c vitamini H2O2 induced transactivation of EGF receptor requires H.pdf

doi: 10.1152/ajprenal.00282.2003286:F858-F865, 2004. First published 10 February 2004;Am J Physiol Renal Physiol

Shougang Zhougang and Rick G. Schnellmanncells

renalSrc and mediates ERK1/2, but not Akt, activation in -induced transactivation of EGF receptor requires2O2H

You might find this additional info useful...

54 articles, 29 of which you can access for free at: This article citeshttp://ajprenal.physiology.org/content/286/5/F858.full#ref-list-1

19 other HighWire-hosted articles: This article has been cited by http://ajprenal.physiology.org/content/286/5/F858#cited-by

including high resolution figures, can be found at: Updated information and serviceshttp://ajprenal.physiology.org/content/286/5/F858.full

found at: can beAmerican Journal of Physiology - Renal Physiology about Additional material and information

http://www.the-aps.org/publications/ajprenal

This information is current as of February 14, 2013.

0363-6127, ESSN: 1522-1466. Visit our website at http://www.the-aps.org/. Rockville Pike, Bethesda MD 20814-3991. Copyright © 2004 the American Physiological Society. ISSN: volume and composition. It is published 12 times a year (monthly) by the American Physiological Society, 9650relating to the kidney, urinary tract, and their respective cells and vasculature, as well as to the control of body fluid

publishes original manuscripts on a broad range of subjectsAmerican Journal of Physiology - Renal Physiology

at Selcuk U

niversity on February 14, 2013

http://ajprenal.physiology.org/D

ownloaded from

http://ajprenal.physiology.org/content/286/5/F858#cited-by

http://ajprenal.physiology.org/

H2O2-induced transactivation of EGF receptor requires Src and mediatesERK1/2, but not Akt, activation in renal cells

Shougang Zhougang and Rick G. SchnellmannDepartment of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425

Submitted 12 August 2003; accepted in final form 25 December 2003

Zhougang, Shougang, and Rick G. Schnellmann. H2O2-inducedtransactivation of EGF receptor requires Src and mediates ERK1/2,but not Akt, activation in renal cells. Am J Physiol Renal Physiol 286:F858–F865, 2004. First published February 10, 2004; 10.1152/ajprenal.00282.2003.—Although oxidative stress activates epidermal growthfactor receptor (EGFR), ERK1/2, and Akt in a number of cell types,the mechanisms by which oxidative stress activates these kinases arenot well defined in renal epithelial cells. Exposure of primary culturesof rabbit renal proximal tubular cells to hydrogen peroxide (H2O2)stimulated Src, EGFR, ERK1/2, and Akt activation in a time-depen-dent manner as determined by the phosphorylation of each protein.The Src inhibitor PP1 completely blocked EGFR, ERK1/2, and Aktphosphorylation following H2O2 exposure. In contrast, blockade ofthe EGFR by AG1478 inhibited phosphorylation of ERK1/2 but notSrc or Akt phosphorylation following H2O2 exposure. ExogenousEGF stimulated EGFR, ERK1/2, and Akt activation and the EGFRinhibitor blocked phorphorylation of ERK1/2 and Akt. The presenceof PP1, but not AG1478, significantly accelerated H2O2-induced celldeath. These results suggest that Src mediates H2O2-induced EGFRtransactivation. H2O2- and EGF-induced ERK1/2 activation is medi-ated by EGFR, whereas Akt is activated by Src independent of EGFRfollowing H2O2 exposure. Src-mediated EGFR transactivation con-tributes to a survival response following oxidative injury.

heparin-binding epidermal growth factor; phosphatidylinositol 3-ki-nase; renal proximal tubule

REACTIVE OXYGEN SPECIES (ROS), including hydrogen peroxide(H2O2), superoxide radical, and hydroxyl radical, have beenimplicated in the pathogenesis of renal ischemia and reperfu-sion injury (28). Although ROS generation following renalischemia and reperfusion injury may induce apoptosis andoncosis in renal proximal tubular cells (RPTC), ROS alsomediate a number of adaptive biological responses and regulatethe expression of a variety of genes that are involved in renalsurvival and regeneration (7). Among intracellular signalingpathways triggered, ERK and Akt have been shown to regulatecell survival in response to oxidative stress (7) and mediate cellproliferation following growth factor stimulation (51).

The molecular mechanisms involved in ERK and Akt acti-vation in response to receptor tyrosine kinases (RTK) havebeen studied extensively. For example, the epidermal growthfactor receptor (EGFR) forms a homodimer or heterodimerwith other EGFR family members on ligand binding. Dimer-ization activates the intrinsic tyrosine kinase activity of theintracellular domain at different residues and, as a result,SH-domain proteins are recruited and trigger downstream sig-naling. 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 resultsin activation of phosphatidylinositol 3-kinase (PI3K) (25),which is an upstream activator of Akt. In addition, these twopathways 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 itsassociation with Grb2, leading to activation of ERK1/2 in anumber of cell types (7). In Hela cells, H2O2-induced activa-tion of EGFR results in the activation of the PI3K/Akt pathway(52). Initially, H2O2-stimulated EGFR activation was proposedto occur through inhibition of EGFR dephosphorylation, theresult of tyrosine phosphatase inhibition (24). However, tworecent reports indicate that activation of this receptor by H2O2

can occur through other mechanisms. Frank et al. (9) demon-strated that metalloprotease-dependent HB-EGF cleavage isrequired for EGFR activation by H2O2 in vascular smoothmuscle cells, and Chen et al. (3) showed that H2O2-stimulatedEGFR activation is dependent on Src in endothelial cells.

Although growth factor receptors are involved in the acti-vation of ERK1/2 and Akt by H2O2, Esposito et al. (8) showedthat ROS-mediated activation of these two kinases was notdependent on RTK phosphorylation but required Src activity.In addition, ROS can stimulate ERK and Akt via focal adhe-sion kinase (FAK) and G proteins (35, 48). Although RPTC aretargeted by ROS generated during renal ischemia-reperfusion,ROS-mediated activation of ERK and Akt in RPTC is poorlycharacterized. In this study, we investigated the mechanismsresponsible for H2O2 activation of Src, EGFR, ERK1/2, andAkt in RPTC.

