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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:489.)
© 2001 American Heart Association, Inc.

Vascular Biology

Epidermal Growth Factor Receptor Transactivation by Angiotensin II Requires Reactive Oxygen Species in Vascular Smooth Muscle Cells

Masuko Ushio-Fukai; Kathy K. Griendling; Peter L. Becker; Lula Hilenski; Sean Halleran; R. Wayne Alexander

From the Division of Cardiology, Department of Medicine (M.U.-F., K.K.G., L.H., S.H., R.W.A.), and the Department of Physiology (P.L.B.), Emory University School of Medicine, Atlanta, Ga.

Correspondence to Masuko Ushio-Fukai, PhD, Emory University School of Medicine, Division of Cardiology, 1639 Pierce Dr, 319 WMB, Atlanta, GA 30322. E-mail mfukai{at}emory.edu

	Abstract

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Abstract—Angiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle cell growth, an important feature of hypertension and atherosclerosis. Many of these effects are dependent on the production of reactive oxygen species (ROS). Ang II induces phosphorylation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules. Here, we provide novel evidence that ROS are critical mediators of EGF-R transactivation by Ang II. Pretreatment of vascular smooth muscle cells with the antioxidants diphenylene iodonium, Tiron, N-acetylcysteine, and ebselen significantly inhibited (80% to 90%) tyrosine phosphorylation of the EGF-R by Ang II but not by EGF. Of the 5 autophosphorylation sites on the EGF-R, Ang II mainly phosphorylated Tyr1068 and Tyr1173 in a redox-sensitive manner. The Src family kinase inhibitor PP1, overexpression of kinase-inactive c-Src, or chelation of intracellular Ca²⁺ attenuated EGF-R transactivation. Although antioxidants had no effects on the Ca²⁺ mobilization or phosphorylation of Ca²⁺-dependent tyrosine kinase Pyk2, they inhibited c-Src activation by Ang II, suggesting that c-Src is 1 signaling molecule that links ROS and EGF-R phosphorylation. Furthermore, Ang II–induced tyrosine phosphorylation of the autophosphorylation site and the SH2 domain of c-Src was redox sensitive. These findings emphasize the importance of ROS in specific Ang II–stimulated growth-related signaling pathways and suggest that redox-sensitive EGF-R transactivation may be a potential target for antioxidant therapy in vascular disease.

Key Words: angiotensin II • vascular smooth muscle • epidermal growth factor receptors • reactive oxygen species

	Introduction

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Angiotensin II (Ang II) is a vasoactive hormone with critical roles in vascular smooth muscle growth, a cardinal feature of hypertension and atherosclerosis. Ang II exerts its effects via activation of a complex set of enzymatic cascades exhibiting considerable cross talk. Recently, attention has focused on the role of reactive oxygen species (ROS) in transducing growth-related molecular signals and cardiovascular disease. Ang II rapidly increases superoxide and hydrogen peroxide (H₂O₂) production in vascular smooth muscle cells (VSMCs) via activation of a p22phox-based NAD(P)H oxidase.^{1 2 3 4} These ROS are required for activation of a specific set of downstream kinases, including p38 mitogen-activated protein kinase (p38 MAPK) and Akt.^{4 5} Importantly, both of these pathways are required for the hypertrophic response by Ang II.^{4 5} The most proximal mechanisms coupling these responses to the Ang II type 1 (AT₁) receptor remain unclear.

