c-Jun N-terminal Kinase Activation by Hydrogen Peroxide in Endothelial Cells Involves Src-dependent Epidermal Growth Factor Receptor Transactivation*

Kai Chen, Joseph A. VitaDagger, Bradford C. Berk§, and John F. Keaney Jr.Dagger

From the Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118 and the § Center for Cardiovascular Research, University of Rochester Medical Center, Rochester, New York 14642

Received for publication, December 28, 2000, and in revised form, February 12, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The phenotypic properties of the endothelium are subject to modulation by oxidative stress, and the c-Jun N-terminal kinase (JNK) pathway is important in mediating cellular responses to stress, although activation of this pathway in endothelial cells has not been fully characterized. Therefore, we exposed endothelial cells to hydrogen peroxide (H2O2) and observed rapid activation of JNK within 15 min that involved phosphorylation of JNK and c-Jun and induction of AP-1 DNA binding activity. Inhibition of protein kinase C and phosphoinositide 3-kinase did not effect JNK activation. In contrast, the tyrosine kinase inhibitors, genistein, herbimycin A, and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) significantly attenuated H2O2-induced JNK activation as did endothelial cell adenoviral transfection with a dominant-negative form of Src, implicating Src as an upstream activator of JNK. Activation of JNK by H2O2 was also inhibited by AG1478 and antisense oligonucleotides directed against the epidermal growth factor receptor (EGFR), implicating the EGFR in this process. Consistent with this observation, H2O2 stimulated EGFR tyrosine phosphorylation and complex formation with Shc-Grb2 that was abolished by PP2, implicating Src in H2O2-induced EGFR activation. Tyrosine phosphorylation of the EGFR by H2O2 did not involve receptor autophosphorylation at Tyr1173 as assessed by an autophosphorylation-specific antibody. These data indicate that H2O2-induced JNK activation in endothelial cells involves the EGFR through an Src-dependent pathway that is distinct from EGFR ligand activation. These data represent one potential pathway for mediating oxidative stress-induced phenotypic changes in the endothelium.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Abundant evidence indicates that cardiovascular disease is characterized by a state of excess oxidative stress and increased production of reactive oxygen species (ROS)1 within the arterial wall. Traditionally, ROS such as superoxide, hydrogen peroxide, and hydroxyl radical have been viewed within the context of inflammation. More recently, however, it has become clear that ROS may mediate specific cellular functions such as the response to growth factors (1), hypertrophy (2), and apoptosis (3). Since blood vessels from atherosclerotic animals exhibit an increased flux of ROS (4), understanding the cellular signals elicited by ROS should provide insight into the pathogenesis of cardiovascular disease.

Among the cellular signals subject to regulation by ROS in the vascular wall, members of the mitogen-activated protein (MAP) kinases are perhaps the best characterized. Three major subfamilies of MAP kinases have been described, the extracellular signal-regulated kinases (ERK1/2), c-Jun NH2-terminal kinases (JNKs), and p38 kinase (5). The ERKs are typically involved in the response to growth factors (i.e. proliferation, hypertrophy, and differentiation) and are activated in response to ROS such as superoxide (6) and hydrogen peroxide (7). The JNKs and p38 kinases are primarily involved in the cellular stress responses (5) and are activated by hydrogen peroxide in smooth muscle cells (8).

The MAP kinase pathway has been implicated in a number of phenomena associated with cardiovascular disease. For example, dual activation of ERK1/2 and p38 kinase is required for the hypertrophic response to angiotensin II in smooth muscle cells (9). Cytokine stimulation of fibroblasts results in activation of both p38 kinase and ERK1/2, either of which is sufficient for up-regulation of matrix metalloproteinase-1 activity (10). Dual localization of p53 and activated JNK in smooth muscle cells within apoptotic regions of atherosclerotic plaque suggests that JNK activation may contribute to plaque rupture (11). Thus, MAP kinase activation in smooth muscle cells and fibroblasts is associated with pathologic changes in the arterial wall.

