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Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
1Correspondence: Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine, 5501 Hopkins Bayview Circle, JHAAC, 4B.77, Baltimore, MD 21224, USA. E-mail: drgarcia{at}jhmi.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: human pulmonary artery endothelium • barrier function • cytoskeleton
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. We have previously shown that thrombin promotes endothelial cell activation and increases vascular permeability (3
4
5
6)
by binding to proteinase-activated receptor-1 (PAR) (2)
, which results in dramatic cytoskeletal rearrangements, phosphorylation of myosin light chains (MLC) (6
, 7)
, and increased intracellular tension. This increase in contractile forces reduces intercellular junctional connections and endothelial cell–extracellular matrix adhesive forces (8
, 9)
.
In contrast to the edemagenic effects of thrombin, phospholipids released after platelet activation may enhance the microvascular barrier properties in vivo and in vitro (10
, 11)
, whereas a reduction in circulating platelets in patients accelerates capillary leakage and tissue edema formation (12
, 13)
. Impaired endothelial barrier function can be reversed with platelet infusions or by the systemic administration of platelet-released products (14)
. Among several platelet-derived lipids, sphingosine 1-phosphate (S1P) is a remarkably effective endothelial cell agonist that induces proliferation, calcium mobilization, adhesion molecule expression, and suppression of apoptosis (15
16
17
18
19)
. S1P is present in human serum (20
21
22)
and is released by stimulated platelets to bind to the endothelial differentiation gene (Edg) family of receptors (19
, 23
, 24)
. We previously reported that the barrier protective effect of SIP on pulmonary endothelial monolayers is associated with the formation of a prominent cortical actin ring, with a potentially key role for the small GTPase Rac in this process (25)
.
Actin-containing cytoskeletal rearrangement is closely related with FA distribution. Recent findings suggest that FA assembly and disassembly result in stress fiber formation and displacement, respectively (26)
, with FAK phosphorylation potentially modulating increased endothelial cell–matrix adhesion. Paxillin is a multidomain adaptor FA protein containing binding sites for various signaling molecules and structural proteins (27
28
29
30
31
32)
. Paxillin facilitates signal transduction from extracellular matrix and receptor-dependent agonists by recruiting specific molecules to FAs, whereas the phosphorylation status of paxillin is believed to be important in determining its binding partners (33
34
35
36)
. For example, the paxillin tyrosine residue Y118 is a major site of FAK-catalyzed phosphorylation (33
, 37)
. FAK and paxillin involvement in FA assembly/disassembly is complex, as fak-/- knockout cell lines and transient transfection techniques indicate FAK involvement in FA disassembly and redistribution (38
, 39)
. Small GTPases (Rac, Rho) also regulate actin cytoskeletal remodeling and FA dynamics via ADP-ribosylation factor GTPase activation factors (ARF GAPs) (40)
, which interact with several signaling and cytoskeletal proteins, including paxillin. Among ARF GAP proteins, G-protein-coupled receptor kinase-interacting proteins GIT 1 and paxillin kinase linker (PKL/GIT2) bind paxillin directly and participate in signaling events at FAs (40
41
42
43)
. GIT1 may be engaged in the regulation of directional cell motility and disassembly of Rho-containing FAs through displacement of paxillin (43)
, while GIT2 may deliver paxillin from cytosol to newly formed focal adhesions via Rac-dependent mechanisms (40
, 41)
. We recently reported that S1P induced the interaction of focal adhesion proteins paxillin and FAK as well as the interaction of paxillin with Rac GTPase, GIT1, and GIT2 (44)
. Our results indicated the active participation of these proteins in FA assembly and demonstrated the novel interaction of FA proteins with members of GIT family. In the current study, we have attempted to extend our understanding of the potential mechanisms involved in SIP-induced FA remodeling and EC barrier enhancement and to elucidate the role of paxillin, FAK, and GITs in the formation of a prominent cortical actin ring after SIP stimulation. Our results suggest the involvement of site-specific FAK phosphorylation and redistribution of FAK, paxillin, GIT1, and GIT2 in thrombin- and S1P-stimulated human endothelial monolayers in the differential FA remodeling and barrier regulation responses.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human pulmonary artery endothelial cell culture
Human pulmonary artery endothelial cells (HPAECs) were obtained from Clonetics and cultured in EBM-2 complete medium containing 10% FBS. Endothelial cell cultures were maintained at 37°C in a humidified atmosphere and grown to contact-inhibited monolayers with typical cobblestone morphology. Cells from each primary flask (passage 5 to 8) were detached with 0.05% trypsin, resuspended in fresh culture medium, and passaged into 100 mm2 dishes for Rho and Rac activity assay, 60 mm2 dishes for Western blot and immunoprecipitation, or a 12-well plate (with cover glasses) for immunofluorescent analysis.