Growing evidence indicates that phosphorylation of the epidermal growth factor (EGF) receptor (EGF-R) is an important step in activation of downstream tyrosine kinases by Ang II⁶ and serves as a scaffold for various signaling molecules in VSMCs. EGF-R phosphorylation by Ang II is required for the activation of extracellular signal–regulated kinase 1/2 (ERK1/2) and Akt, as well as the induction of c-Fos and increased protein synthesis.⁶ Although H₂O₂ phosphorylates the EGF-R in VSMCs, it is not clear whether it directly activates the intrinsic EGF-R kinase or modulates signaling molecules that, in turn, transactivate the receptor. With regard to the mechanisms responsible for Ang II–induced EGF-R phosphorylation, an essential role for Ca²⁺, via activation of a Ca²⁺/calmodulin-dependent kinase or the Ca²⁺-sensitive nonreceptor tyrosine kinase Pyk2, has been demonstrated.⁶ c-Src also binds to and transactivates the EGF-R in response to Ang II.⁶ All of these potential upstream signaling molecules can be activated by ROS.⁷ Taken together, these data suggest that EGF-R transactivation by Ang II may be redox sensitive, either because Ca²⁺, Pyk2, and/or c-Src is redox sensitive or because Ang II–derived ROS can directly activate the EGF-R. The specific tyrosine residues of the EGF-R phosphorylated by Ang II that may be responsible for these interactions and that are potential targets of ROS have not been defined.

In the present study, we examined the hypothesis that transactivation of the EGF-R represents an important and proximal target of Ang II–derived ROS in VSMCs. We found that EGF-R phosphorylation by Ang II at specific tyrosine residues requires ROS and that c-Src, but not Ca²⁺ mobilization or Pyk2, in part contributes to the redox sensitivity of this response. Phosphorylation of the autophosphorylation site and of the SH2 domain of c-Src is also responsive to agonist-induced ROS. Thus, EGF-R transactivation represents one of the most proximal ROS-mediated biochemical pathways activated by Ang II and may be a critical integrator of growth-related signaling that results in redox-sensitive hypertrophy in VSMCs.

	Methods

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Materials
Anti–EGF-R and anti–c-Src antibodies were from Santa Cruz Biotechnology. Human recombinant EGF and anti-Pyk2 monoclonal antibody were from Upstate Biotechnology. Rabbit polyclonal phosphospecific anti–c-Src (pY418 and pY215) antibodies and phosphospecific anti–EGF-R (Tyr1173, Tyr1068, Tyr1148, Tyr1086, and Tyr992) antibodies were obtained from BioSource International. These affinity-purified rabbit polyclonal antibodies are highly selective for the targeted phosphorylation site, as demonstrated by peptide competition studies and/or by analyzing site-directed (Tyr to Phe) mutants at the phosphorylation site of interest. Horseradish peroxidase–conjugated anti-phosphotyrosine antibody (RC20) was from Transduction Laboratories. DMEM with 25 mmol/L HEPES and 4.5 g/L glucose was from Sigma Chemical Co.

Cell Culture
VSMCs were isolated from male Sprague-Dawley rat thoracic aortas by enzymatic digestion and were grown in DMEM, as described previously.^{5 8}

Immunoprecipitation and Immunoblotting
Growth-arrested VSMCs were stimulated with agonist at 37°C, and 600 to 700 µg cell lysates were immunoprecipitated, separated with the use of SDS-PAGE, transferred to nitrocellulose membranes, blocked overnight, and incubated for 1 hour with primary antibodies as described previously.⁴ After incubation with secondary antibodies, proteins were detected by enhanced chemiluminescence.

Measurement of c-Src Activity
Anti–c-Src immunoprecipitates were used for c-Src kinase assays in which ³²P-labeled Sam68 was used as a specific Src substrate, as described previously.⁹

Infection of Adenovirus in VSMCs
The kinase-inactive form of chicken c-Src (KI-Src) adenovirus (Ad.KI-Src) and a control adenovirus (Ad.LacZ) encoding nuclear-targeted ß-galactosidase were kindly provided by Dr Bradford Berk (University of Rochester).¹⁰ VSMCs were incubated with various multiplicities of infection (MOIs) of either Ad.KI-Src or Ad.LacZ in serum-free medium for 72 hours before Ang II stimulation. The MOI was calculated spectrophotometrically.

Measurements of [Ca²⁺]_i
VSMCs grown on coverslips were loaded with fura 2-AM (10 µmol/L) for 45 minutes. Cells were stimulated with 100 nmol/L Ang II, and fluorescence of 5 to 10 adjacent cells was monitored at 520±20 nm when alternately excited with 340- and 380-nm light (at 100 Hz) with a high-time resolution microfluorometer as described previously.¹¹

Statistical Analysis
All values are mean±SE. Statistical significance was assessed by Student paired 2-tailed t test or ANOVA on untransformed data, followed by comparison of group averages by contrast analysis, with use of the SuperANOVA statistical program (Abacus Concepts).