In contrast to fibroblasts and smooth muscle cells, ROS-mediated activation of MAP kinases in endothelial cells is less well characterized. Treatment of endothelial cells with hydrogen peroxide is associated with activation of ERK1/2 (12) and p38 kinase (13); however, the effect on JNK is unclear. Since JNK activation has been observed in other cells in response to ROS (7), we sought to define the effect of ROS on endothelial cell JNK activity and to identify the signaling pathways involved.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Cell culture reagents including medium M-199 and Dulbecco's modified Eagle's medium were obtained from Life Technologies, Inc. Protein A/G-agarose was from Pierce. The EGF receptor-specific inhibitor tyrphostin AG1478, the platelet-derived growth factor receptor-specific inhibitor tyrphostin AG1295, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2), genistein, herbimycin A, LY294002, and calphostin C were obtained from Calbiochem. Anti-phosphotyrosine antibody (clone PY20), anti-Shc, and anti-Grb2 antibodies were from Transduction Laboratories (Lexington, KY). The anti-EGF receptor antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies directed against phospho-JNK, phospho-c-Jun, phospho-ERK1/2, phospho-p38, and secondary peroxidase-labeled antibodies were from New England Biolabs (Beverly, MA). The anti-phospho-EGF receptor (Tyr1173) antibody and anti-Src antibody (GD11) were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). [gamma -32P]ATP was from PerkinElmer Life Sciences. The adenovirus containing kinase-inactive chicken c-Src (Ad.KI-Src; Lys295 to Met mutation) was prepared as described (14). All other reagents were obtained from Sigma or as described.

Cell Culture-- Porcine aortic endothelial cells (PAECs) were harvested and grown up to passage 6 in M-199 supplemented with 15% fetal bovine serum, 10 µg/ml heparin sulfate, 100 units/ml penicillin, and 100 µg/ml streptomycin as described (15). Human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics, Inc., and grown on 0.2% gelatin-coated tissue culture plates in endothelial cell growth medium (Clonetics) and used between passages 2 and 4. COS-7 cells were utilized in some experiments due to their greater EGF receptor density and were purchased from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Prior to experiments, subconfluent cells were cultured in their respective medium containing 1% serum for 16 h and equilibrated for 30 min in HEPES-buffered physiologic salt solution containing 22 mM HEPES (pH 7.4), 124 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 0.16 mM HPO4, 0.4 mM H2PO4, 5 mM NaHCO3, and 5.6 mM glucose in order to minimize extracellular oxidation.

Immunoprecipitation and Western Blotting-- After treatments, cells were washed with ice-cold phosphate-buffered saline twice and incubated in lysis buffer containing 50 mM Tris-HCl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40, 1% Triton X-100, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 100 µg/ml phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1 mM sodium fluoride, and 1 mM EDTA for 30 min on ice followed by brief sonication for 10 s. Cell lysates were then centrifuged at 13,600 × g for 10 min, and the supernatants were incubated with primary antibody for 16 h at 4 °C followed by a 2-h incubation with protein A/G-agarose. Following a brief centrifugation, pellets were washed twice with lysis buffer and twice with phosphate-buffered saline and were resuspended in loading buffer containing 50 mM Tris-HCl (pH 6.8), 2% SDS, 200 mM dithiothreitol, 20% glycerol, and 0.2% bromphenol blue. Western blot analysis was performed as described previously (16). Proteins were detected using an enhanced chemiluminescence detection kit (New England Biolabs, Beverly, MA). Densitometric analysis of Western blots was carried out using the PDI Imageware System (Huntington Station, NY).

Electrophoretic Mobility Shift Assay-- Nuclear extracts were prepared from PAECs using the method of Dignam and colleagues (17). Double-stranded oligonucleotides corresponding to the AP-1 consensus sequence (5'-CGCTTGATGAGTCAGCCGGAA-3') were labeled with [gamma -32P]ATP using a T4 polynucleotide kinase kit (Promega). The binding reactions and electrophoresis of DNA-protein complexes were performed as described previously (16).