Immunofluorescence microscopy
Human lung ECs grown on gelatinized coverslips were rendered quiescent in EBM-2 containing 1% FBS for 20 h, incubated with S1P or vehicle control, fixed in 3.7% paraformaldehyde in PBS for 15 min, washed three times with PBS, permeabilized with 0.25% Triton X-100 and in 0.1% Tween-20 containing TBS (TBS-T) for 15 min, and blocked with 2% BSA in TBS-T for 30 min. Incubations with primary antibodies of interest were performed in blocking solution (2% BSA in TBS-T) for 1 h at room temperature. After three washes with TBS-T, cells were incubated with appropriate secondary antibodies conjugated to immunofluorescent dyes (Alexa 488 for green fluorescence or Alexa 546 for red fluorescence) in blocking solution for 1 h at room temperature. Actin filaments were visualized by staining cells with Texas red-conjugated phalloidin for 1 h at room temperature. After three washes with PBS, the coverslips were mounted using Slow Fade kit (Molecular Probes). Analysis of immunofluorescent staining was performed using a Nikon Eclipse TE 300 microscope with x60 objective lens and Sony Digital Photo camera DKC 5000.
Western blot analysis
Human lung ECs grown on 60 mm2 dishes were rendered quiescent in EBM-2 containing 1% FBS for 20 h, then stimulated with 0.5 µM S1P dissolved in the same medium for the indicated periods. After brief washing with PBS, cells were lysed with 300 µL of cell lysis buffer containing 10 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 0.2 mM EGTA, 0.2 mM vanadate, 0.2 mM PMSF, and 0.5% phosphatase inhibitor cocktail for each dish. Total cell lysates were cleared by centrifugation and boiled with the same amount of 3x SDS sample buffer for 5 min; 15 µL of lysates was then subjected to 7.5% SDS-PAGE (SDS-PAGE). The separated proteins were transferred to PVDF membranes by electrotransfer. The blots were subsequently blocked with 5% FBS in phosphate-buffered saline (PBS) containing 0.1% Tween-20 (PBS-T) at room temperature for 1 h,, then incubated at 4°C overnight with primary rabbit polyclonal anti-FAK [Y397], [Y576], and [Y925] phospho-specific antibodies (1:1000 dilution). After washing three times for 10 min with PBS-T, the membrane was incubated with 1:3000 dilution of HRP-linked anti-rabbit IgG secondary antibody at room temperature for 1 h. The blots were then visualized with the ECL Western blot detection system. To reprove membranes with anti-FAK antibody, membranes were incubated in reproving buffer containing 62.5 mM Tris (pH 6.8), 2% deoxycholate, and 100 mM mercaptoethanol at 4°C for 30 min. After being washed four times with PBS-T for 10 min, membranes were incubated with anti-FAK antibody (1:1000 dilution) at room temperature for 1 h, then visualized again. The amount of detected proteins was analyzed using Image Quant software.
