Focal Adhesion Kinase Is Involved in Angiotensin II–Mediated Protein Synthesis in Cultured Vascular Smooth Muscle Cells
Abstract—The rate of vascular smooth muscle cell protein synthesis and cellular hypertrophy in response to angiotensin II (Ang II) is dependent on activation of protein tyrosine kinases (PTKs) and both the extracellular signal–regulated kinase (ERK) 1/2 and p70S6K pathways. One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions. We used an actin depolymerizing agent, cytochalasin D (Cyt-D), and a replication-defective adenovirus encoding FAK-related nonkinase (FRNK), an inhibitor of FAK-dependent signaling, as tools to assess whether FAK was upstream of the ERK1/2 and/or the p70S6K pathways. Cyt-D reduced basal FAK phosphorylation and blocked Ang II–dependent FAK phosphorylation in a dose-dependent manner. Confocal microscopy indicated that Cyt-D induced actin filament disruption and FAK delocalization from focal adhesions. Cyt-D also reduced Ang II–induced ERK1/2 activation, but p70S6K activation was relatively unaffected. Cyt-D reduced basal protein synthetic rate and substantially reduced the Ang II–induced increase in protein synthesis. Similarly, FRNK overexpression blocked Ang II–induced FAK phosphorylation and ERK1/2 activation, but not p70S6K phosphorylation, and markedly inhibited protein synthesis. This is the first report to demonstrate that FAK is a critical component of the signal transduction pathways that mediate Ang II–induced ERK1/2 activation, c-fos induction, and enhanced protein synthesis in vascular smooth muscle cells.
Angiotensin II (Ang II) plays an important role in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis.1 2 Apart from its vasoconstrictor properties, Ang II is a potent hypertrophic growth factor for vascular smooth muscle cells (VSMCs).3 4 5 Binding of Ang II to AT1s activates several signaling pathways implicated in regulating cellular growth.1
One feature of Ang II–induced VSMC hypertrophy is an increase in protein synthesis. The ERK1/2 family of protein kinases and the ribosomal S6 kinases are key effectors implicated in the regulation of Ang II–induced protein synthesis.6 7 ERK1/2 are serine/threonine protein kinases that are activated by the upstream Ras–Raf–mitogen-activated protein kinase/ERK (MEK) cascade. ERK1/2 modulate gene transcription by induction of transcription factors such as c-fos.8 9 10 The ribosomal p70 S6 kinase (p70S6K) and p90 S6 kinase (p90RSK) families phosphorylate S6 protein, a component of the 40S ribosomal subunit, thereby regulating the protein synthetic machinery.11 p70S6K is activated by numerous extracellular stimuli. Its activation can be blocked by wortmannin and LY294002, suggesting that it is downstream of phosphatidylinositol-3 kinase.12 In contrast, p90RSK is downstream of the ERK1/2 cascade.13 The upstream regulators of both of these pathways have not been clearly defined. Several groups have suggested that 1 or more protein tyrosine kinases (PTKs) are involved, as PTK inhibitors such as genistein completely abolish Ang II–induced protein synthesis and block activation of ERK1/2 and the S6 kinases.14 15
One potential PTK that may regulate these signaling cascades is focal adhesion kinase (FAK), a nonreceptor PTK associated with focal adhesions.16 FAK autophosphorylation in response to agonist-induced integrin clustering can lead to its stable association with Src,17 as well as phosphatidylinositol-3 kinase,18 which is a well-characterized upstream regulator of p70S6K. Src can then tyrosine phosphorylate the C-terminal region of FAK, thereby creating an additional binding site for the adapter protein Grb2.19 Association of Grb2 with the GDP-GTP exchange protein Sos and the small GTP binding protein Ras can activate the ERK1/2 signaling cascade.10 Thus, FAK may potentially regulate both pathways.
