Cross talk between paxillin and Rac is critical for mediation of barrier-protective effects by oxidized phospholipids

Abstract

We previously reported that the barrier-protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cells (ECs) delineate the role of Rac- and Cdc42-dependent mechanisms and described the involvement of the focal adhesion (FA) protein paxillin in enhancement of the EC barrier upon OxPAPC challenge. This study examined a potential role of paxillin in the feedback mechanism of Rac regulation by FAs in OxPAPC-stimulated ECs. Our results demonstrate that OxPAPC induced Rac-dependent, Rho-independent peripheral accumulation of paxillin-containing FAs and time-dependent paxillin phosphorylation. Molecular inhibition of Rac decreased association of paxillin with the Rac-specific guanine nucleotide exchange factor beta-PIX. Molecular inhibition of paxillin also attenuated OxPAPC-induced enhancement of adherens junctions critical for the EC barrier-protective response, accumulation of vascular endothelial cadherin in the membrane fractions, and decreased activation of Rac and its effector p21-activated kinase (PAK1). Expression of paxillin with a mutated PAK1-dependent phosphorylation site (S273A) attenuated OxPAPC-induced PAK1 activation and the EC barrier-protective response. These results suggest that PAK1-specific paxillin phosphorylation at Ser(273) is critically involved in the positive-feedback regulation of the Rac-PAK1 pathway and may contribute to sustained enhancement of the EC barrier caused by oxidized phospholipids.

Fig. 1.

Time-dependent effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) on paxillin redistribution. Endothelial cells (ECs) grown on glass coverslips were stimulated with OxPAPC (20 μg/ml) for 0–45 min. A: ECs were fixed and subjected to dual-immunofluorescence staining with Texas red-phalloidin for detection of F-actin and paxillin antibodies. Arrows, paxillin peripheral distribution after OxPAPC challenge. B: quantitative analysis of OxPAPC-induced peripheral translocation to paxillin-containing focal adhesions (FAs), expressed as arbitrary units of fluorescence intensity. Results are representative of 3–5 independent experiments. *P < 0.05.

Fig. 2.

OxPAPC induces time-dependent site-specific paxillin phosphorylation. Human pulmonary artery ECs were treated with OxPAPC (20 μg/ml) for 0–45 min. Phosphorylation of paxillin was analyzed by immunoblotting of cell lysates with a panel of phosphospecific antibodies. Equal protein loading was verified by membrane reprobing with paxillin antibody. Results of quantitative analysis of site-specific paxillin phosphorylation are shown as relative densitometric units (RDU). Values are means ± SD of 3 independent experiments. *P < 0.05.

Fig. 3.

Involvement of Rac and Rho in regulation of paxillin redistribution. Lung ECs were transiently transfected with constitutively active (CA) Rac (A), CA Rho (B), dominant-negative (DN) Rac (C), or DN Rho (D) bearing hemagglutinin (HA) tags. A and B: ECs were fixed and subjected to dual-immunofluorescence staining with paxillin and HA-tagged antibodies. Insets: areas of cell-cell contacts and differential distribution of paxillin in Rac- and Rho-transfected cells. C and D: ECs were treated with OxPAPC (20 μg/ml) and then dual stained with paxillin and HA-tagged antibodies. E: areas of cell-cell contacts and paxillin accumulation upon OxPAPC stimulation in nontransfected (Non-TF) ECs (1) and ECs transfected with DN Rac [TF-Rac (DN), 2] and Rho [TF-Rho (DN), 3].

Fig. 4.

Role of paxillin regulation of OxPAPC-induced Rac signaling. Human pulmonary ECs were transfected with Rac-specific (A) or paxillin-specific (B–E) small interfering (si) RNA and then stimulated with OxPAPC (20 μg/ml). Control tansfections were performed using nonspecific (ns) RNA. Depletion of target proteins induced by specific siRNA duplexes was confirmed by immunoblotting with appropriate antibodies compared with treatment with nsRNA. Immunoblot with β-tubulin antibodies was used as a normalization control. Veh, vehicle. Results of scanning densitometry of Western blots are shown as RDU. Results are representative of 3 independent experiments. Values are means ± SD. *P < 0.05. A: after cell lysis, protein complexes were immunoprecipitated with paxillin antibodies, and β-PIX was detected by Western blotting. Equal protein loading was confirmed by membrane reprobing with paxillin antibodies. B: Rac activation was determined in control and OxPAPC-stimulated ECs using pull-down assay. Rac content in EC lysates is shown as RDU. C: OxPAPC-induced PAK1 phosphorylation was determined in total lysates using phosphorylated PAK1-specific antibody. D: OxPAPC-induced distribution of vascular endothelial (VE)-cadherin and p115 Rho guanosine nucleotide exchange factor (GEF) in cytosolic and membrane fractions was analyzed by Western blot with corresponding antibodies. E: effects of paxillin knockdown on redistribution of vinculin-containing FAs in control and OxPAPC-treated monolayers were analyzed by immunofluorescence staining with vinculin antibodies. Arrows, areas of peripheral vinculin accumulation.

Fig. 5.

Phosphorylation of paxillin at Ser²⁷³ is critical for OxPAPC-induced EC barrier response. EC monolayers were subjected to nucleofection with the following constructs bearing green fluorescent protein (GFP) tags: empty vector (Em.vector) and wild-type paxillin (PXN-WT, A), paxillin without the LD4 motif (PXN-ΔLD4, B), or phosphorylation-deficient paxillin mutant (PXN-S273A, C). After 24 h of transfection, cells were stimulated with OxPAPC (20 μg/ml). Changes in EC permeability were monitored by measurements of transendothelial electrical resistance. Results are representative of 3 independent experiments.

Fig. 6.

Phosphorylation of paxillin at Ser²⁷³ is critical for OxPAPC-induced PAK1 autophosphorylation, cytoskeletal remodeling, and recovery of EC monolayer integrity. A: EC monolayers were subjected to nucleofection with PXN-wt or PXN-S273A bearing GFP tags. After 24 h of transfection, cells were stimulated with OxPAPC (20 μg/ml). OxPAPC-induced PAK1 phosphorylation was determined in total lysates using phosphorylated PAK1-specific antibody. Results of scanning densitometry of Western blots are shown as RDU. Values are means ± SD of 3 independent experiments. *P < 0.01 vs. nonstimulated control. **P < 0.05 vs. OxPAPC-stimulated EC transfected with PXN-wt. B: subconfluent ECs were transiently transfected with GFP-PXN-S273A and then treated with OxPAPC (20 μg/ml). Cells were fixed and subjected to immunofluorescence staining with Texas red-phalloidin for detection of F-actin. Transfected cells are shown in outlined area. Arrows, remaining gaps between GFP-PXN-S273A-expressing cells after OxPAPC stimulation. Results are representative of 3 independent experiments.

Fig. 7.

Hypothetical scheme of paxillin involvement in OxPAPC-mediated regulation of Rac signaling and endothelial barrier properties. OxPAPC stimulation of EC monolayers leads to Rac/PAK1-dependent translocation of paxillin-containing FA complexes to the cell periphery, which contributes to development of EC barrier-protective response. In turn, paxillin within FAs becomes phosphorylated at Ser²⁷³ contained within the LD4 domain, which results in formation of the paxillin-GIT-β-PIX-PAK complex, activation of Rac-specific GEF β-PIX, additional Rac activation, and further stimulation of Rac-dependent signaling. This positive-feedback mechanism of paxillin-mediated Rac activation contributes to sustained enhancement of the endothelial barrier in response to oxidized phospholipids. AJ, adherens junction.