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College of Physicians and Surgeons, Columbia University, New York, NY 10032
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B-dependent gene expression. TLR2 is apically displayed on airway cells, making it a likely transducer linking bacterial stimuli and kinases that affect Ca2+ release. Using biochemical and genetic approaches, we demonstrate that TLR2 ligands stimulate release of Ca2+ from intracellular stores by activating TLR2 phosphorylation by c-Src, and recruiting PI3K and phospholipase C
to affect Ca2+ release through inositol (1,4,5) trisphosphate receptors. In the absence of TLR2, murine macrophages as well as airway cells do not generate Ca2+ fluxes or induce proinflammatory signaling. Thus, Ca2+ participates as a second messenger in TLR2-dependent signaling and provides another target to modulate proinflammatory responses to bacterial infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B-dependent gene transcription in airway cells, including both muc-2 and cxcl8 by intact bacteria and flagella (6, 7). However, it is unclear exactly how the signal initiated by specific bacterial ligands is linked to the release of Ca2+.
Bacterial components are recognized by TLRs. The major TLRs are expressed in airway cells, but are not necessarily available at the apical surface, where they could be easily activated by bacterial contact (8, 9). Although TLR4 is present in airway cells (10), the involvement of TLR4 in airway signaling is thought to be limited by lack of MD-2 (11) or failure of ligands to access TLR4 (9, 12). Instead, both Gram-negative and Gram-positive pathogens can activate TLR2-mediated signaling at the surface of the airway epithelium (7). Although TLR2 is not abundant on the lumenal surface, TLR2 and glycolipid coreceptors are actively mobilized with appropriate kinases and displayed on the apical surface of airway cells in response to bacteria (13). The association of TLR2 with asialoganglioside gangliotetraosylceramide (Gal
1, 2GalNAc1, 4Gal1, 4-Glc1, 1Cer) (asialoGM1)3 provides a mechanism to signal bacterial lipoproteins that recognize TLR2 binding domains as well as the adhesins, pili, and flagella that bind to the GalNAc-Gal moiety of the glycolipid (14). Distal signaling is accomplished by IL-1R-associated kinase, MyD88, and NF-B (15) and through MAPKs (5). Phosphorylation of TLR2 is required for NF-B signaling, and both Rac1 and PI3K are recruited to the cytoplasmic domain of TLR2 and contribute to NF-B activation (16, 17). Thus, we hypothesized that TLR2 is the likely transducer linking kinases that affect the release of Ca2+ from membrane-bound compartments in response to bacterial stimuli. In the experiments described below, we demonstrate that in response to bacterial ligands, Src family kinases initiate TLR2-associated signaling, followed by recruitment of PI3K and phospholipase C (PLC) to affect the release of Ca2+ from intracellular stores, which is necessary for the downstream activation of proinflammatory gene transcription.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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Pam3Cys-Ser-Lys4 (P3C) was purchased from EMC Microcollections. Thapsigargin, BAPTA-AM, and Fluo-3/AM were purchased from Molecular Probes. EGTA, 2-aminoethoxydiphenyl borate (2-APB), anti-Flag Ab, U-73122, and U-73343 were purchased from Sigma-Aldrich. LY294002 and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine (PP2) were purchased from EMD Biosciences. 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine (PP1) was purchased from BIOMOL. Anti-phosphotyrosine clone 4G10 was purchased from Upstate Biotechnology. Anti-PI3K was purchased from BD Biosciences. Anti-PLC was purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine 783 PLC was purchased from Cell Signaling Technology. The N-terminal Flag-TLR2 construct was obtained from Genentech. The pcDNA 3.1, TLR2 wild-type (WT), and C-terminal truncated TLR2 dominant negative (DN) expression plasmids were gifts from J.-D. Li (House Ear Institute, Los Angeles, CA).
Cell line and bacteria
The 1HAEo-cells, human airway epithelial cell lines (D. Gruenert, California Pacific Medical Center Research Institute, San Francisco, CA), were grown as previously detailed (18). P. aeruginosa PAO1 were grown in Luria-Bertani medium overnight, and then resuspended in MEM (Invitrogen Life Technologies). The 1HAEo-cells were transfected overnight using FuGENE 6 (Roche) and OptiMEM medium (Invitrogen Life Technologies). Transfection reactions were prepared as described in the FuGENE 6 protocol. Forty-eight hours after transfection, pcDNA3.1-containing vectors (TLR2 WT, TLR2 DN, TLR2 Y616A/Y761A) were grown under neomycin selection. Experiments using these cell lines were performed after cells had been grown under selection for at least 2 wk.
