TLR4 signaling is coupled to SRC family kinase activation, tyrosine phosphorylation of zonula adherens proteins, and opening of the paracellular pathway in human lung microvascular endothelia

Abstract

Bacterial lipopolysaccharide (LPS) is a key mediator in the vascular leak syndromes associated with Gram-negative bacterial infections. LPS opens the paracellular pathway in pulmonary vascular endothelia through protein tyrosine phosphorylation. We now have identified the protein-tyrosine kinases (PTKs) and their substrates required for LPS-induced protein tyrosine phosphorylation and opening of the paracellular pathway in human lung microvascular endothelial cells (HMVEC-Ls). LPS disrupted barrier integrity in a dose- and time-dependent manner, and prior broad spectrum PTK inhibition was protective. LPS increased tyrosine phosphorylation of zonula adherens proteins, VE-cadherin, gamma-catenin, and p120(ctn). Two SRC family PTK (SFK)-selective inhibitors, PP2 and SU6656, blocked LPS-induced increments in tyrosine phosphorylation of VE-cadherin and p120(ctn) and paracellular permeability. In HMVEC-Ls, c-SRC, YES, FYN, and LYN were expressed at both mRNA and protein levels. Selective small interfering RNA-induced knockdown of c-SRC, FYN, or YES diminished LPS-induced SRC Tyr(416) phosphorylation, tyrosine phosphorylation of VE-cadherin and p120(ctn), and barrier disruption, whereas knockdown of LYN did not. For VE-cadherin phosphorylation, knockdown of either c-SRC or FYN provided total protection, whereas YES knockdown was only partially protective. For p120(ctn) phosphorylation, knockdown of FYN, c-SRC, or YES each provided comparable but partial protection. Toll-like receptor 4 (TLR4) was expressed both on the surface and intracellular compartment of HMVEC-Ls. Prior knockdown of TLR4 blocked both LPS-induced SFK activation and barrier disruption. These data indicate that LPS recognition by TLR4 activates the SFKs, c-SRC, FYN, and YES, which, in turn, contribute to tyrosine phosphorylation of zonula adherens proteins to open the endothelial paracellular pathway.

FIGURE 1.

LPS increases paracellular permeability across HMVEC-Ls. Postconfluent HMVEC-L monolayers were exposed for 6 h to increasing concentrations of LPS (A) or to one of two fixed concentrations of LPS (10 or 100 ng/ml) or medium alone for increasing exposure times (B). C, in other studies, postconfluent HMVEC-L monolayers were exposed for 6 h to equivalent concentrations of LPS versus protein-free LPS that lacks contaminating TLR2 agonists, increasing concentrations of Pam3cys, an established TLR2 agonist, or medium alone. Vertical bars represent mean ± S.E. transendothelial [¹⁴C]BSA flux in pmol/h immediately after the study period. Mean ± S.E. pretreatment base-line transendothelial [¹⁴C]BSA flux is indicated by the closed bars in A and C, and flux across naked filters is shown by the stippled bar in A. n indicates number of monolayers studied and in A and C is indicated within each bar. In B, the n for each time point within each group was 6. ^*, significantly increased compared with the simultaneous medium control at p < 0.05; ^**, significantly decreased compared with LPS alone at p < 0.05. The data in A and B each were the cumulative result of three independent experiments with 2-6 replicates/treatment/experiment, whereas the data in C were obtained from two experiments with three replicates/treatment/experiment.

FIGURE 2.

