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Am J Physiol Heart Circ Physiol 288: H936-H945, 2005. First published October 7, 2004; doi:10.1152/ajpheart.00519.2004
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Integrin mechanotransduction stimulates caveolin-1 phosphorylation and recruitment of Csk to mediate actin reorganization

C. Radel and V. Rizzo

Center for Cardiovascular Science, Albany Medical College, Albany, New York

Submitted 20 August 2004 ; accepted in final form 22 September 2004


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To identify the role of caveolin-1 in integrin mechanotransduction, we exposed bovine aortic endothelial cells to 10 dyn/cm2 of laminar shear stress. Caveolin-1 was acutely and transiently phosphorylated with shear, occurring downstream of {beta}1-integrin activation as the {beta}1-integrin blocking antibody JB1A was inhibitory. In manipulating Src family kinase (SFK) activity with knockdown of Csk or type 1 protein phosphatase (PP1) treatment, we observed coordinate increase and decrease in shear-induced caveolin-1 phosphorylation, respectively. Hence, shear-stimulated caveolin-1 phosphorylation is regulated by SFKs. Shear-induced recruitment and phosphorylation of caveolin-1 occurred at {beta}1-integrin sites in a {beta}1-integrin- and SFK-dependent manner. Csk, described to interact with pY14-caveolin-1 and integrins, bound to an increased pool of phosphorylated caveolin-1 after shear corresponding with elevated Csk at {beta}1-integrin sites. Like caveolin-1, treatment with JB1A and PP1 attenuated shear-induced Csk association with {beta}1-integrins. Csk function was assayed with transfection of a caveolin-1 phosphorylation domain peptide. The peptide attenuated shear-induced association of Csk at {beta}1-integrin sites, as well as colocalization of Csk with paxillin and phosphorylated caveolin-1. Because integrin and Csk activity regulate cytoskeletal reorganization, we evaluated the role of this mechanism in shear-induced myosin light chain (MLC) phosphorylation. Knockdown of Csk expression was sufficient to reduce MLC diphosphorylation due to shear. Disruption of Csk-integrin association by peptide treatment was also inhibitory of the MLC diphosphorylation response. Together these data indicate that integrin activation with shear stress results in SFK-regulated caveolin-1 phosphorylation that, in turn, mediates Csk association at integrin sites, where it plays a role in downstream, shear-stimulated MLC diphosphorylation.

myosin light chain; caveolae; shear stress

FLUID DYNAMICS CRITICALLY affect the homeostasis of the vascular wall, particularly the endothelium. These fluid forces serve to modulate the intrinsic structure and function of endothelial cells, altering aspects such as genetic phenotype, rate of proliferation, endothelium-dependent vasoresponsiveness, permeability, cell-cell communication, and cytoskeletal organization (11, 13, 14). Many of these responses have been linked to putative mechanotransduction elements such as ion channels (34, 41), tyrosine kinase receptors (7), G proteins (28), junctional proteins such as platelet endothelial cell adhesion molecule 1 (PECAM-1) (23, 36), caveolae (37, 40), and integrin-containing focal adhesions (24).

Focal adhesion sites are perhaps the most widely studied regions for mechanotransduction. Morphologically, these sites undergo significant dynamic remodeling resulting in realignment in the direction of flow (12, 20). Acutely, shear activation of integrins leads to phosphorylation of focal complex proteins such as paxillin and focal adhesion kinase (FAK), which facilitate focal adhesion remodeling (24). Cytosolic proteins such as Fyn and Shc are also recruited to integrin sites to initiate signal transduction cascades, ultimately resulting in downstream transcription (7, 26). These pathways are further potentiated through cross talk with tyrosine kinase receptors and small G proteins coordinating gene transcription and cytoskeletal remodeling (46, 48).

Interestingly, focal adhesion regulation and signaling have recently been linked to caveolin-1, the primary structural protein of caveolae. Caveolin-1 was described to associate with a subset of {alpha}-integrins (49), including the {alpha}1-, {alpha}5-, and {alpha}v-subtypes, and its binding to these subunits was necessary for adhesion-stimulated Shc-Ras-ERK signaling. Reduction of caveolin-1 expression disrupts {beta}1-integrin interaction with Src kinase and induces loss of focal adhesion sites, ligand-induced FAK phosphorylation, and adhesion (52). In addition, osmotic and oxidative stressors induce phosphorylation at tyrosine residue 14 (pY14) of caveolin-1, which localizes with focal adhesion proteins (29). On the basis of these findings, a role for caveolin-1 in regulating integrin-mediated mechanotransduction has been speculated (43); however, experimental evidence confirming such interaction is lacking.

Once caveolin-1 is modified, the pY14 site purportedly functions as an SH2 binding domain that can organize signaling molecules such as Grb7 and Csk (5, 29). The novel observation of Csk binding to caveolin-1 when tyrosine-14 is phosphorylated remains to date an interaction without known functional significance. Csk is a well described Src kinase inhibitor that may play a role in actin remodeling. When overexpressed in HeLa cells, Csk localizes to focal adhesion sites, where it interacts with FAK and {alpha}v{beta}3-integrins to diminish cell adhesion (3). A more recent study found that Rho-dependent, G protein-induced stress fiber formation was blocked in Csk-deficient mouse embryonic fibroblast cells but could be rescued by reexpression of Csk (30). Whether Csk (and its association with pY14) plays a role in shear-induced cytoskeletal reorganization is presently unknown.