TLR2 small interfering RNA (siRNA)
Two pairs of oligonucleotides containing 19 bp of human TLR2 were generated as follows: pair 1, 5'-gatccccatacttggatctcagtggaattcaagagattcactgagatccaagtatttttggaaa-3' and 5'-agcttttccaaaaatacttggatctcagtgaatctcttgaattcactgagatccaagtatggg-3'; pair 2, 5'-gatccccccaacaacaggatcacctattcaagagataggtgatcctgttgttggttttggaaa-3' and 5'-agcttttccaaaaccaacaacaggatcacctatctcttgaataggtgatcctgttgttggggg-3'.
The oligos were annealed and ligated into BglII and HinDIII sites of pRetroSuper vector (pRS). Construct integrity was confirmed by direct sequencing of the plasmid. Packaging of retroviral constructs was conducted in HEK 293 cells (19). The 1HAEo-cells were infected for 18 h in the presence of 4 mg/ml polybrene (Sigma-Aldrich). pBabe-puro-EGFP was used to monitor the efficiency of transfection of HEK 293 cells and infection in 1HAEo-cells. A pRS-scramble plasmid (pRS-Sc) was used as a control by cloning the sequence ggcagttccaccccagtgc into pRS, as described for the TLR2 siRNA.
Ca2+ imaging
The 1HAEo-cells were grown to 80% confluence in coverglass chamberslides and loaded for 1 h at room temperature with 2 µM Fluo-3/AM in the presence of 0.02% pluronic acid in MEM. Cells were washed with PBS and incubated at 37°C for 1 h in MEM. Fluo-3/AM fluorescence imaging was obtained and collected at 6-s intervals using a Zeiss LSM 510 META scanning confocal microscope and analyzed using the ImageJ program.
Mutagenesis
The TLR2 WT plasmid in pcDNA3.1 was used as a template to generate the TLR2 Y616A/Y761A mutants. Mutagenesis reaction was performed as described in the Quick Change II Site Directed Mutagenesis protocol (Stratagene). Primers used for the reaction were as follows: for mutationY616A, 5'-cgtttccatggcctgtgggctatgaaaatgatgtgggcc-3' and 5'-ggcccacatcattttcatagcccacaggccatggaaacg-3'; for mutation Y761A, 5'-cataatgaacaccaagaccgccctggagtggcccatggac-3' and 5'-gtccatgggccactccagggcggtcttggtgttcattatc-3'.
Immunoprecipitations and Western blots
The 1HAEo-cells were grown to confluence on 6-well plates and weaned from serum overnight. The cells were stimulated with 15 µg/ml P3C, and whole cell lysates were made using 60 mM n-octyl--D-glucopyranoside in TBS (0.1 M Tris-HCl and 0.15 M NaCl (pH 7.8)) containing Complete Mini protease inhibitor tablets (Roche), 1 mM sodium orthovanadate, and 100 mM sodium fluoride. Protein concentrations were determined using a micro BCA Protein Assay Kit (Pierce). Immunoprecipitations were performed, as described in the Dynal Bead System (Invitrogen Life Technologies). Briefly, 40 µg of protein lysates was incubated with rabbit anti-Flag Ab for 1 h at 4°C with shaking. A total of 20 µl of protein G Dynal beads was then added to the lysate/Ab mixture and incubated for 1 h at 4°C with shaking. Beads were washed with 2% BSA in PBS and resuspended in NUPAGE sample buffer and reducing agent (Invitrogen Life Technologies). Proteins were separated on 4–12% Bis-Tris NUPAGE gels (Invitrogen Life Technologies), transferred to polyvinylidene difluoride Immobilon P membrane (Millipore), and blocked with 5% milk in TBST (50 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20) for 1 h at room temperature. Immunodetection was performed using 4G10, anti-PLC, anti-Flag, anti-783 phosphotyrosine PLC, anti-TLR2 IMG 410, or anti-PI3K Abs. An anti-mouse or anti-rabbit IgG conjugated to HRP (Santa Cruz Biotechnology) was used as the secondary Abs and detected with LumiGlo Chemiluminescent Substrate System (Kirkegaard & Perry Laboratories).