TLR4 Expression in HMVEC-Ls. A, RNA was isolated from HMVEC-Ls and cDNA generated using oligo(dT) primers and reverse transcriptase. This cDNA was used as a template for amplification with DNA polymerase and primers corresponding to TLR4 (lane 2). Base pairs (bp) and control DNA ladder are indicated on the left (lane 1). PCR mixture without DNA template as a negative control is indicated in lane 3. These RT-PCR experiments were performed twice. B, HMVEC-L lysates were resolved by SDS-PAGE and transferred to PVDF, and blots were probed for TLR4. Molecular masses in kDa are indicated on the left. IB, immunoblot. This blot is representative of three experiments. C, nonpermeabilized (i) and permeabilized (ii) HMVEC-Ls were studied by flow cytometry for TLR4. This study is representative of three experiments. D, postconfluent HMVEC-Ls were fixed and, in selected experiments, were permeabilized, blocked, and incubated with anti-TLR4 antibodies followed by fluoroprobe-labeled secondary antibodies. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. i, surface TLR4; ii, total cell TLR4. Green, TLR4; blue, nuclei. The arrows indicate perinuclear TLR4 staining. Magnification was ×400. These photomicrographs are representative of two experiments.

FIGURE 3.

Role of TLR4 in LPS-induced barrier disruption. HMVEC-Ls were transfected with TLR4-targeting or control siRNAs for 24 h. A, RT-PCR was applied to detect mRNAs for TLR4. B, HMVEC-Ls were processed for immunoblotting for TLR4, and each blot was stripped and reprobed for β-tubulin. IB, immunoblot; IB^*, immunoblot after stripping. These blots are representative of ≥3 experiments. C, for the barrier assay, HMVEC-Ls cultured to 70% confluence in plastic dishes were transfected with siRNA targeting TLR4 or control siRNAs. Transfected cells were seeded onto the filters in assay chambers and cultured for 24 h, after which base-line barrier function was established. Only EC monolayers that retained ≥97% of the tracer molecule were exposed for 6 h to 100 ng/ml LPS or medium alone and again assayed for transendothelial [¹⁴C]BSA flux. Vertical bars represent mean ± S.E. transendothelial [¹⁴C]BSA flux in pmol/h immediately after the 6-h study period. n, the numbers of wells studied, was 6 for each group, and these data were the cumulative result of three independent experiments with two replicates/treatment/experiment. ^*, significantly increased compared with the control siRNA group at p < 0.05; ^**, significantly decreased compared with LPS and control siRNA at p < 0.05.

FIGURE 4.

Role of PTKs in LPS-induced barrier disruption. A, LPS (100 ng/ml) was presented to HMVEC-L monolayers in the presence and absence of either of two broad spectrum PTK inhibitors, herbimycin A (HerbA) (1 μm) or geldanamycin (geld) (1 μm), or monolayers were treated with each agent or medium alone. B, postconfluent HMVEC-Ls cultured in barrier assay chambers were exposed for 6 h to LPS (100 ng/ml) or medium alone in the presence or absence of PP2 (5 μm). Vertical bars represent mean ± S.E. transendothelial [¹⁴C]BSA flux in pmol/h immediately after the 6-h study period. Mean ± S.E. pretreatment base-line transendothelial [¹⁴C]BSA flux is indicated by the closed bars. n indicates the number of monolayers studied and is indicated within each bar. Data were generated from ≥3 independent experiments with 2-3 replicates/treatment/experiment. ^*, significantly increased compared with the medium control at p < 0.05; ^**, significantly decreased compared with LPS alone at p < 0.05.

FIGURE 5.

SFK expression in HMVEC-Ls. A, RT-PCR was used to detect mRNAs for the eight human SFKs, c-SRC, YES, FYN, LYN, BLK, FGR, HCK, and LCK, as well as GAPDH (lane 10) as a housekeeping control. Base pairs (bp) with control DNA ladder are shown on the left (lane 1). B, HMVEC-L lysates were resolved by SDS-PAGE and transferred to PVDF, and blots were probed with antibodies raised against the four SFKs, expressed in HMVEC-Ls at the mRNA levels, c-SRC, YES, FYN, and LYN. Molecular mass in kDa is indicated on the left. The arrow on the right indicates SFKs. Each of these two blots is representative of two experiments.

FIGURE 6.