Kinase Assay-- After various treatments, cells were lysed as described above, and JNK activity was determined using a commercially available assay kit (New England Biolabs). Briefly, the cell lysate was precipitated with GST-c-Jun-(1-89) fusion protein bound to glutathione-Sepharose beads overnight at 4 °C. Washed immunoprecipitates were then incubated for 30 min at 30 °C with 100 µM ATP in 30 µl of assay buffer. Reactions were stopped by the addition of 3× gel loading buffer (150 mM Tris-HCl (pH 6.8), 6% SDS, 600 mM dithiothreitol, 30% glycerol, and 0.6% bromphenol blue), and samples were boiled for 3 min prior to SDS-polyacrylamide gel electrophoresis. Phosphorylation of c-Jun was determined by Western blotting (as described above) with antibody specific for the phosphorylated form of c-Jun.

Transfections-- The protocol for HUVEC transfection with KI-Src adenovirus (Ad.KI-Src) and a control adenovirus (Ad.LacZ) encoding beta -galactosidase was described previously (14). Antisense and control oligonucleotides directed against human EGF receptor (catalog no. S12116) were obtained from Sequitur (Natick, MA). Cells were seeded (2 × 105/well) in six-well plates and allowed to reach 70-80% confluence (~16 h). Cells were washed with fresh Dulbecco's modified Eagle's medium, and oligonucleotides were introduced by incubation in 2 ml of Dulbecco's modified Eagle's medium containing 6.6 µl of oligofectin I (Sequitur, Natick, MA) and 100 nM oligonucleotides for 4 h. Cells were then washed and cultured in endothelial cell growth medium (Clonetics) for 48 h before experiments. Under these conditions, transfection efficiency was ~70% as judged by parallel controls with fluorescent oligonucleotides.

Reverse Transcription and Polymerase Chain Reaction-- The procedures for semiquantitative reverse transcription-polymerase chain reaction were essentially as described (18) using forward (5'-CAGCCTCCAGAGGATGTTCA-3') and reverse (5'-GGTGGCACCAAAGCTGTATT-3') primers corresponding to human EGF receptor mRNA. Constitutively expressed beta -actin mRNA was amplified with forward (5'-TCACCCTGAAGTACCCCATC-3') and reverse (5'-CACACGCAGCTCATTGTAGA-3') primers in a similar fashion.

Statistical Analysis-- All blots are representative of three or four experiments. Comparisons among treatment groups were performed with one-way analysis of variance and a post hoc Dunnett's or Student's Newman-Keuls comparison as appropriate. Statistical significance was accepted if the null hypothesis was rejected with p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

H2O2-induced JNK Activation in PAECs-- We observed time-dependent JNK and c-Jun phosphorylation in response to H2O2 (Fig. 1, A and B). We also observed concentration-dependent c-Jun phosphorylation beginning with only 50 µM H2O2 (Fig. 1C). We found that H2O2 concentrations of >= 500 µM were toxic and thus employed only nontoxic H2O2 concentrations in subsequent studies. As expected, c-Jun phosphorylation (Fig. 1B) correlated closely with H2O2-induced JNK activity by immune complex kinase assay (Figs. 2, A and B) and AP-1 DNA binding activity (Fig. 2C). These data confirm intact activation of the JNK signaling cascade in response to H2O2 in endothelial cells.


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Fig. 1.   H2O2 stimulates JNK and c-Jun phosphorylation. PAECs were treated with 200 µM H2O2 for the indicated periods of time, and total cell lysates were subjected to immunoblotting with phospho-JNK (A) and phospho-c-Jun antibodies (B), respectively. C, PAECs were treated with the indicated concentration of H2O2 for 30 min, and cell lysates were analyzed by immunoblotting with phospho-c-Jun antibody as in B. Data are representative of three independent experiments.


