The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chun, J.
Right arrow Articles by Prince, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chun, J.
Right arrow Articles by Prince, A.
The Journal of Immunology, 2006, 177: 1330-1337.
Copyright © 2006 by The American Association of Immunologists

Activation of Ca2+-Dependent Signaling by TLR21

Jarin Chun and Alice Prince2

College of Physicians and Surgeons, Columbia University, New York, NY 10032


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Upon contact with airway epithelial cells, bacterial products activate Ca2+ fluxes that are required for induction of NF-{kappa}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{gamma} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The participation of Ca2+ as a second messenger is an essential component of many cellular signaling cascades. Ca2+ fluxes are especially important in mediating responses to extracellular stimuli, such as the activation of G protein-coupled receptors (1) and the TCR (2, 3, 4). The canonical pathway generally involves the perturbation of membrane inositol phospholipids that link a ligand-receptor interaction and release of Ca2+ from an intracellular compartment. The common airway pathogens Staphylococcus aureus and Pseudomonas aeruginosa activate 100 nM Ca2+ fluxes immediately upon contact with airway epithelial cells, and this Ca2+ signal initiates CXCL8 production (5). Mucosal epithelial cells are actively involved in responding to bacterial contamination of the respiratory tract and express both chemokines and cytokines (CXCL8, IL-6, GM-CSF) to recruit and activate phagocytic cells. The generation of Ca2+ fluxes has been implicated in the activation of NF-{kappa}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 (Galbeta1, 2GalNAcbeta1, 4Galbeta1, 4-Glcbeta1, 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-{kappa}B (15) and through MAPKs (5). Phosphorylation of TLR2 is required for NF-{kappa}B signaling, and both Rac1 and PI3K are recruited to the cytoplasmic domain of TLR2 and contribute to NF-{kappa}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{gamma} (PLC{gamma}) to affect the release of Ca2+ from intracellular stores, which is necessary for the downstream activation of proinflammatory gene transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

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{gamma} was purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine 783 PLC{gamma} 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-beta-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{gamma}, anti-Flag, anti-783 phosphotyrosine PLC{gamma}, 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-{kappa}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-{kappa}B-luciferase plasmid DNA (Stratagene), which contains five NF-{kappa}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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Activation of TLR2 induces Ca2+ release from intracellular stores

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).


Figure 1
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 1. TLR2 ligand induces Ca2+ release. The 1HAEo-cells loaded with Fluo-3/AM and stimulated with 15 µg/ml P3C (P) or 1 µM thapsigargin (T) in the presence of control (A), 6 µM BAPTA-AM (B), 1 mM EGTA (C), or 100 µM 2-APB (D) were imaged by confocal microscopy. Adjacent graphs show changes in fluorescence intensities in individual cells over time. Data are representative of at least three separate experiments.

 
The importance of Ca2+ release in the activation of TLR2-associated signaling cascade was examined in parallel, demonstrating that BAPTA-AM (Fig. 2, A and B) significantly inhibited NF-{kappa}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-{kappa}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).


Figure 2
View larger version (23K):
[in this window]
[in a new window]
  		FIGURE 2. TLR2-mediated Ca2+ release signals proinflammatory responses. The 1HAEo-cells were stimulated with 15 µg/ml P3C, 1 µM thapsigargin (Thaps), 100 µg/ml S. aureus protein A, or 108 CFU P. aeruginosa PAO1, as indicated, in the presence or absence of BAPTA-AM (6 µM), EGTA (0.5 or 1 mM), or 2-APB (75 or 100 µM), and NF-{kappa}B luciferase activity (A) or CXCL8 expression (B–D) was measured. Data are mean ± SD of sextuplicate samples of one representative from three independent experiments. *, p < 0.01; **, p < 0.001; ***, p < 0.0001 compared with no inhibitor (Student’s t test).

 
TLR2 is required to generate Ca2+ fluxes

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.


