H 2 O 2 -induced transactivation of EGF receptor requires Src and mediates ERK1/ - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 08:14 |只看该作者 |倒序浏览 |打印

作者：Shougang Zhougang and Rick G. Schnellmann作者单位：Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425 5 [+ M0 s1 [- C  L. G; |




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      【摘要】" {+ ~: f* F& N8 L
   Although oxidative stress activates epidermal growth factor receptor (EGFR), ERK1/2, and Akt in a number of cell types, the mechanisms by which oxidative stress activates these kinases are not well defined in renal epithelial cells. Exposure of primary cultures of rabbit renal proximal tubular cells to hydrogen peroxide (H 2 O 2 ) stimulated Src, EGFR, ERK1/2, and Akt activation in a time-dependent manner as determined by the phosphorylation of each protein. The Src inhibitor PP1 completely blocked EGFR, ERK1/2, and Akt phosphorylation following H 2 O 2 exposure. In contrast, blockade of the EGFR by AG1478 inhibited phosphorylation of ERK1/2 but not Src or Akt phosphorylation following H 2 O 2 exposure. Exogenous EGF stimulated EGFR, ERK1/2, and Akt activation and the EGFR inhibitor blocked phorphorylation of ERK1/2 and Akt. The presence of PP1, but not AG1478, significantly accelerated H 2 O 2 -induced cell death. These results suggest that Src mediates H 2 O 2 -induced EGFR transactivation. H 2 O 2 - and EGF-induced ERK1/2 activation is mediated by EGFR, whereas Akt is activated by Src independent of EGFR following H 2 O 2 exposure. Src-mediated EGFR transactivation contributes to a survival response following oxidative injury. / Z4 g. G+ M% e  o& z/ }  I# b: f
      【关键词】 heparinbinding epidermal growth factor phosphatidylinositol kinase renal proximal tubule; {& n9 @0 y9 K- X8 H. ~
   REACTIVE OXYGEN SPECIES (ROS), including hydrogen peroxide (H 2 O 2 ), superoxide radical, and hydroxyl radical, have been implicated in the pathogenesis of renal ischemia and reperfusion injury ( 28 ). Although ROS generation following renal ischemia and reperfusion injury may induce apoptosis and oncosis in renal proximal tubular cells (RPTC), ROS also mediate a number of adaptive biological responses and regulate the expression of a variety of genes that are involved in renal survival and regeneration ( 7 ). Among intracellular signaling pathways triggered, ERK and Akt have been shown to regulate cell survival in response to oxidative stress ( 7 ) and mediate cell proliferation following growth factor stimulation ( 51 ).6 R# X  Z1 @1 \+ a$ T+ c& Q

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 ).
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ROS have been shown to activate several RTK ( 31, 36 ). H 2 O 2 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, H 2 O 2 -induced activation of EGFR results in the activation of the PI3K/Akt pathway ( 52 ). Initially, H 2 O 2 -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 H 2 O 2 can occur through other mechanisms. Frank et al. ( 9 ) demonstrated that metalloprotease-dependent HB-EGF cleavage is required for EGFR activation by H 2 O 2 in vascular smooth muscle cells, and Chen et al. ( 3 ) showed that H 2 O 2 -stimulated EGFR activation is dependent on Src in endothelial cells.
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Although growth factor receptors are involved in the activation of ERK1/2 and Akt by H 2 O 2, 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 H 2 O 2 activation of Src, EGFR, ERK1/2, and Akt in RPTC.! y9 o* o* ^1 k7 Z3 F" a  g

MATERIALS AND METHODS" {7 W0 _  E: K( t$ F

Chemicals and regents. GM6001, 4-(4'-biphenyl)-4-hydroxyimino-butyric acid, and GF109203X were obtained from Calbiochem (San Diego, CA). Human-recombinant EGF was purchased from R&D Systems (Minneapolis, MN). LY-294002 was obtained from Cell Signaling Technology (Beverly, MA). AG1478 and BAPTA-AM were obtained from Biomol (Plymouth Meeting, PA). All other chemicals 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) were obtained from Cell Signaling Technology. Antibodies to ERK1/2 (no. 06-182) and EGFR (Sc-03-G) were purchased from BD Laboratories (San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. All antibodies were used at 1:1,000 for immunoblotting.# p  P2 J! O- E

