Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425
Submitted 12 August 2003 ; accepted in final form 25 December 2003
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ABSTRACT |
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heparin-binding epidermal growth factor; phosphatidylinositol 3-kinase; renal proximal tubule
The molecular mechanisms involved in ERK and Akt activation in response to receptor tyrosine kinases (RTK) have been studied extensively. For example, the epidermal growth factor receptor (EGFR) forms a homodimer or heterodimer with other EGFR family members on ligand binding. Dimerization activates the intrinsic tyrosine kinase activity of the intracellular domain at different residues and, as a result, SH-domain proteins are recruited and trigger downstream signaling. Phosphorylation of EGFR tyrosine 1068 recruits Crb2, an adaptor protein, and initiates a series of events leading to ERK1/2 activation. Interaction of Gab with the EGFR results in activation of phosphatidylinositol 3-kinase (PI3K) (25), which is an upstream activator of Akt. In addition, these two pathways can be stimulated by other agents such as G protein-coupled receptor agonists and environmental stress (41).
ROS have been shown to activate several RTK (31, 36). H2O2 stimulates tyrosine phosphorylation of EGFR and its association with Grb2, leading to activation of ERK1/2 in a number of cell types (7). In Hela cells, H2O2-induced activation of EGFR results in the activation of the PI3K/Akt pathway (52). Initially, H2O2-stimulated EGFR activation was proposed to occur through inhibition of EGFR dephosphorylation, the result of tyrosine phosphatase inhibition (24). However, two recent reports indicate that activation of this receptor by H2O2 can occur through other mechanisms. Frank et al. (9) demonstrated that metalloprotease-dependent HB-EGF cleavage is required for EGFR activation by H2O2 in vascular smooth muscle cells, and Chen et al. (3) showed that H2O2-stimulated EGFR activation is dependent on Src in endothelial cells.
Although growth factor receptors are involved in the activation of ERK1/2 and Akt by H2O2, Esposito et al. (8) showed that ROS-mediated activation of these two kinases was not dependent on RTK phosphorylation but required Src activity. In addition, ROS can stimulate ERK and Akt via focal adhesion kinase (FAK) and G proteins (35, 48). Although RPTC are targeted by ROS generated during renal ischemia-reperfusion, ROS-mediated activation of ERK and Akt in RPTC is poorly characterized. In this study, we investigated the mechanisms responsible for H2O2 activation of Src, EGFR, ERK1/2, and Akt in RPTC.
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MATERIALS AND METHODS |
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Isolation and culture of renal proximal tubules. Female New Zealand White rabbits were purchased from Myrtle's Rabbitry (Thompson Station, TN). RPTC were isolated using the iron oxide perfusion method and grown in six-well or 35-mm tissue culture dishes under improved conditions as previously described (37). The culture medium was a 1:1 mixture of DMEM/Ham's F-12 (without glucose, phenol red, or sodium pyruvate) supplemented with 15 mM HEPES buffer, 2.5 mM L-glutamine, 1 µM pyridoxine HCl, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 nM), selenium (5 ng/ml), human transferrin (5 µg/ml), bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (50 µM) were added daily to fresh culture medium.
Preparation of cell lysates and immunoblot analysis. Confluent RPTC were used for all experiments. After treatment with inhibitors and/or H2O2 for various times, RPTC were washed twice with PBS without Ca2+ and Mg2+ and harvested in lysis buffer (0.25 M Tris·HCl, pH 6.8, 4% SDS, 10% glycerol, 1 mg/ml bromophenol blue, and 0.5% 2-mercaptoethanol). Cells were disrupted by sonication for 15 s. Whole cell lysates were stored at -20°C.
Equal amounts of cellular protein lysates were separated on 10% polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. After treatment with 5% skim milk at 4° C overnight, membranes were incubated with various antibodies for 1 h and then incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Amersham, Piscataway, NJ). Bound antibodies were visualized following chemiluminescence detection on autoradiographic film.
MTT assay. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. After a 6-h exposure to H2O2, MTT was added (final concentration of 0.5 mg/ml), and RPTC were incubated for additional 30 min and tetrazolium was released by dimethyl sulfoxide. Optical density was determined with a spectrophotometer (570 nm).
RPTC isolated from a single rabbit equals an n of 1, and each experiment was repeated a minimum of three times (n = 3).