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Fig. 2.   H2O2 activates c-Jun kinase activity and enhances AP-1 DNA binding. PAECs were treated with H2O2 (200 µM) for the indicated periods of time. A, cell lysates were immunoprecipitated with GST-c-Jun fusion protein bound to Sepharose beads. The immunoprecipitates were then subjected to kinase activity assay, followed by Western blot analysis using anti-phospho-c-Jun antibody. B, composite JNK activity determined by densitometric analysis of c-Jun phosphorylation normalized to 0 µM H2O2. Results represent mean ± S.D. from three independent experiments. *, p < 0.05 compared with 0 µM H2O2 by analysis of variance with post hoc Dunnett's test. C, PAECs were treated as in A, and electrophoretic mobility shift assay was performed as described under "Experimental Procedures." Data are representative of three independent experiments.

H2O2-induced JNK Activation Involves Src Tyrosine Kinase Activity-- Although JNK activation has been linked to the activity of protein kinase C (19) or PI 3-kinase (20) in some systems, we did not observe any effect of protein kinase C or PI 3-kinase inhibition with calphostin C and LY294002, respectively, on H2O2-induced JNK activation (data not shown). Similarly, the inhibitor of growth factor receptor ligand binding suramin had no effect (Figs. 3, A and B). In contrast, general tyrosine kinase inhibition with genistein or herbimycin A significantly attenuated H2O2-induced c-Jun phosphorylation as did the specific Src family kinase inhibitor PP2 (Fig. 3, A and B). We also found that PP2 inhibited the entire time course of JNK activation (up to 2 h, data not shown), indicating that Src activation is an early event in this process. To examine the role of Src specifically, we overexpressed kinase-inactive c-Src in endothelial cells and found an inverse relation between KI-Src overexpression and H2O2-induced JNK activation (Fig. 3C), consistent with this being a Src-dependent process.


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Fig. 3.   H2O2-induced c-Jun phosphorylation involves Src tyrosine kinase activity. PAECs were pretreated with suramin (0.3 mM), genistein (50 µM), herbimycin A (10 µM), PP2 (25 µM), or catalase (300 units/ml) for 30 min prior to stimulation with H2O2 (200 µM) for 30 min (A). Immunoblot (IB) was performed on cell lysates using anti-phospho-c-Jun antibody. B, composite densitometric analysis of phospho-c-Jun from three independent experiments. Data are presented as mean ± S.E.; *, p < 0.05 versus 0 µM H2O2 by analysis of variance and post hoc Dunnett's comparison. C, HUVECs were transfected with adenovirus encoding beta -galactosidase (Ad.Ctl) or the avian kinase-inactive c-Src (Ad.KI-Src) for 48 h and then stimulated with H2O2 (200 µM) for 30 min. Cell lysates were resolved with SDS-polyacrylamide gel electrophoresis, transferred to membranes, and probed with an antibody against phospho-c-Jun and c-Src.

The EGF Receptor Is Involved in H2O2-induced JNK Activation-- There is increasing evidence that cellular stresses such as UV irradiation and osmotic shock can transactivate growth factor receptors as a component of their cellular signaling (21). Accordingly, we investigated receptor involvement in H2O2-induced endothelial cell JNK activation using the receptor tyrosine kinase inhibitors AG1478 (for the EGF receptor) and AG1295 (for the platelet-derived growth factor receptor). As shown in Fig. 4, AG1478 inhibited H2O2-induced c-Jun phosphorylation in a dose-dependent manner, whereas AG1295 did not, implicating the EGF receptor in JNK activation induced by H2O2. We found that AG1478 inhibited the entire time course of JNK activation (up to 2 h, data not shown), indicating that the EGF receptor is an early event in H2O2-induced JNK activation. To confirm a role for the EGF receptor in this process, we inhibited EGF receptor mRNA expression in HUVECs with antisense oligonucleotides (Fig. 5A), and this also attenuated H2O2-induced JNK activation (Fig. 5B). We were able to demonstrate a reduction in EGF receptor expression with antisense oligonucleotide treatment in COS-7 cells (due to higher EGF receptor density) that also inhibited JNK activation by H2O2 (Fig. 5C). These data indicate that the EGF receptor is involved in H2O2-induced JNK activation.