Figure 3
View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 3. TLR2 is necessary to generate Ca2+ fluxes and CXCL8 expression. A, 1HAEo-cell lines expressing scrambled siRNA or TLR2 siRNA were analyzed for TLR2 expression by Western hybridization. Adjacent graph shows the adjusted volume for each band, determined by densitometry and standardized by actin expression. B, Scrambled control or TLR2 siRNA cell lines were loaded with Fluo-3/AM, stimulated with 15 µg/ml P3C (P) and 1 µM thapsigargin (T), and imaged by confocal microscopy. Adjacent graphs show changes in fluorescence intensities in individual cells over time. Data are representative of at least three separate experiments. C, Fold increase in CXCL8 expression was determined for scrambled or TLR2 siRNA cell lines stimulated with 15 µg/ml P3C. Data are a mean ± SD of sextuplicate samples of one representative from three independent experiments. *, p < 0.01 compared with scramble infected cells (Student’s t test). D, Peritoneal macrophages from WT or TLR2 KO mice were loaded with Fluo-3/AM, and flow cytometry was used to detect mean fluorescence intensity (MFI) before (shaded gray) and after (black line) stimulation with 15 µg/ml (P3C) or 1 µM thapsigargin. {Delta}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.

 
To confirm that TLR2-dependent changes in Ca2+ are also important in immune cells, we compared responses to P3C in macrophages harvested by peritoneal lavage from WT and TLR2 null mice. Macrophages were loaded with Fluo-3/AM, and fluorescence was measured by flow cytometry. In WT cells, P3C induced a shift in mean fluorescence intensity that was absent in the TLR2 null cells (Fig. 3D). P3C also induced secretion of the murine chemokine, KC, in WT, but not TLR2 null macrophages (Fig. 3E). As in the airway cells, TLR2 was necessary for Ca2+ signaling and chemokine expression in murine peritoneal macrophages.

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).


Figure 4
View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 4. TLR2 tyrosine phosphorylation is required for Ca2+ fluxes. A, 1HAEo-cells transfected with pFlag-TLR2 were stimulated with 15 µg/ml P3C for the indicated times (s). Whole cell lysates from 1HAEo-cells were immunoprecipitated with anti-Flag and immunoblotted with anti-phosphotyrosine 4G10 and anti-Flag. Data are representative of three separate experiments. B, 1HAEo-stable cell lines containing vector alone pcDNA3.1 as control, TLR2 WT, TLR2DN, and TLR2 Y616A/Y761A expression constructs were stimulated with 15 µg/ml P3C (P) or 1 µM thapsigargin (T), as indicated by the arrows, and imaged by confocal microscopy. Adjacent graphs show changes in fluorescence intensities in individual cells over time. Data are representative of at least three separate experiments. C, Fold increase in NF-{kappa}B luciferase activity of TLR2 WT-, TLR2 DN-, and TLR2 Y616A/Y761A-transfected cells stimulated with 15 µg/ml P3C. Data are a mean ± SD of triplicate samples of one representative from three independent experiments. *, p < 0.05 compared with TLR2 WT-transfected cells (Student’s t test).

 
A likely candidate for this phosphorylation event is c-Src, which was previously shown to be associated with TLR2 (13). Cells treated with the c-Src inhibitor PP1 had substantially decreased TLR2 tyrosine phosphorylation (Fig. 5A). P3C-induced Ca2+ fluxes and CXCL8 expression were significantly inhibited in the presence of the c-Src inhibitors, PP1 and PP2 (Fig. 5, B and C).


Figure 5
View larger version (33K):
[in this window]
[in a new window]
  		FIGURE 5. c-Src phosphorylates TLR2. A, 1HAEo-cells transfected with pFlag-TLR2 were stimulated with 15 µg/ml P3C with or without 25 µM PP1 for the indicated times (s). B, 1HAEo-cells stimulated with 15 µg/ml P3C (P) or 1 µM thapsigargin (T) in the presence or absence of 10 µM PP1 or 10 µM PP2 were imaged by confocal microscopy. Adjacent graphs show changes in fluorescence intensities in individual cells over time. Data are representative of at least three separate experiments. C, Supernatants from 1HAEo-cells were stimulated with 15 µg/ml P3C with or without 5 or 25 µM PP1 or PP2 and assayed for CXCL8. Data are a mean ± SD of sextuplicate samples of one representative from three independent experiments. *, p < 0.05; **, p < 0.001 compared with cells stimulated with no inhibitor (Student’s t test).

 
PI3K is required for TLR2-mediated Ca2+ flux

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.