	Results

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To confirm previous observations that Ang II transactivates the EGF-R,¹² we incubated VSMCs with Ang II (100 nmol/L) for various times and measured EGF-R phosphorylation. As shown in Figure 1, Ang II induced a rapid tyrosine phosphorylation of the EGF-R that peaked at 1 minute (7.7±2.0-fold increase) and remained above baseline for up to 15 minutes (P<0.05).

View larger version (80K): [in this window] [in a new window]   Figure 1. Time course of Ang II–induced EGF-R phosphorylation in VSMCs. VSMCs were stimulated with 100 nmol/L Ang II for the indicated times, and lysates were immunoprecipitated with anti–EGF-R antibody, followed by immunoblotting (IB) with phosphotyrosine antibody (pTyr, top) or the EGF-R antibody (middle). EGF-R-PY indicates EGF-R tyrosine phosphorylation; IP, immunoprecipitation. Bottom panel represents averaged data, expressed as fold increase in phosphorylation over control. Values are mean±SE for 3 independent experiments. *P<0.05 vs control.

We then investigated the role of ROS in Ang II–stimulated EGF-R phosphorylation. As shown in Figure 2A, pretreatment of VSMCs with various antioxidants (diphenylene iodonium [DPI], an inhibitor of flavin-containing enzymes; Tiron, a scavenger of superoxide; N-acetylcysteine [NAC]; and ebselen, a glutathione peroxidase mimetic) caused significant inhibition of Ang II–stimulated tyrosine phosphorylation of the EGF-R (75±9%, 85±8%, 90±6%, and 93±5%, respectively). These data indicate that Ang II–induced EGF-R transactivation is mediated by ROS.

View larger version (55K): [in this window] [in a new window]   Figure 2. ROS mediate Ang II–induced, but not EGF-induced, EGF-R phosphorylation in VSMCs. A, VSMCs were preincubated with vehicle, DPI (10 µmol/L), Tiron (10 mmol/L), NAC (10 mmol/L), or ebselen (40 µmol/L) for 30 to 60 minutes before treatment with 100 nmol/L Ang II for 1 minute. B, VSMCs were treated with H2O2 (200 µmol/L) or the superoxide-generating compound LY83583 (10 µmol/L) for the indicated times. Phosphorylation in response to Ang II is included for comparison. Results are representative of 2 or 3 experiments. C, VSMCs, treated with inhibitors as described in panel A, were stimulated with (+) or without (-) EGF (100 ng/mL) for 30 seconds. Lysates were used for the measurement of EGF-R-PY, as described in the legend to Figure 1. In panels A and C, values are mean±SE for 3 to 5 independent experiments. *P<0.05 for increase in EGF-R phosphorylation by agonist in the presence of inhibitor vs agonist alone.

To examine whether ROS mediate coupling of the AT₁ receptor to the EGF-R or the activity of the EGF-R intrinsic tyrosine kinase, we used 2 approaches. First, we tested the ability of exogenous ROS to phosphorylate the EGF-R, and second, we assessed the effect of antioxidants on EGF-induced EGF-R activation. H₂O₂ and the superoxide-generating compound LY83583¹³ time-dependently increased EGF-R phosphorylation (Figure 2B). However, none of the antioxidants tested had any effect on the ability of EGF (100 ng/mL) to phosphorylate its own receptor (Figure 2C). Taken together, these results suggest that the target of ROS is not the EGF-R intrinsic tyrosine kinase but an enzyme or protein linking AT₁ receptor activation to the EGF-R.