Immunoprecipitation analysis
For immunoprecipitation, 70 µL of cell lysates was diluted with 100 µL of the same buffer, then incubated with 2 µg of the appropriate antibody (anti-paxillin or anti-FAK antibody) at 4°C for 1 h, followed by incubation with protein G-Agarose 4B for 1 h. Agarose beads were collected by centrifugation, washed three times with the same buffer, resuspended in 20 µL of 3x SDS sample buffer, then boiled for 5 min. Proteins were separated and incubated with anti-phosphotyrosine antibody (PY20, 1:1000 dilution) as described for Western blot analysis. For coimmunoprecipitation analysis, cell lysis buffer was substituted with coprecipitation buffer containing 50 mM Tris (pH 7.8), 1% NP-40 and 20 mM EDTA, 0.2 mM vanadate, and 0.2 mM PMSF (45)
; paxillin was immunoprecipitated and proteins were separated as described for Western blot analysis. Resulted membranes were blotted with appropriate antibodies (anti-GIT1 (1:250 dilution), anti-GIT-2 (1:500 dilution), and anti-FAK (1:1000 dilution) antibody) and the amount of coprecipitated proteins was analyzed using Image Quant software.
Statistical analysis
Results are expressed as means ± SD of three to five independent experiments. Stimulated samples were compared with controls by unpaired Student’s t test. For multiple group comparisons, one-way ANOVA (ANOVA), followed by the post hoc Fisher’s test, was used. P< 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B, D), suggesting partial localization at focal adhesions. Thrombin (100 nM) induced formation of massive stress fibers and intercellular gaps with redistribution of FAK to the ends of stress fibers (Fig. 1A, B
), which contrasted with redistribution of FAK to the cell periphery in conjunction with SIP-induced enhancement of the cortical actin ring (Fig. 1
, insets), consistent with our previous report (44)
. Thrombin-mediated alterations in FAK distribution and stress fiber formation persisted for up to 30 min (Fig. 1A, B
). Thrombin-induced paxillin redistribution was similar to FAK (Fig. 2
C) and occurred in a dot-like pattern, suggesting the formation of FAK- and paxillin-containing focal adhesions. A merged image of F-actin and FAK staining further demonstrated FAK positioning at the stress fiber attachment sites in thrombin-stimulated HPAEC whereas FAK colocalized with the peripheral actin ring in response to S1P stimulation (Fig. 1D
).
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Thrombin-induced redistribution of GIT1 and FIT2
The small GTPases Rac and Rho play an important role in the regulation of cytoskeletal remodeling and cell contact rearrangements (25
, 46)
. A new group of small GTPase regulators, G-protein interacting (GIT) proteins, have been implicated in disassembly of preexisting focal adhesions (40
, 43)
. We recently reported the role of the two members of GIT family, GIT1 and GIT2, in SIP-mediated focal adhesion remodeling (44)
. With this background, we next examined the redistribution of GIT1 and GIT2 in response to thrombin. Thrombin induced the redistribution of GIT1 both at the ends of stress fibers, where GIT1 colocalized with paxillin, also consistent with results of the GIT1/paxillin coimmunoprecipitation assay (see Fig. 3
), and along stress fibers (Fig. 2A
); this pattern persisted for up to 60 min. These results show that thrombin stimulation induces GIT1 association with two distinct protein partners, paxillin and F-actin, and indicate a novel GIT1 interaction with actin cytoskeleton. The role of GIT2 in FA dynamics differs from the role of GIT1, as GIT2 is tightly bound with paxillin and may be engaged in the delivery of paxillin from cytosol to focal adhesions (40
, 41)
. GIT2 stained diffusely in quiescent HPAECs (Fig. 2B
). Thrombin induced the redistribution of GIT2 similar to the pattern of FAK and paxillin staining (Fig. 1)
, suggesting GIT2 localization at newly formed focal adhesions (Fig. 2B
). The effect of thrombin on GIT1 and GIT2 redistribution contrasted with the effects of SIP, which induced predominant localization of GIT1 and GIT2 at the cell cortical area without noticeable GIT1 colocalization with actin filaments (Fig. 2
, insets).