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Fig. 4.   Effect of EGF receptor and platelet-derived growth factor receptor tyrosine kinase inhibitors on H2O2-induced c-Jun phosphorylation. PAECs were pretreated (30 min) with the EGF receptor tyrosine kinase inhibitor AG1478 (A) or the platelet-derived growth factor receptor tyrosine kinase inhibitor AG1295 (B) at the indicated concentrations prior to stimulation with H2O2 (200 µM) for 30 min. Cell lysates were subjected to immunoblot analysis (IB) with anti-phospho-c-Jun antibody. Composite densitometric analysis is presented in (C) as mean ± S.D. from three independent experiments. *, p < 0.05 versus 200 µM H2O2 alone-treated group by analysis of variance with a post hoc Dunnett's test.


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Fig. 5.   EGF receptor down-regulation inhibits H2O2-stimulated c-Jun phosphorylation. A, HUVECs were treated with vehicle alone or with antisense or control oligonucleotides directed against the EGF receptor as described under "Experimental Procedures." After 24 h, total RNA was extracted, and reverse transcription-polymerase chain reaction was performed to estimate EGF receptor and beta -actin mRNA expression. B, HUVECs were treated as in A, but after 48 h cells were exposed to the indicated concentration of H2O2 and lysed, and resolved proteins were probed with antibodies directed at either phospho-c-Jun or beta -actin as a loading control. C, COS-7 cells were transfected with antisense or control oligonucleotides as above and treated with H2O2 (200 µM) for 30 min. Cell lysates were resolved with SDS-polyacrylamide gel electrophoresis and subjected to immunoblot analysis with antibodies specific for the EGF receptor, phospho-c-Jun, and beta -actin antibodies in the same blots. Data are representative of three independent experiments.

H2O2 Stimulates ERK1/2, but Not p38, via an Src- and EGF Receptor-dependent Mechanism-- To determine the involvement of Src and the EGF receptor in H2O2-induced activation of other MAP kinase pathways, we investigated H2O2-induced stimulation of ERK1/2 and p38 in PAECs. We found that H2O2 stimulated activation of both ERK1/2 (Fig. 6, A and B, p < 0.05) and p38 (Figs. 6, C and D, p < 0.05), although only the former was inhibited by PP2 and AG1478. Substituting COS-7 cells for PAECs yielded similar results (data not shown). Thus, H2O2-induced activation of ERK1/2 but not p38 involves both Src and the EGF receptor.


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Fig. 6.   H2O2-stimulated ERK1/2 activation involves Src and the EGF receptor. PAECs were pretreated with vehicle alone (control), PP2 (25 µM), AG1478 (1 or 50 µM) or catalase (300 units/ml) for 30 min prior to stimulation with H2O2 (200 µM) for 15 (A and B) or 30 min (C and D). After stimulation, cells were lysed, proteins were resolved by SDS-polyacrylamide gel electrophoresis, and activation of ERK1/2 (A) or p38 (C) was assessed by immunoblot (IB) with phosphospecific antibodies as described under "Experimental Procedures." Immunoblots for ERK1/2 (A) and p38 (C) served as loading controls. Composite densitometric analysis of phospho-ERK1/2 (B) and phospho-p38 (D) normalized to control are also shown and represent mean ± S.E. from three independent experiments (*, p < 0.05 versus 200 µM H2O2 by analysis of variance and post hoc Dunnett's comparison).