Figure 6
View larger version (24K):
[in this window]
[in a new window]
  		FIGURE 6. PI3K is required for TLR2-mediated Ca2+ fluxes. The 1HAEo-cells transfected with pFlag-TLR2 were stimulated with 15 µg/ml P3C (A) and 25 µM PP1 (B) for the indicated times (s). Whole cell lysates were immunoprecipitated with anti-Flag and immunoblotted for PI3K and Flag. C, 1HAEo-cells treated with 10 µM LY294002 and stimulated with 15 µg/ml P3C (P) and 1 µM thapsigargin (T) were imaged by confocal microscopy. D, Supernatants from 1HAEo-cells treated with LY294002 (5 or 10 µM) and stimulated with either 15 µg/ml P3C or 108 CFU PAO1 were assayed for CXCL8. Data are a mean ± SD of sextuplicate samples of one representative from three independent experiments. *, p < 0.05; **, p < 0.001 compared with cells stimulated with no inhibitor (Student’s t test).

 
PLC{gamma} acts downstream of TLR2 and PI3K

The generation of phosphatidylinositol (3,4,5) trisphosphate (PI(3,4,5)P3) by PI3K activates PLC{gamma} and is involved in intracellular Ca2+release by generating inositol (1,4,5) trisphosphate (Ins(1, 4, 5)P3) (21). PLC{gamma} activity is regulated by localization to the plasma membrane and tyrosine phosphorylation. PLC{gamma} was shown to be associated with TLR2 in response to the TLR2 agonist (Fig. 7A). Tyrosine phosphorylation of PLC{gamma} occurred within 60 s (Fig. 7A) and was inhibited by the PI3K inhibitor, LY294002. An inhibitor of PLC{gamma}, 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{gamma} activation, we tested whether this phosphorylation event was mediated by TLR2 stimulation. PLC{gamma} 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{gamma}, which in turn mediate the TLR2-associated Ca2+ fluxes, resulting in downstream CXCL8 expression.


Figure 7
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7. PLC{gamma} is required for TLR2-mediated Ca2+ flux. A, 1HAEo-cells transfected with pFlag-TLR2 were stimulated with 15 µg/ml P3C in the presence of absence of 10 µM LY294002 for the indicated times (s). Whole cell lysates were immunoprecipitated with anti-Flag and immunoblotted for anti-phosphotyrosine 4G10, anti-PLC{gamma}, and anti-Flag Abs. Data are representative of at least three separate experiments. B, 1HAEo-cells treated with 1.5 µM U-73122 and 1.5 µM U-73343 were stimulated with 15 µg/ml P3C (P) and 1 µM thapsigargin (T) and imaged by confocal microscopy. Adjacent graphs show changes in fluorescence intensities over time obtained from individual cells in the corresponding confocal images. C, Supernatants from 1HAEo-cells treated with 1.5 µM U-73122 or U-73323 and stimulated with 15 µg/ml P3C or 108 CFU PAO1 were assayed for CXCL8. Data are a mean ± SD of sextuplicate samples of one representative from three independent experiments. *, p < 0.05; **, p < 0.01 compared with cells stimulated with no inhibitor (Student’s t test). D, Lysates from scrambled or TLR2 siRNA cells stimulated with 15 µg/ml P3C for times (s) indicated were immunoblotted for PLC{gamma} phosphotyrosine-783 and actin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
There is a substantial literature demonstrating that various bacterial products induce Ca2+ fluxes and proinflammatory gene expression in mucosal cells (22). Several different mechanisms have been implicated, including direct effects of bacterial toxins (23), activation of purinergic signaling by bacterial adhesins that bind asialoGM1 (24), as well as activation of NF-{kappa}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{gamma}, 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-{kappa}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{gamma} suggested that Ca2+ release follows TLR2 phosphorylation. Several studies examining Ca2+-dependent responses to bacteria also implicated PI3K or PLC{gamma}, 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{gamma} 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 {alpha} and beta, which are activated by Ca2+, have been shown to be downstream of TLR2-mediated NF-{kappa}B activation (37, 38). Alternatively, Ca2+ calmodulin-dependent protein kinase could activate NF-{kappa}B through I{kappa}K 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-{kappa}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.