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Effect of thrombin on paxillin association with FAK, GIT1, and GIT2
Confluent HPAECs were next stimulated with thrombin (100 nM), followed by immunoprecipitation of the individual FA component. Thrombin promoted the association of FAK and GIT1 with paxillin in a time-dependent manner (Fig. 3
A1, A2, B), whereas the amounts of GIT2 coprecipitated with paxillin did not significantly change (Fig. 3
A3, B).
Analysis of site-specific FAK phosphorylation in thrombin-stimulated HPAECs
As shown in Fig. 1
, thrombin induces redistribution of FAK and paxillin, which is distinct from the S1P response. This dissimilarity led us to hypothesize that the differential activation of FAK may be a major mechanism of the S1P- and thrombin-induced focal adhesion remodeling. We previously reported that S1P (0.5 µM) induces FAK [Y576] whereas [Y397] and [Y925] phosphorylation sites are not influenced by S1P challenge (44)
. To compare differential site-specific phosphorylation of FAK, confluent and static HPAECs were stimulated with S1P and thrombin and total cell lysates were subjected to electrophoresis and blotted with site-specific anti-phospho-FAK antibodies. While both thrombin and S1P activated Y576 phosphorylation, thrombin induced additional phosphorylation of FAK at tyrosine residues Y 397 and Y925 (Fig. 4
A, C).
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Differential effect of PP2 on site-specific FAK tyrosine phosphorylation induced by S1P and thrombin
Differences in the phosphorylation profile of FAK by S1P and thrombin (Fig. 4)
suggest a potentially important role for FAK in the mechanisms underlying the differential dynamics of focal adhesion remodeling and F-actin rearrangement (Figs. 1
2
3)
. Src is a known binding partner of FAK, and the close relationship between FAK and Src activation has been described (37)
. To elucidate the involvement of Src in S1P- and thrombin-induced FAK tyrosine phosphorylation, quiescent HPAEC monolayers were stimulated with 0.5 µM S1P (Fig. 5
A) or 100 nM thrombin (Fig. 5B1, 2, 3
) for 30 min after incubation with 5 µM PP2 (60 min), a selective Src inhibitor. Total HPAEC lysates were probed with three site-specific anti-phospho-FAK antibodies as indicated. Basal levels of phosphorylation at Y397, Y576, and Y925 in nonstimulated cells were taken as 100% for each individual experiment. PP2 (5 µM) treatment did not significantly affect the basal level of phosphorylation at Y397, Y576, and Y925, but abolished S1P-induced FAK phosphorylation at Y576 (Fig. 5A
) as well as thrombin-induced FAK phosphorylation at Y397 (Fig. 5B1
). An inhibitory effect of PP2 on FAK Y576 and Y925 phosphorylation evoked by thrombin was expressed to a lesser extent (Fig. 5B2, B3
), suggesting potential involvement of other tyrosine kinases in thrombin-mediated EC responses.