H2O2-stimulated JNK Activation Involves Src-mediated EGF Receptor Transactivation-- To investigate ligand-independent EGF receptor activation, we treated HUVECs (available EGFR antibodies are unreactive in PAECs) with H2O2 and observed rapid tyrosine phosphorylation (2 min) of the EGF receptor (Fig. 7A) that was accompanied by phosphorylation of the adapter protein Shc (Fig. 7B). Using COS-7 cells, we found that H2O2-induced EGF receptor tyrosine phosphorylation produced receptor activation manifested as complex formation with the adapter proteins Shc and Grb2, similar to that observed with authentic EGF (Fig. 7C). This EGF receptor activation by H2O2 was inhibited by AG1478 only at high concentrations (50 µM) but readily inhibited by PP2 (Fig. 7C), consistent with Src-mediated EGF receptor activation. In contrast, EGF-induced EGFR-Shc-Grb2 complex formation was inhibited by AG1478 at a conventional concentration of 1 µM.


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Fig. 7.   H2O2-stimulated EGF receptor tyrosine phosphorylation and Shc-Grb2 complex formation. A, HUVECs were treated with H2O2 (200 µM) for the indicated periods of time. Cell lysates were then immunoprecipitated (IP) with anti-EGF receptor antibody, and immunoblots (IB) were performed with anti-phosphotyrosine antibody (PY20). B, cell lysates from ECs treated as in A were immunoprecipitated (IP) with PY20 and immunoblotted (IB) with anti-Shc antibody. C, COS-7 cells were stimulated with H2O2 (200 µM) or EGF (50 ng/ml) for 5 min with or without pretreatment (30 min) with AG1478 (1 or 50 µM) or PP2 (25 µM). Cell lysates were subjected to immunoprecipitation with anti-EGF receptor antibody and immunoblotting with phosphotyrosine, Shc, and Grb2 antibodies, respectively. Data are representative of three independent experiments.

EGF Receptor Autophosphorylation Is Not Involved in H2O2-mediated JNK Activation-- Activation of the EGF receptor typically involves receptor tyrosine kinase activity and autophosphorylation of the EGF receptor at tyrosine residue 1173 (22). To examine this process in H2O2-mediated EGF receptor activation, we employed an antibody specific for the autophosphorylation site (Tyr1173) of the activated EGF receptor. We did not observe EGF receptor autophosphorylation in response to H2O2 despite significant tyrosine phosphorylation of the receptor (Fig. 8). In contrast, both antibodies detected a strong signal from EGF-stimulated cells (Fig. 8). Therefore, these data confirm that H2O2-mediated JNK activation involves an autophosphorylation-independent mechanism that is distinct from EGF receptor signaling itself.


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Fig. 8.   H2O2-mediated tyrosine phosphorylation of the EGF receptor is distinct from ligand-induced autophosphorylation. COS-7 cells were stimulated with H2O2 (200 µM) or EGF (50 ng/ml) for 5 min. Cell lysates were subjected to immunoprecipitation with anti-EGF receptor antibody. The immunoprecipitates were immunoblotted with phospho-EGF receptor (Tyr1173) antibody and reprobed with anti-phosphotyrosine and EGF receptor antibodies. Data are indicative of three independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The principal finding of this study is that H2O2 activates JNK in endothelial cells through Src-dependent EGF receptor transactivation. We found that nontoxic concentrations of H2O2 rapidly activate the JNK pathway in endothelial cells, leading to c-Jun phosphorylation and activation of the transcription factor, AP-1. Endothelial cell JNK activation by H2O2 involved tyrosine kinase activity but not protein kinase C or phosphoinositide 3-kinase. The specific tyrosine kinase(s) involved in H2O2-mediated JNK activation were Src and the EGF receptor. Moreover, we found that H2O2-induced EGF receptor activation in endothelial cells was dependent upon Src and could be inhibited by AG1478 at concentrations of >= 10 µM. Taken together, these data are consistent with Src as an upstream signal for EGF receptor activation in response to H2O2.