MATERIALS AND METHODS

Chemicals and regents. GM6001, 4-(4�-biphenyl)-4-hydroxy-imino-butyric acid, and GF109203X were obtained from Calbiochem(San Diego, CA). Human-recombinant EGF was purchased fromR&D Systems (Minneapolis, MN). LY-294002 was obtained fromCell Signaling Technology (Beverly, MA). AG1478 and BAPTA-AMwere obtained from Biomol (Plymouth Meeting, PA). All otherchemicals were from Sigma (St. Louis, MO). Antibodies to phospho-EGFR (no. 2236), phospho-Akt (no. 9271), Akt (no. 2966), phospho-Src (no. 2101), Src (no. 2102), and phospho-ERK1/2 (no. 9101) wereobtained from Cell Signaling Technology. Antibodies to ERK1/2(no. 06–182) and EGFR (Sc-03-G) were purchased from BD Labo-ratories (San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz,CA), respectively. All antibodies were used at 1:1,000 for immuno-blotting.

Isolation and culture of renal proximal tubules. Female NewZealand White rabbits were purchased from Myrtle’s Rabbitry(Thompson Station, TN). RPTC were isolated using the iron oxide

Address for reprint requests and other correspondence: R. G. Schnellmann,Dept. of Pharmaceutical Sciences, Medical Univ. of South Carolina, 280Calhoun St., POB 250140, Charleston, SC 29425 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Renal Physiol 286: F858–F865, 2004.First published February 10, 2004; 10.1152/ajprenal.00282.2003.

0363-6127/04 $5.00 Copyright © 2004 the American Physiological Society http://www.ajprenal.orgF858

at Selcuk U



ownloaded from


perfusion method and grown in six-well or 35-mm tissue culturedishes under improved conditions as previously described (37). Theculture medium was a 1:1 mixture of DMEM/Ham’s F-12 (withoutglucose, phenol red, or sodium pyruvate) supplemented with 15 mMHEPES buffer, 2.5 mM L-glutamine, 1 �M pyridoxine HCl, 15 mMsodium bicarbonate, and 6 mM lactate. Hydrocortisone (50 nM),selenium (5 ng/ml), human transferrin (5 �g/ml), bovine insulin (10nM), and L-ascorbic acid-2-phosphate (50 �M) were added daily tofresh culture medium.

Preparation of cell lysates and immunoblot analysis. ConfluentRPTC were used for all experiments. After treatment with inhibitorsand/or H2O2 for various times, RPTC were washed twice with PBSwithout Ca2� and Mg2� and harvested in lysis buffer (0.25 MTris�HCl, pH 6.8, 4% SDS, 10% glycerol, 1 mg/ml bromophenol blue,and 0.5% 2-mercaptoethanol). Cells were disrupted by sonication for15 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 nitrocellu-lose membranes. After treatment with 5% skim milk at 4°C overnight,membranes were incubated with various antibodies for 1 h and thenincubated with an appropriate horseradish peroxidase-conjugated sec-ondary antibody (Amersham, Piscataway, NJ). Bound antibodies werevisualized following chemiluminescence detection on autoradio-graphic film.

MTT assay. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. After a6-h exposure to H2O2, MTT was added (final concentration of 0.5mg/ml), and RPTC were incubated for additional 30 min and tetra-zolium was released by dimethyl sulfoxide. Optical density wasdetermined with a spectrophotometer (570 nm).

RPTC isolated from a single rabbit equals an n of 1, and eachexperiment was repeated a minimum of three times (n � 3).

RESULTS

To determine the effect of H2O2 on ERK1/2 and Aktactivation in RPTC, we examined the phosphorylation of Aktand ERK1/2 using phospho-specific antibodies and immuno-blot analysis. As shown in Fig. 1A, 1 mM H2O2 stimulatedphosphorylation of ERK1/2; stimulation occurred within 5min, reached a maximum at 30 min, and was sustained through120 min of treatment. Akt phosphorylation also increasedwithin 5 min of H2O2 exposure and increased over the 120-minincubation period. Increasing concentrations of H2O2 inducedERK1/2 and Akt phosphorylation with a maximal effect at 250�M for ERK1/2 and 1 mM for Akt (Fig. 1B). Total ERK1/2and Akt levels were determined using antibodies that recognizethese proteins, independent of their phosphorylation state.Total ERK1/2 and Akt levels did not change over the 120-minexposure period.

EGF (10 ng/ml) increased ERK1/2 and Akt phosphorylationto a maximum level within 5 min. After 120 min of incubation,EGF-induced Akt phosphorylation returned to the control lev-els, whereas ERK1/2 phosphorylation decreased but remainedelevated (Fig. 1C). Densitometry confirmed the time-depen-dent changes in p-Akt levels following EGF and H2O2 treat-ment. These data demonstrate that the ERK1/2 and Akt sig-naling pathways are activated in response to H2O2 and EGF inRPTC, but the kinetics of ERK1/2 and Akt activation by EGFare transient compared with H2O2.

To evaluate the role of EGFR in H2O2- and EGF-inducedERK1/2 and Akt activation in RPTC, we first determined thephosphorylation of EGFR tyrosine 1068 by H2O2 and EGFusing 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 mannerwith an initial increase observed within 5 min and a maximalincrease occurring at 30 min. In contrast, 10 ng/ml EGFinduced maximal phosphorylation of EGFR at 5 min andEGFR phosphorylation returned to control levels by 60 min(Fig. 2, B and C).