FAK is highly expressed in the medial layer of intact arteries, and high levels of FAK were detected in cultured VSMCs in vitro.20 Okuda et al21 have shown that Ang II acutely induces FAK tyrosine phosphorylation in cultured VSMCs. The purpose of this study was to determine whether FAK is indeed an upstream PTK regulating ERK1/2 and p70S6K activation, as well as enhanced protein synthesis in response to Ang II in VSMCs.
Materials and Methods
Cytochalasin D (Cyt-D) was obtained from Calbiochem. Polyclonal anti-FAK and p70S6K antibodies were from Upstate Biotechnologies, and anti-PYK2 monoclonal and anti-phosphotyrosine (pTyr) polyclonal antibodies were from Transduction Laboratories. Phosphospecific polyclonal ERK1/2 antibody was from New England Biolabs. c-fos oligodeoxynucleotide probe was from Oncogene Science. Horseradish peroxidase–conjugated goat anti-rabbit IgG was from Bio-Rad. Angiotensin II and protein A and G beads were from Sigma. [3H]Phenylalanine and [32P]ATP were from Amersham.
Rat aortic VSMCs were isolated from 10- to 12-week-old Sprague-Dawley rats and cultured on uncoated, plastic dishes, as previously described.22
Protein Synthesis Measurements
VSMCs were maintained in serum-free medium or stimulated with Ang II (100 nmol/L) for 24 hours. Cells were labeled with [3H]phenylalanine (2 μCi/mL) during the last 6 hours of treatment. Cells were rinsed with PBS and harvested with 10% trichloroacetic acid (TCA) on ice. Samples were centrifuged (25 000g, 5 minutes), washed with TCA, and recentrifuged. Pellets were solubilized in 0.2N NaOH (500 μL, 20 minutes, 60°C). A portion of the sample was analyzed for total protein using a bicinchonic acid protein assay (Pierce Chemical Co). Radioactivity was measured by liquid scintillation spectroscopy. Results are means of triplicate dishes and are expressed as disintegrations per minute (dpm)/μg protein.
Western Blot Analysis
Cell lysates were prepared as previously described.22 Equal amounts of protein (40 μg) were resolved by SDS-PAGE and Western blotting. Primary antibody binding was visualized by enhanced chemiluminescence (Amersham). Band intensity was quantified by laser densitometry.
Lysates were prepared as previously described.22 Equal amounts of protein (500 μg) were treated overnight with antibodies against phosphotyrosine residues (pTyr) or FAK. Immune complexes were then precipitated using protein A or protein G Sepharose (2 hours; 4°C). Beads were centrifuged, washed, resuspended in sample buffer, and analyzed by SDS-PAGE and Western blotting as described above.
Cells were fixed, permeabilized, and stained with anti-FAK antibody (to visualize FAK and FAK-related nonkinase [FRNK]) and rhodamine-conjugated phalloidin (to visualize actin filaments).23 Cells were viewed using a Zeiss model LSM 510 confocal microscope.
Total cellular RNA was isolated, size fractionated, and transferred to nylon membranes. c-fos mRNA and 18S rRNA were detected by hybridization to 32P-labeled oligodeoxynucleotide probes, as previously described.24
A replication-defective adenovirus encoding FRNK (Adv-FRNK) was constructed as previously described.23 A replication-defective adenovirus containing the β-galactosidase gene, LacZ (Adv-LacZ), was used to control for the nonspecific effects of viral infection. Adenoviruses were amplified and purified using HEK293 cells, as previously described.23 Viral titers were estimated by absorbance at 260 nm as follows: viral particles per milliliter=A260×dilution×1010. Preliminary experiments determined that a concentration of 500 particles of Adv-FRNK or Adv-LacZ per cell (ppc) produced readily detectable overexpression of these proteins within 48 hours (Western blot analysis; see Figure 5A⇓) and infected most VSMCs (X-gal staining) (data not shown).