The activation of JNK by H2O2 has been observed in a number of cell types and transformed cell lines (7, 23-25). However, little is known about oxidative stress-induced JNK activation in endothelial cells. Leptin binding to HUVECs is associated with ROS production and activation of the JNK pathway (26). Laminar shear stress on endothelial cells is associated with peroxynitrite-dependent activation of JNK (20). However, neither of these studies provided insight into the mechanisms of JNK activation. The data presented here extend these previous studies to H2O2-induced JNK activation in endothelial cells. The precise role of the JNK pathway in modulating the endothelial cell phenotype in vascular disease is not completely clear. Overexpression of c-Jun in cultured endothelial cells results in apoptosis (27), and human atherosclerotic plaques contain apoptotic endothelial cells (28). Although the functional consequences of JNK activation have thus far focused on cell death and apoptosis (29), it is reasonable to speculate that JNK may play some role in nonlethal phenotypic changes in the endothelial cell.

Activation of the JNK group of MAP kinases by cytokines has been examined in some detail. Tumor necrosis factor binding to the tumor necrosis factor receptor-1 induces receptor aggregation and recruitment of TRADD (tumor necrosis factor receptor-1 death domain protein) and TRAF2 (tumor necrosis factor receptor-associated factor 2) (30). Activation of JNK by TRAF2 may be mediated by either MAP kinase kinase 4 or 7 through pathways that may involve MAP kinase/ERK kinase 1 or apoptosis signal-regulating kinase (reviewed in Ref. 31). In contrast to cytokines, the upstream signals linking environmental stress to JNK activation are less well characterized. In the present study, we found that H2O2-induced JNK activation was dependent upon Src. This finding is in keeping with observations that Src tyrosine kinases are activated by oxidative events (32-34) and that Src is involved in JNK activation (19, 35). Yoshizumi et al. (36) have recently demonstrated that H2O2-mediated JNK activation in fibroblasts is dependent upon Src and Cas. The data presented here extend these findings to involve the EGF receptor in Src-dependent signaling. The precise role of Cas in this scheme vis-à-vis the EGF receptor will require further investigation.

Previous studies in vascular smooth muscle cells and fibroblasts indicated that H2O2-induced JNK activation involved Src, whereas activation of ERK1/2 and p38 did not (36). In contrast, mucoepidermoid carcinoma cells treated with H2O2 exhibit EGF receptor activation that is manifest as stimulation of ERK1/2 (37). The data presented here suggest that endothelial cells respond to H2O2 in a manner similar to the mucoepidermoid cells in that H2O2-induced ERK1/2 stimulation was significantly inhibited by both PP2 and AG1478, implicating both Src and EGF in this process. The mechanism(s) responsible for this distinct pattern of responses between endothelial and smooth muscle cells is not clear but certainly warrants further investigation.

A novel aspect of this work is the involvement of the EGF receptor in H2O2-induced JNK activation. We found that H2O2-mediated c-Jun phosphorylation was significantly inhibited by the EGF receptor tyrosine kinase inhibitor AG1478 and down-regulation of the EGF receptor by antisense oligonucleotides. Although EGF has been shown previously to activate the JNK pathway (35), our results indicate that H2O2 activates the EGF receptor via a mechanism that is distinct from EGF receptor autophosphorylation (Fig. 8) and requires Src-mediated tyrosine phosphorylation (Fig. 7). Such ligand-independent "transactivation" of the EGF receptor has been described with respect to a number of diverse stimuli including G-protein-coupled receptors, cytokines, and cellular stress (38). The data presented here are in agreement with one other report of H2O2-induced EGF receptor transactivation (37). In that study, EGF receptor transactivation by peroxide was found to mediate ERK1/2-dependent mucin synthesis in NCI-H292 cells, a mucoepidermoid carcinoma cell line. However, there was no attempt in that study to investigate the effect of H2O2 on nonreceptor tyrosine kinases or the JNK family of MAP kinases.