   Acknowledgments
 
Confocal microscopy was performed at the Herbert Irving Optical Microscopy facility at Columbia University.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant RO1 HL73989. Back

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 Back

3 Abbreviations used in this paper: asialoGM1, asialoganglioside gangliotetraosylceramide (Galbeta1, 2GalNAcbeta1, 4Galbeta1, 4-Glcbeta1, 1Cer); 2-APB, 2-aminoethoxydiphenyl borate; DN, dominant negative; IP3R, inositol (1,4,5) triphosphate receptor; KO, knockout; P3C, Pam3Cys-Ser-Lys4; PLC{gamma}, phospholipase C{gamma}; 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. Back

Received for publication February 27, 2006. Accepted for publication May 1, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Werry, T. D., G. F. Wilkinson, G. B. Willars. 2003. Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+. Biochem. J. 374: 281-296. [Medline]
  2. Gallo, E. M., K. Cante-Barrett, G. R. Crabtree. 2006. Lymphocyte calcium signaling from membrane to nucleus. Nat. Immunol. 7: 25-32. [Medline]
  3. Gardner, P.. 1989. Calcium and T lymphocyte activation. Cell 59: 15-20. [Medline]
  4. Wang, C., J. L. Mooney, R. Meza-Romero, Y. K. Chou, J. Huan, A. A. Vandenbark, H. Offner, G. G. Burrows. 2003. Recombinant TCR ligand induces early TCR signaling and a unique pattern of downstream activation. J. Immunol. 171: 1934-1940. [Abstract/Free Full Text]
  5. Ratner, A. J., R. Bryan, A. Weber, S. Nguyen, D. Barnes, A. Pitt, S. Gelber, A. Cheung, A. Prince. 2001. Cystic fibrosis pathogens activate Ca2+-dependent mitogen-activated protein kinase signaling pathways in airway epithelial cells. J. Biol. Chem. 276: 19267-19275. [Abstract/Free Full Text]
  6. McNamara, N., M. Gallup, A. Khong, A. Sucher, I. Maltseva, J. Fahy, C. Basbaum. 2004. Adenosine up-regulation of the mucin gene, MUC2, in asthma. FASEB J. 18: 1770-1772. [Abstract/Free Full Text]
  7. Adamo, R., S. Sokol, G. Soong, M. I. Gomez, A. Prince. 2004. Pseudomonas aeruginosa flagella activate airway epithelial cells through asialoGM1 and Toll-like receptor 2 as well as Toll-like receptor 5. Am. J. Respir. Cell Mol. Biol. 30: 627-634. [Abstract/Free Full Text]
  8. Gewirtz, A. T., T. A. Navas, S. Lyons, P. J. Godowski, J. L. Madara. 2001. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J. Immunol. 167: 1882-1885. [Abstract/Free Full Text]
  9. Zhang, Z., J. P. Louboutin, D. J. Weiner, J. B. Goldberg, J. M. Wilson. 2005. Human airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of flagellin by Toll-like receptor 5. Infect. Immun. 73: 7151-7160. [Abstract/Free Full Text]
  10. Muir, A., G. Soong, S. Sokol, B. Reddy, M. I. Gomez, A. Van Heeckeren, A. Prince. 2004. Toll-like receptors in normal and cystic fibrosis airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 30: 777-783. [Abstract/Free Full Text]
  11. Jia, H. P., J. N. Kline, A. Penisten, M. A. Apicella, T. L. Gioannini, J. Weiss, P. B. McCray, Jr. 2004. Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2. Am. J. Physiol. 287: L428-L437.
  12. Guillot, L., S. Medjane, K. Le-Barillec, V. Balloy, C. Danel, M. Chignard, M. Si-Tahar. 2004. Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4. J. Biol. Chem. 279: 2712-2718. [Abstract/Free Full Text]
  13. Soong, G., B. Reddy, S. Sokol, R. Adamo, A. Prince. 2004. TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells. J. Clin. Invest. 113: 1482-1489. [Medline]
  14. DiMango, E., H. J. Zar, R. Bryan, A. Prince. 1995. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J. Clin. Invest. 96: 2204-2210. [Medline]
  15. Akira, S., K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Immunol. 4: 499-511. [Medline]
  16. Arbibe, L., J. P. Mira, N. Teusch, L. Kline, M. Guha, N. Mackman, P. J. Godowski, R. J. Ulevitch, U. G. Knaus. 2000. Toll-like receptor 2-mediated NF-{kappa}B activation requires a Rac1-dependent pathway. Nat. Immunol. 1: 533-540. [Medline]
  17. Schmeck, B., S. Huber, K. Moog, J. Zahlten, A. C. Hocke, B. Opitz, S. Hammerschmidt, T. J. Mitchell, M. Kracht, S. Rosseau, et al 2005. Pneumococci induced TLR- and Rac1-dependent NF-{kappa}B recruitment to the IL-8 promoter in lung epithelial cells. Am. J. Physiol. 290: L730-L737.
  18. Rajan, S., G. Cacalano, R. Bryan, A. J. Ratner, C. U. Sontich, A. van Heerckeren, P. Davis, A. Prince. 2000. Pseudomonas aeruginosa induction of apoptosis in respiratory epithelial cells: analysis of the effects of cystic fibrosis transmembrane conductance regulator dysfunction and bacterial virulence factors. Am. J. Respir. Cell Mol. Biol. 23: 304-312. [Abstract/Free Full Text]
  19. Pearson, J. P., M. Feldman, B. H. Iglewski, A. Prince. 2000. Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection. Infect. Immun. 68: 4331-4334. [Abstract/Free Full Text]