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.+ [5 E4 p/ Q/ E" ]1 R0 G

Preparation of cell lysates and immunoblot analysis. Confluent RPTC were used for all experiments. After treatment with inhibitors and/or H 2 O 2 for various times, RPTC were washed twice with PBS without Ca 2  and Mg 2  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.- ]$ T6 l/ R' s! u) `
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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 H 2 O 2, 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).

RESULTS- w6 b2 F/ U6 @9 ^9 M& ~
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To determine the effect of H 2 O 2 on ERK1/2 and Akt activation in RPTC, we examined the phosphorylation of Akt and ERK1/2 using phospho-specific antibodies and immunoblot analysis. As shown in Fig. 1 A, 1 mM H 2 O 2 stimulated phosphorylation of ERK1/2; stimulation occurred within 5 min, reached a maximum at 30 min, and was sustained through 120 min of treatment. Akt phosphorylation also increased within 5 min of H 2 O 2 exposure and increased over the 120-min incubation period. Increasing concentrations of H 2 O 2 induced ERK1/2 and Akt phosphorylation with a maximal effect at 250 µM for ERK1/2 and 1 mM for Akt ( Fig. 1 B ). Total ERK1/2 and Akt levels were determined using antibodies that recognize these proteins, independent of their phosphorylation state. Total ERK1/2 and Akt levels did not change over the 120-min exposure period.- D" q- D* d, X& ?8 Z

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

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. 1 C ). Densitometry confirmed the time-dependent changes in p-Akt levels following EGF and H 2 O 2 treatment. These data demonstrate that the ERK1/2 and Akt signaling pathways are activated in response to H 2 O 2 and EGF in RPTC, but the kinetics of ERK1/2 and Akt activation by EGF are transient compared with H 2 O 2., i+ C( }% x6 v- G7 |7 y8 q

To evaluate the role of EGFR in H 2 O 2 - and EGF-induced ERK1/2 and Akt activation in RPTC, we first determined the phosphorylation of EGFR tyrosine 1068 by H 2 O 2 and EGF using immunoblot analysis and a phospho-specific antibody for EGFR tyrosine 1068. As shown in Fig. 2, A and C, 1 mM H 2 O 2 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 ).' M+ s0 m2 l6 d
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Fig. 2. H 2 O 2 and EGF stimulate phosphorylation of EGF receptor (EGFR). Confluent RPTC were stimulated with 1 mM H 2 O 2 ( A ) or 10 ng/ml EGF ( B ) for the indicated time periods. Cell lysates were separated by SDS-PAGE and immunoblotted 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 the percentage of expression relative to that in control. Values are means ± SE of 3 independent experiments.2 K. W0 A4 ]% _9 c! i8 T3 k

To determine whether H 2 O 2 -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 H 2 O 2 -induced tyrosine 1068 phosphorylation of EGFR in a concentration-dependent manner (0.1-10 µM), with complete inhibition at 1 µM ( Fig. 3 A ). AG1478 also decreased H 2 O 2 -induced ERK1/2 phosphorylation to basal levels ( Fig. 3 A ). In contrast, H 2 O 2 -induced activation of Akt was not affected by AG1478 ( Fig. 3 A ). Similar results were observed when RPTC were exposed to a lower concentration of H 2 O 2 (0.25 mM) in the presence of AG1478 ( Fig. 3 B ). 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. 3 B ). 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 H 2 O 2 is dependent on the EGFR, whereas H 2 O 2 -induced Akt activation is not EGFR dependent. In contrast, ERK1/2 and Akt activation following EGF exposure is EGFR dependent.
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Fig. 3. Effects of AG1478 on H 2 O 2 and EGF-induced ERK1/2 and Akt phosphorylation. Confluent RPTC were pretreated with 0.1-10 µM AG1478 for 1 h and then exposed to 1 mM H 2 O 2 for 10 min ( A ), or 10 µM AG1478 for 1 h and then 0.25 mM H 2 O 2 ( B ), or 10 ng/ml EGF for 10 min ( C ). Cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-Tyr1068 EGFR, anti-EGFR, anti-phospo-ERK1/2, anti-ERK1/2, anti-phospho-Akt, and anti-Akt antibodies. Representative immunoblots from 3 or more experiments are shown.( Y+ J8 |) G' S- Z" Y) g