CXCL8 assay
The 1HAEo-cells were grown to confluence on 96-well plates and weaned from serum overnight. Cells were preincubated with inhibitors for 30 min and stimulated with 15 µg/ml P3C, 108 CFU P. aeruginosa PAO1, or 1 µM thapsigargin for 30 min in the presence or absence of inhibitor. Cells were incubated with fresh MEM containing 100 µg/ml gentamicin for 3 h, and supernatants were collected for CXCL8 ELISA (BD Pharmingen). Data for each condition were plotted as the mean of sextuplicate samples and are representative of at least three independent experiments.
NF-B luciferase assay
The 1HAEo-cells were grown on 24-well plates to 70% confluence and transiently transfected for 24 h using FuGENE 6 (Roche), OptiMEM (Invitrogen Life Technologies), and 1 ng of pNF-B-luciferase plasmid DNA (Stratagene), which contains five NF-B binding sites upstream of a luciferase reporter gene. Cells were washed with PBS, and selected wells were pretreated with 6 µM BAPTA-AM for 30 min. Cells were stimulated with 5 µg/ml P3C, or 1 µM thapsigargin for 30 min and incubated in fresh MEM for 3 h. Lysis and luciferase assays were performed using the reagents and protocol for the dual luciferase reporter assay system (Promega) and analyzed with a luminometer. Luciferase activity was standardized by protein concentration; data were plotted as a mean of quadruplicate samples and are representative of at least three independent experiments.
Flow cytometry of peritoneal macrophages
C57BL/6 WT or TLR2 knockout (KO) mice were euthanized, and their peritoneal contents were collected by flushing with 10 ml RPMI 1640 (Invitrogen Life Technologies) with 10% FCS. Cells were washed with RPMI 1640, loaded with Fluo-3/AM for 1 h at room temperature in the dark, washed again with RPMI 1640, incubated at 37°C for 1 h, and analyzed on a FACSCalibur using CellQuest software (BD Biosciences). Mouse protocol number AAAA1718 was approved by the Institutional Animal Care and Use Committee at Columbia University.
Statistical analysis
Data are expressed as mean ± SD. Statistical significance between groups was evaluated by Student’s t test using GraphPad Instat version 3.0 (GraphPad). Differences between groups were considered significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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To test the hypothesis that TLR2 ligands initiate Ca2+ fluxes, 1HAEo-cells were loaded with Fluo-3/AM and stimulated with P3C, a TLR2- specific agonist (Fig. 1). A 3- to 4-fold increase in fluorescence intensity was noted by 60 s, which returned to baseline by 5 min poststimulation (Fig. 1A). In the presence of the intracellular Ca2+ chelator, BAPTA-AM, the P3C or thapsigargin-induced Ca2+ signal was blocked, whereas EGTA had no effect (Fig. 1, B and C). P3C stimulation of airway cells treated with the inositol (1, 4, 5) trisphosphate receptor (IP3R) antagonist, 2-APB, failed to generate Ca2+ fluxes, indicating that the source of Ca2+ released was from internal stores (Fig. 1D).
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B and CXCL8 expression. The specificity of the effect of BAPTA-AM in these same cells was confirmed by documenting CXCL8 induction by S. aureus protein A, which activates NF-B through the TNFR1 pathway (20) (Fig. 2B). The extracellular Ca2+ chelator, EGTA, did not block CXCL8 expression, demonstrating that extracellular Ca2+ entry is not critical for this signaling event (Fig. 2C), whereas the IP3R inhibitor, 2-APB, had a dose-dependent effect on bacterial-induced CXCL8 expression (Fig. 2D).
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The requirement for TLR2 in activating Ca2+ was tested in 1HAEo-cell lines in which TLR2 expression was silenced by retroviral delivery of siRNA. The 1HAEo-cells infected with TLR2-targeted siRNA sequences had a >80% decrease in TLR2 expression compared with cells infected with scrambled oligonucleotides (Fig. 3A). Ca2+ fluxes were not generated in TLR2 siRNA cell lines in response to P3C, but did occur in response to thapsigargin. Control cells expressing the scrambled oligonucleotides were capable of generating Ca2+ fluxes in response to P3C (Fig. 3B). CXCL8 expression in response to P3C was also significantly decreased in TLR2 siRNA cells (Fig. 3C), indicating that P3C-induced Ca2+ fluxes and CXCL8 expression are mediated by TLR2.