To determine whether H2O2-induced transactivation ofEGFR is responsible for activation of ERK1/2 and Akt, weused AG1478, a potent and selective inhibitor of EGFR (38).AG1478 inhibited H2O2-induced tyrosine 1068 phosphoryla-tion of EGFR in a concentration-dependent manner (0.1–10�M), with complete inhibition at 1 �M (Fig. 3A). AG1478 alsodecreased H2O2-induced ERK1/2 phosphorylation to basallevels (Fig. 3A). In contrast, H2O2-induced activation of Aktwas not affected by AG1478 (Fig. 3A). Similar results wereobserved when RPTC were exposed to a lower concentration

Fig. 1. H2O2 and epidermal growth factor (EGF) stimulate phosphorylation ofERK1/2 and Akt. Confluent renal proximal tubular cells (RPTC) were exposedto 1 mM H2O2 or 10 ng/ml EGF for the indicated time periods (A and C) orfor 30 min at the indicated concentrations (B). Cell lysates were separated bySDS-PAGE and immunoblotted with antibodies to phospho(p)-ERK1/2,ERK1/2, phospho-Akt, and Akt. Representative immunoblots from 3 or moreexperiments are shown. D: densitometry of phospho-Akt data in A and C. Dataare expressed as the percentage of expression relative to that in control. Valuesare means � SE of 3 independent experiments.

F859SRC-MEDIATED TRANSACTIVATION OF EGFR BY OXIDATIVE STRESS

AJP-Renal Physiol • VOL 286 • MAY 2004 • www.ajprenal.org

at Selcuk U



ownloaded from


of H2O2 (0.25 mM) in the presence of AG1478 (Fig. 3B). In acomparison, the effect of AG1478 on EGF-induced phosphor-ylation of Akt and ERK1/2 was determined. The addition ofEGF (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 com-pletely blocked. These data suggest that the activation ofERK1/2 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 EGFRdependent.

PI3K mediates Akt phosphorylation following growth factoror oxidant stimulation (22, 47). PI3K has also been reported tomediate ERK1/2 activation by some stimuli such as insulin (5),lysophosphatidic acid, and thrombin (16). To determinewhether H2O2-mediated ERK1/2 activation requires PI3K inRPTC, we measured H2O2-stimulated ERK1/2 phosphoryla-tion in the presence of the PI3K inhibitor LY-294002. Treat-ment of RPTC with LY-294002 decreased, but did not block,H2O2-induced Akt phosphorylation; ERK1/2 phosphorylationwas not affected by LY-294002 (Fig. 4). In contrast, LY-294002 completely blocked EGF-induced Akt phosphorylationin RPTC (data not shown). These data suggest that H2O2-stimulated ERK1/2 activation does not require PI3K and fur-ther support the dissociation of PI3K/Akt from the transacti-vated EGFR and ERK1/2 cascade in RPTC exposed to H2O2.Unlike EGF-mediated Akt phosphorylation, it is possible thatAkt phosphorylation is also mediated by PI3K-independentmechanisms in H2O2-treated cells.

It has been reported that transactivation of EGFR can occurthrough 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 determinedwhether HB-EGF shedding contributes to H2O2-inducedERGF activation using CRM 197, a diphtheria toxin mutantthat specifically blocks the action of HB-EGF (32). As shownin Fig. 5A, H2O2-induced EGFR phosphorylation was notaffected by CRM 197. Consistent with this result, CRM 197did not have an effect on ERK1/2 (data not shown).

In addition to HB-EGF, the EGFR can be activated by otherEGF-like ligands and the ADAM (a disintegrin and metallo-protease) family of metalloproteases is believed to mediateproteolysis of EGFR ligand precursors (1). Among the family,

Fig. 3. Effects of AG1478 on H2O2 and EGF-induced ERK1/2 and Aktphosphorylation. Confluent RPTC were pretreated with 0.1–10 �M AG1478for 1 h and then exposed to 1 mM H2O2 for 10 min (A), or 10 �M AG1478 for1 h and then 0.25 mM H2O2 (B), or 10 ng/ml EGF for 10 min (C). Cell lysateswere separated by SDS-PAGE and immunoblotted with anti-phospho-Tyr1068EGFR, anti-EGFR, anti-phospo-ERK1/2, anti-ERK1/2, anti-phospho-Akt, andanti-Akt antibodies. Representative immunoblots from 3 or more experimentsare shown.

Fig. 2. H2O2 and EGF stimulate phosphorylation of EGF receptor (EGFR).Confluent RPTC were stimulated with 1 mM H2O2 (A) or 10 ng/ml EGF (B)for the indicated time periods. Cell lysates were separated by SDS-PAGE andimmunoblotted with antibodies to phospho-EGFR (Try1068) and EGFR.Representative immunoblots from 3 or more experiments are shown. C:densitometry of phospho-EGFR in A and B. Data are expressed as thepercentage of expression relative to that in control. Values are means � SE of3 independent experiments.

F860 SRC-MEDIATED TRANSACTIVATION OF EGFR BY OXIDATIVE STRESS


at Selcuk U



ownloaded from


ADAM17/TACE and ADAM9/MDC9 can be inhibited byhydroxamic acid-based metalloprotease inhibitors (43). There-fore, we evaluated the role of metalloproteases in H2O2-induced EGFR phosphorylation using GM6001 (10 �M), abroad-spectrum metalloprotease inhibitor and [4-(4�-biphe-nyl)-4-hydroxyimino-butyric acid], a metalloprotease III inhib-itor (20 �M) (21). As shown in Fig. 5B, treatment with eitherinhibitor did not alter EGFR phosphorylation by H2O2. H2O2-induced ERK1/2 activation was also not affected by theseinhibitors (data not shown). These results suggest that H2O2-induced EGFR transactivation is not the result of metallopro-tease-dependent EGF ligand generation.