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 H 2 O 2 -mediated ERK1/2 activation requires PI3K in RPTC, we measured H 2 O 2 -stimulated ERK1/2 phosphorylation in the presence of the PI3K inhibitor LY-294002. Treatment of RPTC with LY-294002 decreased, but did not block, H 2 O 2 -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 H 2 O 2 -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 H 2 O 2. Unlike EGF-mediated Akt phosphorylation, it is possible that Akt phosphorylation is also mediated by PI3K-independent mechanisms in H 2 O 2 -treated cells.

Fig. 4. Effect of LY-294002 on H 2 O 2 -induced phosphorylation of Akt and ERK1/2. Confluent RPTC were pretreated with 20 µM LY-294002 for 15 min and exposed to 1 mM H 2 O 2 for 10 min. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, and anti-Akt antibodies. Representative immunoblots from 3 or more experiments are shown.' L- M4 P# V- @2 o& @
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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 H 2 O 2 -induced ERGF activation using CRM 197, a diphtheria toxin mutant that specifically blocks the action of HB-EGF ( 32 ). As shown in Fig. 5 A, H 2 O 2 -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).# G! r6 `! \2 y" b8 ]6 T

Fig. 5. H 2 O 2 -induced EGFR phosphorylation (Try 1068) does not require HB-EGF or a metalloprotease-dependent mechanism. Confluent RPTC were pretreated with 10 µM CRM 197 ( A ), 10 µM GM 6001, or 20 µM [4-(4'-biphenyl)-4-hydroxyimino-butyric acid (HBA); B ] for 1 h and exposed to 1 mM H 2 O 2 for 10 min. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho EGFR (Try1068) and anti-EGF receptor antibodies. Representative immunoblots from 3 or more experiments are shown.0 E1 K# `8 d7 L  o7 z8 l

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 H 2 O 2 -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. 5 B, treatment with either inhibitor did not alter EGFR phosphorylation by H 2 O 2. H 2 O 2 -induced ERK1/2 activation was also not affected by these inhibitors (data not shown). These results suggest that H 2 O 2 -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 H 2 O 2 exposure, we assessed the role of intracellular Ca 2 , PKC, and Src in H 2 O 2 -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 Ca 2 , or GF109203X (10 µM), an inhibitor of conventional and novel PKC ( 50 ), did not affect H 2 O 2 -induced EGFR phosphorylation at Tyr1068. In contrast, H 2 O 2 -induced phosphorylation of EGFR at this residue was abolished by PP1, a selective inhibitor of Src ( Fig. 6 C ). However, PP1 did not effect EGFR phosphorylation following EGF exposure ( Fig. 6 D ). These data suggest that H 2 O 2 -induced EGFR activation is dependent on Src, but not Ca 2  or conventional and novel PKC. In contrast, EGF activation of the EGFR is not Src mediated.5 J( i/ C" b2 V3 Y! d! F& M
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Fig. 6. Effects of an intracellular calcium chelator, PKC inhibitor, and Src inhibitor on H 2 O 2 -induced EGFR phosphorylation. Confluent RPTC were pretreated with 10 µM BAPTA-AM ( A ), 10 µM GF109203X (GF; B ), or 10 µM PP1 ( C and D ) for 1 h and exposed to 1 mM H 2 O 2 for 10 min. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho EGFR (Tyr1068) antibody ( A, B, and D ), anti-phospho EGFR (Tyr845; C and D ), and anti-EGFR ( A - D ) antibodies. Representative immunoblots from 3 or more experiments are shown.' N0 Q4 w7 C- d5 _. Z* |9 X
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Because Src can activate the EGFR by direct phosphorylation of tyrosine 845 ( 46 ), we evaluated the effect of H 2 O 2 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 H 2 O 2 exposure and that this response was blocked by PP1 ( Fig. 6 C ). 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.8 x* a. k* _4 W' L7 ?8 u( N6 P