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The differential effect of PP2 on the morphological changes induced by S1P and thrombin
To investigate the role of Src in S1P- and thrombin-induced redistribution of F-actin and focal adhesion-related proteins, quiescent HPAEC monolayers were stimulated with 0.5 µM S1P (Fig. 6
) or 100 nM thrombin (Fig. 7
) for 30 min preceded by 60 min incubation with PP2 (5 µM). Endothelial cells were then double-stained with both anti-FAK monoclonal antibody and Texas red phalloidin to detect F-actin (Figs. 6
, 7
, left two columns). HPAEC monolayers were also stained with anti-paxillin, GIT1, and GIT2 antibodies, as indicated. PP2 inhibited the S1P-mediated cortical actin ring formation and redistribution of FAK, paxillin, GIT1, and GIT2, although some random and insignificant stress fiber formation was observed (Fig. 6)
, consistent with a recent report describing involvement of Src in S1P-mediated cytoskeletal responses in HUVEC (47)
. In contrast, PP2 failed to influence thrombin-induced stress fiber formation or the redistribution of FA proteins (FAK, paxillin, GIT1, and GIT2) to the newly formed focal adhesions (Fig. 7)
. PP2 pretreatment appeared to selectively abolish the redistribution of GIT1 not to focal adhesions but to stress fibers after thrombin challenge (Fig. 7)
. Consistent with PP2 effects on FAK site-specific phosphorylation profiles (Fig. 5)
, these results further suggest involvement of Src-independent mechanisms in thrombin-induced FA remodeling and FAK tyrosine phosphorylation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 9)
associated with dramatic cytoskeletal rearrangements (8
, 48)
. Several reports suggest that FA assembly in response to integrin binding promotes stress fiber formation, whereas FA disassembly leads to stress fiber dissolution (26)
. These findings strongly suggest the role of FA remodeling in endothelial cell cytoskeletal rearrangement and therefore barrier regulation. Paxillin, a major component of focal adhesions, plays a pivotal role facilitating signal transduction from both the extracellular matrix and soluble agonists by recruiting specific molecules to FA, with the phosphorylation status of paxillin apparently important for the interaction with its binding partners (33
34
35
36)
. Paxillin contains a docking site for FAK with Y118 in paxillin, a major site of FAK-catalyzed phosphorylation (33
, 37)
. Furthermore, gene knockout experiments using fak-/- fibroblast cell lines demonstrate an important role of FAK in FA disassembly (38
, 39)
.
We have investigated the dynamics of FAK and paxillin interaction and their redistribution induced by thrombin. Thrombin induced formation of massive stress fibers and intercellular gaps and the stable redistribution of FAK to the newly formed, enlarged FAs at the ends of stress fibers, observed at 10 and 30 min of stimulation (Fig. 1)
. Thrombin induced similar patterns of FAK and paxillin redistribution (Fig. 1)
. Conversely, a coimmunoprecipitation study revealed the difference in the effect of thrombin and S1P on the association of FAK with paxillin. In contrast to our previously described dissociation of FAK from paxillin at later times of S1P stimulation (44)
, thrombin induced stable association of FAK with paxillin that persisted for at least 60 min (Fig. 3
A1, B).
In this study, we examined thrombin-mediated redistribution of GIT1. In contrast to S1P-induced transient localization of GIT1 to the cell cortical area described in our earlier studies (44)
, thrombin stimulates GIT1 redistribution not only at the ends of stress fibers, but also along stress fibers, a pattern observed within 60 min poststimulation (Fig. 2A
). In addition, GIT1/paxillin coimmunoprecipitation assay revealed that thrombin caused association of GIT1 with paxillin at later times (30–60 min) (Fig. 3)
. The finding that GIT1 is redistributed along with stress fibers suggests a novel role for GIT1 (either direct or indirect) in cytoskeletal rearrangement and indicates the participation of other regulatory proteins in this novel interaction of GIT1 with actin cytoskeleton.
Analysis of GIT2 localization and association with FA proteins revealed that despite the differential localization after S1P and thrombin stimulation, GIT2 interaction with its protein partner, paxillin, was not affected. Taking into account proposed role for GIT2 in paxillin trafficking to the areas of de novo focal adhesions, we speculate that differential GIT2 patterns in S1P- and thrombin-stimulated EC depict the patterns of novel focal FA induced by thrombin and S1P and demonstrate GIT2 involvement in both these processes. Our results also suggest that intracellular targeting of GIT2-paxillin complexes is mediated by as yet unknown mechanisms, although one potential mechanism may involve Src and FAK tyrosine kinases (Fig. 6)
.