To determine whether ligand-independent mechanisms areinvolved in EGFR activation following H2O2 exposure, weassessed the role of intracellular Ca2�, PKC, and Src inH2O2-induced phosphorylation of EGFR. These signaling mol-ecules have been implicated previously in the transactivation ofEGFR by different stimuli (39). As shown in Fig. 6, treatmentof cells with 10 �M BAPTA-AM, a chelator of intracellular

Ca2�, or GF109203X (10 �M), an inhibitor of conventionaland novel PKC (50), did not affect H2O2-induced EGFRphosphorylation at Tyr1068. In contrast, H2O2-induced phos-phorylation of EGFR at this residue was abolished by PP1, aselective inhibitor of Src (Fig. 6C). However, PP1 did noteffect EGFR phosphorylation following EGF exposure (Fig.6D). These data suggest that H2O2-induced EGFR activation isdependent on Src, but not Ca2� or conventional and novelPKC. In contrast, EGF activation of the EGFR is not Srcmediated.

Because Src can activate the EGFR by direct phosphoryla-tion of tyrosine 845 (46), we evaluated the effect of H2O2 onthe phosphorylation of this residue and the effect of PP1.Immunoblot analysis using a phospho-specific EGFR tyrosine845 antibody revealed tyrosine 845 phosphorylation of EGFRon H2O2 exposure and that this response was blocked by PP1(Fig. 6C). In contrast, EGF-induced EGFR phosphorylation attyrosine 845 was not sensitive to this inhibitor. These resultsreflect differences in EGFR activation by ligands and nonli-gands.

Src activity is regulated mainly by phosphorylation of dif-ferent tyrosine sites with phosphorylation at tyrosine 416 in the

Fig. 4. Effect of LY-294002 on H2O2-induced phosphorylation of Akt andERK1/2. Confluent RPTC were pretreated with 20 �M LY-294002 for 15 minand exposed to 1 mM H2O2 for 10 min. Cell lysates were separated bySDS-PAGE and immunoblotted with anti-phospho-ERK1/2, anti-ERK1/2,anti-phospho-Akt, and anti-Akt antibodies. Representative immunoblots from3 or more experiments are shown.

Fig. 5. H2O2-induced EGFR phosphorylation (Try 1068) does not requireHB-EGF or a metalloprotease-dependent mechanism. Confluent RPTC werepretreated with 10 �M CRM 197 (A), 10 �M GM 6001, or 20 �M [4-(4�-biphenyl)-4-hydroxyimino-butyric acid (HBA); B] for 1 h and exposed to 1mM H2O2 for 10 min. Cell lysates were separated by SDS-PAGE andimmunoblotted with anti-phospho EGFR (Try1068) and anti-EGF receptorantibodies. Representative immunoblots from 3 or more experiments areshown.

Fig. 6. Effects of an intracellular calcium chelator, PKC inhibitor, and Srcinhibitor on H2O2-induced EGFR phosphorylation. Confluent RPTC werepretreated with 10 �M BAPTA-AM (A), 10 �M GF109203X (GF; B), or 10�M PP1 (C and D) for 1 h and exposed to 1 mM H2O2 for 10 min. Cell lysateswere separated by SDS-PAGE and immunoblotted with anti-phospho EGFR(Tyr1068) antibody (A, B, and D), anti-phospho EGFR (Tyr845; C and D), andanti-EGFR (A-D) antibodies. Representative immunoblots from 3 or moreexperiments are shown.



at Selcuk U



ownloaded from


catalytic domain as an activating signal (27). We examined theeffect of H2O2 on Src phosphorylation and demonstrated thatH2O2 stimulated Src tyrosine 416 phosphorylation within 5min and the response was sustained through 120 min oftreatment (Fig. 7A). The Src inhibitor PP1 blocked the increasein Src tyrosine 416 phosphorylation (Fig. 7B). These resultssuggest that Src is an early target of H2O2.

The above results suggest that Src acts upstream of EGFR;consequently, it would be predicted that inhibition of theEGFR using AG1478 would not have an effect on Src phos-phorylation. Indeed, treatment of RPTC with AG1478 did notresult in inhibition of Src phosphorylation induced by H2O2

(Fig. 7C). The next series of experiments determined the effectof Src inhibition on H2O2-induced ERK1/2 and Akt phosphor-ylation. The Src inhibitor PP1 completely inhibited basal andH2O2-induced ERK1/2 phosphorylation (Fig. 7B). PP1 blockedH2O2-induced Akt phosphorylation but not basal Akt phos-phorylation. As described above, Akt activation was not asso-ciated with the EGFR (Fig. 3A). These data strongly suggestthat Src functions as an upstream activator of Akt and ERK1/2via distinct mechanisms.

Because FAK and G proteins have also been reported to beinvolved 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 toselectively disrupt the network of actin filaments and inhibitFAK phosphorylation (4, 33, 54). Preincubation with cytocha-lasin D reduced ERK1/2 phosphorylation following H2O2

treatment. In contrast, H2O2-induced Akt phosphorylation wasnot 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-inducedphosphorylation of ERK1/2 or Akt in RPTC (Fig. 8B). Collec-tively, these data reveal that H2O2-induced ERK1/2 and Aktare differently regulated; ERK1/2 phosphorylation is partiallymediated by FAK, whereas Akt activation is not regulated byeither FAK or Gi/o proteins in RPTC following H2O2 exposure.

Previous reports showed that activation of Akt and ERK1/2by H2O2 is associated with protection from apoptosis in avariety of other cell types (15, 19, 52). Because EGFR and Srcfunction as upstream activators of ERK1/2 or Akt and ERK1/2following H2O2 exposure, we investigated the influence ofEGF and Src activation on H2O2-induced cell death. Exposureof RPTC to H2O2 for 6 h induced reduced cell viability to 72%of controls (Fig. 9). Pretreatment of RPTC with AG1478 hadno effect on the decrease in viability following H2O2 exposure.In contrast, PP1 pretreatment resulted in a further decrease inviability following H2O2 exposure. These agents were notcytotoxic when given alone (data not shown). These resultsreveal that Src activation, but not EGFR, contributes to RPTCsurvival in response to H2O2.