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c-Src-dependent phosphorylation of TLR2 is necessary to induce Ca2+ fluxes
Tyrosine phosphorylation of TLR2 is rapidly induced by P3C (Fig. 4A). As the sites of TLR2 phosphorylation have been identified (16), stable cell lines were generated expressing the TLR2 Y616A/Y761A mutations, and responses to P3C were tested (Fig. 4B). P3C stimulation failed to activate Ca2+ fluxes in cell lines expressing the tyrosine mutations or in a DN TLR2 mutant in contrast to cells expressing WT TLR2 (Fig. 4B).
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PI3K is known to be recruited to the plasma membrane, and is a likely candidate to link TLR2 and proteins involved in Ca2+ release. The requirement for TLR2 phosphorylation in this association with PI3K is shown (Fig. 6A). PI3K was not coimmunoprecipitated with TLR2 in cells treated with the c-Src inhibitor PP1 (Fig. 6A). In addition, treatment of the cells with LY294002, an inhibitor of PI3K, blocked P3C-induced Ca2+ flux (Fig. 6B) and CXCL8 expression (Fig. 6C), confirming the association of PI3K and TLR2 signaling.
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acts downstream of TLR2 and PI3K
The generation of phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3) by PI3K activates PLC and is involved in intracellular Ca2+release by generating inositol (1,4,5) trisphosphate (Ins(1, 4, 5)P3) (21). PLC activity is regulated by localization to the plasma membrane and tyrosine phosphorylation. PLC was shown to be associated with TLR2 in response to the TLR2 agonist (Fig. 7A). Tyrosine phosphorylation of PLC occurred within 60 s (Fig. 7A) and was inhibited by the PI3K inhibitor, LY294002. An inhibitor of PLC, U-73122, but not the inactive analog U-73343, blocked Ca2+ release (Fig. 7B), as well as P3C and P. aeruginosa PAO1-induced CXCL8 expression in a dose-dependent manner (Fig. 7C). Because phosphorylation of tyrosine 783 is required for PLC activation, we tested whether this phosphorylation event was mediated by TLR2 stimulation. PLC tyrosine 783 phosphorylation was absent in TLR2 siRNA cells, while cells expressing scrambled control oligonucleotides displayed rapid phosphorylation at this residue (Fig. 7D). Thus, stimulation of TLR2 initiates recruitment and activation of PI3K and PLC, which in turn mediate the TLR2-associated Ca2+ fluxes, resulting in downstream CXCL8 expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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B-dependent gene expression by S. aureus and P. aeruginosa (5, 20), Salmonella (25), and Chlamydophilia pneumonia (26). We have demonstrated that bacterial ligands interacting with TLR2 or asialoGM1 linked to TLR2, activate c-Src, followed by the recruitment of PI3K and PLC, which in turn stimulate the release of Ca2+ through IP3R channels. This Ca2+ flux is essential to activate distal proinflammatory responses, as measured by either activation of NF-B or production of CXCL8. In the experiments presented, we focused on superficial bacterial stimuli that are sufficient to initiate proinflammatory signaling in the airway. The mucosal cells of the lung protect a normally sterile airway and thus have a relatively low threshold for proinflammatory activation. However, because recruitment of polymorphonuclear leukocytes into the airway lumen impedes the primary role of the respiratory tract in facilitating air exchange, inflammation must be limited. Bacterial ligands interact with TLRs and TLR-associated glycolipid (7 , 27). TLR2 presented on the apical surface of airway cells can signal not only the bacterial lipoproteins specific for its own exposed residues, but bacterial adhesins such as pili and flagella, which bind to asialoGM1 (7, 28). Although TLR4 (LPS) is present in airway cells, it is not involved in the immediate responses to bacterial ligands (7, 9). Limited responses to LPS by airway cells suggest that TLRs other than TLR4 predominate in surveillance functions. TLR2-mediated Ca2+ signaling provides an immediate and transient mechanism to respond to the perceived threat of infection.
This Ca2+-dependent signaling cascade in airway epithelial cells shares major elements with the well-characterized pathways associated with G protein-coupled receptors (1, 29, 30). The recruitment of PI3K to TLR2 (16) and activation of PLC suggested that Ca2+ release follows TLR2 phosphorylation. Several studies examining Ca2+-dependent responses to bacteria also implicated PI3K or PLC, as might be expected (31). The importance of the class I PI3Ks in immune signaling is well established (32). PI3Ks are involved in mediating signals generated by bacterial recognition of various TLR, besides TLR2, and have been implicated in both pro- and anti-inflammatory pathways (33, 34). As the link between PI3K and specific TLRs is suggested to be tissue specific, we have focused on TLR2 expression in airway cells. However, as murine peritoneal macrophages similarly generate Ca2+ fluxes following stimulation with TLR2 ligands, and the recruitment of PI3K and PLC to TLR2 has been described in immune cells, it seems likely that the pathway described is a generally conserved signaling mechanism.
