The Journal of Immunology Caveolin-1 Tyr14 Phosphorylation Induces Interaction with TLR4 in Endothelial Cells and Mediates MyD88-Dependent Signaling and Sepsis-Induced Lung Inflammation Hao Jiao,*,†,1 Yang Zhang,*,†,1 Zhibo Yan,* Zhen-Guo Wang,* Gongjian Liu,† Richard D. Minshall,*,‡ Asrar B. Malik,‡ and Guochang Hu*,‡ Activation of TLR4 by the endotoxin LPS is a critical event in the pathogenesis of Gram-negative sepsis. Caveolin-1, the signaling protein associated with caveolae, is implicated in regulating the lung inflammatory response to LPS; however, the mechanism is not understood. In this study, we investigated the role of caveolin-1 in regulating TLR4 signaling in endothelial cells. We observed that LPS interaction with CD14 in endothelial cells induced Src-dependent caveolin-1 phosphorylation at Tyr14. Using a TLR4-MD2- CD14–transfected HEK-293 cell line and caveolin-1–deficient (cav-12/2) mouse lung microvascular endothelial cells, we demon- strated that caveolin-1 phosphorylation at Tyr14 following LPS exposure induced caveolin-1 and TLR4 interaction and, thereby, TLR4 activation of MyD88, leading to NF-kB activation and generation of proinflammatory cytokines. Exogenous expression of phosphorylation-deficient Y14F caveolin-1 mutant in cav-12/2 mouse pulmonary vasculature rendered the mice resistant to LPS compared with reintroduction of wild-type caveolin-1. Thus, caveolin-1 Y14 phosphorylation was required for the interaction with TLR4 and activation of TLR4-MyD88 signaling and sepsis-induced lung inflammation. Inhibiting caveolin-1 Tyr14 phosphoryla- tion and resultant inactivation of TLR4 signaling in pulmonary vascular endothelial cells represent a novel strategy for preventing sepsis-induced lung inflammation and injury. The Journal of Immunology, 2013, 191: 6191–6199. epsis from severe bacterial infection is the major cause of recruits a number of cytoplasmic adaptor proteins containing TIR morbidity and mortality in critically ill patients (1). LPS domains, including MyD88, TIR-associated protein/MyD88 adaptor- S (also known as endotoxin), the key structural component like, TRIF, and TLR-associated molecule (9, 10). Recruitment of the of the cell wall of Gram-negative bacteria, is a highly potent adaptor protein MyD88 initiates early activation of NF-kB, whereas trigger of cytokine release through TLR4 (2). Release of LPS into recruitment of TRIF activates a MyD88-independent pathway, the blood activates a generalized inflammatory response, leading leading to delayed activation of NF-kB (11) and activation of IFN to multiple organ dysfunction syndrome (2–4). The binding of regulatoryfactor3(12,13).UponactivationofTLR4withLPS, LPS to TLR4 in endothelial cells plays a particularly important MyD88 induces the association with IRAK-4 and IRAK-1 and re- role in the inflammatory response. LPS activates endothelial cells cruitment of TNFR-associated factor 6 (TRAF6) to IRAK-1 (14-16). through a receptor complex consisting of TLR4, CD14, and MD-2 The IRAK-4/IRAK-1/TRAF6 complex dissociates from TLR4 and (5, 6). The outcome of activation of this signaling pathway is the interacts with the TGF-b–activated kinase 1 complex, which, in turn, production of proinflammatory mediators by endothelial cells, activates IkB kinases, leading to phosphorylation and degradation of upregulation of endothelial adhesion molecules, loss of endothe- IkB and release and translocation of NF-kB to the nucleus, as well as lial barrier integrity, leukocyte recruitment, and, ultimately, cel- induces expression of proinflammatory genes (9). lular and organ injury (7, 8). LPS engagement of TLR4 also Caveolin-1 (Cav-1), a 21–24-kDa protein, is the principal struc- tural and signaling component of caveolae that oligomerizes, and the oligomers are inserted into caveolae membrane (17, 18). Cav-1 *Department of Anesthesiology, University of Illinois at Chicago College of Medi- functions as a scaffolding protein and interacts with multiple mole- cine, Chicago, IL 60612; †Department of Anesthesiology, Xuzhou Medical Col- cules (19, 20). Recent studies implicated a key role for Cav-1 in ‡ lege, Xuzhou 221002, China; and Department of Pharmacology, University of regulating innate immunity and inflammation (21–23), but the results Illinois at Chicago College of Medicine, Chicago, IL 60612 have been inconclusive as to how Cav-1 functions. Downregulation 1H.J. and Y.Z. equally contributed to this work. of Cav-1 expression in murine alveolar and peritoneal macrophages Received for publication April 2, 2013. Accepted for publication October 15, 2013. using small interfering RNAs (siRNAs) increased LPS-induced This work was supported by National Science Foundation of China Grant 81070058 production of proinflammatory cytokines (TNF-a and IL-6) (24, and National Institutes of Health/National Heart, Lung, and Blood Institute Grant HL104092 (both to G.H.). 