However, intrinsic tyrosine kinase activity has been shown to be required for EGF-R transactivation by Ang II,¹² suggesting that only certain of the autophosphorylation sites are targets of ROS or that phosphorylation of these sites can be modulated by a redox-sensitive intermediary enzyme. We verified that Ang II–induced EGF-R phosphorylation requires intrinsic kinase activity by using AG1478, an EGF-R kinase inhibitor (100% inhibition). We then determined which autophosphorylation sites of the EGF-R are phosphorylated by Ang II and examined their redox sensitivity. Five autophosphorylation sites have been identified in the EGF-R: 3 major sites (Tyr1173, Tyr1068, and Tyr1148) and 2 minor sites (Tyr992 and Tyr1086).^{14 15 16} Using EGF-R site-specific and phosphospecific antibodies, we found that Ang II mainly phosphorylates Tyr1173 and Tyr1068 (Figure 3A), although EGF was able to phosphorylate Tyr1086, Tyr1148, and Tyr992 as well (data not shown). Importantly, phosphorylation of Tyr1173 and Tyr1068 was significantly inhibited by NAC (Figure 3B). Similar results were found with the use of ebselen as an antioxidant (72±6% and 75±8% inhibition for Tyr1173 and Tyr1068 phosphorylation, respectively).

View larger version (50K): [in this window] [in a new window]   Figure 3. Redox sensitivity of specific autophosphorylation sites of EGF-Rs in Ang II–stimulated VSMCs. A, VSMCs were stimulated with or without Ang II (100 nmol/L) for 1 minute. Lysates were used for measurement of EGF-R-PY at autophosphorylation sites (pY1173, pY1068, pY1148, pY1086, and pY992). C indicates control. Blots are representative of 3 independent experiments. B, Averaged data for phosphorylation of EGF-R (pY1173 and pY1068) by Ang II (100 nmol/L, 1 minute) or EGF (100 ng/mL, 30 seconds) in VSMCs pretreated with or without NAC (10 mmol/L, 1 hour). Densitometric data were quantified as described above. Values are mean±SE for 3 independent experiments. *P<0.05 for increase in phosphorylation by Ang II in the presence of NAC vs Ang II alone.

We next examined the upstream signaling pathways that mediate EGF-R transactivation to determine the target(s) of ROS. Inhibition of tyrosine kinases by genistein, Src-family kinases by PP1, or intracellular Ca²⁺ chelation by BAPTA-AM significantly inhibited Ang II–induced EGF-R phosphorylation (Figure 4A). In contrast, neither inhibition of Janus kinase (JAK)-2 by AG490 (10 µmol/L, a concentration previously shown to be maximally effective¹⁷ ) nor phosphatidylinositol 3-kinase by wortmannin and LY294002 had any effect on this response (data not shown). To further specifically evaluate the role of c-Src in Ang II–induced EGF-R phosphorylation, we tested the effect of overexpression of KI-Src on this response. At 600 to 2000 MOI, KI-Src was significantly expressed in VSMCs (2.5:1 and 7.5:1, respectively, for ratio of KI-Src to endogenous Src) as determined by Western analysis (data not shown). As shown in Figure 4B, Ang II–induced EGF-R phosphorylation was partially inhibited by KI-Src overexpression, whereas a control virus, Ad.LacZ, had no effect. These results suggest that c-Src and Ca²⁺ are the major signaling pathways involved in EGF-R transactivation by Ang II.

View larger version (37K): [in this window] [in a new window]   Figure 4. Ang II–induced EGF-R transactivation is mediated by Ca2+ and c-Src in VSMCs. VSMCs were preincubated for 30 minutes with vehicle, 10 µmol/L of the tyrosine kinase inhibitor genistein, 20 µmol/L of the Src family kinase inhibitor PP1, or 20 µmol/L of the intracellular Ca2+ chelator BAPTA-AM (A) or infected with the indicated concentrations of either Ad.KI-Src or Ad.LacZ (B) and stimulated with (+) or without (-) 100 nmol/L Ang II for 1 minute. Lysates were used for the measurement of EGF-R-PY, as described in the legend to Figure 1. Values are mean±SE for 3 or 4 independent experiments. *P<0.05 for increase in EGF-R phosphorylation by Ang II in the presence of inhibitor vs Ang II alone.