The involvement of Src family kinases in the initial phosphorylation of TLR2 is consistent with several published studies demonstrating the requirement for Src activity in cellular responses to bacterial ligands (35, 36), for cyclooxygenase-2 activation by Helicobacter pylori (37), for MUC-2 activation (35), and CXCL8 expression in response to P. aeruginosa ligands (7). Previously, c-Src had been shown to coimmunoprecipitate with TLR2 in airway cells following bacterial stimulation (13).
Having identified the more proximal elements of the TLR2-Ca2+-dependent signaling pathway, the distal target for the Ca2+ transient is unknown. Conventional protein kinase C 
and , which are activated by Ca2+, have been shown to be downstream of TLR2-mediated NF-B activation (37, 38). Alternatively, Ca2+ calmodulin-dependent protein kinase could activate NF-B through IK activation (39). However, the specific Ca2+-dependent kinase(s) remains to be identified. The multiply phosphorylated IL-1R-associated kinase is a possible candidate, but it is not established whether other TLRs exhibit a similar Ca2+ dependency, as would be expected if a shared kinase is a target for a Ca2+ enzyme. Although our data were generated primarily with airway epithelial cell lines, the confirmatory results obtained with murine macrophages suggest that TLR2 signaling is Ca2+ dependent in other cell types as well.
It is likely that many different types of bacterial pathogens are signaled through this pathway. In addition to the airway pathogens P. aeruginosa and S. aureus that have already been demonstrated to activate epithelial NF-B signaling through the generation of Ca2+ fluxes, Mycobacterium tuberculosis, a classic intracellular pathogen that signals through TLR2, is also known to activate Ca2+-dependent responses (40). In the gastrointestinal tract, H. pylori similarly induces CXCL8 and cyclooxygenase-2 expression through Ca2+-dependent cascades (37). Thus, TLR2 appears to be broadly reactive and able to mediate responses to diverse types of pathogens. The participation of Ca2+ as a second messenger in the host response to pathogens seems consistent with its central role in signal transduction. The inclusion of Ca2+-dependent signaling to TLR2-mediated responses adds yet another degree of complexity, as well as another potential target to modulate the host response to bacterial pathogens.
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Acknowledgments |
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Disclosures |
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Footnotes |
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1 This work was supported by National Institutes of Health Grant RO1 HL73989. 
2 Address correspondence and reprint requests to Dr. Alice Prince, College of Physicians and Surgeons, Columbia University, 650 West 168th Street BB4-416, New York, NY 10032. E-mail address: asp7{at}columbia.edu
3 Abbreviations used in this paper: asialoGM1, asialoganglioside gangliotetraosylceramide (Gal1, 2GalNAc1, 4Gal1, 4-Glc1, 1Cer); 2-APB, 2-aminoethoxydiphenyl borate; DN, dominant negative; IP3R, inositol (1,4,5) triphosphate receptor; KO, knockout; P3C, Pam3Cys-Ser-Lys4; PLC, phospholipase C; siRNA, small interfering RNA; pRS, pRetroSuper vector; WT, wild type; PP1 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine; PP2 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine.
Received for publication February 27, 2006. Accepted for publication May 1, 2006.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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 H. G. Kim, J. Y. Kim, M. G. Gim, J. M. Lee, and D. K. Chung Mechanical stress induces tumor necrosis factor-{alpha} production through Ca2+ release-dependent TLR2 signaling Am J Physiol Cell Physiol, August 1, 2008; 295(2): C432 - C439. [Abstract] [Full Text] [PDF]
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S.-K. Heo, M.-A Yoon, S.-C. Lee, S.-A Ju, J.-H. Choi, P.-G. Suh, B. S. Kwon, and B.-S. Kim HVEM Signaling in Monocytes Is Mediated by Intracellular Calcium Mobilization J. Immunol., November 1, 2007; 179(9): 6305 - 6310. [Abstract] [Full Text] [PDF]
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MFI: MFI in untreated cells subtracted from MFI in treated cells. E, WT and TLR2 KO peritoneal macrophages were stimulated with 15 µg/ml P3C and assayed for KC.