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RESULTS |
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EGF (10 ng/ml) increased ERK1/2 and Akt phosphorylation to a maximum level within 5 min. After 120 min of incubation, EGF-induced Akt phosphorylation returned to the control levels, whereas ERK1/2 phosphorylation decreased but remained elevated (Fig. 1C). Densitometry confirmed the time-dependent changes in p-Akt levels following EGF and H2O2 treatment. These data demonstrate that the ERK1/2 and Akt signaling pathways are activated in response to H2O2 and EGF in RPTC, but the kinetics of ERK1/2 and Akt activation by EGF are transient compared with H2O2.
To evaluate the role of EGFR in H2O2- and EGF-induced ERK1/2 and Akt activation in RPTC, we first determined the phosphorylation of EGFR tyrosine 1068 by H2O2 and EGF using immunoblot analysis and a phospho-specific antibody for EGFR tyrosine 1068. As shown in Fig. 2, A and C, 1 mM H2O2 stimulated EGFR phosphorylation in a time-dependent manner with an initial increase observed within 5 min and a maximal increase occurring at 30 min. In contrast, 10 ng/ml EGF induced maximal phosphorylation of EGFR at 5 min and EGFR phosphorylation returned to control levels by 60 min (Fig. 2, B and C).
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To determine whether H2O2-induced transactivation of EGFR is responsible for activation of ERK1/2 and Akt, we used AG1478, a potent and selective inhibitor of EGFR (38). AG1478 inhibited H2O2-induced tyrosine 1068 phosphorylation of EGFR in a concentration-dependent manner (0.110 µM), with complete inhibition at 1 µM (Fig. 3A). AG1478 also decreased H2O2-induced ERK1/2 phosphorylation to basal levels (Fig. 3A). In contrast, H2O2-induced activation of Akt was not affected by AG1478 (Fig. 3A). Similar results were observed when RPTC were exposed to a lower concentration of H2O2 (0.25 mM) in the presence of AG1478 (Fig. 3B). In a comparison, the effect of AG1478 on EGF-induced phosphorylation of Akt and ERK1/2 was determined. The addition of EGF (10 ng/ml) resulted in the phosphorylation of EGFR, ERK1/2, and Akt (Fig. 3B). In the presence of AG1478, EGF-mediated EGFR, ERK1/2, and Akt activation was completely blocked. These data suggest that the activation of ERK1/2 by H2O2 is dependent on the EGFR, whereas H2O2-induced Akt activation is not EGFR dependent. In contrast, ERK1/2 and Akt activation following EGF exposure is EGFR dependent.
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PI3K mediates Akt phosphorylation following growth factor or oxidant stimulation (22, 47). PI3K has also been reported to mediate ERK1/2 activation by some stimuli such as insulin (5), lysophosphatidic acid, and thrombin (16). To determine whether H2O2-mediated ERK1/2 activation requires PI3K in RPTC, we measured H2O2-stimulated ERK1/2 phosphorylation in the presence of the PI3K inhibitor LY-294002. Treatment of RPTC with LY-294002 decreased, but did not block, H2O2-induced Akt phosphorylation; ERK1/2 phosphorylation was not affected by LY-294002 (Fig. 4). In contrast, LY-294002 completely blocked EGF-induced Akt phosphorylation in RPTC (data not shown). These data suggest that H2O2-stimulated ERK1/2 activation does not require PI3K and further support the dissociation of PI3K/Akt from the transactivated EGFR and ERK1/2 cascade in RPTC exposed to H2O2. Unlike EGF-mediated Akt phosphorylation, it is possible that Akt phosphorylation is also mediated by PI3K-independent mechanisms in H2O2-treated cells.
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It has been reported that transactivation of EGFR can occur through the release of membrane-anchored EGFR ligands (40). HB-EGF is a peptide mitogen of the EGF family that is expressed in RPTC (34, 42). Therefore, we determined whether HB-EGF shedding contributes to H2O2-induced ERGF activation using CRM 197, a diphtheria toxin mutant that specifically blocks the action of HB-EGF (32). As shown in Fig. 5A, H2O2-induced EGFR phosphorylation was not affected by CRM 197. Consistent with this result, CRM 197 did not have an effect on ERK1/2 (data not shown).