Here, we show that caveolin-1 is tyrosine phosphorylated in response to acute shear stress applied to cultured bovine aortic endothelial cells (BAEC). We propose that the functional result of this phosphorylation event has significance in shear-induced integrin signaling through recruitment of SH2-containing molecules to focal adhesion sites. Ultimately, these events culminate in a downstream signal to initiate cytoskeletal rearrangement, a phenomenon commonly observed in the early adaptation response of endothelial cells to shear stress.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies. The following primary antibodies were obtained from commercial sources: Csk, paxillin, and pY14 caveolin-1 MAbs, and caveolin-1 polyclonal antibody (PAb) (Transduction Labs); pY14-caveolin-1 PAb, c-Src PAb, and Csk PAb (Santa Cruz); {beta}1-integrin PAb and MAbs JB1A and P4G11 (Chemicon); myosin light chain (MLC) and {beta}-actin MAb (Sigma); diphosphorylated MLC PAb (Strategic Biosolutions); pY416 Src PAb (Biosource); horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies (Amersham).

Cell culture. Bovine thoracic aortas were procured from a local slaughterhouse (Greenville, NY) and BAEC harvested by digestion with 0.1% collagenase type II (Sigma) at 37°C for 10–30 min. Isolated cells were verified as endothelial by positive staining for PECAM-1 (CD31) and uptake of acetylated LDL. Cells were grown in MCDB-131 culture medium (Life Technologies) supplemented with 20% FBS (Atlanta Biologicals) and 0.05 mg/ml gentamicin (Sigma) and maintained at 37°C, 97% humidity, and 5% CO2. For shear stress experiments, cells were plated on 30 µg/ml Vitrogen (Cohesion)-coated glass slides and grown for 2–3 days until confluent.

Flow experiments. Laminar shear stress was applied to confluent monolayers of BAEC with a parallel plate flow apparatus (Flexcell). Before shear, cultures were serum deprived for 2 h and then either subjected to 10 dyn/cm2 shear stress or kept static. In some experiments, cells were preincubated with either 10 µM type protein phosphatase (PP1; Biochemica), the {beta}1-integrin-blocking MAb clone JB1A (1 µg/ml; Chemicon), or the {beta}1-integrin-activating MAb clone P4G11 (1 µg/ml; Chemicon) for 1 h. After shear experiments, cells were washed with ice-cold PBS and scraped into lysis buffer (50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 60 mM {beta}-octylglucoside, and phosphatase and protease inhibitors) for preparation of whole cell lysates.

Immunoprecipitation. Poly- and monoclonal primary antibodies were conjugated to sheep anti-rabbit- or sheep anti-mouse-coated paramagnetic Dynabeads (Dynal), respectively, according to the manufacturer's protocol, and in some cases streptavidin-coated Dynabeads (Dynal) were utilized. Cell lysates (400 µg) were incubated with the antibody-bead conjugates for 2 h at room temperature. The bound fraction was separated from the unbound material, washed three times with lysis buffer, and processed for SDS-PAGE. Isotype-matched control immunoprecipitations lacking primary antibody were performed on lysates and indicated no detectable background binding (data not shown).

Immunofluorescence. After static or shear conditions, BAEC were processed for immunofluorescence with a modified protocol described previously (44). Briefly, cells were washed three times with PBS and fixed with 4% paraformaldehyde in a cytoskeleton-stabilizing buffer for 30 min at 20°C. Monolayers were permeabilized with 0.2% Triton X-100 for 20 min, blocked for 1 h in 0.5% fish gel (Sigma), and then incubated with either pY14 or Csk primary antibody diluted in 0.5% fish gel for 2 h at 20°C. The cells were washed again in PBS and incubated for 1 h with tetramethylrhodamine isothiocyanate (TRITC)-conjugated paxillin (1:500; Transduction Labs) and the isotype matched secondary antibodies conjugated to Alexa 488 and Alexa 633 (1:500; Molecular Probes). Slides were mounted with antifade aqueous mounting medium (Biomeda). Cells were visualized with a Zeiss 510META confocal microscope, and image analysis was performed with Adobe Photoshop 6.0.

Western blotting. Protein content of the various samples was determined by bicinchoninic acid analysis (Pierce). Equivalent amounts of protein from each sample were prepared and separated by SDS-PAGE followed by electrotransfer to nitrocellulose filters (Bio-Rad). SDS electrophoresis was performed as described previously (39). MLC phosphorylation assays were conducted as described previously (18) with slight modification. Briefly, cells were scraped into 10% TCA and 10 mM DTT, washed with 100% acetone, and then allowed to dry. Pellets were reconstituted in Tris-urea buffer containing 0.01% Triton X-100 and DTT, electrophoresed on a non-SDS 10% polyacrylamide gel, and transferred to nitrocellulose. Immunoblotting included incubation with the primary antibodies indicated and species-matched secondary antibodies conjugated with horseradish peroxidase. Proteins of interest were detected with enhanced chemiluminescence substrate (ECL, Amersham). Autoradiograms were scanned and digitized (Molecular Dynamics). Densitometric quantification of immunoblots with Imagequant software (Molecular Dynamics) allowed direct comparisons between experimental sets.