We then tested whether activation of either of these signaling pathways by Ang II was sensitive to antioxidant treatment. Ang II induced a biphasic increase in [Ca²⁺]_i, consisting of an initial peak due to intracellular Ca²⁺ release followed by a plateau phase due to extracellular Ca²⁺ influx. None of the antioxidants tested had a substantial effect on either phase of the Ca²⁺ response, and exogenous application of H₂O₂ had no effect on [Ca²⁺]_i in these cells (data not shown). However, it is possible that a Ca²⁺-dependent tyrosine kinase such as Pyk2, which has previously been implicated in EGF-R transactivation by Ang II, may itself be redox sensitive.¹⁸ However, Ang II–induced Pyk2 tyrosine phosphorylation, which was sensitive to thapsigargin, an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase, was not affected by antioxidants (Figure 5).

View larger version (67K): [in this window] [in a new window]   Figure 5. Antioxidants have no effect on Ang II–induced Pyk2 phosphorylation in VSMCs. VSMCs were preincubated with various antioxidants, as described in the legend to Figure 2A, or thapsigargin (10 µmol/L) for 30 minutes before treatment with 100 nmol/L Ang II for 1 minute. Lysates were immunoprecipitated with anti-Pyk2 antibody and used for the measurement of Pyk2 tyrosine phosphorylation (Pyk2-PY). Values are mean±SE for 3 independent experiments. *P<0.05 for increase in phosphorylation by Ang II in the presence of inhibitor vs Ang II alone.

In contrast, Ang II–induced c-Src phosphorylation at the autophosphorylation site (Tyr418) was partially inhibited by various antioxidants (Figure 6A). This result was confirmed by the observation that Ang II–induced c-Src activity, as measured by a specific phosphorylation of the c-Src substrate Sam68 in c-Src immunoprecipitates, was inhibited by DPI, Tiron, NAC, or ebselen (93±9%, 95±8%, 99±6%, and 91±5% inhibition, respectively; n=2). Furthermore, c-Src phosphorylation in the SH2 domain (Tyr215) was dramatically inhibited by all the antioxidants (Figure 6B). The redox sensitivity of c-Src was consistent with the observation that exogenously applied H₂O₂ activates c-Src with a time course similar to EGF-R phosphorylation by H₂O₂ (data not shown). Taken together, these results suggest not only that c-Src is a target of ROS but also that the interaction of c-Src is with other signaling molecules may be redox sensitive.

View larger version (37K): [in this window] [in a new window]   Figure 6. ROS mediate c-Src activation by Ang II in VSMCs. VSMCs were preincubated with various antioxidants as described in the legend to Figure 2A and stimulated with 100 nmol/L Ang II for 1 minute. Lysates were immunoblotted with anti–phospho-c-Src (pY418) antibody (A, top), anti–phospho-c-Src (pY215) antibody (B, top), or c-Src antibody (A and B, middle). Graphs at the bottom represent averaged data, expressed as fold increase in c-Src phosphorylation over that in unstimulated cells without inhibitor. Values are mean±SE for 3 independent experiments. *P<0.05 for increase in c-Src phosphorylation by Ang II in the presence of inhibitor vs Ang II alone.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Ang II is an important mediator of VSMC growth, and its effects are dependent, in part, on the production of ROS. The most proximal molecular target(s) of ROS has not been defined. It has been demonstrated that Ang II induces Ca²⁺-dependent transactivation of the EGF-R that serves as a scaffold for various signaling molecules in VSMCs.⁶ In the present study, we provided novel evidence that ROS are critical mediators of EGF-R transactivation by Ang II. We identified 2 major autophosphorylation sites on EGF-R, Tyr1173 and Tyr1068, as redox sensitive. In addition, we determined which of several candidate redox-sensitive proteins or pathways (Ca²⁺, Pyk2, or c-Src) were responsible for this sensitivity to oxidative stress and clearly demonstrated that c-Src is a signaling molecule that links ROS and EGF-R phosphorylation. Moreover, we showed that Ang II induces tyrosine phosphorylation of the autophosphorylation site and SH2 domain of c-Src, which reflects its activity, in a redox-sensitive manner.