Fig. 8. Effect of cytochalasin D and pertussis toxin on H2O2-induced ERK1/2and Akt phosphorylation. Confluent RPTC were pretreated with 5 �M cy-tochalasin D (A) or 100 ng/ml pertussis toxin (B) and exposed to 1 mM H2O2

for 10 min. Cell lysates were separated by SDS-PAGE and immunoblottedwith anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, or anti-Akt anti-bodies. Representative immunoblots from 3 or more experiments are shown.

Fig. 7. Effect of PP1 and AG1478 on H2O2-induced Src, ERK1/2, and Aktphosphorylation. A: confluent RPTC were exposesd to 1 mM H2O2 for theindicated time periods. B: confluent RPTC were pretreated with 10 �M PP1 for1 h and then exposed to 1 mM H2O2 for 5 min. C: confluent RPTC werepretreated with 10 �M AG1478 for 1 h and then exposed to 1 mM H2O2 for5 min. Cell lysates were separated by SDS-PAGE and immunoblotted withanti-phospho Src (Try 416), anti-Src, anti-phospho-ERK1/2, anti-ERK1/2,anti-phospho-Akt, or anti-Akt antibodies. Representative immunoblots from 3or more experiments are shown.



at Selcuk U



ownloaded from


DISCUSSION

Stimulation of the EGFR by ligand binding initiates activa-tion of ERK and Akt, two important signaling molecules.Oxidative stress also activates EGFR, Akt, and ERK1/2; how-ever, the mechanisms by which oxidative stress regulatesactivation of these two signaling molecules are incompletelyunderstood. In this study, we demonstrate that Src mediatesH2O2-induced EGFR transactivation, which is required forERK1/2 activation, whereas H2O2-induced Akt activation ismediated through Src, independent of EGFR transactivation.These findings provide new insights into the molecular mech-anisms by which H2O2 activates separate signaling pathwaysdownstream of Src, leading to the activation of survival mol-ecules in renal epithelial cells.

EGFR transactivation can be induced by many stimuli andoccurs through different pathways (2, 13). EGFR transactiva-tion can occur through intracellular signaling pathways such asPKC, Ca2�, and Src (39). In this study, we demonstrate thatH2O2-induced EGFR transactivation is dependent on the acti-vation of Src, whereas chelation of intracellular Ca2� orinhibition of conventional and novel PKC had no affect onH2O2-induced EGFR transactivation in RPTC. H2O2 inducedthe phosphorylation of Src tyrosine 416, which is required forSrc activity, and the phosphorylation of EGFR tyrosine 845,the Src-mediated phosphorylation site. Furthermore, the inhi-bition of the EGFR did not interfere with H2O2-induced Srcactivation. These data support the concept that Src acts up-stream of the EGFR in H2O2-treated cells. Remarkably, it hasbeen reported that phosphorylation of EGFR tyrosine 845 isable to stabilize the activation loop of EGFR, maintaining theenzyme in the active state and providing a binding surface forprotein substrates (27).

EGFR activation can also occur through autocrine/paracrinerelease of soluble EGF ligands (2). For example, Frank et al.(10) showed that H2O2-stimulated EGFR activation is pro-duced by metalloprotease-dependent HB-EGF cleavage in vas-cular smooth muscle cells. HB-EGF is expressed in RPTC(34), and we examined whether this mechanism is applicable inEGFR activation by H2O2 in RPTC. The use of two metallo-protease inhibitors [GM6001 and 4-(4�-biphenyl)-4-hydroxy-imino-butyric acid] and an HB-EGF inhibitor (CRM 197) did

not reveal that H2O2-induced EGFR transactivation is throughshedding of pro-HB-EGF by metalloproteases in RPTC. Al-though it is possible that other EGFR ligands such as TGF-�,amphiregulin, and betacellulin may be involved in transactiva-tion of EGFR in H2O2-treated cells, it is unlikely because themetalloprotease inhibitors had no effect on the EGFR phos-phorylation induced by H2O2 and it is generally believed thatrelease of the endogenous ligands from their membrane pre-cursor requires metalloprotease activity (39).

The finding that H2O2-stimulated phosphorylation ofERK1/2, but not Akt, was completely blocked by the EGFRinhibitor AG1478 shows that EGFR mediates activation ofERK1/2, but not Akt in RPTC. Consistent with the concept thattransactivation of EGFR involves Src in H2O2-treated RPTC,the Src inhibitor PP1, at a concentration that inhibits the EGFRphosphorylation, also blocked ERK1/2 phosphorylation inH2O2-treated cells. Stress-induced ERK1/2 activation via Srcand EGFR activation is not restricted to H2O2 because UV-induced ERK1/2 activation was also completely abolished byAG1478 and the PP1 (23).

Activation of Akt by H2O2, independent of EGFR, in RPTCis an interesting observation. It is well established that Akt isone of the downstream intracellular signaling molecules ofEGFR on ligand binding (6), and inhibition of the RPTC EGFRalso blocked EGF-stimulated Akt phosphorylation, suggestingthat this pathway is intact in this cell type. Thus the failure ofAG1478 to inhibit H2O2-mediated Akt phosphorylation sug-gests that the transactivated EGFR does not contribute toH2O2-stimulated Akt phosphorylation in RPTC. To our knowl-edge, this is the first example of different requirements for theEGFR in H2O2-induced activation of ERK1/2 and Akt in thesame cell type. Similar to our observation, Roudabush et al.(44) demonstrated that transactivation of EGFR is required forphosphorylation of ERK1/2, but not Akt, in insulin-like growthfactor-treated cells. Our results are in contrast to a previousstudy in which EGFR activation was coupled to the PI3K/Aktsignaling cascade in H2O2-treated Hela cells (52). The reasonfor these differences in cellular response is not known but maybe related to difference in cell types.