LPS Activates SFK(s) through TLR4. Postconfluent HMVEC-Ls were exposed to increasing concentrations of LPS or medium alone for increasing exposure times and processed for a cell-based ELISA that detects phosphorylation of Tyr⁴¹⁶, the activation site conserved across SFKs. The assay normalizes phospho-Tyr⁴¹⁶ to total SFK and total cellular protein. SFK activation is expressed as mean ± S.E. -fold increase relative to the simultaneous medium control. A, ECs exposed for 10 min to increasing concentrations of LPS (n = 6). B, ECs exposed for increasing times to LPS (100 ng/ml) or medium alone (n = 6). ^*, significantly increased compared with the simultaneous medium control at p < 0.01. C, HMVEC-Ls were transfected with TLR4-targeting, or control siRNAs for 24 h and processed for the same ELISA that detects Tyr⁴¹⁶ phosphorylation. n = 9 for each control and experimental group. ^*, significantly increased compared with the control siRNA group at p < 0.05; ^**, significantly decreased compared with LPS and control siRNA at p < 0.05. The data in A, B, and C each were generated from ≥3 independent experiments with 2-3 replicates/treatment/experiment.

FIGURE 7.

Identification of ZA proteins as substrates for LPS-induced tyrosine phosphorylation. Postconfluent HMVEC-L monolayers were exposed for varying times to LPS (100 ng/ml) or medium alone. A, HMVEC-Ls were fixed, incubated with FITC-conjugated antiphosphotyrosine antibody, and analyzed by fluorescence microscopy. i, medium control (1 h); ii, LPS (1 h). The arrows indicate phosphotyrosine signal at intercellular boundaries. Magnification was ×400. These photomicrographs are representative of two experiments. B, in other studies, HMVEC-Ls were exposed to LPS (100 ng/ml) or medium alone in the presence of vanadate (200 μm) and phenylarsine oxide (1.0 μm), only during the last 0.25 h of incubation. HMVEC-L lysates were resolved by SDS-PAGE and transferred to PVDF membranes, and the blots were probed with antiphosphotyrosine antibody. To confirm equivalent protein loading and transfer, blots were stripped and reprobed for β-tubulin. Molecular masses in kDa are indicated on the left. The arrows on the right indicate bands with altered phosphotyrosine signal in response to LPS. This blot is representative of three experiments. C, lysates of LPS-treated and medium control HMVEC-Ls were immunoprecipitated with antibodies raised against VE-cadherin (lanes 1 and 2), β-catenin (lanes 3 and 4), γ-catenin (lanes 5 and 6), and p120^ctn (lanes 7 and 8). The immunoprecipitates were resolved by SDS-PAGE and transferred onto PVDF, and the blots were probed with antiphosphotyrosine antibody. For normalization of phosphotyrosine signal to the immunoprecipitated protein, blots were stripped and reprobed with each immunoprecipitating antibody. Each blot is representative of ≥3 experiments. IP, immunoprecipitate; IB, immunoblot; IB^*, immunoblot after stripping.

FIGURE 8.

LPS increases tyrosine phosphorylation of ZA proteins and endothelial paracellular permeability through SFK(s) activation. A, ECs exposed to LPS (100 ng/ml) or medium alone in the presence or absence of either of two SFK-selective inhibitors, PP2 or SU6656 (10 μm), were processed for a cell-based ELISA that detects Tyr⁴¹⁶ phosphorylation. SFK activation is expressed as mean ± S.E. -fold increase relative to the simultaneous medium control. n, the number of monolayers studied, is indicated within each bar. B, HMVEC-Ls were exposed for 4 h to LPS (100 ng/ml) or medium alone in the presence or absence of PP2 (5 μm) or SU6656 (10 μm) as well as in the presence of vanadate (200 μm) and phenylarsine oxide (1.0 μm), only during the last 0.25 h of incubation. ECs were lysed and immunoprecipitated with antibodies against VE-cadherin (lanes 1-3), γ-catenin (lanes 4-6), and p120^ctn (lanes 7-9). The immunoprecipitates were resolved by SDS-PAGE and transferred to PVDF, and the blots were probed with antiphosphotyrosine antibody. Blots were stripped and reprobed with the respective immunoprecipitating antibodies. IP, immunoprecipitate; IB, immunoblot; IB^*, immunoblot after stripping. These blots are representative of ≥3 experiments. C, postconfluent human pulmonary artery ECs cultured in barrier assay chambers were exposed for 6 h to LPS (100 ng/ml) or medium alone in the presence or absence of increasing concentrations of PP2 or SU6656. Vertical bars represent mean ± S.E. transendothelial [¹⁴C]BSA flux in pmol/h immediately after the 6-h study period. The mean ± S.E. pretreatment base lines are indicated in C by the closed bar. In C, the number of monolayers studied was six for each condition. ^*, significantly increased compared with the simultaneous medium control at p < 0.05; ^**, significantly decreased compared with LPS alone at p < 0.05. The data in A and C each were generated from three independent experiments with 2-3 replicates/treatment/experiment.