The precise mechanism of H2O2-induced EGF receptor transactivation is not clear, although receptor modification by H2O2 has been observed. Gamou and Shimizu (39) found that H2O2 induced EGF receptor tyrosine phosphorylation in both intact cells and isolated membranes with the major site of phosphorylation being Tyr1173. In contrast, EGF induced both tyrosine and serine phosphorylation in its receptor, resulting in quantitatively twice the phosphorylation as that observed with H2O2 (39). Goldkorn et al. (40) also observed that H2O2 preferentially induced EGF receptor tyrosine phosphorylation, and this effect was inhibited by genistein, implicating a nonreceptor protein-tyrosine kinase. Our data extend these findings and indicate that H2O2-induced EGF tyrosine phosphorylation is mediated by Src (Fig. 8C) but does not appear to involve Tyr1173 under our experimental conditions. This contention is not without precedent, since Src has been shown to mediate phosphorylation of the EGF receptor at tyrosine residues 845 and 1101, the former residue being critical for EGF-mediated stimulation of DNA synthesis (41). These two Src phosphorylation sites are not among the autophosphorylation sites in the carboxyl terminus of the protein (41), consistent with the results presented here (Fig. 8). With this in mind, if H2O2-induced EGF receptor tyrosine phosphorylation were exclusively mediated by Src, one would not expect AG1478 to inhibit H2O2-induced receptor tyrosine phosphorylation as reported here (Fig. 7C). It is worth noting that AG1478 was only effective against H2O2-induced responses at a concentration 50-fold higher than required to inhibit EGF-mediated receptor activation (Fig. 8C). As yet undescribed actions of AG 1478 against Src kinase activity or EGFR-Src complex formation merit consideration as potential explanations for these observations.

The nature of Src stimulation by oxidative stress is not known. In this study, we used H2O2, a two-electron oxidant, as our source of oxidative stress. Once formed, H2O2 participates in two major oxidation reactions relevant to biologic systems. In the first of these reactions, H2O2 may oxidize sulfur atoms to the corresponding sulfoxide via SN2 reaction of the sulfur with the O-O bond of H2O2 (42). If the sulfur atom is reduced, H2O2 can oxidize the thiols to the corresponding disulfide or sulfenic acid (42). This latter activity may be particularly germane to the activation of protein-tyrosine kinases such as Src. There is a growing appreciation that protein-tyrosine kinases are under a tonic inhibition by protein tyrosine phosphatases that typically contain thiol groups critical to their activity. Moreover, H2O2 has been shown to reversibly inactivate protein tyrosine phosphatases both in vitro (43, 44) and in cells (45) by virtue of thiol oxidation. Based upon these reports, it is attractive to speculate that H2O2 signaling is mediated through the inactivation of protein-tyrosine phosphatases, and such speculation could easily apply to Src family kinases. However, since all protein phosphatases share a similar active site motif consisting of a cysteine and an arginine separated by five residues (CXXXXXR), it is difficult to understand why H2O2 demonstrates relative signal specificity in many systems. It is also not clear if other single-electron (e.g. superoxide, lipid peroxyl radicals, hydroxyl) or two-electron (e.g. peroxynitrite, hypochlorous acid) oxidants will produce similar or diverse patterns of signal transduction.

In summary, the data presented here indicate that H2O2 readily induces JNK activation in endothelial cells. This H2O2-mediated JNK activation involves both Src family kinases and transactivation of the EGF receptor. Given the emerging importance of EGF receptor transactivation in a number of heterologous signaling systems, this may represent a new pathway for JNK activation in the setting of oxidative stress.

    FOOTNOTES

* This work was supported by National Institute of Health Grants HL53398 and HL52936 (to J. A. V.); HL49192 and HL18645 (to B. C. B.); and HL59346, HL55854, and DK55656 (to J. F. K).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Established Investigator of the American Heart Association.

To whom correspondence should be addressed: Boston University School of Medicine, Whitaker Cardiovascular Institute, 715 Albany St., Rm. W507, Boston, MA 02118. Tel.: 617-638-4894; Fax: 617-638-5437; E-mail: jkeaney@bu.edu.

Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M011766200

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; MAP, mitogen-activated protein; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; EGFR, EGF receptor; PAEC, porcine aortic endothelial cell; HUVEC, human umbilical vein endothelial cell; PP2, 4-amino-5-(4-chlorophenyl)-7- (t-butyl)pyrazolo[3,4-D]pyrimidine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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