors abolished ated RhoA activation was not altered by Syk inhibition (data not poly(I:C)-induced IFN-␣␤ production (Fig. 4G). Supernatants de- shown). The Syk inhibitor piceatannol showed strong autofluores- rived from unstimulated, piceatannol-treated SALE cells reduced cence, thus precluding imaging studies and confirmation of the baseline transcriptional activity in L929-ISRE cells, which changes in biosensor FRET in the presence or absence of this could indicate some unspecific effect of this compound. compound. Src family kinases are upstream of TLR2- and TLR3-induced RhoA activation Discussion Previous studies indicated that the TLR2-RhoA pathway may di- Rho GTPases are master regulators of many immunoreceptors, verge from the canonical TLR2-MyD88 pathway (20). Src family ranging from receptors required for differentiation and maturation kinases have been implicated in TLR-TIR domain phosphoryla- of immune cells and for pathogen recognition to receptors in- tion, thus providing initial binding sites for association of signaling volved in pathogen uptake and the subsequent host signaling re- complexes. Src-mediated phosphorylations are also often prereq- sponses. Several TLRs induce the activation of the Rho family uisite for GEF and thus GTPase activation, placing Src upstream of GTPases Rac, Rho, and Cdc42. Almost every cell type, including GTPases such as RhoA. Preincubation of SALE cells with the Src professional innate immune cells such as macrophages, neutro- kinase inhibitors SI and PP2 abolished RhoA activation by phils, and dendritic cells, responds to TLR stimulation by activat- MALP-2 and by poly(I:C) when pull-down assays were analyzed ing Rho GTPases rapidly (8, 20, 31). Similarly, lung epithelial (Fig. 5A). The effect of Src kinase inhibitors on RhoA activation cells induce Rho GTPase activation when they encounter TLR was also studied by FRET using biosensor-expressing SALE/ ligands. It seems apparent that RhoA activation depends on the RhoA cells. As shown in Fig. 5B, MALP-2-stimulated RhoA ac- interaction of a specific TLR ligand with its cognate TLR, inde- tivity was reduced in the presence of SI. Analysis of the whole-cell pendently of the connecting adapters or the cellular compartment emission ratios revealed a significant FRET reduction upon Src where TLR signaling occurs. A RhoA FRET probe was used to 3528 RhoA IN TLR SIGNALING examine localized RhoA activity at early time points after initia- TLR2 and AKAP13 was observed (supplemental Fig. 3), the pres- tion of TLR2 or TLR3 signaling. After 3–5 min of treatment with ence of RhoA in this complex could not be confirmed. It seems the TLR2 ligand MALP-2, RhoA was rapidly activated at ruffling likely that rapid association and dissociation of RhoA from its areas at the cell surface. Later on, RhoA activity was detected in GEF takes place. In general, the well-characterized role of Rho vesicular structures, which moved to the perinuclear region and GTPases in controlling cytoskeletal remodeling may provide a may contain internalized TLR2. To visualize colocalization of platform for constant assembly and disassembly of the TLR sig- TLR3 ligands with RhoA-YFP and with areas of high FRET, nalosome. Receptor dimerization, coreceptor usage, and involve- which indicate high RhoA activity, a labeled dsRNA ligand was ment of cooperating receptors and integrins may be needed for used. Areas of colocalization and FRET were predominantly de- association of recruited signaling components. During these tected in perinuclear, speckle-like structures. These structures are events, Rho GTPases may participate in the dynamic assembly of reminiscent of TRIF speckles, which formed in HeLa cells in re- signaling platforms at specific cellular locations. sponse to poly(I:C) stimulation (32). These speckles contained the receptor-interacting protein 1, a signaling molecule connecting Acknowledgments TRIF to NF-␬B activation, and NF-␬B-activating kinase-associ- We thank Dr. W. Kiosses for advice, Katrina Schreiber for graphic assis- ated protein 1, which links TRIF to TANK-binding kinase 1 and tance, and Monica Ruse for critical reading of the manuscript. the IRF3-IFN-␣␤ pathway. Energy transfer from TRIF to NAP1 was not efficient, whereas RIP1 was tightly associated with TRIF Disclosures (32). Our data suggest that RhoA is part of this putative speckle The authors have no financial conflict of interest. signalosome, and that incorporation of RhoA into the TRIF sig- References naling complex is essential for NF-␬B activation. On the other 1. Guillot, L., S. Medjane, K. Le-Barillec, V. Balloy, C. Danel, M. Chignard, and hand, RhoA activation occurs also rapidly when TLR2-connected M. Si-Tahar. 2004. Response of human pulmonary epithelial cells to lipopoly- adapter signaling is triggered at the plasma membrane. saccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4. J. Biol. Chem. 279: Recent reports (5, 11–14, 22, 33, 34) connect the Src family 2712–2718. kinases c-Src, Hck, Lyn, Fyn, Yes, and Fgr to TLR3, TLR2, TLR4, 2. Hippenstiel, S., B. Opitz, B. Schmeck, and N. Suttorp. 2006. 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