Results were expressed as mean±SEM. Data were compared by ANOVA followed by the Dunnet test or Student-Newman-Keuls test using SigmaStat, version 1.0 (Jandel Scientific).
Effects of Protein Kinase Inhibitors on Ang II–Induced Protein Synthesis
Using pharmacological inhibitors, we first examined the relative contribution of the ERK1/2 and p70S6K pathways to Ang II–mediated increases in protein synthesis. As seen in Figure 1A⇓, Ang II (100 nmol/L) caused a 57±5% increase in [3H]phenylalanine incorporation into total protein. Pretreatment with PD98059 (10 to 30 μmol/L; 30 minutes), a highly specific inhibitor of ERK1/2 activation, resulted in a dose-dependent decrease in Ang II–dependent [3H]phenylalanine incorporation, with a 38% reduction in protein synthesis at the 30 μmol/L concentration. Of note, this concentration of PD98059 completely inhibited Ang II–mediated ERK1/2 phosphorylation, as assessed by Western blotting with phospho-ERK–specific antibody (data not shown). Similarly, pretreatment of cells with rapamycin (1 to 10 nmol/L; 45 minutes), a p70S6K inhibitor, also significantly reduced the rate of Ang II–induced protein synthesis (Figure 1B⇓). However, rapamycin had no effect on ERK1/2 phosphorylation, as detected by Western blotting (data not shown). Similar results have been obtained by other investigators.7 25
Genistein, a PTK inhibitor, was then used to determine whether 1 or more PTKs were upstream regulators of these parallel pathways. Pretreatment with genistein (10 to 100 μmol/L; 60 minutes) caused a dose-dependent inhibition of Ang II–induced protein synthesis (Figure 1C⇑). However, daidzein (100 μmol/L, 60 minutes), an inactive analog of genistein, had no effect on Ang II–induced protein synthesis. Herbimycin A (0.1 to 0.5 μmol/L), another PTK inhibitor, produced similar results (data not shown).
Ang II Induces FAK Phosphorylation
We next examined whether Ang II activated FAK. As seen in Figure 2A⇓, VSMCs demonstrated a substantial degree of basal FAK phosphorylation. However, Ang II (100 nmol/L; 2 to 60 minutes) substantially increased FAK phosphorylation in a time- and concentration-dependent manner. FAK activation was maximal at 5 minutes and returned to basal levels by 60 minutes. Ang II–induced FAK phosphorylation was detectable with as little as 10–12 mol/L and reached a maximum with 10–7 mol/L of agonist (Figure 2B⇓). These results were obtained by immunoprecipitating cell extracts with anti-pTyr and probing the resulting Western blot with anti-FAK antibody. However, similar results were obtained when the same cell extracts were immunoprecipitated with anti-FAK, and the resulting Western blot was probed with anti-pTyr (data not shown). This time- and dose-dependent response of FAK activation are consistent with previous studies.21 22
In light of the effects of genistein on Ang II–induced protein synthesis (Figure 1C⇑), we then examined whether genistein inhibited Ang II–mediated FAK phosphorylation. As seen in Figure 2C⇑, genistein caused a dose-dependent decrease in basal FAK phosphorylation and also significantly inhibited Ang II–mediated FAK phosphorylation at concentrations that also inhibited protein synthesis.
FAK Localization and Ang II–Induced FAK Phosphorylation Are Dependent on the Actin Cytoskeleton
We next examined FAK localization and the effects of cytoskeletal disassembly with Cyt-D on Ang II–mediated FAK phosphorylation. In initial experiments, control VSMCs and VSMCs pretreated with Cyt-D were stained with rhodamine-phalloidin and anti-FAK polyclonal antibody. As seen in Figure 3A⇓, actin stress fibers traversed the length and breadth of untreated, control cells, colocalizing with FAK at focal adhesions. However, pretreatment of VSMCs with Cyt-D (10 μmol/L) resulted in the complete disruption of the actin cytoskeleton and the loss of punctate FAK staining, suggesting delocalization of FAK from focal adhesions (Figure 3D⇓). Lower concentrations of Cyt-D (Figures 3B⇓ and 3C⇓) produced less dramatic alterations in FAK localization and stress fiber assembly. However, acute stimulation of VSMCs with Ang II did not result in any obvious differences in FAK localization or cytoskeletal organization in either control or Cyt-D–treated cells (data not shown).