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In addition to HB-EGF, the EGFR can be activated by other EGF-like ligands and the ADAM (a disintegrin and metalloprotease) family of metalloproteases is believed to mediate proteolysis of EGFR ligand precursors (1). Among the family, ADAM17/TACE and ADAM9/MDC9 can be inhibited by hydroxamic acid-based metalloprotease inhibitors (43). Therefore, we evaluated the role of metalloproteases in H2O2-induced EGFR phosphorylation using GM6001 (10 µM), a broad-spectrum metalloprotease inhibitor and [4-(4'-biphenyl)-4-hydroxyimino-butyric acid], a metalloprotease III inhibitor (20 µM) (21). As shown in Fig. 5B, treatment with either inhibitor did not alter EGFR phosphorylation by H2O2. H2O2-induced ERK1/2 activation was also not affected by these inhibitors (data not shown). These results suggest that H2O2-induced EGFR transactivation is not the result of metalloprotease-dependent EGF ligand generation.
To determine whether ligand-independent mechanisms are involved in EGFR activation following H2O2 exposure, we assessed the role of intracellular Ca2+, PKC, and Src in H2O2-induced phosphorylation of EGFR. These signaling molecules have been implicated previously in the transactivation of EGFR by different stimuli (39). As shown in Fig. 6, treatment of cells with 10 µM BAPTA-AM, a chelator of intracellular Ca2+, or GF109203X (10 µM), an inhibitor of conventional and novel PKC (50), did not affect H2O2-induced EGFR phosphorylation at Tyr1068. In contrast, H2O2-induced phosphorylation of EGFR at this residue was abolished by PP1, a selective inhibitor of Src (Fig. 6C). However, PP1 did not effect EGFR phosphorylation following EGF exposure (Fig. 6D). These data suggest that H2O2-induced EGFR activation is dependent on Src, but not Ca2+ or conventional and novel PKC. In contrast, EGF activation of the EGFR is not Src mediated.
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Because Src can activate the EGFR by direct phosphorylation of tyrosine 845 (46), we evaluated the effect of H2O2 on the phosphorylation of this residue and the effect of PP1. Immunoblot analysis using a phospho-specific EGFR tyrosine 845 antibody revealed tyrosine 845 phosphorylation of EGFR on H2O2 exposure and that this response was blocked by PP1 (Fig. 6C). In contrast, EGF-induced EGFR phosphorylation at tyrosine 845 was not sensitive to this inhibitor. These results reflect differences in EGFR activation by ligands and nonligands.
Src activity is regulated mainly by phosphorylation of different tyrosine sites with phosphorylation at tyrosine 416 in the catalytic domain as an activating signal (27). We examined the effect of H2O2 on Src phosphorylation and demonstrated that H2O2 stimulated Src tyrosine 416 phosphorylation within 5 min and the response was sustained through 120 min of treatment (Fig. 7A). The Src inhibitor PP1 blocked the increase in Src tyrosine 416 phosphorylation (Fig. 7B). These results suggest that Src is an early target of H2O2.
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The above results suggest that Src acts upstream of EGFR; consequently, it would be predicted that inhibition of the EGFR using AG1478 would not have an effect on Src phosphorylation. Indeed, treatment of RPTC with AG1478 did not result in inhibition of Src phosphorylation induced by H2O2 (Fig. 7C). The next series of experiments determined the effect of Src inhibition on H2O2-induced ERK1/2 and Akt phosphorylation. The Src inhibitor PP1 completely inhibited basal and H2O2-induced ERK1/2 phosphorylation (Fig. 7B). PP1 blocked H2O2-induced Akt phosphorylation but not basal Akt phosphorylation. As described above, Akt activation was not associated with the EGFR (Fig. 3A). These data strongly suggest that Src functions as an upstream activator of Akt and ERK1/2 via distinct mechanisms.
Because FAK and G proteins have also been reported to be involved in the activation of Akt or ERK1/2 by ROS (35, 48), we assessed the roles of these proteins in mediating H2O2-induced activation of Akt and ERK1/2 using cytochalasin D and pertussis toxin. Cytochalasin D has been reported to selectively disrupt the network of actin filaments and inhibit FAK phosphorylation (4, 33, 54). Preincubation with cytochalasin D reduced ERK1/2 phosphorylation following H2O2 treatment. In contrast, H2O2-induced Akt phosphorylation was not affected by cytochalasin D. Pertussis toxin inactivates Gi/o proteins. Previous studies showed that treatment with H2O2 directly activates purified heterotrimeric Gi and Go but not Gs in vitro (35). Pertussis toxin did not attenuate H2O2-induced phosphorylation of ERK1/2 or Akt in RPTC (Fig. 8B). Collectively, these data reveal that H2O2-induced ERK1/2 and Akt are differently regulated; ERK1/2 phosphorylation is partially mediated by FAK, whereas Akt activation is not regulated by either FAK or Gi/o proteins in RPTC following H2O2 exposure.