FIGURE 9.

Knockdown of SFKs in HMVEC-Ls through siRNA. HMVEC-Ls were transfected with siRNAs targeting the four SFKs expressed in HMVEC-Ls, c-SRC, YES, FYN, and LYN, or control siRNAs. A, after 72 h, ECs were processed for immunoblotting with antibodies against each of these four SFK proteins. Blots were stripped and reprobed for β-tubulin. These blots are representative of three experiments. B, ECs were processed for a cell-based ELISA to detect phospho-Tyr⁴¹⁶ as a measure of SFK activation. The phospho-Tyr⁴¹⁶ was normalized to both total SFK and cellular protein and expressed as mean ± S.E. -fold increase relative to the simultaneous medium control. n, the numbers of wells studied, was 6 for each group. C, HMVEC-Ls were transfected with siRNAs specifically targeting c-SRC, YES, FYN, or control siRNAs. After 72 h, ECs were exposed for 4 h with LPS (100 ng/ml) or medium alone and lysed, and the lysates were immunoprecipitated with anti-VE-cadherin or anti-p120^ctn antibodies. The immunoprecipitates were resolved by SDS-PAGE and transferred to PVDF, and the blots were probed with anti-phosphotyrosine antibodies. To normalize phosphotyrosine signal to immunoprecipitated protein, the immunoblots were stripped and reprobed with the immunoprecipitating antibodies raised against VE-cadherin and p120^ctn. These blots are representative of four experiments. D, on each immunoblot, densitometric quantification of phosphotyrosine signal of VE-cadherin and p120^ctn immunoprecipitates each were normalized to total VE-cadherin and p120^ctn signal, respectively. Vertical bars represent mean ± S.E. -fold increase of arbitrary densitometry units of phosphotyrosine signal normalized to arbitrary densitometry units of total signal relative to the simultaneous control. n = 4. E, for the barrier assay, HMVEC-Ls cultured to 70% confluence in plastic dishes were transfected with siRNA targeting FYN, c-SRC, LYN, YES, or control siRNAs. After 24 h, transfected cells were seeded onto the filters in assay chambers and cultured for 48 h, after which base line barrier function was established. Only EC monolayers that retained ≥97% of the tracer molecule were exposed for 6 h to 100 ng/ml LPS or medium alone and again assayed for transendothelial [¹⁴C]BSA flux. Vertical bars represent mean ± S.E. transendothelial [¹⁴C]BSA flux in pmol/h immediately after the 6-h study period. n indicates the number of monolayers studied and is indicated within each bar. In A and C, IP, immunoprecipitate; IB, immunoblot; IB^*, immunoblot after stripping. In B, D, and E, ^*, significantly increased compared with the simultaneous medium with control siRNA at p < 0.05; ^**, significantly decreased compared with LPS and control siRNA at p < 0.05. In B and E, the data sets were generated from three independent experiments with 2-4 replicates/treatment/experiment. In C, the data were generated from four independent experiments, and the mean ± S.E. changes of the combined data are displayed in D.