Immunoprecipitation studies substantiated these findings. As seen in Figure 3E⇑, dose-dependent disruption of the actin cytoskeleton resulted in a significant decrease in basal FAK phosphorylation. In addition, Ang II–induced FAK phosphorylation was inhibited with as little as 1 μmol/L of Cyt-D and was completely abrogated with 10 μmol/L of the drug. Similar results were obtained using latrunculin A, another cytoskeleton-disrupting agent (data not shown).
Ang II–Induced ERK Phosphorylation, but Not p70S6K Phosphorylation, Is Affected by Cytoskeletal Disruption
Cyt-D was then used to determine whether FAK was upstream of either ERK1/2 or p70S6K activation or both. As seen in Figure 4A⇓, pretreatment of VSMCs with Cyt-D had no effect on basal ERK1/2 phosphorylation as detected by Western blotting with phosphospecific ERK1/2 antibody. However, Ang II–induced ERK1/2 phosphorylation was completely abrogated at similar Cyt-D concentrations at which FAK phosphorylation was inhibited, suggesting that FAK activation was upstream of ERK1/2. In contrast, p70S6K phosphorylation, as detected by a band shift on Ang II stimulation, remained unaffected even in the presence of higher doses of Cyt-D than those that inhibited both FAK and ERK1/2 phosphorylation (Figure 4B⇓).
Ang II–Induced Protein Synthesis Is Inhibited by Cytoskeletal Disruption
We then examined whether interfering with FAK phosphorylation with Cyt-D affected protein synthetic rates. As seen in Figure 4C⇑, Cyt-D had only a modest, nonsignificant effect on basal protein synthetic rate. However, Cyt-D produced a dose-dependent decrease in Ang II–induced protein synthesis. Ang II increased protein synthetic rate by only 22% at the highest concentration of Cyt-D tested, as compared with 48% in untreated, control cells. Thus, the ability of Ang II to increase protein synthesis was reduced by cytoskeletal disruption, which is consistent with the effects of Cyt-D on FAK phosphorylation (Figure 3E⇑) and the ERK1/2 signaling cascade (Figure 4A⇑).
Effect of FRNK on FAK Localization and Cytoskeletal Architecture
Although these pharmacological studies provided circumstantial evidence for a role for FAK in regulating Ang II–induced protein synthesis, we sought to more specifically target FAK-dependent signaling using a replication-defective adenovirus encoding FRNK.26 As seen in Figure 5A⇓, 100 to 500 ppc of Adv-FRNK caused the dose-dependent expression of a 41-/43-kDa polypeptide that was recognized by an antibody raised against the C-terminal portion of FAK. Traces of this polypeptide were also observed in uninfected and Adv-LacZ-infected VSMCs. Higher concentrations of virus above 500 particles per cell (ppc) did not further increase FRNK overexpression. Furthermore, FRNK overexpression appeared to have no significant effect on endogenous FAK levels.
We then examined the effect of FRNK overexpression on cytoskeletal architecture. As seen in Figure 5D⇑, FRNK overexpression was inhomogeneous, with some cells nearly completely filled with immunoreactive protein, whereas others displayed much less FAK/FRNK staining. Most VSMCs, however, showed enhanced focal adhesion immunofluorescence, suggesting that FRNK indeed localized to these structures. Despite this inhomogeneity of expression, we noted the complete loss of actin stress fibers in almost all of the Adv-FRNK–infected cells, which suggested that nearly all cells were expressing some level of transgene. The markedly reduced f-actin staining also suggested that FRNK interfered with actin filament assembly. This was not due to a nonspecific effect of adenoviral infection, as both uninfected and β-galactosidase–overexpressing cells stained normally for actin filaments and FAK (Figures 5B⇑ and 5C⇑, respectively).