  20. Gomez, M. I., A. Lee, B. Reddy, A. Muir, G. Soong, A. Pitt, A. Cheung, A. Prince. 2004. Staphylococcus aureus protein A induces airway epithelial inflammatory responses by activating TNFR1. Nat. Med. 10: 842-848. [Medline]
  21. Bae, Y. S., L. G. Cantley, C. S. Chen, S. R. Kim, K. S. Kwon, S. G. Rhee. 1998. Activation of phospholipase C-{gamma} by phosphatidylinositol 3,4,5-trisphosphate. J. Biol. Chem. 273: 4465-4469. [Abstract/Free Full Text]
  22. TranVan Nhieu, G., C. Clair, G. Grompone, P. Sansonetti. 2004. Calcium signalling during cell interactions with bacterial pathogens. Biol. Cell 96: 93-101. [Medline]
  23. Ganguly, U., A. G. Chaudhury, A. Basu, P. C. Sen. 2001. STa-induced translocation of protein kinase C from cytosol to membrane in rat enterocytes. FEMS Microbiol. Lett. 204: 65-69. [Medline]
  24. McNamara, N., A. Khong, D. McKemy, M. Caterina, J. Boyer, D. Julius, C. Basbaum. 2001. ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor. Proc. Natl. Acad. Sci. USA 98: 9086-9091. [Abstract/Free Full Text]
  25. Gewirtz, A. T., A. S. Rao, P. O. Simon, Jr, D. Merlin, D. Carnes, J. L. Madara, A. S. Neish. 2000. Salmonella typhimurium induces epithelial IL-8 expression via Ca2+-mediated activation of the NF-{kappa}B pathway. J. Clin. Invest. 105: 79-92. [Medline]
  26. Wissel, H., T. Muller, M. Rudiger, M. Krull, R. R. Wauer. 2005. Contact of Chlamydophila pneumoniae with type II cell triggers activation of calcium-mediated NF-{kappa}B pathway. Biochim. Biophys. Acta 1743: 37-48. [Medline]
  27. Triantafilou, M., S. Morath, A. Mackie, T. Hartung, K. Triantafilou. 2004. Lateral diffusion of Toll-like receptors reveals that they are transiently confined within lipid rafts on the plasma membrane. J. Cell Sci. 117: 4007-4014. [Abstract/Free Full Text]
  28. DiMango, E., A. J. Ratner, R. Bryan, S. Tabibi, A. Prince. 1998. Activation of NF-{kappa}B by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells. J. Clin. Invest. 101: 2598-2605. [Medline]
  29. Bony, C., S. Roche, U. Shuichi, T. Sasaki, M. A. Crackower, J. Penninger, H. Mano, M. Puceat. 2001. A specific role of phosphatidylinositol 3-kinase {gamma}A regulation of autonomic Ca2+ oscillations in cardiac cells. J. Cell Biol. 152: 717-728. [Abstract/Free Full Text]
  30. Naga Prasad, S. V., G. Esposito, L. Mao, W. J. Koch, H. A. Rockman. 2000. Gbeta{gamma}-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy. J. Biol. Chem. 275: 4693-4698. [Abstract/Free Full Text]
  31. Sukumaran, S. K., G. McNamara, N. V. Prasadarao. 2003. Escherichia coli K-1 interaction with human brain micro-vascular endothelial cells triggers phospholipase C-{gamma}1 activation downstream of phosphatidylinositol 3-kinase. J. Biol. Chem. 278: 45753-45762. [Abstract/Free Full Text]
  32. Koyasu, S.. 2003. The role of PI3K in immune cells. Nat. Immunol. 4: 313-319. [Medline]
  33. Fukao, T., S. Koyasu. 2003. PI3K and negative regulation of TLR signaling. Trends Immunol. 24: 358-363. [Medline]
  34. Strassheim, D., K. Asehnoune, J. S. Park, J. Y. Kim, Q. He, D. Richter, K. Kuhn, S. Mitra, E. Abraham. 2004. Phosphoinositide 3-kinase and Akt occupy central roles in inflammatory responses of Toll-like receptor 2-stimulated neutrophils. J. Immunol. 172: 5727-5733. [Abstract/Free Full Text]
  35. Li, J. D., A. F. Dohrman, M. Gallup, S. Miyata, J. R. Gum, Y. S. Kim, J. A. Nadel, A. Prince, C. B. Basbaum. 1997. Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease. Proc. Natl. Acad. Sci. USA 94: 967-972. [Abstract/Free Full Text]
  36. David, M. D., C. L. Cochrane, S. K. Duncan, J. W. Schrader. 2005. Pure lipopolysaccharide or synthetic lipid A induces activation of p21Ras in primary macrophages through a pathway dependent on Src family kinases and PI3K. J. Immunol. 175: 8236-8241. [Abstract/Free Full Text]
  37. Chang, Y. J., M. S. Wu, J. T. Lin, B. S. Sheu, T. Muta, H. Inoue, C. C. Chen. 2004. Induction of cyclooxygenase-2 overexpression in human gastric epithelial cells by Helicobacter pylori involves TLR2/TLR9 and c-Src-dependent nuclear factor-{kappa}B activation. Mol. Pharmacol. 66: 1465-1477. [Abstract/Free Full Text]
  38. Asehnoune, K., D. Strassheim, S. Mitra, J. Yeol Kim, E. Abraham. 2005. Involvement of PKC{alpha}beta in TLR4 and TLR2 dependent activation of NF-{kappa}B. Cell. Signal. 17: 385-394. [Medline]
  39. Hughes, K., S. Edin, A. Antonsson, T. Grundstrom. 2001. Calmodulin-dependent kinase II mediates T cell receptor/CD3- and phorbol ester-induced activation of I{kappa}B kinase. J. Biol. Chem. 276: 36008-36013. [Abstract/Free Full Text]
  40. Malik, Z. A., G. M. Denning, D. J. Kusner. 2000. Inhibition of Ca2+ signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages. J. Exp. Med. 191: 287-302. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
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]