Although the RPTC EGFR is not involved in the activationof Akt by H2O2, it seems that Src still functions as the upstreammediator of Akt activation. This is clearly indicated by ourobservation 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 pro-duced by diethylmaleate, a glutathione-depleting agent, wasindependent of RTK phosphorylation and dependent on Srcactivity. The mechanisms by which the Src is coupled to Aktfollowing H2O2 treatment remain clear. One possibility is thatSrc operates as a regulator in other signaling pathways thatmediate activation of Akt. In this regard, it has been reportedthat FAK mediates activation of PI3K/Akt pathways in re-sponse 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 FAKby cytochalasin D did not affect H2O2-induced Akt phosphor-ylation (Fig. 8A), indicating that FAK does not act as amediator of Src in activation of Akt by H2O2 in RPTC.Although H2O2 has been reported to activate Gab1, an adaptorprotein of PI3K, and its activation is sensitive to a Src inhibitor(18), Gab1 is not expressed in kidney (17). Another possibility

Fig. 9. Effect of AG1478 and PP1 on H2O2-induced cell death in RPTC.Confluent RPTC were pretreated with 10 �M AG1478 or 10 �M PP1 for 1 h,exposed to 1 mM H2O2 for 6 h, and cell survival was determined by the MTTassay. Cell viability was expressed as the percentage of the control. Values aremeans � SE from 3 independent experiments.



at Selcuk U



ownloaded from


is that Src regulates Akt activation via altering the function ofPTEN. PTEN is a PI3K-phosphatase that antagonizes PI3Kaction (30). It has been reported that activated Src can inhibitPTEN function, leading to upregulation of Akt activity (29).This finding, in conjunction with a recent observation thatinactivation of cellular PTEN activity by H2O2 results inactivation of Akt (26), suggests that regulation of PTEN by Srcmay be involved in Akt activation in response to oxidativestress.

In addition to PI3K-mediated Akt activation, H2O2-inducedAkt activation can also occur independently of PI3K in ratprimary astrocytes (45). Consequently, alternative mechanismsfor H2O2-induced Akt activation may involve the direct inter-action of Src with Akt, thereby triggering its activity. Thispossibility is suggested by our observations that partial block-ade of H2O2 induced Akt phosphorylation by a Src inhibitor(Fig. 7B) and partial inhibition of Akt phosphorylation by thePI3K inhibitor LY-294002 (Fig. 4). It was reported that Src candirectly regulate Akt activity by phosphorylating tyrosine 315and tyrosine 326 in the activation loop of Akt (20). Thus it islikely that multiple mechanisms are involved in Src-mediatedAkt activation following H2O2 treatment and will be thesubject of future studies.

We also investigated the biological roles of Src and EGFR inresponse to H2O2 injury and demonstrated that inhibition ofSrc further reduced cell viability in H2O2-treated RPTC (Fig.9). This finding is consistent with a previous observation thatSrc mediates the protective action of nitric oxide in cell deathinduced by serum deprivation (49). The effect of Src may bethrough the activation of Akt, as this signaling pathway hasbeen shown to mediate a survival response that counteracts celldeath after H2O2-induced injury in a variety of cell typesincluding renal epithelial cells (14, 19, 52). In contrast, inhi-bition of EGFR did not show a protective effect followingoxidative injury, suggesting that EGFR-mediated signalingpathways are not associated with cytoprotection in renal cells.Two studies demonstrated that the activation of Akt, but notERK, is required for EGF-stimulated protection of embryonickidney epithelial (HEK293) cells from apoptosis induced byFas (12) and tumor necrosis factor-related apoptosis-inducingligand (11).

In summary, the data presented here reveal that H2O2 acti-vation of ERK1/2 and Akt is through different mechanisms inRPTC. Activation of both kinases by H2O2 occurs through aSrc-dependent mechanism. However, activation of ERK1/2,but not Akt, is mediated by EGFR transactivation. Src-medi-ated signaling pathways play an important cytoprotective re-sponse after oxidative injury.

REFERENCES

1. Blobel CP. Metalloprotease-disintegrins: links to cell adhesion and cleav-age of TNF alpha and Notch. Cell 90: 589–592, 1997.

2. Carpenter G. EGF receptor transactivation mediated by the proteolyticproduction of EGF-like agonists. Sci STKE 15: PE1, 2000.

3. Chen K, Vita JA, Berk BC, and Keaney JF Jr. c-Jun N-terminal kinaseactivation by hydrogen peroxide in endothelial cells involves SRC-depen-dent epidermal growth factor receptor transactivation. J Biol Chem 276:16045–16050, 2001.

4. Chen Q, Kinch MS, Lin TH, Burridge K, and Juliano RL. Integrin-mediated cell adhesion activates mitogen-activated protein kinases. J BiolChem 269: 26602–26605, 1994.

5. Cross DA, Alessi DR, Vandenheede JR, McDowell HE, Hundal HS,and Cohen P. The inhibition of glycogen synthase kinase-3 by insulin or

insulin-like growth factor 1 in the rat skeletal muscle cell line L6 isblocked by wortmannin, but not by rapamycin: evidence that wortmanninblocks activation of the mitogen-activated protein kinase pathway in L6cells between Ras and Raf. Biochem J 303: 21–26, 1994.

6. Danielsen AJ and Maihle NJ. The EGF/ErbB receptor family andapoptosis. Growth Factors 20: 1–15, 2002.

7. Droge W. Free radicals in the physiological control of cell function.Physiol Rev 82: 47–95, 2002.

8. Esposito F, Chirico G, Gesualdi NM, Posadas I, Ammendola R, RussoT, Cirino G, and Cimino F. Protein kinase B activation by reactiveoxygen species is independent of tyrosine kinase receptor phosphorylationand requires SRC activity. J Biol Chem 278: 20828–20834, 2003.

9. Frank GD, Eguchi S, Yamakawa T, Tanaka S, Inagami T, and MotleyED. Involvement of reactive oxygen species in the activation of tyrosinekinase and extracellular signal-regulated kinase by angiotensin II. Endo-crinology 141: 3120–3126, 2000.