Peptide experiments. Peptides were constructed consisting of amino acids 1–27 (MSGGKYVDSEGHLY*TVPIREQCNIYKPNNC; Genscript) of caveolin-1 with tyrosine-14 (*) either phosphorylated or unmodified. The peptides were individually combined with an equimolar amount of biotinylated Penetratin (Qbiogene) in a 2-h coupling reaction at 20°C per the manufacturer's instructions. Before shear exposure, 120 nM Penetratin coupled to either the control peptide or the phosphopeptide was incubated with BAEC for 2 h and then sheared and processed as described above.

Csk small interfering RNA experiments. Csk Smartpool or control Smartpool small interfering RNAs (siRNAs) (1 nmol; Upstate) were complexed with the lipofection agents Targefect F-2 and Virofect (Targeting Systems) by incubation in starvation medium at 37°C for 25 min. The complex was added to BAEC for 2 h and then supplemented with medium containing 20% FBS. The cells were then cultured for 48 h, exposed to shear, lysed, and analyzed for Csk expression and MLC phosphorylation.

Statistical analysis. Differences between groups were determined by unpaired two-tailed Student's t-test with Microsoft Excel software (Microsoft). Differences between control and experimental groups were significant at P < 0.05, with all n values at least equal to three separate experiments. All data are presented as average values ± SE.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Acute shear stress results in transient phosphorylation of caveolin-1. Given the observation that a portion of phosphorylated caveolin-1 colocalizes with focal adhesion proteins (29) and that focal adhesions are critical mechanotransduction sites (10), we tested whether caveolin-1 phosphorylation occurs in shear-stimulated endothelial cells within the same time course as shear-induced integrin activation (i.e., 1–10 min) (46). BAEC were subjected to laminar shear stress at 10 dyn/cm2 for 1, 5, and 10 min, and caveolin-1 phosphorylation at tyrosine 14 (pY14) was evaluated with a pY14-specific antibody. Shear stress transiently increased caveolin-1 phosphorylation by approximately twofold, peaking at 5 min and beginning to return to baseline by 10 min with all time points significantly increased over the nonsheared control (Fig. 1).



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  		Fig. 1. Shear stress induces acute and transient caveolin-1 phosphorylation. Bovine aortic endothelial cells (BAEC) were either kept static or exposed to shear stress (SS) for 1, 5, or 10 min at 10 dyn/cm2. Samples were resolved by SDS-PAGE, Western blotted (IB) with a phosphorylated tyrosine residue 14 (pY14) caveolin-1-specific antibody, stripped, and reprobed for caveolin-1. Densitometric quantification showed that caveolin-1 phosphorylation increased ~2-fold with shear stress, peaking at 5 min (*P < 0.05).

 
{beta}1-Integrin activation is necessary and sufficient for caveolin-1 phosphorylation. To test whether shear-induced caveolin-1 phosphorylation is dependent on integrin activation, we pretreated BAEC monolayers with the {beta}1-integrin-inhibiting antibody JB1A (1 µg/ml). As illustrated in Fig. 2, pretreatment with JB1A completely blocked the shear-induced increase in caveolin-1 phosphorylation compared with nonsheared cells. Additionally, incubation with a {beta}1-integrin-activating antibody, P4G11 (1 µg/ml; Ref. 50), was sufficient to cause a 2.25 ± 0.45-fold increase in caveolin-1 phosphorylation, whereas a nonmodulating {beta}1-integrin antibody had no effect on caveolin-1 phosphorylation (data not shown). Thus caveolin-1 phosphorylation is dependent on {beta}1-integrin activation induced by either shear stress or antibody stimulation of {beta}1-integrins.



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  		Fig. 2. Shear-induced caveolin-1 phosphorylation is dependent on {beta}1-integrin activation. BAEC were either untreated or treated with the {beta}1-integrin-activating antibody P4G11 (1 µg/ml) or the {beta}1-integrin-inhibitory antibody JB1A (1 µg/ml) for 1 h before shear stress. After the indicated shear time points, cells were lysed, resolved by SDS-PAGE, and Western blotted for pY14 caveolin-1 and caveolin-1. Densitometric quantification showed that JB1A (#P < 0.05) blocked shear-induced caveolin-1 phosphorylation (*P < 0.05), whereas incubation with {beta}1-integrin stimulatory antibody alone resulted in an ~2.5-fold increase in caveolin-1 phosphorylation (^P < 0.05).

 
Src family kinases facilitate shear-induced phosphorylation of caveolin-1. To determine the role of Src family kinase (SFK) in shear-induced caveolin-1 phosphorylation, we first evaluated the time course of SFK signaling in our system. BAEC were exposed to shear stress for 1, 5, or 10 min at 10 dyn/cm2. SFK activity was detected by measuring the extent of autophosphorylation at tyrosine 416 (pY416), a residue present in the enzyme's activation loop and an event that strongly correlates with enzyme activity (32). We detected a 50% increase over baseline in pY416 signal after only 1-min exposure to shear stress followed by a rapid decrease in SFK activity by 5 and 10 min (Fig. 3A).



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  		Fig. 3. Shear-stimulated caveolin-1 phosphorylation depends on Src family kinase (SFK) activity. BAEC were kept static or subjected to shear stress for 5 min and then lysed and processed for Western blot analysis. A: the phosphorylation state of the SFK activity in the cell was determined by immunodetection of SFK phosphorylated on tyrosine 416 (pY416). Phosphorylation was seen to increase ~50% by 1 min of shear, decreasing thereafter (*P < 0.05). B: BAEC were transfected with either 1 nmol Csk small interfering RNA (siRNA) or control siRNA 48 h before shear stress. Expression levels of Csk after siRNA treatment were determined by immunodetection. Quantitation via densitometry and normalization to {beta}-actin levels showed an ~70% reduction in Csk expression by 48 h, with no change seen in control siRNA-treated cells. C: BAEC were pretreated with Csk siRNA and then sheared for 5 and 10 min. Evaluation of Y416 SFK levels showed that knockdown of Csk resulted in maintenance of SFK phosphorylation at 5 and 10 min of shear treatment (#P < 0.05) but no change in basal SFK activity. D: the phosphorylation state of caveolin-1 was determined by immunodetection of pY14 caveolin-1 levels normalized to caveolin-1 after either Csk knockdown or SFK inhibition by pretreatment with 10 µM type 1 protein phosphatase (PP1). Csk siRNA-treated cells showed increased caveolin-1 phosphorylation with shear without changes in basal phosphorylation (#P < 0.05). Inhibition of SFK with PP1 treatment was sufficient to inhibit caveolin-1 phosphorylation (^P < 0.05) induced by shear (*P < 0.05).

 
SFK activity was indirectly manipulated by genetic knockdown of the SFK inhibitor Csk. Pretreatment with Csk-specific siRNA effectively reduced Csk expression by 70%, whereas an engineered control pool siRNA had no effect (Fig. 3B). The consequence of Csk reduction in our cell system was maintenance of SFK activity through 5 and 10 min of shear treatment, where typically it would be decreasing. Interestingly, no significant difference in SFK activity was seen between siRNA-treated and nontreated cells under basal conditions. Therefore, BAEC with reduced Csk levels displayed constitutively activated SFK activity after exposure to shear stress (Fig. 3C).

To identify whether the increased activity of SFK directly altered caveolin-1 phosphorylation due to shear, pY14 levels were evaluated in Csk siRNA-treated BAEC. Additionally, as a second independent method of manipulating SFK activity, monolayers were pretreated with 10 µM PP1, a global SFK inhibitor. Loss of SFK regulation by Csk resulted in caveolin-1 phosphorylation similar to SFK activity seen in Fig. 3C. Mirroring the maintenance of SFK activity through 5 and 10 min, caveolin-1 displayed increased phosphorylation at 5 and 10 min, with the transient nature of the typical shear response lost. Moreover, we found that the shear-induced increase of pY14 was completely blocked with the PP1 pretreatment, indicating that activation of SFKs is critical in mediating shear-induced caveolin-1 phosphorylation (Fig. 3D).

Shear-stimulated integrin and SFK activity are necessary for recruitment and phosphorylation of caveolin-1 at {beta}1-integrin sites. {beta}1-Integrin immunoprecipitations were conducted to determine whether shear-induced caveolin-1 phosphorylation localizes specifically at integrin sites. We found that caveolin-1 associated with {beta}1-integrin was phosphorylated by greater than fourfold compared with static controls after 5 min of shear stress (Fig. 4). This increase was inhibited by 1 µg/ml JB1A or 10 µM PP1 pretreatment. Thus shear-stimulated caveolin-1 phosphorylation occurs at {beta}1-integrin sites and is facilitated by {beta}1-integrin and SFK activation.



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  		Fig. 4. Caveolin-1 is recruited and phosphorylated at integrin complexes with shear in an integrin- and SFK-dependent manner. After pretreatment with either 1 µg/ml JB1A or 10 µM PP1 for 1 h, BAEC were kept static or exposed to shear stress for 5 min. {beta}1-Integrin was immunoprecipitated (IP) from cell lysates and resolved with SDS-PAGE. A: Western blot analysis for pY14 caveolin-1, caveolin-1, and {beta}1-integrin. B: histograms showing that when normalized to {beta}1-integrin levels, phosphorylated caveolin-1 and caveolin-1 were increased by 5 min at {beta}1-integrin sites (*P < 0.05). Recruitment and phosphorylation was regulated by {beta}1-integrin (#P < 0.05) and SFK activation (^P < 0.05).

 
Interestingly, caveolin-1 association with the {beta}1-integrin complex was increased with shear stress coordinate with the increased caveolin-1 phosphorylation (Fig. 4). The shear-induced increase in caveolin-1 binding to {beta}1-integrins, however, was significantly reduced with 1 µg/ml JB1A or 10 µM PP1 pretreatment. This suggests that caveolin-1 phosphorylation may be an important step for its recruitment to the integrin complex.

Shear causes increased Csk binding to phosphorylated caveolin-1 and Csk recruitment to {beta}1-integrin sites in integrin- and SFK-dependent manner. To identify a function for the shear-induced increase in phosphorylated caveolin-1, we examined a recently described interaction involving direct association between pY14 caveolin-1 and Csk (5). Csk immunoprecipitated from static and sheared BAEC revealed that Csk was constitutively bound to caveolin-1; however, after a 5-min exposure to shear stress, the pool of phosphorylated caveolin-1 associated with Csk was elevated (Fig. 5A).



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  		Fig. 5. Shear stress enhances Csk association with pY14 caveolin-1 and {beta}1-integrin. A: BAEC were sheared for the indicated times, lysates were prepared, and Csk immunoprecipitations were conducted. The bound fraction was resolved with SDS-PAGE and Western blotted for Csk, pY14 caveolin-1, and caveolin-1. The results show that 5-min shear stress induced a >2-fold increase in Csk association with phosphorylated caveolin-1 (*P < 0.05) and the association was maintained but decreasing by 10 min. Caveolin-1 association with Csk was unaffected by shear. B: BAEC were either left untreated or pretreated with 1 µg/ml JB1A or 10 µM PP1 and then sheared for 5 min. {beta}1-Integrin immunoprecipitations were conducted and Western blotted for Csk and {beta}1-integrin. Densitometric quantification indicated that Csk was recruited to {beta}1-integrin sites by 5 min (*P < 0.05) and that shear-induced Csk recruitment to {beta}1-integrin sites was effectively blocked by inhibition of both {beta}1-integrin (#P < 0.05) and SFK activation (^P < 0.05).

 
Consistent with shear stimulation of phosphorylated caveolin-1 at {beta}1-integrin sites (Fig. 4), 5 min of shear stress also induced an approximately twofold increase in Csk binding to {beta}1-integrin complexes. Pretreatment of BAEC with JB1A or PP1 was sufficient to block the shear-induced increase in Csk association with {beta}1-integrins (Fig. 5B). As with caveolin-1, shear activation of integrins and SFKs appears to regulate Csk association with {beta}1-integrin sites.

Phosphorylated Y14 caveolin-1 peptide binds shear-activated Csk, blocking its translocation to {beta}1-integrins and paxillin/pY14 caveolin-1-containing focal adhesions. To directly identify the importance of the shear-induced Csk-phospho-caveolin-1 interaction at {beta}1-integrin sites, caveolin-1 peptides spanning the pY14 region were constructed with the tyrosine residue 14 either phosphorylated (pY14) or unmodified (Y14). These peptides were then coupled to a biotinylated Penetratin delivery agent that allowed for ~70% transfection rate of the endothelial monolayers (data not shown). Streptavidin precipitations showed that the nonphosphorylated peptide was unable to associate with Csk, whereas Csk was readily detected in the phosphorylated peptide pull-downs (Fig. 6A). Interestingly, little Csk was bound to the peptide in nonsheared cells compared with sheared cells, suggesting that the pY14-Csk interaction requires Csk activation. Biochemical analysis after peptide treatment indicated that shear-induced Csk recruitment to {beta}1-integrins was blocked with the pY14 peptide but not with the Y14 peptide (Fig. 6B). These data suggest that shear stimulates Csk to bind directly to the Y14 phosphorylation site of caveolin-1, potentially at integrin sites.



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  		Fig. 6. Transfection of a caveolin-1 peptide mimicking the phosphorylation domain (pY14) binds to Csk and prevents its shear-induced redistribution to {beta}1-integrin sites. Cell monolayers were treated for 2 h with 120 nM biotinylated Penetratin coupled with a caveolin-1 peptide corresponding to amino acids 1–27 with tyrosine residue 14 either unmodified (Y14) or phosphorylated (pY14) A: streptavidin pull-downs of lysates from static or 5-min shear-exposed cells were probed for binding of the peptides to Csk with standard Western blotting techniques. No background binding of Csk was detectable in the Y14-treated cells; however, Csk association was detectable in the pY14-treated cells, but only at the 5 min shear time point. B: {beta}1-integrin immunoprecipitations probed for Csk association indicated that Y14 did not effect Csk recruitment to {beta}1-integrins with 5 min of shear (*P < 0.05), whereas the pY14 peptide resulted in a significant inhibition of Csk binding (#P < 0.05).

 
We next evaluated the distribution of Csk within the cell under static and sheared conditions and determined the specific effects of the pY14 peptide by confocal microscopy. Triple immunostaining was performed, labeling Csk with an Alexa 488 fluorochrome (green), paxillin with TRITC (red), and pY14-caveolin with an Alexa 633 fluorochrome (far red). Images were obtained for static, 5-min shear, and 5-min shear after pY14 peptide treatment (Fig. 7). Under static conditions, Csk immunostaining showed that the protein localized predominantly to perinuclear regions of the cell. Paxillin and pY14-caveolin-1 showed characteristic distribution with apparent localization to focal contacts. Overlay of the individual signals revealed that Csk has a low level of colocalization with paxillin or pY14-caveolin-1 (observed as yellow); however, paxillin and pY14-caveolin-1 showed significant colocalization. After 5 min of shear, Csk underwent a dramatic change in localization, spreading throughout the cell. Additionally, paxillin and pY14-caveolin-1 immunostaining appeared more dense compared with static cells. Merging the images revealed significant colocalization of Csk with paxillin, consistent with a redistribution to focal contacts after activation and with the biochemical observation of increased Csk association with {beta}1-integrin (Fig. 5). To a lesser degree, Csk also displayed colocalization to sites containing pY14-caveolin-1, and pY14 caveolin-1 and paxillin maintained a high level of colocalization. These observations are highlighted in selected 50% magnifications of 5-min shear images (Fig. 7, J-L). Merging of all three signals showed that these molecules all colocalize (seen as white pixels in Fig. 7M) in a subset of focal contacts in the cell. Pretreatment of the monolayers with the pY14 peptide and subsequent immunostaining showed that Csk translocation is inhibited after shear. Colocalization of Csk with paxillin and pY14-caveolin seen in the 5-min sheared cells was nearly abrogated, although the peptide had no effect on the distribution of pY14 caveolin-1 to focal sites marked by paxillin staining. These data provide strong evidence for the shear-stimulated association of Csk to pY14 caveolin-1-containing focal adhesions and verify the biochemical observation of the inhibitory effects of the pY14 peptide for Csk recruitment to {beta}1-integrin complexes.



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  		Fig. 7. Shear stimulates Csk to colocalize with pY14 caveolin-1 and paxillin. BAEC were kept static or exposed to shear stress for 5 min at 10 dyn/cm2. Some cells were pretreated with 120 nM pY14 peptide conjugated to Penetratin for cellular delivery. After shear, cells were processed for immunofluorescence and confocal microscopic images were taken under x63 magnification. Csk was labeled with Alexa 488, paxillin with a tetramethylrhodamine isothiocyanate label, and pY14 caveolin-1 with Alexa 633. Under static conditions, Csk displayed a perinuclear localization (A, B), whereas paxillin (A, C) was visible at the basal cell surface in regions assumed to be focal contacts. The pattern of pY14 caveolin-1 was similar to that of paxillin (B, C). Merging images showed colocalization (yellow) for paxillin and pY14 caveolin-1 (C) but no significant colocalization of Csk with paxillin (B) or pY14 caveolin-1 (C). After 5 min of shear stress, Csk showed marked redistribution (D) and more dense focal structures consisting of paxillin (A, C) and pY14 caveolin-1 (B, C). Significant colocalization for Csk and paxillin (D), Csk and pY14 caveolin-1 (E), and paxillin and pY14 caveolin-1 (F) was visible in merged images. Confocal images of cells pretreated with the pY14 peptide before and after 5 min of shear stress are depicted in G-I. Distribution of Csk (G, H) was similar to static Csk immunostaining, whereas paxillin (G, I) and pY14 caveolin-1 (H, I) were unchanged by the peptide. Shear-induced colocalization of Csk with paxillin (G) and pY14 caveolin-1 (I) was nearly eliminated by pY14 peptide treatment. Paxillin and pY14 caveolin-1 colocalization was, however, unaffected by the peptide (I). J-L: 50% magnification of the boxed regions in the 5-min shear images (D-F), highlighting the regions of colocalization. A triple merge of these images (M) shows regions of Csk (green), paxillin (red), and pY14 caveolin-1 (blue) as white pixels.

 
Caveolin-1 phosphorylation-dependent interaction with Csk at {beta}1-integrin sites is critical for mediating shear-induced MLC diphosphorylation. As integrin mechanosignaling has been implicated in regulation of actin dynamics (20), we tested the role of Csk in regulating shear-induced actin reorganization in BAEC by using MLC diphosphorylation as an indicator of early cytoskeletal remodeling. Monolayers were sheared for 5 and 10 min, and the level of diphosphorylated MLC was quantified relative to total MLC. The data indicate an ~30% increase in MLC diphosphorylation both at 5 and 10 min of shear (Fig. 8).



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  		Fig. 8. Csk expression and localization to integrin sites via the caveolin-1 phosphorylation site is required to regulate shear induced myosin light chain (MLC) diphosphorylation (diP-MLC). A: BAEC were left untreated or transfected with either Csk siRNA or control siRNA before shear stress. After shear exposure, lysates were immunoblotted for diP-MLC and total MLC. Densitometric analysis showed that nontreated and control siRNA had no effect (*P > 0.05), whereas reduction of Csk protein expression significantly decreased shear-induced MLC diphosphorylation (#P < 0.05). B: BAEC were pretreated with either 120 nM Y14 peptide or pY14 peptide coupled to Penetratin. Although the Y14 peptide had little effect on shear-induced diP-MLC response (*P > 0.05), the pY14 peptide was found to attenuate the diP-MLC response at both 5- and 10-min durations of shear stress (^P < 0.05).

 
To test the hypothesis that Csk is a critical link in the acute actin reorganization due to shear, we introduced Csk siRNA into BAEC to reduce cellular levels of Csk and evaluated the extent of shear-induced MLC diphosphorylation. Decreased Csk expression in the cells was sufficient to attenuate the shear-induced increase of MLC diphosphorylation at 5 and 10 min, whereas control siRNA had no effect (Fig. 8A). These findings suggest that Csk may be a crucial mediator for proper actin adaptation in the endothelial response to shear stress.

Finally, we evaluated the role of caveolin-1 phosphorylation in regard to shear-induced stress fiber formation. Figure 8B demonstrates that blockade of the recruitment of Csk to {beta}1-integrin sites with the pY14 peptide resulted in attenuation of the downstream MLC diphosphorylation at 5 and 10 min compared with control and Y14-treated cells. These data indicate that localization of Csk to integrin sites via interaction with the caveolin-1 pY14 domain facilitates actin remodeling due to shear stress.


   DISCUSSION
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
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Here we show a novel pathway that adds to our knowledge of integrin-related mechanosignaling. We found that caveolin-1 is phosphorylated and recruited to integrin sites with shear stress and that this is dependent on integrin activation and SFK activity stimulated by flow. This acute response was found to be important in facilitating Csk association with a subset of {beta}1-integrin-containing focal adhesions. Furthermore, we observed that Csk expression and association with focal adhesions was critical in mediating shear-induced MLC phosphorylation.

Integrin sensitivity to shear stress is evidenced by a net increase in integrin adhesion to the extracellular matrix (46) and clustering that results in the formation of large focal complexes organized in the longitudinal direction of flow (12). These focal adhesions are interconnected by bundles of F-actin organized in stress fibers that also align in the direction of flow. In addition, shear-induced transcriptional activity depends partially on integrin-mediated mechanotransduction. These events include integrin-dependent activation of ERK1/2 via direct recruitment of Fyn and Shc to the integrin complex and also through cross talk interactions with the VEGF-2 receptor (7, 48).

In cellular adhesion studies, Fyn and Shc association with integrin subunits is a caveolin-1-mediated process (49). Under both oxidative and osmotic stress, caveolin-1 can be phosphorylated on tyrosine residue 14 and appears to concentrate at focal adhesions (29). These observations suggest that caveolin-1 may be important for integrin mechanosignaling. However, studies examining the specific effects of shear stress on caveolin-1 phosphorylation have found that extended exposure (>24 h) of endothelial monolayers to shear stress does not result in phosphorylation of caveolin-1 (1, 47). By focusing on the acute shear responses, we found that caveolin-1 is in fact phosphorylated at tyrosine-14. The response is sensitive to changes in shear stress whether in static-to-shear cultures or in shear-adapted cells where monolayers were exposed to an additional step increase in laminar shear stress (39). We show further that shear-induced caveolin-1 phosphorylation is rapid and transient, peaking at 5 min and decreasing thereafter (Fig. 1).

To examine whether caveolin-1 phosphorylation is regulated by integrin mechanotransduction, BAEC were plated on a collagen substrate and cultured in serum containing fibronectin; thus integrin association with the matrix was limited to {beta}1- and {beta}3-type matrix interactions. Given that {beta}1-integrin has a prominent role in mechanotransduction (24, 46) and was highly expressed in these cells (data not shown), we utilized both {beta}1-integrin-activating and -inhibiting antibodies. Our findings show for the first time that integrin activation alone is sufficient to stimulate caveolin-1 phosphorylation and that the shear-stimulated phosphorylation of caveolin-1 is dependent on {beta}1-integrin activation (Fig. 2). Additionally, {beta}1-integrin immunoprecipitation demonstrated that shear stress enhanced caveolin-1 association with {beta}1-integrins (Fig. 4). The recruitment and phosphorylation of caveolin to integrin sites may serve multiple roles in regulating integrin function. Caveolin-1 clustering has been hypothesized to promote integrin clustering, permitting heightened avidity for ligand binding (6). In adhesion studies, caveolin-1 functioned as a key adapter molecule, bringing signaling elements to the relatively inert cytoplasmic tails of the integrin heterodimer (49). The caveolin-1 phosphorylation site may additionally be important as a mediator of SH2-SH3 interactions, a concept supported by our studies and discussed below.

Because caveolin-1 was first identified as a v-Src target (21) and recent evidence points to the activity of an additional nonreceptor tyrosine kinase, Abl (17), in caveolin-1 phosphorylation, we explored whether SFK plays a role in caveolin-1 phosphorylation induced by shear activation of integrins. We found that activation state of SFKs, as measured by pY416 phosphorylation, was significantly increased after 1 min and declined thereafter (Fig. 3A). This time frame for SFK activity corroborates previous reports of shear-induced SFK activity (26) and is in accordance with the observed caveolin-1 phosphorylation, which rises at 1 min and begins to fall after 5 min (Fig. 1). By removing the negative regulator of SFK activity through knockdown of Csk, we found that SFKs were not hyperactivated under basal conditions but maintained activity through 10 min of shear (Fig. 3C). Under these conditions, caveolin-1 phosphorylation was again found to correlate with SFK activity. Additionally, using a general inhibitor of SFKs, PP1, we showed that caveolin-1 phosphorylation induced by shear stress is blocked (Fig. 3D). As with inhibition of integrin activity, loss of SFK function resulted in the loss of caveolin-1 recruitment and phosphorylation specifically at {beta}1-integrin sites (Fig. 4).

Physically, phosphorylated caveolin-1 maintains characteristic localization to caveolae and association with caveolin-2 as determined by subcellular fractionation and electron microscopic studies (29, 33). Interestingly, despite a lack of changes in characteristic distribution, phosphorylated caveolin-1 does appear to be uniquely concentrated at focal adhesion sites compared with atypical caveolin-1 localization (29). Here the phosphorylation domain is postulated to serve as an SH3 site for Src and Abl targeting and to function as an SH2 site for protein recruitment. To date, only two molecules, Csk and Grb7, have been demonstrated to associate with the pY14 site of caveolin-1 (5, 29). Both proteins have been implicated in actin remodeling, although little has been done to identify the mechanisms of action. Grb7 seems to be critical for migration responses (22), whereas Csk is an important regulator of stress fiber formation (30).