FRNK Abrogates FAK Phosphorylation
We examined the effect of FRNK overexpression on FAK phosphorylation. As seen in Figures 6A⇓ and 6B⇓, basal FAK phosphorylation was reduced by 28±8% by FRNK. FRNK also completely blocked the Ang II–induced increase in FAK phosphorylation, as compared with cells infected with Adv-LacZ. Of note, Adv-LacZ–infected cells displayed somewhat reduced Ang II–induced FAK phosphorylation as compared with uninfected VSMCs (see Figure 2⇑). However, total FAK levels were similar in Adv-FRNK– and Adv-LacZ-infected cells (Figure 6A⇓). Similar immunoprecipitation experiments were then performed to examine the effects of FRNK overexpression on PYK2, the other member of the FAK family of PTKs. As seen in Figure 6C⇓, Ang II–induced PYK2 phosphorylation was also partially blocked by FRNK overexpression, as compared with uninfected cells, or cells infected with Adv-LacZ. Taken together, these results indicate that FRNK indeed interferes with FAK phosphorylation in VSMCs, but there is considerable interaction between FAK, PYK2, and cytoskeleton-dependent signaling in these cells.
FRNK Overexpression Blocks Ang II–Mediated ERK1/2 Phosphorylation and Protein Synthesis
As expected from the results obtained with Cyt-D (Figure 4⇑), FRNK overexpression markedly suppressed Ang II–induced ERK1/2 phosphorylation but had relatively little effect on p70S6K activation. As seen in Figure 7A⇓, Ang II–mediated ERK1/2 activation was relatively unaffected by adenoviral infection with Adv-LacZ, as compared with uninfected VSMCs. However, Ang II–stimulated ERK1/2 activation was substantially reduced in Adv-FRNK–infected cells. In contrast, neither Adv-LacZ nor Adv-FRNK infection affected p70S6K phosphorylation (Figure 7B⇓). Of note, the total amount of p70S6K was relatively unaffected in either Adv-LacZ– or Adv-FRNK–infected cells, as compared with uninfected, control VSMCs.
To further substantiate the role of FAK in the ERK1/2 signaling cascade, we examined c-fos induction in FRNK-overexpressing cells. As seen in Figure 7C⇑, Ang II markedly increased c-fos mRNA levels in uninfected and Adv-LacZ–infected VSMCs. However, FRNK overexpression abolished c-fos induction, which correlated with the inhibition of ERK1/2 activation (Figure 7A⇑). Finally, FRNK overexpression had no significant effect on basal rates of protein synthesis but completely abrogated the Ang II–induced increase in [3H]phenylalanine incorporation (Figure 7D⇑). The loss of Ang II responsiveness in Adv-FRNK–infected cells was not due to a nonspecific effect of adenoviral infection, as Ang II stimulated protein synthesis to nearly the same extent in Adv-LacZ–infected and uninfected VSMCs.
There is accumulating evidence to indicate that both the ERK1/2 and p70S6K pathways are regulated by 1 or more upstream PTKs. Indeed, Ang II stimulation in VSMCs leads to the activation of a number of different nonreceptor PTKs, including Src,27 FAK,20 21 and PYK2,22 28 29 which may all be involved in regulating specific aspects of cellular growth. This study demonstrates for the first time that FAK is a critical component of the signal transduction pathway that may regulate at least 1 of these downstream pathways.