10. Frank GD, Mifune M, Inagami T, Ohba M, Sasaki T, Higashiyama S,Dempsey PJ, and Eguchi S. Distinct mechanisms of receptor and non-receptor tyrosine kinase activation by reactive oxygen species in vascularsmooth muscle cells: role of metalloprotease and protein kinase C-delta.Mol Cell Biol 23: 1581–1589, 2003.

11. Gibson EM, Henson ES, Haney N, Villanueva J, and Gibson SB.Epidermal growth factor protects epithelial-derived cells from tumornecrosis factor-related apoptosis-inducing ligand-induced apoptosis byinhibiting cytochrome c release. Cancer Res 62: 488–496, 2002.

12. Gibson S, Tu S, Oyer R, Anderson SM, and Johnson GL. Epidermalgrowth factor protects epithelial cells against Fas-induced apoptosis.Requirement for Akt activation. J Biol Chem 274: 17612–17618, 1999.

13. Gschwind A, Zwick E, Prenzel N, Leserer M, and Ullrich A. Cellcommunication networks: epidermal growth factor receptor transactiva-tion as the paradigm for interreceptor signal transmission. Oncogene 20:1594–1600, 2001.

14. Guyton KZ, Gorospe M, Kensler TW, and Holbrook NJ. Mitogen-activated protein kinase (MAPK) activation by butylated hydroxytoluenehydroperoxide: implications for cellular survival and tumor promotion.Cancer Res 56: 3480–3485, 1996.

15. Guyton KZ, Liu Y, Gorospe M, Xu Q, and Holbrook NJ. Activation ofmitogen-activated protein kinase by H2O2. Role in cell survival followingoxidant injury. J Biol Chem 271: 4138–4142, 1996.

16. Hawes BE, Luttrell LM, van Biesen T, and Lefkowitz RJ. Phosphati-dylinositol 3-kinase is an early intermediate in the G�-mediated mitogen-activated protein kinase signaling pathway. J Biol Chem 271: 12133–12136, 1996.

17. Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK, andWong AJ. A Grb2-associated docking protein in EGF- and insulin-receptor signalling. Nature 379: 560–564, 1996.

18. Holgado-Madruga M and Wong AJ. Gab1 is an integrator of cell deathversus cell survival signals in oxidative stress. Mol Cell Biol 23: 4471–4484, 2003.

19. Hung CC, Ichimura T, Stevens JL, and Bonventre JV. Protection ofrenal epithelial cells against oxidative injury by endoplasmic reticulumstress preconditioning is mediated by ERK1/2 activation. J Biol Chem278: 29317–29326, 2003.

20. Jiang T and Qiu Y. Interaction between Src and a C-terminal proline-richmotif of Akt is required for Akt activation. J Biol Chem 278: 15789–15793, 2003.

21. Johnson LL, Bornemeier DA, Janowicz JA, Chen J, Pavlovsky AG,and Ortwine DF. Effect of species differences on stromelysin-1 (MMP-3)inhibitor potency. An explanation of inhibitor selectivity using homologymodeling and chimeric proteins. J Biol Chem 274: 24881–24887, 1999.

22. Kandel ES and Hay N. The regulation and activities of the multifunc-tional serine/threonine kinase Akt/PKB. Exp Cell Res 253: 210–229, 1999.

23. Kitagawa D, Tanemura S, Ohata S, Shimizu N, Seo J, Nishitai G,Watanabe T, Nakagawa K, Kishimoto H, Wada T, Tezuka T,Yamamoto T, Nishina H, and Katada T. Activation of extracellularsignal-regulated kinase by ultraviolet is mediated through Src-dependentepidermal growth factor receptor phosphorylation. Its implication in ananti-apoptotic function. J Biol Chem 277: 366–371, 2002.

24. Knebel A, Rahmsdorf HJ, Ullrich A, and Herrlich P. Dephosphoryla-tion of receptor tyrosine kinases as target of regulation by radiation,oxidants or alkylating agents. EMBO J 15: 5314–5325, 1996.

25. Lehr S, Kotzka J, Herkner A, Sikmann A, Meyer HE, Krone W, andMuller-Wieland D. Identification of major tyrosine phosphorylation sites



at Selcuk U



ownloaded from


in the human insulin receptor substrate Gab-1 by insulin receptor kinase invitro. Biochemistry 39: 10898–10907, 2000.

26. Leslie NR, Bennett D, Lindsay YE, Stewart H, Gray A, and DownesCP. Redox regulation of PI 3-kinase signalling via inactivation of PTEN.EMBO J 22: 5501–5510, 2003.

27. Leu TH and Maa MC. Functional implication of the interaction betweenEGF receptor and C-SRC. Front Biosci 1: S28–S38, 2003.

28. Li C and Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 282: C227–C241, 2002.

29. Lu Y, Yu Q, Liu JH, Zhang J, Wang H, Koul D, McMurray JS, FangX, Yung WK, Siminovitch KA, and Mills GB. Src family protein-tyrosine kinases alter the function of PTEN to regulate phosphatidylino-sitol 3-kinase/AKT cascades. J Biol Chem 278: 40057–40066, 2003.

30. Maehama T and Dixon JE. PTEN: a tumour suppressor that functions asa phospholipid phosphatase. Trends Cell Biol 9: 125–128, 1999.

31. Martindale JL and Holbrook NJ. Cellular response to oxidative stress:signaling for suicide and survival. J Cell Physiol 192: 1–15, 2002.

32. Mitamura T, Higashiyama S, Taniguchi N, Klagsbrun M, andMekada E. Diphtheria toxin binds to the epidermal growth factor (EGF)-like domain of human heparin-binding EGF-like growth factor/diphtheriatoxin receptor and inhibits specifically its mitogenic activity. J Biol Chem270: 1015–1019, 1995.

33. Morino N, Mimura T, Hamasaki K, Tobe K, Ueki K, Kikuchi K,Takehara K, Kadowaki T, Yazaki Y, and Nojima Y. Matrix/integrininteraction activates the mitogen-activated protein kinase, p44erk-1 andp42erk-2. J Biol Chem 270: 269–273, 1995.