Transactivation of the EGF-R is essential for activation of G protein–coupled receptors by agonists to produce responses attributed to tyrosine kinase receptors.^{6 19} In the present study, we provide the first evidence that Ang II–induced tyrosine phosphorylation of the EGF-R is mediated through ROS. Consistent with these results, lysophosphatidic acid–stimulated EGF-R phosphorylation in HeLa cells has been shown to require ROS.²⁰ Redox sensitivity of EGF-R phosphorylation was further confirmed by the finding that exogenous ROS increased EGF-R phosphorylation in VSMCs (Figure 2B), which agrees with other reports.^{21 22} The slower activation of EGF-R phosphorylation by exogenous oxidants is most likely due to barriers to diffusion presented by different cellular compartments. Receptor-mediated ROS production is more compartmentalized than that produced by the exogenous addition of H₂O₂, so that activation of signaling molecules occurs more rapidly and efficiently. In addition, Ang II also activates other signaling pathways (eg, Ca²⁺) that may enhance the rate of EGF-R phosphorylation. Our previous work shows that Ang II activates redox-sensitive kinases such as Akt and p38 MAPK, which contribute to Ang II–induced hypertrophy.^{4 5} Interestingly, Ang II–induced Akt phosphorylation has been shown to occur through the EGF-R,⁶ raising the possibility that the redox sensitivity of Akt⁵ is transduced via ROS-dependent EGF-R activation in VSMCs.

Because intrinsic tyrosine kinase activity has been shown to be required for EGF-R transactivation by Ang II, it is possible that either the autophosphorylation sites are targets of ROS or that phosphorylation of these sites can be modulated by a redox-sensitive intermediary enzyme. Antioxidants had no effect on the EGF-induced autophosphorylation of EGF-R (Figure 2C), indicating that the direct target of ROS is unlikely to be the EGF-R kinase itself. These results are in contrast to those of Bae et al,²³ who showed that addition of catalase to A431 cells inhibited EGF-induced autophosphorylation of EGF-R. This discrepancy may be due to a difference in the concentration of EGF used (500 ng/mL [Bae et al²³ ] versus 100 ng/mL [present study]) or to the cell types. We also determined that only 2 sites (Tyr1068 and Tyr1173) of the reported 5 autophosphorylation sites were activated by Ang II in a redox-sensitive manner, whereas EGF-induced responses were redox insensitive. These results strongly suggest that ROS modulate an enzyme or protein linking AT₁ receptor activation to the EGF-R. Interestingly, Tyr1068 is the Grb2 binding site that leads to activation of Ras/Raf/ERK1/2,²⁴ whereas Tyr1173 binds SHP-1 and negatively regulates ERK1/2 activation.²⁵ These opposing effects would thus be expected to offset each other, so that ERK1/2 activation by Ang II would not appear to be redox sensitive, even though it is downstream from the EGF-R, a finding that we have in fact previously reported.⁴ The identification and possible redox sensitivity of EGF-R phosphorylation sites other than autophosphorylation sites modulated by Ang II require further investigation.

We also investigated the identity of proteins and enzymes intermediary between the AT₁ receptor and the EGF-R that might be targets of ROS. Although phosphorylation of the EGF-R by other agonists has been shown to be mediated by JAK-2,²⁶ we found no evidence for involvement of JAK-2 in Ang II–stimulated VSMCs. Similarly, phosphatidylinositol 3-kinase inhibitors had no effect on EGF-R transactivation at concentrations that block Ang II–induced Akt activation.⁵ Ca²⁺ has been shown to mediate Ang II–induced EGF-R transactivation, possibly via activation of the Ca²⁺-dependent tyrosine kinase Pyk2 and/or c-Src.⁶ We confirmed an essential role for Ca²⁺ in Ang II–stimulated EGF-R phosphorylation in our system (Figure 4A). The previous assertion that c-Src mediates EGF-R transactivation by Ang II in VSMCs was based on indirect evidence that active c-Src inducibly associates with the EGF-R on Ang II stimulation and is insensitive to the EGF-R kinase inhibitor AG1478.⁶ In the present study, using the Src-family kinase inhibitor PP1 and overexpression of KI-Src, we directly demonstrate that c-Src is involved in EGF-R phosphorylation by Ang II (Figure 4B). The role of c-Src in EGF-R transactivation by lysophosphatidic acid or Gß has similarly been demonstrated in COS cells.²⁷ However, in VSMCs, inhibition by PP1 and KI-Src was partial, suggesting that another unidentified tyrosine kinase might be involved in this activation.