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Previous reports showed that activation of Akt and ERK1/2 by H2O2 is associated with protection from apoptosis in a variety of other cell types (15, 19, 52). Because EGFR and Src function as upstream activators of ERK1/2 or Akt and ERK1/2 following H2O2 exposure, we investigated the influence of EGF and Src activation on H2O2-induced cell death. Exposure of RPTC to H2O2 for 6 h induced reduced cell viability to 72% of controls (Fig. 9). Pretreatment of RPTC with AG1478 had no effect on the decrease in viability following H2O2 exposure. In contrast, PP1 pretreatment resulted in a further decrease in viability following H2O2 exposure. These agents were not cytotoxic when given alone (data not shown). These results reveal that Src activation, but not EGFR, contributes to RPTC survival in response to H2O2.
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DISCUSSION |
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EGFR transactivation can be induced by many stimuli and occurs through different pathways (2, 13). EGFR transactivation can occur through intracellular signaling pathways such as PKC, Ca2+, and Src (39). In this study, we demonstrate that H2O2-induced EGFR transactivation is dependent on the activation of Src, whereas chelation of intracellular Ca2+ or inhibition of conventional and novel PKC had no affect on H2O2-induced EGFR transactivation in RPTC. H2O2 induced the phosphorylation of Src tyrosine 416, which is required for Src activity, and the phosphorylation of EGFR tyrosine 845, the Src-mediated phosphorylation site. Furthermore, the inhibition of the EGFR did not interfere with H2O2-induced Src activation. These data support the concept that Src acts upstream of the EGFR in H2O2-treated cells. Remarkably, it has been reported that phosphorylation of EGFR tyrosine 845 is able to stabilize the activation loop of EGFR, maintaining the enzyme in the active state and providing a binding surface for protein substrates (27).
EGFR activation can also occur through autocrine/paracrine release of soluble EGF ligands (2). For example, Frank et al. (10) showed that H2O2-stimulated EGFR activation is produced by metalloprotease-dependent HB-EGF cleavage in vascular smooth muscle cells. HB-EGF is expressed in RPTC (34), and we examined whether this mechanism is applicable in EGFR activation by H2O2 in RPTC. The use of two metalloprotease inhibitors [GM6001 and 4-(4'-biphenyl)-4-hydroxyimino-butyric acid] and an HB-EGF inhibitor (CRM 197) did not reveal that H2O2-induced EGFR transactivation is through shedding of pro-HB-EGF by metalloproteases in RPTC. Although it is possible that other EGFR ligands such as TGF-, amphiregulin, and betacellulin may be involved in transactivation of EGFR in H2O2-treated cells, it is unlikely because the metalloprotease inhibitors had no effect on the EGFR phosphorylation induced by H2O2 and it is generally believed that release of the endogenous ligands from their membrane precursor requires metalloprotease activity (39).
The finding that H2O2-stimulated phosphorylation of ERK1/2, but not Akt, was completely blocked by the EGFR inhibitor AG1478 shows that EGFR mediates activation of ERK1/2, but not Akt in RPTC. Consistent with the concept that transactivation of EGFR involves Src in H2O2-treated RPTC, the Src inhibitor PP1, at a concentration that inhibits the EGFR phosphorylation, also blocked ERK1/2 phosphorylation in H2O2-treated cells. Stress-induced ERK1/2 activation via Src and EGFR activation is not restricted to H2O2 because UV-induced ERK1/2 activation was also completely abolished by AG1478 and the PP1 (23).