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 H 2 O 2 on Src phosphorylation and demonstrated that H 2 O 2 stimulated Src tyrosine 416 phosphorylation within 5 min and the response was sustained through 120 min of treatment ( Fig. 7 A ). The Src inhibitor PP1 blocked the increase in Src tyrosine 416 phosphorylation ( Fig. 7 B ). These results suggest that Src is an early target of H 2 O 2.' z6 g: o% i1 Y. u

Fig. 7. Effect of PP1 and AG1478 on H 2 O 2 -induced Src, ERK1/2, and Akt phosphorylation. A : confluent RPTC were exposesd to 1 mM H 2 O 2 for the indicated time periods. B : confluent RPTC were pretreated with 10 µM PP1 for 1 h and then exposed to 1 mM H 2 O 2 for 5 min. C : confluent RPTC were pretreated with 10 µM AG1478 for 1 h and then exposed to 1 mM H 2 O 2 for 5 min. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho Src (Try 416), anti-Src, anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, or anti-Akt antibodies. Representative immunoblots from 3 or more experiments are shown.% n6 d, p1 ^% Z
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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 H 2 O 2 ( Fig. 7 C ). The next series of experiments determined the effect of Src inhibition on H 2 O 2 -induced ERK1/2 and Akt phosphorylation. The Src inhibitor PP1 completely inhibited basal and H 2 O 2 -induced ERK1/2 phosphorylation ( Fig. 7 B ). PP1 blocked H 2 O 2 -induced Akt phosphorylation but not basal Akt phosphorylation. As described above, Akt activation was not associated with the EGFR ( Fig. 3 A ). These data strongly suggest that Src functions as an upstream activator of Akt and ERK1/2 via distinct mechanisms.# h/ Y$ m& E4 A: \% }( p! d
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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 H 2 O 2 -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 H 2 O 2 treatment. In contrast, H 2 O 2 -induced Akt phosphorylation was not affected by cytochalasin D. Pertussis toxin inactivates G i/o proteins. Previous studies showed that treatment with H 2 O 2 directly activates purified heterotrimeric G i and G o but not G s in vitro ( 35 ). Pertussis toxin did not attenuate H 2 O 2 -induced phosphorylation of ERK1/2 or Akt in RPTC ( Fig. 8 B ). Collectively, these data reveal that H 2 O 2 -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 G i/o proteins in RPTC following H 2 O 2 exposure.# \) L; U1 f9 ?2 C& G2 M
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Fig. 8. Effect of cytochalasin D and pertussis toxin on H 2 O 2 -induced ERK1/2 and Akt phosphorylation. Confluent RPTC were pretreated with 5 µM cytochalasin D ( A ) or 100 ng/ml pertussis toxin ( B ) and exposed to 1 mM H 2 O 2 for 10 min. Cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-Akt, or anti-Akt antibodies. Representative immunoblots from 3 or more experiments are shown./ \) ]0 b2 C( Z* T% q( j
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Previous reports showed that activation of Akt and ERK1/2 by H 2 O 2 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 H 2 O 2 exposure, we investigated the influence of EGF and Src activation on H 2 O 2 -induced cell death. Exposure of RPTC to H 2 O 2 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 H 2 O 2 exposure. In contrast, PP1 pretreatment resulted in a further decrease in viability following H 2 O 2 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 H 2 O 2.5 b) B3 x% {5 D( h
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Fig. 9. Effect of AG1478 and PP1 on H 2 O 2 -induced cell death in RPTC. Confluent RPTC were pretreated with 10 µM AG1478 or 10 µM PP1 for 1 h, exposed to 1 mM H 2 O 2 for 6 h, and cell survival was determined by the MTT assay. Cell viability was expressed as the percentage of the control. Values are means ± SE from 3 independent experiments.) g' G, k0 z; d$ ?9 ^