The clear involvement of Ca²⁺ and c-Src in Ang II–induced EGF-R transactivation suggested that 1 of these pathways might be the redox-sensitive targets that link AT₁ receptors to EGF-R phosphorylation. Several other groups have shown that Ca²⁺ can be mobilized by ROS²⁸ ; however, we found that the increase in [Ca²⁺]_i by Ang II is not redox sensitive and that exogenous application of H₂O₂ has no effect on [Ca²⁺]_I in VSMCs. It appears instead that the increase in [Ca²⁺]_i may be required for ROS generation by Ang II in VSMCs (authors’ unpublished data, 2000), raising the possibility that the redox-sensitive step occurs downstream from Ca²⁺ mobilization.

We observed divergent effects with regard to the redox sensitivity of 2 immediate targets of Ca²⁺, Pyk2 and c-Src.⁶ Antioxidants had no effect on Ang II–induced Pyk2 phosphorylation at 1 minute, the time of peak EGF-R transactivation. Although Frank et al²⁹ showed that Pyk2 is redox sensitive, only late (>2-minute) activation of Pyk2 by Ang II was inhibited by NAC. In contrast, antioxidants partially, but significantly, inhibited c-Src activation, as assessed by c-Src phosphorylation at the autophosphorylation site (Tyr418, Figure 6A) or measurement of c-Src kinase activity. It has been reported that c-Src mediates H₂O₂-induced activation of MAPK families and that other Src-family kinases (Fyn and Lyn) can be activated by H₂O₂.³⁰ The present study provides direct evidence that c-Src activation by endogenous agonists is partially mediated through ROS. Moreover, antioxidants dramatically inhibited c-Src phosphorylation at the SH2 domain (Tyr215, Figure 6B), which is thought to enhance c-Src kinase activity³¹ and association with other signaling molecules.³² These data suggest not only that c-Src is a target of ROS but also that the interaction of c-Src with other signaling molecules may be redox sensitive.

Recently, Prenzel et al³³ implicated metalloproteinase-mediated cleavage of pro–heparin-binding EGF in transfected cells to produce heparin-binding EGF and induce EGF-R transactivation by the G-protein–coupled muscarinic cholinergic receptors. Because matrix metalloproteinases can be activated by ROS,⁷ this raises the interesting possibility that activation of a cell surface metalloproteinase may represent an additional redox-sensitive step in EGF-R transactivation by Ang II.

The redox sensitivity of EGF-R transactivation by Ang II has important implications for understanding the control of VSMC growth by ROS. We have previously demonstrated that Ang II–induced hypertrophy is inhibited by antioxidants, antisense to p22phox (a component of the NAD(P)H oxidase), and overexpression of catalase, proving that ROS are required in the hypertrophic response.^{1 3 7} Ang II–induced c-Fos expression and protein synthesis in VSMCs are markedly attenuated by the EGF-R kinase inhibitor AG1478,⁶ confirming that this kinase is integral to the growth response. Because of the proximal nature of EGF-R–mediated signaling, these results suggest that the redox sensitivity of the hypertrophic response may be conferred in part at the level of EGF-R transactivation, presenting a new potential therapeutic target in atherosclerosis and hypertension.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL-38206, HL-60728, and HL-58000. We thank Dr Bradford C. Berk for the generous gift of kinase-inactive Src adenovirus, Mike Davis for amplifying the virus, and Carolyn Morris for excellent secretarial assistance.

Received November 27, 2000; accepted December 18, 2000.

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