Activation of Akt by H2O2, independent of EGFR, in RPTC is an interesting observation. It is well established that Akt is one of the downstream intracellular signaling molecules of EGFR on ligand binding (6), and inhibition of the RPTC EGFR also blocked EGF-stimulated Akt phosphorylation, suggesting that this pathway is intact in this cell type. Thus the failure of AG1478 to inhibit H2O2-mediated Akt phosphorylation suggests that the transactivated EGFR does not contribute to H2O2-stimulated Akt phosphorylation in RPTC. To our knowledge, this is the first example of different requirements for the EGFR in H2O2-induced activation of ERK1/2 and Akt in the same cell type. Similar to our observation, Roudabush et al. (44) demonstrated that transactivation of EGFR is required for phosphorylation of ERK1/2, but not Akt, in insulin-like growth factor-treated cells. Our results are in contrast to a previous study in which EGFR activation was coupled to the PI3K/Akt signaling cascade in H2O2-treated Hela cells (52). The reason for these differences in cellular response is not known but may be related to difference in cell types.
Although the RPTC EGFR is not involved in the activation of Akt by H2O2, it seems that Src still functions as the upstream mediator of Akt activation. This is clearly indicated by our observation that inhibition of Src by PP1 abolished the H2O2-induced Akt phosphorylation. Supporting this observation, Esposito et al. (8) reported that Akt activation by ROS produced by diethylmaleate, a glutathione-depleting agent, was independent of RTK phosphorylation and dependent on Src activity. The mechanisms by which the Src is coupled to Akt following H2O2 treatment remain clear. One possibility is that Src operates as a regulator in other signaling pathways that mediate activation of Akt. In this regard, it has been reported that FAK mediates activation of PI3K/Akt pathways in response to ROS (48) and is subjected to regulation by Src (53). We examined the possible involvement of FAK in H2O2-stimulated Akt phosphorylation. However, inhibition of FAK by cytochalasin D did not affect H2O2-induced Akt phosphorylation (Fig. 8A), indicating that FAK does not act as a mediator of Src in activation of Akt by H2O2 in RPTC. Although H2O2 has been reported to activate Gab1, an adaptor protein of PI3K, and its activation is sensitive to a Src inhibitor (18), Gab1 is not expressed in kidney (17). Another possibility is that Src regulates Akt activation via altering the function of PTEN. PTEN is a PI3K-phosphatase that antagonizes PI3K action (30). It has been reported that activated Src can inhibit PTEN function, leading to upregulation of Akt activity (29). This finding, in conjunction with a recent observation that inactivation of cellular PTEN activity by H2O2 results in activation of Akt (26), suggests that regulation of PTEN by Src may be involved in Akt activation in response to oxidative stress.
In addition to PI3K-mediated Akt activation, H2O2-induced Akt activation can also occur independently of PI3K in rat primary astrocytes (45). Consequently, alternative mechanisms for H2O2-induced Akt activation may involve the direct interaction of Src with Akt, thereby triggering its activity. This possibility is suggested by our observations that partial blockade of H2O2 induced Akt phosphorylation by a Src inhibitor (Fig. 7B) and partial inhibition of Akt phosphorylation by the PI3K inhibitor LY-294002 (Fig. 4). It was reported that Src can directly regulate Akt activity by phosphorylating tyrosine 315 and tyrosine 326 in the activation loop of Akt (20). Thus it is likely that multiple mechanisms are involved in Src-mediated Akt activation following H2O2 treatment and will be the subject of future studies.
We also investigated the biological roles of Src and EGFR in response to H2O2 injury and demonstrated that inhibition of Src further reduced cell viability in H2O2-treated RPTC (Fig. 9). This finding is consistent with a previous observation that Src mediates the protective action of nitric oxide in cell death induced by serum deprivation (49). The effect of Src may be through the activation of Akt, as this signaling pathway has been shown to mediate a survival response that counteracts cell death after H2O2-induced injury in a variety of cell types including renal epithelial cells (14, 19, 52). In contrast, inhibition of EGFR did not show a protective effect following oxidative injury, suggesting that EGFR-mediated signaling pathways are not associated with cytoprotection in renal cells. Two studies demonstrated that the activation of Akt, but not ERK, is required for EGF-stimulated protection of embryonic kidney epithelial (HEK293) cells from apoptosis induced by Fas (12) and tumor necrosis factor-related apoptosis-inducing ligand (11).
In summary, the data presented here reveal that H2O2 activation of ERK1/2 and Akt is through different mechanisms in RPTC. Activation of both kinases by H2O2 occurs through a Src-dependent mechanism. However, activation of ERK1/2, but not Akt, is mediated by EGFR transactivation. Src-mediated signaling pathways play an important cytoprotective response after oxidative injury.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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