Our results establish the interdependence of FAK and the actin cytoskeleton in Ang II–dependent signal transduction. Disruption of actin stress fibers with Cyt-D resulted in FAK delocalization and dephosphorylation, and inhibition of ERK1/2 phosphorylation and Ang II–mediated protein synthesis. This requirement for actin filaments may be related to their role in maintaining the association of FAK with cell-surface integrins and other components of the cytoskeleton. These components appear necessary for agonist-induced local tyrosine phosphorylation by the signaling molecules that accumulate at focal adhesions.30 It should be pointed out, however, that Cyt-D treatment potently inhibited FAK phosphorylation but only partially blocked Ang II–induced increases in protein synthetic rate. In contrast, FRNK overexpression had a lesser effect on FAK tyrosine phosphorylation but completely abrogated Ang II–induced protein synthesis. The reason for these differences is unclear, but they suggest that phosphorylation-independent determinants on FAK may play a role in regulating protein synthesis in VSMCs.
Similarly, interfering with FAK phosphorylation by FRNK overexpression resulted in actin cytoskeletal disassembly. We targeted FAK by overexpressing FRNK, which not only resulted in the inhibition of Ang II–induced FAK phosphorylation but also significantly reduced Ang II–induced ERK1/2 activation and c-fos induction. FRNK, which contains the focal adhesion targeting sequence of FAK but is devoid of the Y-397 autophosphorylation site and kinase domain, appeared to localize in high concentrations within focal adhesions. Although the mechanism whereby FRNK interferes with FAK-dependent signal transduction is not known, it seems reasonable to conclude that FRNK competed with endogenous FAK for specific binding sites present on other focal adhesion components. The displacement of FAK may in turn have resulted in the loss of signals necessary for the maintenance of actin stress fiber assembly and for other downstream signaling events leading to the ERK1/2 cascade.31
In contrast, cytoskeletal disruption by Cyt-D and FRNK did not significantly affect Ang II–induced p70S6K activation. These results indicate that the effects of Cyt-D and FRNK overexpression on ERK1/2 phosphorylation were not just the result of nonspecific effects on cell viability. This was confirmed by the fact that basal rates of protein synthesis were relatively similar in control, Cyt-D–treated, and FRNK-overexpressing cells. It is conceivable that the residual degree of FAK phosphorylation in Cyt-D and FRNK-overexpressing cells was sufficient to maintain agonist-induced downstream signaling to p70S6K. Also likely, however, is the possibility that p70S6K activation occurred independently of FAK activation by a different, upstream, PTK. Graves et al14 previously demonstrated a critical role for a Ca2+-sensitive PTK upstream of p70S6K, but not p90RSK, in rat liver epithelial cells. They postulated that PYK2, the other member of the FAK family of nonreceptor PTKs, was responsible. Indeed, our previous study22 demonstrated that PYK2 is activated in VSMCs in response to similar concentrations of Ang II and over a similar time period. We found that PYK2 was predominantly localized to the cytoplasm,22 which is quite distinct from the focal adhesion staining of FAK demonstrated in the present report. However, we found that FRNK overexpression partially blocked Ang II–induced PYK2 phosphorylation in VSMCs, suggesting a close interaction between both members of this kinase family and the cytoskeleton. Although it is interesting to speculate that PYK2 and FAK may regulate different components of the signaling pathways involved in Ang II–induced protein synthesis, future studies using more specific antagonists of FAK- and PYK2-dependent signaling will be necessary to confirm this speculation.
These studies were supported by NIH Grants RO1 HL34328 (to A.M.S.), HL63711 (to A.M.S.), and HL56046 (to P.A.L.) and by gifts from Mr. and Mrs. James Beck, the Nalco Foundation, and the Ralph and Marian Falk Trust for Medical Research. G.G. is a recipient of an American Heart Association-Midwest Affiliate Predoctoral Fellowship. We thank Alan G. Ferguson for excellent technical assistance.
- Received April 26, 2000.
- Revision received August 14, 2000.
- Accepted August 21, 2000.
- © 2000 American Heart Association, Inc.
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