25), indicating an anti-inflammatory role for Cav-1. Studies using 2/2 Address correspondence and reprint requests to Dr. Guochang Hu, Department of Cav-1–deficient (cav-1 ) mice demonstrated that Cav-1 induced Pharmacology (m/c 868), University of Illinois at Chicago College of Medicine, 835 upregulation of NF-kBactivityandincreasedlunginflammatory South Wolcott Avenue, Chicago, IL 60612. E-mail address: [email protected] response following LPS stimulation (26). NF-kB activity and lung The online version of this article contains supplemental material. inflammation and injury following LPS challenge in cav-12/2 mouse Abbreviations used in this article: Cav-1, caveolin-1; eNOS, endothelial NO syn- lungs were reduced compared with controls (26). This was ascribed thase; HEK-TLR4, HEK-293 cell line stably expressing TLR4 and MD2 in combi- nation with CD14; m, mouse; MLMVEC, mouse lung microvascular endothelial cell; to greater production of NO due to deinhibition of endothelial NO ROS, reactive oxygen species; SEAP, secreted embryonic alkaline phosphatase; synthase (eNOS) in the Cav-1–deleted mice. eNOS activation sec- siRNA, small interfering RNA; TRAF6, TNFR-associated factor 6; WT, wild-type; ondary to Cav-1 deficiency also impaired IRAK-4 activity through Y14F–Cav-1, nonphosphorylatable caveolin-1 Y14F mutant. nitration of IRAK-4 in response to LPS (27). Although the above Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 studies described a role for increased eNOS activity following the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300873 6192 CAVEOLIN-1 PHOSPHORYLATION ENHANCES TLR4 SIGNALING deletion of Cav-1 in attenuating inflammatory signaling pathways, (Cell Biologics, Chicago IL) (28). Briefly, lung tissues were minced and they did not address the important signaling function of Cav-1, which digested with collagenase A (1.0 mg/ml) for 45–60 min at 37˚C. Endo- is regulated by phosphorylation at Tyr14 (21–23), or how Cav-1 thelial cells were purified using an anti–PECAM-1 mAb magnetic bead 14 (BD Pharmingen, San Diego, CA) separation technique and allowed to signaling by Tyr phosphorylation influences TLR4 activation by grow for 3–4 d in growth medium. The purity of endothelial cells reached LPS. In the current study, we explored the role of Cav-1 phosphor- .95%. For monolayer cultures, the cells were plated on fibronectin-coated ylation at Tyr14 in activating the LPS–TLR4–NF-kB–signaling dishes in endothelial cell growth medium 2 supplemented with the Bullet pathway in endothelial cells. We found that Cav-1 Tyr14 phosphor- Kit (BioWhittaker, Walkersville, MD) and 10% FBS, incubated (37˚C) under a humidified atmosphere of 5% CO2–95% air, and used at passages ylation was essential for TLR4 signaling and, hence, mediated the three to five. inflammatory response in endothelial cells. These results raise the The HEK-293 cell line stably expressing TLR4 and MD2 in combination possibility that blockade of Cav-1 phosphorylation at Tyr14 in lung with CD14 (293-hTLR4A-MD2-CD14; referred to as “HEK-TLR4” in this vascular endothelial cells would be beneficial in preventing lung article) was purchased from InvivoGen (San Diego, CA). HEK-TLR4 cells inflammatory injury during sepsis. were maintained in high-glucose DMEM (Cellgro, Manassas, VA) sup- plemented with penicillin, streptomycin, and 10% FBS. Methods and Materials Treatment with LPS and Src inhibitor Reagents and chemicals LPS was diluted with the appropriate basal culture media without sup- plements and added to the cells, which were preincubated in serum-deprived LPS (Escherichia coli 0127:B8; purity . 99%) and PP2 (purity $ 98%) media (without LPS-binding protein) for 2 h. Confluent MLMVEC mon- were obtained from Sigma-Aldrich. MyD88 siRNA (mouse [m]), TRIF olayers were incubated with Src inhibitor PP2 (15 mM) in HBSS for siRNA (m), CD14 siRNA (m), and control siRNA, anti–a-tubulin, anti– 15 min at 37˚C, followed by two washes with HBSS. IRAK-1, anti-TLR4, and anti–IkB-a mAbs were obtained from Santa Cruz Biotechnology. Cav-1 and b-actin mAbs were purchased from BD Bio- sciences. Anti-MyD88 mAb and anti–pY418-Src polyclonal Ab were ob- Cav-1 cDNA transfection tained from Abcam and Cell Signaling, respectively. Lipofectamine 2000 HEK-TLR4 cells and cav-12/2 MLMVECs were transfected with pcDNA6 was purchased from Invitrogen. plasmid vector containing cDNAs of Myc-tagged WT Cav-1 or non- phosphorylatable Cav-1 Y14F mutant (Y14F–Cav-1). HEK-TLR4 cells Mice and lung inflammatory injury were transfected using a mixture of 4 mg cDNA and 10 ml Lipofectamine 2/2 Wild-type (WT) B6/129SJ (cav-1+/+) and cav-12/2 mice were purchased 2000 (Invitrogen) in each well of a six-well plate. Cav-1 MLMVECs from The Jackson Laboratory (Bar Harbor, ME). Mice were housed in were transfected using a Nucleofector Kit (Lonza, Cologne, Germany), microisolator cages under specific pathogen–free conditions, fed with according to the manufacturer’s protocol. Successful expression of exog- autoclaved food, and used in experiments at 8–12 wk of age. All studies enous Cav-1 was confirmed by Western blot analysis of cell lysates. using mice were approved by the University of Illinois Institutional Animal Care and Use Committee.