The differential FAK activation by S1P and thrombin may be one of the mechanisms underlying differential redistribution of FA-related proteins and EC cytoskeletal remodeling. Src is a major binding partner of FAK, and a close relationship exists between the mechanisms of FAK and Src activation (37)
. There are three proposed models for the activation of FAK and Src and their assembly into a signaling complex (37
, 49
, 50)
. One model proposes that FAK clustering might be sufficient to induce trans-phosphorylation at Y397 and the inactive Src protein might be recruited into complex with FAK, resulting in the activation of Src. A second model suggests that Src is initially activated by a phosphatase and activated Src phosphorylates FAK on the activation loop residues such as Y576 and Y577, with activated Src recruited into complex with activated FAK. Finally, there is a possibility that Src and FAK are independently activated with activated Src and FAK subsequently assembled into a signaling complex.
PP2 pretreatment abolished FAK Y576 phosphorylation in S1P-stimulated EC and FAK Y397 phosphorylation in thrombin-stimulated EC, whereas FAK Y576 and Y925 phosphorylation induced by thrombin was only partially attenuated (Fig. 5)
. Furthermore, morphological studies revealed that PP2 inhibited cortical actin ring formation and redistribution of FAK, paxillin, GIT1, and GIT2 by S1P (Fig. 6)
, consistent with a previous report (47)
. In contrast, PP2 did not influence thrombin-induced stress fiber formation or the redistribution of FAK, paxillin, GIT1, and GIT2 to the newly formed FAs (Fig. 7)
. PP2 pretreatment selectively abolished the redistribution of GIT1 to stress fibers, not to FAs, after thrombin challenge (Fig. 7)
. The mechanisms underlying this phenomenon remain to be elucidated but suggest the involvement of Src in GIT1 targeting to stress fibers.
Based on time course analysis of FA redistribution and on intracellular localization and interaction of specific FA proteins, we propose a model of SIP- and thrombin-induced FA remodeling (Fig. 8
). In quiescent cells, FAK and paxillin are both distributed in the cytosol and in preexisting FAs. Physiological concentrations of S1P induce partial disassembly of preexisting FAs coincident with the transient association of GIT1 with FAK, followed by translocation of FAK and paxillin from cytosol to the cell periphery. FAK, paxillin, and GIT2 are redistributed to the cell peripheral area within newly formed FAs linked to the newly formed cortical actin ring. FAK and paxillin may not be associated directly with each other in these protein complexes (Fig. 8
, left). In contrast to S1P, thrombin induces novel FA formation associated with massive stress fiber formation, increased association of GIT1, GIT2, and FAK with paxillin, prominent FAK phosphorylation at Y397, Y576, and Y925, and partial localization of GIT1 along the actin stress fibers, which was attenuated by pharmacological inhibition of Src (Fig. 8
, right). As previously reported, FAs in adherent cells undergo basal turnover regardless of cell activation state (39)
. Therefore, elevated and sustained association of GIT1 with paxillin may reflect the accelerated FA turnover in SIP- and thrombin-stimulated EC because GIT1 is engaged in FA disassembly (Fig. 8)
. It is possible that paxillin migration to the cell periphery in SIP-stimulated EC and paxillin accumulation in the newly formed random FAs in thrombin-stimulated EC is mediated by GIT2. Finally, our results suggest a distinct role for Src-kinase in SIP- and thrombin-induced FA remodeling indicating different mechanisms underlying FA remodeling induced by barrier protective and disruptive agonists.
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Taken together, our earlier findings (25
, 44)
and present data suggest a potential involvement of the S1P-Edg receptor–Rac-Src signaling pathway in selective FAK Y576 phosphorylation resulting in the peripheral focal adhesion formation, redistribution of FAK and paxillin to the cell periphery, and cortical actin ring formation. In contrast, thrombin stimulation triggers multiple signaling pathways to phosphorylate several FAK tyrosine residues, resulting in the massive stress fiber formation and intracellular gap formation. Our data suggest GITs as important targets mediating differential FA remodeling and demonstrate that agonist-specific FA remodeling may play an essential role in the pulmonary endothelial cell barrier regulation by barrier protective and disruptive stimuli.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication March 19, 2003. Accepted for publication August 5, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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