Home page
 J. Immunol.Home page
F. J. Martin and A. S. Prince
TLR2 Regulates Gap Junction Intercellular Communication in Airway Cells
J. Immunol., April 1, 2008; 180(7): 4986 - 4993.
[Abstract] [Full Text] [PDF]

Home page
 GENES CELLSHome page
D. Aki, Y. Minoda, H. Yoshida, S. Watanabe, R. Yoshida, G. Takaesu, T. Chinen, T. Inaba, M. Hikida, T. Kurosaki, et al.
Peptidoglycan and lipopolysaccharide activate PLCgamma2, leading to enhanced cytokine production in macrophages and dendritic cells.
Genes Cells, February 1, 2008; 13(2): 199 - 208.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
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]

Home page
 J. Biol. Chem.Home page
T. Into, Y. Kanno, J.-i. Dohkan, M. Nakashima, M. Inomata, K.-i. Shibata, C. J. Lowenstein, and K. Matsushita
Pathogen Recognition by Toll-like Receptor 2 Activates Weibel-Palade Body Exocytosis in Human Aortic Endothelial Cells
J. Biol. Chem., March 16, 2007; 282(11): 8134 - 8141.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chun, J.
Right arrow Articles by Prince, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chun, J.
Right arrow Articles by Prince, A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS