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Circulation Research. 1999;84:1194-1202

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(Circulation Research. 1999;84:1194-1202.)
© 1999 American Heart Association, Inc.

Original Contribution

Vascular Endothelial Growth Factor Induces Activation and Subcellular Translocation of Focal Adhesion Kinase (p125FAK) in Cultured Rat Cardiac Myocytes

Naoyuki Takahashi, Yoshinori Seko, Eisei Noiri, Kazuyuki Tobe, Takashi Kadowaki, Hisataka Sabe, Yoshio Yazaki

From the Departments of Cardiovascular Medicine (N.T., Y.S., K.T., T.K., Y.Y.) and Nephrology and Endocrinology (E.N.), Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo; Institute for Adult Diseases (N.T., Y.S.), Asahi Life Foundation, Shinjuku-ku, Tokyo; Department of Immunology (Y.S.), School of Medicine, Juntendo University, Bunkyo-ku, Tokyo; and Department of Molecular Biology (H.S.), Osaka BioScience Institute, Suita, Osaka, Japan.

Correspondence to Naoyuki Takahashi, Department of Cardiovascular Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail takan-tky{at}umin.ac.jp


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract—Vascular endothelial growth factor (VEGF) has been proposed to be among the candidate factors with the most potential to play a role in ischemia-induced collateral vessel formation. Recently, we found that VEGF activated the mitogen-activated protein kinase cascade in cultured rat cardiac myocytes. To elucidate how VEGF affects adhesive interaction of cardiac myocytes with the extracellular matrix (ECM), one of the important cell functions, we investigated the molecular mechanism of activation of focal adhesion-related proteins, especially focal adhesion kinase (p125FAK), in cultured rat cardiac myocytes. We found that the 2 VEGF receptors, KDR/Flk-1 and Flt-1, were expressed in cardiac myocytes and that KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation. VEGF induced tyrosine phosphorylation and activation of p125FAK as well as tyrosine phosphorylation of paxillin; this was accompanied by subcellular translocation of p125FAK from perinuclear sites to the focal adhesions. This VEGF-induced activation of p125FAK was inhibited partially by the tyrosine kinase inhibitors genistein and tyrphostin. Activation of p125FAK was accompanied by its increased association with adapter proteins GRB2, Shc, and nonreceptor type tyrosine kinase p60c-src. Furthermore, we confirmed that VEGF induced a significant increase in adhesive interaction between cardiac myocytes and ECM using an electric cell-substrate impedance sensor. These results strongly suggest that p125FAK is one of the most important components in VEGF-induced signaling in cardiac myocytes, playing a critical role in adhesive interaction between cardiac myocytes and ECM.


Key Words: signal transduction • growth substance • cardiac myocyte • cell adhesion


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is an important regulator of endothelial cell proliferation, migration, and permeability and is secreted from tumor cells and cells exposed to hypoxia.1 2 3 4 VEGF has been proposed to be among the candidate factors with the most potential to play a role in ischemia-induced collateral vessel formation, as well as in tumor neovascularization.5 VEGF has been reported to be induced by hypoxia in several cell types in vitro,4 6 including cardiac myocytes.7 8 Levy et al7 demonstrated that cardiac myocytes synthesize and secrete VEGF in response to hypoxia in vitro. We previously reported that serum level of VEGF was markedly elevated in patients with acute myocardial infarction and it rapidly returned to basal level after reperfusion therapy.9 This indicates that most of the tissue cells, including cardiac myocytes, are exposed to high levels of VEGF in conditions such as acute myocardial infarction, as well as that VEGF is one of the most sensitive indicators of hypoxia. Recently, we found that VEGF activated mitogen-activated protein kinases (MAPKs), S6 kinase (p90rsk), and Raf-1 in cultured rat cardiac myocytes.10 This indicates that cardiac myocytes themselves are also target cells for VEGF.

Adhesive interactions between cells and the extracellular matrix (ECM) are known to be mediated by the integrin family.11 The cell-ECM interaction plays a fundamental role in regulating cellular behaviors such as migration, proliferation, and differentiation, especially for cardiac myocytes to perform continuously repetitive contractions and to adapt to external stresses such as hypoxia.12 Recent studies suggest that integrins transduce extracellular signals across the plasma membrane.11 13 The identification of focal adhesion kinase (p125FAK) provided the first evidence for the activation of an intracellular signaling molecule by integrins.14 15 16 p125FAK is a nonreceptor protein tyrosine kinase that is widely expressed in different cell types and phosphorylated on tyrosine residues accompanied with formation of focal adhesions. p125FAK has also been proved to be involved in signal transduction from cell surface receptors for neuropeptides and growth factors.17 18 19 20 21 Recently, Abedi and Zachary22 have reported that VEGF induces tyrosine phosphorylation of p125FAK and paxillin, another focal adhesion protein, in endothelial cells, as well as increases immunofluorescent staining of them in focal adhesions, which suggests that adhesion between endothelial cells and ECM may be modulated by VEGF stimulation. In this study, we investigated whether VEGF modulates adhesion between cardiac myocytes and ECM, as is the case with endothelial cells, analyzing the activation of focal adhesion-related proteins, especially p125FAK, in cultured rat cardiac myocytes. To confirm that VEGF modulates cell adhesion, we evaluated the state of cell adhesion to ECM using an electric impedance sensor. Here we show that VEGF induces activation of p125FAK, accompanied by subcellular translocation of p125FAK from perinuclear sites to focal adhesions, as well as its increased association with adapter proteins GRB2-, Shc-, and nonreceptor-type tyrosine kinase p60c-src. VEGF also induced tyrosine phosphorylation of paxillin, a cytoskeletal component that localizes to the focal adhesions at the ends of actin stress fibers. Furthermore, VEGF definitely accelerated the adhesion of cardiac myocytes to the ECM.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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Cell Culture
Primary culture of ventricular cardiac myocytes were prepared from neonatal rats as previously described.23 Briefly, heart ventricles were aseptically removed from neonatal Wistar rats, minced in calcium-free PBS, and digested with 0.025% trypsin-EDTA in PBS. The isolated cells were washed in DMEM (GIBCO Laboratories) containing 10% FBS and preplated onto plastic dishes for 1 hour to selectively remove fibroblasts. Nonadherent cells (enriched in cardiac myocytes) were collected and replated onto gelatin-coated culture dishes. They were cultured for 2 days in DMEM supplemented with 10% FBS, 50 U/mL penicillin, and 34 µmol/L streptomycin until they were confluent. They were starved for 24 hours before stimulation with VEGF.

The percentage of myocytes was estimated to be {approx}90%, as judged by immunoperoxidase staining with a mouse anti-cardiac myosin heavy chain monoclonal antibody (mAb) derived from the CMA-19 clone,24 followed by counterstaining with hematoxylin. Preparation of mouse anti-cardiac myosin heavy chain mAb (CMA19) was previously described.24 Contamination by endothelial cells was estimated to be <1%, as judged by immunoperoxidase staining with mouse monoclonal anti–factor VIII–related antigen antibody (Z002; Zymed Laboratories) (data not shown). Therefore, we concluded that the rest of the contaminating nonmuscle cells ({approx}10% of total cells) mostly consisted of fibroblasts.

Analyses of Phosphotyrosine Content of KDR/Flk-1, p125FAK, and Paxillin
The cells were treated with 22.8 pmol/L (1.0 ng/mL) recombinant human VEGF (rhVEGF; Upstate Biotechnology, Inc) for various times as indicated, and then they were frozen in liquid nitrogen and lysed on ice with NP-40 buffer containing (in mmol/L) Tris/HCl 25 (pH 7.6), NaCl 25, Na3VO4 1, sodium pyrophosphate 10, EGTA 0.5, and phenylmethylsulfonyl fluoride 1; 10 nmol/L okadaic acid; and 1% NP-40. The cell lysates were centrifuged, and the supernatants containing detergent-soluble proteins were collected. Proteins were immunoprecipitated at 4°C overnight with mouse anti-phosphotyrosine mAb (PY20; Transduction Laboratories) and protein G–Sepharose (Pharmacia LKB). The immunoprecipitates were subjected to SDS-PAGE and then transferred onto polyvinylidene difluoride transfer membranes (NEN Research Products). Polyvinylidene difluoride membranes were blocked with 1% BSA in PBS and then incubated overnight at 4°C with rabbit polyclonal anti-KDR/Flk-1 antibody (C20; Santa Cruz Biotechnology), rabbit polyclonal anti-p125FAK antibody,25 or mouse anti-paxillin mAb (349; Transduction Laboratories). The preparation of rabbit polyclonal anti-p125FAK antibody was previously described.25 After incubation with alkaline phosphatase–conjugated anti-rabbit or anti-mouse IgG antibody (both antibodies from Vector Laboratories), the blots were developed with a chemiluminescence detection kit (New England Biolabs). Tyrosine phosphorylation of p125FAK was confirmed by immunoprecipitation using anti-p125FAK polyclonal antibody followed by Western blotting with anti-phosphotyrosine mAb (4G10; Upstate Biotechnology, Inc) or anti-p125FAK polyclonal antibody. The increase in tyrosine phosphorylation of KDR/Flk-1 was quantified by scanning densitometry. For analysis of expression of VEGF receptors, total cell lysates in Laemmli sample buffer were electrophoresed and immunoblotted using rabbit polyclonal anti-KDR/Flk-1 antibody or rabbit polyclonal anti-Flt-1 antibody (C-17; Santa Cruz Biotechnology).

Kinase Assay of p125FAK
Cardiac myocytes were treated with rhVEGF (22.8 pmol/L) for various times as indicated, and then they were frozen in liquid nitrogen. Immune complex tyrosine kinase assays of p125FAK were performed with a nonradioactive isotope solid-phase ELISA kit using the exogenous substrate poly(Glu-Tyr)15 26 27 (Universal Tyrosine Kinase Assay Kit, Takara Shuzou Co Ltd) according to the manufacturer's instructions. Briefly, the cell lysates were centrifuged and the supernatants were immunoprecipitated with rabbit polyclonal anti-p125FAK antibody. The immunoprecipitates were incubated with ATP in the microtiter plate onto which poly(Glu-Tyr) had been precoated. The amount of phosphorylated poly(Glu-Tyr) was measured by ELISA using a horseradish peroxidase–linked anti-phosphotyrosine (PY20) antibody.

Analyses of the Effects of Tyrosine Kinase Inhibitors on Phosphorylation of p125FAK
Cells were preincubated for 30 minutes with or without either of the tyrosine kinase inhibitors genistein28 (37 µmol/L) and tyrphostin29 (50 µmol/L) (both from GIBCO-BRL), and then they were stimulated with 22.8 pmol/L rhVEGF. The cell lysates were immunoprecipitated with mouse anti-phosphotyrosine mAb (PY20) and subjected to Western analysis using rabbit polyclonal anti-p125FAK antibody. The increase in tyrosine phosphorylation at the 125-kDa band was quantified by scanning densitometry.

Analyses of Association of p125FAK With p60c-src, GRB2, or Shc
After stimulation with 22.8 pmol/L rhVEGF for various times as indicated, the cell lysates were immunoprecipitated with mouse anti-v-Src mAb 2-17 (LA074; Quality Biotech), rabbit polyclonal anti-GRB2 antibody (Santa Cruz Biotechnology), or mouse monoclonal anti-Shc antibody (PG-797; Santa Cruz Biotechnology) along with protein G–Sepharose and subjected to Western analysis using rabbit polyclonal anti-p125FAK antibody.

Subcellular Fractionation
After stimulation with 22.8 pmol/L rhVEGF for various times as indicated, cytosolic and membrane fractions were prepared from cardiac myocytes according to modified methods previously described.30 31 Briefly, cells were washed twice with PBS and then harvested in a buffer containing 20 mmol/L HEPES and 250 mmol/L sucrose (pH 7.4) followed by centrifuging at 1000g for 3 minutes. The supernatant was discarded, and the pellet was suspended in hypotonic Tris buffer containing (in mmol/L) Tris (pH 7.5) 10, MgCl2 1, phenylmethylsulfonyl fluoride 1, and Na3VO4 1, and 100 µmol/L leupeptin. The pellet was homogenized by 80 strokes of Dounce homogenizer. The homogenate was centrifuged at 1000g for 15 minutes to remove nuclei and debris. The supernatant was centrifuged at 48 000g for 30 minutes, resulting in a pellet, which was resuspended in hypotonic Tris buffer containing 1% NP-40 and stored as a membrane fraction, and the supernatant, which was recentrifuged at 246 000g for 90 minutes to remove the microsome-rich fraction. The resulting supernatant was stored as a cytosolic fraction. All of the above steps were carried out at 4°C unless otherwise indicated. Protein estimation was carried out using the BCA protein assay reagent (Pierce). The fractions were suspended in Laemmli sample buffer and incubated at 37°C for 30 minutes, and then their aliquots were subjected to SDS-PAGE followed by immunoblotting with polyclonal anti-p125FAK antibody or rabbit polyclonal anti–rat Na,K-ATPase {alpha}1 fusion protein (Upstate Biotechnology, Inc).

Immunocytochemistry
To investigate whether subcellular distribution of p125FAK in cardiac myocytes and fibroblasts is altered in response to VEGF, we performed double staining for p125FAK and cardiac myosin heavy chain. rhVEGF-treated (60 minutes) or untreated cardiac myocytes were fixed in acetone for 5 minutes at room temperature. The cells were incubated first with rabbit polyclonal anti-p125FAK antibody for 1 hour at 37°C and then incubated sequentially with biotinylated anti-rabbit IgG (Vector Laboratories) for 1 hour at 37°C and FITC-conjugated avidin D (Vector Laboratories) for 30 minutes at 37°C. The cells were then incubated sequentially with mouse anti-cardiac myosin heavy chain mAb (CMA-19) for 1 hour at 37°C and TRITC-conjugated anti-mouse IgG1 antibody (Chemicon International, Inc) for 30 minutes at 37°C. To investigate the relationship between the immunofluorescent staining of p125FAK and focal adhesions in rhVEGF-treated (60 minutes) cells, we performed double staining for p125FAK and vinculin. rhVEGF-treated (60 minutes) cells were first stained with rabbit polyclonal anti-p125FAK antibody and then were stained sequentially with biotinylated anti-rabbit IgG and FITC-conjugated avidin D as described above. The cells were then incubated with mouse anti-vinculin mAb (V-4505; Sigma) for 1 hour at 37°C and TRITC-conjugated anti-mouse IgG1 antibody for 30 minutes at 37°C. The sections were examined and photographed under a fluorescence microscope (Microphoto-FX, Nikon).

Cell-Substrate Adhesion Assays
Cell-substrate adhesion was measured using the electric cell-substrate impedance sensing system (Applied BioPhysics, Inc) previously reported by Giaever and Keese32 33 and Ghosh et al.34 In this system, the cells were cultured on a small gold electrode (area, {approx}10–4 cm2) deposited on the bottom of tissue culture vessels. A small alternating current signal (1 µA) at a frequency of 4000 Hz was passed between the small electrode and a larger counter electrode (area, {approx}10–1 cm2) placed at a distance. The voltage between the small and large electrodes was monitored. As cell membranes have very high impedance, the attachment of cells to the small electrode blocks the current, forcing it to flow under the cells, causing an increase in the impedance. Primary cultures of ventricular cardiac myocytes were prepared as above. Cardiac myocytes were seeded on gelatin-precoated electrodes and cultured for 2 days in DMEM supplemented with 10% FBS, 50 U/mL penicillin, and 34 µmol/L streptomycin. They were starved for 24 hours and then stimulated with rhVEGF (34.2 pmol/L). The resistance, which reflects the extent of cell-substrate adhesion, was monitored as described previously.35 36


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Cardiac Myocytes Express KDR/Flk-1 and Flt-1
It has been shown that VEGF binds to 2 structurally related tyrosine kinase receptors, Flt-137 38 39 and KDR/Flk-1.40 41 42 43 We examined whether cardiac myocytes expressed these receptors by Western analysis. As shown in Figure 1Down, KDR/Flk-1 and Flt-1 were expressed in the cultured cardiac myocytes. To examine tyrosine phosphorylation of VEGF receptors, the cell lysates from rhVEGF-treated and untreated myocytes were immunoprecipitated with anti-phosphotyrosine mAb (PY20) followed by Western blotting using anti-KDR/Flk-1 or anti-Flt-1 antibodies. KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation, with phosphorylation reaching a maximum level at 5 to 10 minutes with rhVEGF (Figure 2ADown), whereas Flt-1 was not (data not shown). The increase in tyrosine phosphorylation of KDR/Flk-1 was quantified by scanning densitometry of the autoradiogram and expressed as a ratio of the increase to the initial phosphorylation before addition of rhVEGF. As shown in Figure 2BDown, tyrosine phosphorylation of KDR/Flk-1 was significantly increased at 5 to 10 minutes after rhVEGF treatment as compared with the initial value.



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  		Figure 1. KDR/Flk-1 and Flt-1 are expressed in cardiac myocytes. To investigate whether cardiac myocytes express VEGF receptors KDR/Flk-1 and Flt-1, total cell lysates in Laemmli sample buffer were electrophoresed and immunoblotted using polyclonal anti-KDR/Flk-1 antibody or polyclonal anti-Flt-1 antibody. Mr indicates molecular weight.



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  		Figure 2. KDR/Flk-1 is tyrosine phosphorylated in response to VEGF. Cultured rat cardiac myocytes were exposed to rhVEGF (22.8 pmol/L) for the indicated periods, and the cell lysates were immunoprecipitated with an anti-phosphotyrosine mAb (PY20) and then immunoblotted with anti-KDR/Flk-1 antibody (A). In addition, the increase in tyrosine phosphorylation of KDR/Flk-1 was quantified by scanning densitometry of the autoradiograms (B). The initial phosphorylation of KDR/Flk-1 before addition of rhVEGF at 0 minutes is defined as 1.0. Fold induction values represent average of 5 independent experiments. Ab indicates antibody; anti-pTyr I.P., anti-phosphotyrosine immunoprecipitation. *P<0.05, **P<0.01 vs initial value. Results shown are mean±SD.

VEGF Phosphorylates p125FAK and Paxillin
To examine whether VEGF can stimulate tyrosine phosphorylation of a focal adhesion-associated protein tyrosine kinase p125FAK, quiescent rat cardiac myocytes were treated with rhVEGF for various time periods and lysed. The cell lysates from the treated myocytes were immunoprecipitated with anti-p125FAK polyclonal antibody followed by Western blotting with anti-phosphotyrosine mAb (4G10) or anti-p125FAK polyclonal antibody. Conversely, immunoprecipitates with anti-phosphotyrosine mAb (PY20) were also analyzed by Western blotting with anti-p125FAK antibody. As shown in Figure 3ADown and 3BDown (top panel), VEGF significantly increased tyrosine phosphorylation of p125FAK, which peaked at 5 to 10 minutes after addition of rhVEGF. We confirmed that almost equal amounts of p125FAK protein were electrophoresed in each reaction (Figure 3ADown, bottom panel). We also investigated whether VEGF caused tyrosine phosphorylation of paxillin, another focal adhesion-associated protein, which interacts with several proteins, including p125FAK, members of the src family of tyrosine kinases, the transforming protein v-crk, and the cytoskeletal protein vinculin. Immunoprecipitates with anti-phosphotyrosine mAb (PY20) from the stimulated cardiac myocytes were analyzed by Western blotting with anti-paxillin mAb. As shown in Figure 3BDown (bottom panel), VEGF also caused a significant increase in the tyrosine phosphorylation of paxillin. The tyrosine phosphorylation of paxillin reached a maximum level at 5 to 10 minutes after addition of VEGF and decreased subsequently.



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  		Figure 3. VEGF causes tyrosine phosphorylation of p125FAK and paxillin. Serum-starved cardiac myocytes were treated for the indicated periods with 22.8 pmol/L rhVEGF and lysed with NP-40 buffer. Tyrosine phosphorylation of p125FAK was analyzed either by immunoprecipitation (I.P.) using anti-p125FAK polyclonal antibody (Ab) followed by Western blotting with anti–phosphotyrosine (pTyr) mAb (4G10) (A, top panel) or by immunoprecipitation using anti-phosphotyrosine mAb (PY20) followed by Western blotting with anti-p125FAK antibody (B, top panel). To ascertain equal loading of p125FAK protein, immunoprecipitates with anti-p125FAK antibody were analyzed by anti-p125FAK Western blotting (A, bottom panel). Tyrosine phosphorylation of paxillin was analyzed by immunoprecipitation with anti-phosphotyrosine mAb (PY20) followed by Western blotting using anti-paxillin mAb (B, bottom panel). Arrows indicate positions of p125FAK and paxillin. Experiments were performed in triplicate. Results shown are representative of 3 independent experiments.

VEGF Stimulates Protein Tyrosine Kinase Activity of p125FAK
To examine whether the catalytic activity of p125FAK is stimulated by VEGF, kinase activity of p125FAK immunoprecipitates from untreated or rhVEGF-treated cardiac myocytes was assayed using poly(Glu-Tyr) as substrate. As shown in Figure 4Down, VEGF induced activation of p125FAK as early as 2 minutes, peaking at 5 minutes, which paralleled its tyrosine phosphorylation.



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  		Figure 4. VEGF stimulates protein tyrosine kinase activity of p125FAK. Protein tyrosine kinase activity of p125FAK immunoprecipitates from untreated or 22.8 pmol/L rhVEGF-treated cardiac myocytes were assayed with a nonradioactive isotope solid-phase ELISA kit using poly(Glu-Tyr) as substrate. The immunoprecipitates were incubated with ATP in the microtiter plate onto which poly(Glu-Tyr) had been precoated. The amount of phosphorylated poly(Glu-Tyr) was measured by ELISA using a horseradish peroxidase–linked anti-phosphotyrosine (PY20) antibody. *P<0.001, **P<0.01 vs control. Results shown are mean±SD.

Tyrosine Kinase Inhibitors Partially Inhibit VEGF-Induced Activation of p125FAK
Next, to examine whether VEGF-induced tyrosine phosphorylation of p125FAK was dependent on tyrosine kinase activity, the cardiac myocytes were pretreated with or without either of the tyrosine kinase inhibitors genistein (37 µmol/L) and tyrphostin (50 µmol/L) before addition of VEGF. Representative results of 1 of 3 independent experiments are shown in Figure 5Down. Both genistein and tyrphostin at least partially inhibited VEGF-induced increase in tyrosine phosphorylation of p125FAK at 5 minutes.



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  		Figure 5. Effects of tyrosine kinase inhibitors genistein and tyrphostin on VEGF-induced tyrosine phosphorylation of p125FAK. Serum-starved cardiac myocytes were pretreated for 30 minutes with or without 37 µmol/L genistein or 50 µmol/L tyrphostin and were then challenged with 22.8 pmol/L rhVEGF for the indicated periods. Cells were subsequently lysed with NP-40 buffer, and lysates were immunoprecipitated with anti-phosphotyrosine antibody (PY20) and further analyzed by anti-p125FAK Western blotting. Arrow indicates position of p125FAK. The increase in tyrosine phosphorylation of p125FAK was quantified by scanning densitometry of the autoradiogram (the corresponding figures underneath the panel). Initial phosphorylation of p125FAK before addition of rhVEGF at 0 minutes is defined as 1.0. Fold induction values represent average of 3 independent experiments.

VEGF Induces Association of p125FAK With Shc, GRB2, and p60c-src
To determine whether c-Src plays a role in VEGF-induced tyrosine phosphorylation of p125FAK, immunoprecipitates with anti-v-Src mAb were subjected to SDS-PAGE followed by immunoblotting with anti-p125FAK antibody. As shown in Figure 6aDown, VEGF caused a significant increase in the association of p60c-src with p125FAK, which peaked at 5 to 10 minutes after addition of VEGF. To determine whether the VEGF-activated p125FAK signaling complexes contained other known Src homology (SH) 2–containing proteins, such as the SH2/SH3 adapter proteins GRB2 and Shc, we examined the VEGF-induced association of p125FAK with these adapter proteins. Immunoprecipitates with either anti-GRB2 polyclonal antibody or anti-Shc mAb from VEGF-stimulated cardiac myocytes were subjected to SDS-PAGE followed by immunoblotting with anti-p125FAK polyclonal antibody. As shown in Figure 6bDown and 6cDown, VEGF caused a significant increase in the association of Shc and GRB2 with p125FAK. The association peaked at 5 to 10 minutes after addition of VEGF.



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  		Figure 6. VEGF induces association of p125FAK with p60c-src, GRB2, or Shc. Quiescent cardiac myocytes were treated with 22.8 pmol/L rhVEGF for the indicated periods. Cells were subsequently lysed with NP-40 buffer and immunoprecipitated with anti-v-Src mAb 2-17 (LA074) (a), polyclonal anti-GRB2 antibody (b), or anti-Shc mAb (PG-797) (c). Immunoprecipitates were analyzed by Western blotting with anti-p125FAK polyclonal antibody. Arrows indicate positions of p125FAK. Results shown are representative of 3 independent experiments.

VEGF Induces Subcellular Translocation of p125FAK in Cardiac Myocytes and Fibroblasts
It has been shown that cell adhesion to ECM, such as fibronectin, through integrins causes increased tyrosine phosphorylation of p125FAK as well as accumulation of p125FAK in focal adhesions.13 44 45 46 We examined the effects of VEGF on subcellular localization of p125FAK in cardiac myocytes and nonmuscle cells (which mostly consisted of fibroblasts). To distinguish cardiac myocytes from nonmuscle cells, we performed double staining for cardiac myosin heavy chain and p125FAK by immunofluorescence. Figures 7BDown and 7DDown show that myofibrils of cardiac myocytes were strongly stained, making it easy to distinguish cardiac myocytes from fibroblasts. As shown in Figure 7ADown, p125FAK predominantly localized in the perinuclear region in nonstimulated cardiac myocytes. Only weak fluorescent dots of p125FAK were seen in some nonstimulated fibroblasts (Figure 7ADown, arrows). Fluorescent dots of p125FAK, which mostly localized in the central regions at a higher density, appeared to scatter to the peripheral cytoplasm in cardiac myocytes stimulated with rhVEGF for 60 minutes (Figure 7CDown, arrowheads). In fibroblasts stimulated with VEGF for 60 minutes, fluorescent dots of p125FAK were observed to be concentrated in the patchy arrowhead-like structures at the peripheral cytoplasm reminiscent of focal adhesions (Figure 7CDown, arrow). To compare the localization of p125FAK staining to focal adhesions in VEGF-treated cells, we performed double staining for p125FAK (Figure 7EDown) and vinculin (Figure 7FDown) by immunofluorescence. Fluorescent dots of p125FAK showed localization similar to that of vinculin in cardiac myocytes (Figure 7EDown and 7FDown, arrowheads), which suggests that p125FAK was localized to focal adhesions in response to VEGF, although the rest of the p125FAK remained in cytoplasm. In fibroblasts, the patchy arrowhead-like fluorescent dots of p125FAK were clearly observed with higher resolution (Figure 7EDown, arrow). Incubation of cardiac myocytes with control nonimmune rabbit serum yielded no significant staining (data not shown).



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  		Figure 7. VEGF alters the subcellular localization of p125FAK in cardiac myocytes and fibroblasts. Quiescent cardiac myocytes were given no treatment (A and B) or were stimulated with 22.8 pmol/L rhVEGF for 60 minutes (C and D) and fixed in acetone. To distinguish cardiac myocytes from contaminating fibroblasts, we performed double staining for cardiac myosin heavy chain and p125FAK by immunofluorescence. Cells were incubated with rabbit polyclonal anti-p125FAK antibody and then sequentially incubated with biotinylated anti-rabbit IgG and FITC-conjugated avidin D (A and C). Cells were then incubated sequentially with mouse monoclonal anti-cardiac myosin heavy chain antibody (CMA-19) and TRITC-conjugated anti-mouse IgG1 antibody (B and D). For the rhVEGF-treated (60 minutes) cells, we also performed double staining for p125FAK (E) and vinculin (F) by immunofluorescence. Cells were first incubated sequentially with rabbit polyclonal anti-p125FAK antibody, biotinylated anti-rabbit IgG, and FITC-conjugated avidin D as above and then incubated with mouse anti-vinculin mAb followed by TRITC-conjugated anti-mouse IgG1 antibody. Arrowheads indicate localization of p125FAK (C and E) and that of vinculin (F) in cardiac myocytes treated with rhVEGF for 60 minutes. Arrows indicate localization of p125FAK in fibroblasts, either not treated (A) or treated (C and E) with rhVEGF for 60 minutes. An arrow in panel F indicates corresponding localization of vinculin in a fibroblast treated with rhVEGF for 60 minutes. Bars=10 µm.

Next, to confirm that VEGF induces subcellular translocation of p125FAK, we analyzed p125FAK content in subcellular fractions by Western analysis with and without VEGF stimulation. As shown in Figure 8Down, the amount of p125FAK in the membrane fraction was increased in response to rhVEGF (Figure 8bDown, top panel), whereas that in the cytosolic fraction remained almost unchanged (Figure 8aDown). We confirmed that almost equal amounts of the membrane fraction were electrophoresed in each reaction by Western analysis using anti–Na,K ATPase {alpha}1 subunit antibody (Figure 8bDown, lower panel).



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  		Figure 8. Effect of VEGF on subcellular distribution of p125FAK. After stimulation with 22.8 pmol/L rhVEGF for the indicated periods, cytosolic and membrane fractions were prepared from cardiac myocytes as described in Materials and Methods and subjected to Western analysis using anti-p125FAK antibody (a and b, top panels). To confirm that almost equal amounts of membrane fraction were electrophoresed in each reaction, the same amounts of membrane fractions were subjected to Western analysis using anti–Na,K ATPase {alpha}1 subunit (isoform-specific) antibody (b, bottom panel).

VEGF Strengthens Adhesion of Cardiac Myocytes to the ECM
To assess the effects of VEGF on the extent of cell-substrate adhesion, we monitored changes in cell-substrate resistance after addition of rhVEGF, using an electric cell-substrate impedance sensor. Typical results are shown in Figure 9ADown. The extent of cell-substrate adhesion is expressed as "normalized" resistance, which was defined as a ratio of the resistance to the initial value before addition of rhVEGF. VEGF induced a significant increase in normalized resistance, indicating strengthening of the cell-substrate adhesion at 3 hours after addition of rhVEGF (Figure 9BDown).



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  		Figure 9. Effects of VEGF on adhesion of cardiac myocytes to the ECM. Cardiac myocytes were cultured on the surface of gelatin-coated wells manufactured especially for measurement of resistance. After starvation for 24 hours, they were stimulated with rhVEGF (34.2 pmol/L). A, Typical tracing representative of 4 replicate experiments. In all of these experiments, initial resistance of cardiac myocytes was within the range of 5000 to 7000 {Omega}. To simplify the comparison, ordinate represents normalized resistance defined as a fraction of the initial resistance before addition of rhVEGF. B, Comparison of percentage increase in normalized resistance at 3 hours after addition of rhVEGF, VEGF(+), with control, VEGF(–). Results shown are mean±SD from 4 independent experiments.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The biological effects of VEGF are mediated by specific cell surface receptors. It has been shown that VEGF binds with high affinity to 2 structurally related tyrosine kinase receptors, Flt-137 38 39 and KDR/Flk-1.40 41 42 43 Studies on signal transduction from Flt-1 and KDR showed that these 2 receptor tyrosine kinases have different signal transduction properties.47 48 It has been reported that KDR-expressing cells showed striking changes in cell morphology, actin reorganization and membrane ruffling, chemotaxis, and mitogenicity on VEGF stimulation, whereas Flt-1–expressing cells lacked such responses in a cultured human umbilical vein endothelial cell population.47 We confirmed by Western blot analysis that KDR/Flk-1, as well as Flt-1, was expressed in the cultured cardiac myocytes and that KDR/Flk-1 was significantly tyrosine phosphorylated on VEGF stimulation, whereas Flt-1 was not. Regarding the capacity for autophosphorylation, KDR has been reported to undergo autophosphorylation much more efficiently than Flt-1 in response to VEGF, on the basis of a comparison between Flt-1–transfected porcine aortic endothelial cells and KDR-transfected ones.47 By analogy, it is possible that detectable tyrosine phosphorylation was not induced for Flt-1 protein in response to VEGF in cardiac myocytes at least at the concentration used in this study, although KDR/Flk-1 protein was efficiently tyrosine phosphorylated. It might also in part reflect the difference in the sensitivity between the anti-KDR/Flk-1 antibody and the anti-Flt-1 antibody used in the present study. These results suggest that the VEGF-induced signaling pathway may be more dependent on KDR/Flk-1 than Flt-1 or that it requires formation of heterodimeric complexes between KDR and Flt-1. To clarify which of the 2 receptor tyrosine kinases (or both) mediates VEGF-induced activation in cardiac myocytes, further investigation is needed.

p125FAK is a widely expressed nonreceptor protein tyrosine kinase that localizes to focal adhesion structures. p125FAK is thought to be one of the key elements in the signal transduction pathway underlying changes in cell behavior induced by diverse stimuli, including integrin engagement; oncogenic transformation; and mitogenic neuropeptides such as bombesin, endothelin, vasopressin, angiotensin, platelet-derived growth factor, and lysophosphatidic acid.14 15 17 18 19 20 21 49 It was shown that tyrosine-phosphorylated pp125FAK directly interacts with pp60c-src and pp59fyn as one of their major substrates.27 50 Indeed, tyrosine phosphorylation of p125FAK by Src-family kinases has been shown to be directly correlated with increased protein tyrosine kinase activity, which is an important step in the formation of an active signaling complex.15 Paxillin is a cytoskeletal protein involved in actin-membrane attachment at sites of cell adhesion to the ECM, which has also been demonstrated to be one of the major substrates of pp60c-src in Rous sarcoma virus–transformed cells as p125FAK.51 It becomes tyrosine phosphorylated concomitantly with p125FAK in response to multiple stimuli, including integrin-mediated cell adhesion, several neuropeptide growth factors, and platelet-derived growth factor.52 53 54

In the present study, we have shown that VEGF stimulated tyrosine phosphorylation and activation of p125FAK in cultured cardiac myocytes, peaking at 2 to 10 minutes. Although the time courses of the change in tyrosine phosphorylation and the activation of the kinase activity were similar, the extent of p125FAK activation seemed to be smaller than that expected from the increase in its tyrosine phosphorylation. The kinase activity assayed with exogenous substrate poly(Glu-Tyr) reflected only the ability to phosphorylate downstream substrates, including other kinases. On the other hand, tyrosine phosphorylation of p125FAK results not only from autophosphorylation but also from phosphorylation by other upstream protein kinases, including Src family kinases.27 50 The contribution from the latter might be relatively large compared with that from the former. This may be the reason for the discrepancy between the extent of its activation measured with the exogenous substrate and tyrosine phosphorylation.

We also showed that VEGF also stimulated tyrosine phosphorylation of paxillin, and VEGF-induced tyrosine phosphorylation of p125FAK was inhibited at least partially by the tyrosine kinase inhibitors genistein and tyrphostin. Moreover, VEGF caused increased association of p125FAK with pp60c-src concomitantly with increased tyrosine phosphorylation of p125FAK. Recently, Abedi and Zachary22 have reported that VEGF induces tyrosine phosphorylation of p125FAK and paxillin in endothelial cells, suggesting that they are components in a VEGF-stimulated signaling pathway. Our results indicate that tyrosine phosphorylation of p125FAK and paxillin is also a part of the signal transduction by VEGF in cardiac myocytes as in endothelial cells, resulting in organization of the cytoskeleton. Tyrosine kinases, especially p60c-src, might take some part in VEGF-induced tyrosine phosphorylation and activation of p125FAK in cardiac myocytes.

Transduction of various mitogenic signals from the cell membrane to the nucleus involves the adapter proteins Shc and GRB2, which mediate activation of the Ras/MAPK pathway.55 56 57 Shc is an immediate substrate of receptor tyrosine kinase activity and serves to physically link activated receptors to downstream signaling components.58 59 GRB2 is a ubiquitously expressed-24 kDa mammalian protein, which directly binds autophosphorylated tyrosine kinase receptors as well as phosphorylated Shc proteins and p125FAK through its SH2 domain.55 56 60 61 It is well known that activation of insulin receptor results in interaction of the GRB2-Sos complex with insulin receptor substrate-1 and Shc via the SH2 domain of GRB2.59 62 The tyrosine kinase–Shc–GRB2–Sos pathway was shown also to be involved in signal transduction from Gq protein–coupled angiotensin II receptor leading to activation of p21ras in cardiac myocytes.63 It has also been demonstrated that adhesion of fibroblasts to fibronectin promotes SH2-domain–mediated association of GRB2 and p60c-src with p125FAK in vivo, resulting in activation of MAPK.60 In the present study, we demonstrated VEGF-induced association of p125FAK with GRB2 and Shc, occurring concomitantly with increased tyrosine phosphorylation of p125FAK. This strongly suggests that 2 ubiquitously expressed adapter proteins, GRB2 and Shc, play a role in VEGF-induced signal transduction involving the MAPK pathway in cardiac myocytes, as we previously reported.10

Furthermore, the immunocytochemical study demonstrated that VEGF stimulation significantly altered the subcellular localization of p125FAK from the perinuclear region to the peripheral cytoplasm in cardiac myocytes and increased accumulation of p125FAK in the patchy arrowhead-like structures at the peripheral cytoplasm (ie, focal adhesions) in cardiac myocytes as well as in fibroblasts. In addition, we confirmed quantitatively by Western analysis that the amount of p125FAK in the membrane-rich fraction significantly increased in response to VEGF. These results indicate that p125FAK translocates to focal adhesions in response to VEGF. Taken together, VEGF causes activation as well as subcellular translocation of p125FAK to focal adhesions, where it works. Activation and accumulation of p125FAK in focal adhesions strongly suggests that adhesive interaction between cardiac myocytes and ECM may be strengthened in response to VEGF. Using an electric cell-substrate impedance sensor, we confirmed that VEGF induced significant increase in adhesion between cardiac myocytes and ECM. Whether this may reflect one of the cardiac adaptive responses in vivo in situations such as acute ischemia, in which cardiac myocytes are exposed to high levels of VEGF, is unknown and remains to be clarified.


*    Acknowledgments
 
This work was supported by a grant for cardiomyopathy from the Ministry of Health and Welfare, Japan; a grant for scientific research from the Ministry of Education, Science and Culture, Japan; a grant from Sankyo Foundation of Life Science; Japan Heart Foundation-Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease; and a grant from the Study Group of Molecular Cardiology. We thank Kaori Takahashi for excellent technical assistance.


*    Footnotes
 
This manuscript was sent to Michael R. Rosen, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received June 8, 1998; accepted February 11, 1999.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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