DISCUSSION

Stimulation of the EGFR by ligand binding initiates activation of ERK and Akt, two important signaling molecules. Oxidative stress also activates EGFR, Akt, and ERK1/2; however, the mechanisms by which oxidative stress regulates activation of these two signaling molecules are incompletely understood. In this study, we demonstrate that Src mediates H 2 O 2 -induced EGFR transactivation, which is required for ERK1/2 activation, whereas H 2 O 2 -induced Akt activation is mediated through Src, independent of EGFR transactivation. These findings provide new insights into the molecular mechanisms by which H 2 O 2 activates separate signaling pathways downstream of Src, leading to the activation of survival molecules in renal epithelial cells.8 O3 W7 R) K# l" q) c# F

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, Ca 2 , and Src ( 39 ). In this study, we demonstrate that H 2 O 2 -induced EGFR transactivation is dependent on the activation of Src, whereas chelation of intracellular Ca 2  or inhibition of conventional and novel PKC had no affect on H 2 O 2 -induced EGFR transactivation in RPTC. H 2 O 2 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 H 2 O 2 -induced Src activation. These data support the concept that Src acts upstream of the EGFR in H 2 O 2 -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 H 2 O 2 -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 H 2 O 2 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 H 2 O 2 -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 H 2 O 2 -treated cells, it is unlikely because the metalloprotease inhibitors had no effect on the EGFR phosphorylation induced by H 2 O 2 and it is generally believed that release of the endogenous ligands from their membrane precursor requires metalloprotease activity ( 39 ).- n, D! n$ Z' ~4 i

The finding that H 2 O 2 -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 H 2 O 2 -treated RPTC, the Src inhibitor PP1, at a concentration that inhibits the EGFR phosphorylation, also blocked ERK1/2 phosphorylation in H 2 O 2 -treated cells. Stress-induced ERK1/2 activation via Src and EGFR activation is not restricted to H 2 O 2 because UV-induced ERK1/2 activation was also completely abolished by AG1478 and the PP1 ( 23 ).

Activation of Akt by H 2 O 2, 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 H 2 O 2 -mediated Akt phosphorylation suggests that the transactivated EGFR does not contribute to H 2 O 2 -stimulated Akt phosphorylation in RPTC. To our knowledge, this is the first example of different requirements for the EGFR in H 2 O 2 -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 H 2 O 2 -treated Hela cells ( 52 ). The reason for these differences in cellular response is not known but may be related to difference in cell types.. y$ g1 N5 t0 s  B% P2 Q% S

Although the RPTC EGFR is not involved in the activation of Akt by H 2 O 2, 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 H 2 O 2 -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 H 2 O 2 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 H 2 O 2 -stimulated Akt phosphorylation. However, inhibition of FAK by cytochalasin D did not affect H 2 O 2 -induced Akt phosphorylation ( Fig. 8 A ), indicating that FAK does not act as a mediator of Src in activation of Akt by H 2 O 2 in RPTC. Although H 2 O 2 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 H 2 O 2 results in activation of Akt ( 26 ), suggests that regulation of PTEN by Src may be involved in Akt activation in response to oxidative stress.
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In addition to PI3K-mediated Akt activation, H 2 O 2 -induced Akt activation can also occur independently of PI3K in rat primary astrocytes ( 45 ). Consequently, alternative mechanisms for H 2 O 2 -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 H 2 O 2 induced Akt phosphorylation by a Src inhibitor ( Fig. 7 B ) 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 H 2 O 2 treatment and will be the subject of future studies.# i  Q/ I/ U0 |1 }# j+ X
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We also investigated the biological roles of Src and EGFR in response to H 2 O 2 injury and demonstrated that inhibition of Src further reduced cell viability in H 2 O 2 -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 H 2 O 2 -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 ).8 L( ~+ s, ?2 R0 [/ O) s

In summary, the data presented here reveal that H 2 O 2 activation of ERK1/2 and Akt is through different mechanisms in RPTC. Activation of both kinases by H 2 O 2 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.- N% O' |+ J5 f
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