A recent study by Cao and coworkers (42) demonstrated that a possible function for the phospho-caveolin-1-Csk interaction may involve the positioning of Csk to act as a negative-feedback regulator of SFK signaling. Oxidative and osmotic stress-induced phosphorylation required activation of Fyn, which was in turn negatively regulated by recruitment of Csk to the caveolin-1 phosphorylation site. Consistent with their observations, we found that Csk and pY14 caveolin-1 association was significantly increased after shear stimulation (Fig. 5A). Interestingly, Csk association with caveolin-1 was constitutive, in contrast to findings in mouse embryonic fibroblasts, where Csk immunoprecipitated with caveolin-1 only when stimulated by 2 mM hydrogen peroxide treatment (42). Differences in these observations likely arise from cell-type variability and levels of caveolin-1 expression. Although Csk has not been shown definitively to localize to caveolae subdomains or interact directly with the caveolin-1 scaffolding domain like other SFK members (9), Csk does contain the conserved WSFGILLW sequence that has been shown to mediate SFK protein association with the caveolin-1 scaffolding site. This may explain the constitutive association and possibly maintain Csk in an inactive state because the binding sequence is located in the kinase domain similar to other SFK members (17). Once activated, Csk might then bind to the phosphorylation domain of caveolin-1. This concept is supported by Csk's increased association with {beta}1-integrin sites due to shear stimulation (Fig. 5B). Csk recruitment corresponds with increased caveolin-1 association; however, under conditions that block caveolin-1 phosphorylation, i.e., JB1A and PP1 treatment, shear-induced association of Csk with {beta}1-integrins is lost (Fig. 5B). Moreover, specific interaction of Csk with the caveolin-1 phosphorylation domain at integrin sites was demonstrated by addition of a caveolin-1 phosphorylated peptide that under shear conditions competitively bound Csk away from {beta}1-integrin sites. Confocal immunomicroscopy confirmed the increased and rapid recruitment of Csk to focal adhesion sites containing pY14 caveolin-1 with shear and confirmed that addition of the phosphorylated peptide could successfully inhibit this translocation event (Fig. 7). These data in total suggest a mechanism where shear stimulates {beta}1-integrin and SFKs within a complex to recruit and phosphorylate caveolin-1, providing a binding site for Csk where it potentially negatively regulates SFK activity.

The purpose of such a mechanism is not immediately clear, although, as indicated above, Csk has been identified as a key player in stress fiber formation at least in response to G protein stimulation of fibroblasts (30). One possible mechanism for Csk regulation of actin reorganization may arise from its classic negative regulation of SFK activity, as mounting evidence implicates a role for SFKs in cytoskeletal rearrangement.

In endothelial cells, SFK activity seems particularly important in promoting cortical actin rearrangement. Recent studies show that peripheral band formation stimulated by sphingosine-1-phosphate- and MLC kinase (MLCK)-1-dependent cortical actin remodeling requires SFK activity (4, 16). In examining cytoskeletal responses of endothelial cells exposed to shear, phosphorylation of Cas and cortactin, two key molecules in actin regulation, was shown to be downstream of Src (19, 35). Cas phosphorylation induces Crk binding, an interaction implicated in Rac-mediated lamellipodial extension and membrane ruffling (8, 27), whereas cortactin activity, important for actin nucleation and filament branching at cortical regions of the cell, is inhibited by Src phosphorylation, potentially through direct association with MLCK (15). In contrast to SFK activity in promoting cortical actin remodeling, negative regulation of SFKs may be necessary for internal stress fiber formation. This occurs through direct phosphorylation of the Rho GTPase-activating protein p190RhoGAP, which, when phosphorylated, inactivates Rho/Rho kinase (ROCK) signaling to the actin cytoskeleton (2).

In endothelial cells, early shear-stimulated actin reorganization within the time frame of our described interaction includes both the remodeling of cortical actin and the appearance of preliminary stress fibers (51). The activities of MLCK and ROCK were shown to be critical in mediating acute shear-induced MLC diphosphorylation (4), an event that promotes myosin filament assembly and myosin ATPase activity to bundle actin (31, 45). We showed acute diphosphorylation of MLC with shear stress at 5 and 10 min, and in experiments designed to explore the connection between our described pathway and MLC phosphorylation, we found that both loss of Csk expression and targeting to integrins were sufficient to abrogate shear-induced MLC diphosphorylation (Fig. 8). Although we cannot eliminate the possibility that Csk is directly affecting unknown target proteins, our own observations of p190RhoGAP after shear suggest an important role for Csk in removing SFK inhibition of Rho signaling to ROCK and MLC. We found that exposing BAEC to shear stress decreased p190RhoGAP phosphorylation, which was corollary with increasing Rho activity. However, both Csk knockdown and loss of Csk targeting resulted in maintenance of p190RhoGAP phosphorylation (data not shown). Therefore, an interesting conceptualization of these findings might include the recruitment of Csk to integrins via caveolin-1. Once associated, it then functions as a switch to remove negative SFK regulation of RhoA/ROCK, allowing progression of actin cytoskeletal reorganization.

Further work should focus on detailing the mechanism of action that links the {beta}1-integrin-caveolin-1-Csk complex to the observed actin cytoskeletal signaling. Particular emphasis should be placed on ascertaining the spatiotemporal aspect of this pathway in regulating the overall remodeling process of the cell. Our previous work has identified caveolae as important mechanotransduction sites, as increasing flow and pressure in situ stimulated protein tyrosine phosphorylation within caveolae (40) and stimulated NO production from caveolae-associated endothelial nitric oxide synthase (38). As caveolin-1, the primary coat protein of caveolae, appears to be a key regulator of mechanosignaling pathways within both caveolae and integrins, there is an interesting potential for coregulation between focal adhesions and caveolae, both previously identified as independent mechanotransducing elements.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded in part by National Heart, Lung, and Blood Institute Grants HL-66301-2 (V. Rizzo) and T32-HL-07194 and American Heart Association Grant AHA0030300T (V. Rizzo).


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Rizzo, Cardiovascular Research Center and Dept. of Anatomy and Cell Biology, Temple Univ. School of Medicine, 3420 North Broad St., Philadelphia, PA 19140 (E-mail: rizzov{at}temple.edu)

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


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 DISCUSSION
 GRANTS
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00519.2004v1
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