34. Nakagawa T, Hayase Y, Sasahara M, Haneda M, Kikkawa R, Higash-iyama S, Taniguchi N, and Hazama F. Distribution of heparin-bindingEGF-like growth factor protein and mRNA in the normal rat kidneys.Kidney Int 51: 1774–1779, 1997.

35. Nishida M, Maruyama Y, Tanaka R, Kontani K, Nagao T, andKurose H. G�i and G�o are target proteins of reactive oxygen species.Nature 408: 492–495, 2000.

36. Nishinaka T and Yabe-Nishimura C. EGF receptor-ERK pathway is themajor signaling pathway that mediates upregulation of aldose reductaseexpression under oxidative stress. Free Radic Biol Med 31: 205–216,2001.

37. Nowak G and Schnellmann RG. Improved culture conditions stimulategluconeogenesis in primary cultures of renal proximal tubule cells. Am JPhysiol Cell Physiol 268: C1053–C1061, 1995.

38. Partik G, Hochegger K, Schorkhuber M, and Marian B. Inhibition ofepidermal growth factor receptor-dependent signalling by tyrphostins A25and AG1478 blocks growth and induces apoptosis in colorectal tumor cellsin vitro. J Cancer Res Clin Oncol 125: 379–388, 1999.

39. Prenzel N, Fischer OM, Streit S, Hart S, and Ullrich A. The epidermalgrowth factor receptor family as a central element for cellular signaltransduction and diversification. Endocr Relat Cancer 8: 11–31, 2001.

40. Prenzel N, Zwick E, Daub H, Leserer M, Abraham R, Wallasch C,and Ullrich A. EGF receptor transactivation by G protein-coupled recep-

tors requires metalloproteinase cleavage of proHB-EGF. Nature 402:884–888, 1999.

41. Prenzel N, Zwick E, Leserer M, and Ullrich A. Tyrosine kinasesignalling in breast cancer. Epidermal growth factor receptor: convergencepoint for signal integration and diversification. Breast Cancer Res 2:184–190, 2000.

42. Raab G and Klagsbrun M. Heparin-binding EGF-like growth factor.Biochim Biophys Acta 9: F179–F199, 1997.

43. Roghani M, Becherer JD, Moss ML, Atherton RE, Erdjument-Bro-mage H, Arribas J, Blackburn RK, Weskamp G, Tempst P, andBlobel CP. Metalloprotease-disintegrin MDC9: intracellular maturationand catalytic activity. J Biol Chem 274: 3531–3540, 1999.

44. Roudabush FL, Pierce KL, Maudsley S, Khan KD, and Luttrell LM.Transactivation of the EGF receptor mediates IGF-1-stimulated shc phos-phorylation and ERK1/2 activation in COS-7 cells. J Biol Chem 275:22583–22589, 2000.

45. Salsman S, Felts N, Pye QN, Floyd RA, and Hensley K. Induction ofAkt phosphorylation in rat primary astrocytes by H2O2 occurs upstream ofphosphatidylinositol 3-kinase: no evidence for oxidative inhibition ofPTEN. Arch Biochem Biophys 386: 275–280, 2001.

46. Sato K, Sato A, Aoto M, and Fukami Y. c-Src phosphorylates epidermalgrowth factor receptor on tyrosine 845. Biochem Biophys Res Commun215: 1078–1087, 1995.

47. Shaw M, Cohen P, and Alessi DR. The activation of protein kinase B byH2O2 or heat shock is mediated by phosphoinositide 3-kinase and not bymitogen-activated protein kinase-activated protein kinase-2. Biochem J336: 241–246, 1998.

48. Sonoda Y, Watanabe S, Matsumoto Y, Aizu-Yokota E, and KasaharaT. FAK is the upstream signal protein of the phosphatidylinositol 3-ki-nase-Akt survival pathway in hydrogen peroxide-induced apoptosis of ahuman glioblastoma cell line. J Biol Chem 274: 10566–10570, 1999.

49. Tejedo JR, Ramirez R, Cahuana GM, Rincon P, Sobrino F, andBedoya FJ. Evidence for involvement of c-Src in the anti-apoptotic actionof nitric oxide in serum-deprived RINm5F cells. Cell Signal 13: 809–817,2001.

50. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T,Ajakane M, Baudet V, Boissin P, Boursier E, and Loriolle F. Thebisindolylmaleimide GF 109203X is a potent and selective inhibitor ofprotein kinase C. J Biol Chem 266: 15771–15781, 1991.

51. Vojtek AB and Der CJ. Increasing complexity of the Ras signalingpathway. J Biol Chem 273: 19925–19928, 1998.

52. Wang X, McCullough KD, Franke TF, and Holbrook NJ. Epidermalgrowth factor receptor-dependent Akt activation by oxidative stress en-hances cell survival. J Biol Chem 275: 14624–14631, 2000.

53. Zachary I. Focal adhesion kinase. Int J Biochem Cell Biol 29: 929–934,1997.

54. Zhu X and Assoian RK. Integrin-dependent activation of MAP kinase: alink to shape-dependent cell proliferation. Mol Biol Cell 6: 273–282, 1995.



at Selcuk U



ownloaded from


Corrigendum

Volume 286, May 2004Volume 55, May 2004

Pages F858–F865: Shougang Zhougang and Rick G. Schnellmann. “H2O2-induced transactivationof EGF receptor requires Src and mediates ERK1/2, but not Akt, activation in renal cells.” On pageF858, the first author’s last name was misspelled. The first author’s name should have appeared asShougang Zhuang.

Am J Physiol Renal Physiol 287: F857, 2004;10.1152/ajprenal.00256.2004.

0363-6127/04 $5.00 Copyright© 2004 the American Physiological Societyhttp://www.ajprenal.org F857

at Selcuk U



ownloaded from


Documents

c vitamini H2O2 induced transactivation of EGF receptor requires H.pdf