AJP - Renal Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Renal Physiol 293: F895-F903, 2007. First published July 11, 2007; doi:10.1152/ajprenal.00227.2007
0363-6127/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/F895    most recent
00227.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kiley, S. C.
Right arrow Articles by Chevalier, R. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kiley, S. C.
Right arrow Articles by Chevalier, R. L.

Species differences in renal Src activity direct EGF receptor regulation in life or death response to EGF

Susan C. Kiley and Robert L. Chevalier

Department of Pediatrics, University of Virginia School of Medicine, Charlottesville, Virginia

Submitted 15 May 2007 ; accepted in final form 28 June 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 GRANTS
 REFERENCES
 
In rodent models of obstructive nephropathy, exogenous epidermal growth factor (EGF) attenuates tubule cell death in rats and exacerbates cell death in mice. To identify species differences in EGF receptor (EGFR) regulation and signaling, cell lysates were prepared from rat, mouse, and human proximal tubule cells (PTC) and compared by immunoblot analysis for expression and phosphorylation of Src and EGFR. Frozen kidney tissue was also analyzed. Results indicate mouse PTC have constitutive Src- and EGFR-kinase activities not detected in rat or human PTC. Immunoblots of rat, mouse, and human kidney homogenates confirmed this finding in vivo. Src-specific inhibitor PP2 and EGFR kinase inhibitor AG1478 decreased EGF-induced apoptosis in mouse PTC by 74% (P < 0.001) and 70% (P < 0.001), respectively. Expression of a constitutive Src mutant cDNA in rat PTC rendered cells susceptible to EGF-induced death. EGF decreased stretch-induced apoptosis by 66% (P < 0.001) relative to vehicle control in human PTC, similar to rat PTC response. We conclude that elevated Src activity in mouse tubular cells alters downstream EGFR signaling and increases susceptibility to EGF-induced cell death. The unexpected finding that a therapeutic agent (EGF) in rats is detrimental in mice underscores the importance of determining which animal best represents the response of human kidneys to a given agent.

activated Src; mouse proximal tubule cells; apoptosis; tubule injury

EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) regulation and signaling are dictated by ligand binding, dimerization partners, and phosphorylation of specific tyrosine residues. The EGFR family consists of four closely related receptor tyrosine kinases (Human ErbB Receptors 1-4, HER 1-4) and each HER can be activated by several ligands (36). The EGFR (HER1) can pair with itself to form a homodimer, or with all three remaining HERs to form a heterodimer. Ligand binding to EGFR triggers receptor dimerization and ligand availability most likely dictates dimer composition (23, 25, 26). Receptor dimerization triggers trans-autophosphorylation of several tyrosines (Y) that function as docking sites for Src homology 2 domain signaling proteins; PLC{gamma} [Y992], GRB1/2, and GAB1 [Y1068] (14, 28). Phosphorylation of Y845 in the activation loop of the kinase domain is critical for maintaining EGFR activity and function (6, 32), as well as transactivation by G protein-coupled receptors (8). Thus upstream regulation of the EGFR influences signaling through multiple downstream pathways leading to diverse biological responses that include cell proliferation, differentiation, tumor progression, and apoptosis.

The EGFR has been implicated in the progression of renal injury and disease characterized by profound epithelial cell proliferation. For this reason, a number of therapeutic approaches have been advanced to limit EGFR activity. Terzi et al. (31) took a genetic approach using transgenic mice with functionally inactive EGFR in two models of renal injury. Inactivation of the EGFR attenuated tubule cell injury following renal ablation and prolonged renal ischemia (31). In polycystic kidney disease (PKD), EGFR overexpression, mislocalization, and receptor tyrosine kinase activity in the collecting ducts drive epithelial cell proliferation in cyst formation and enlargement. Richards et al. (27) crossed mice with the cystic orpk mutation and mice with the waved-2 EGFR mutation that reduces EGFR activity. Mice expressing both mutant genes (orpk+/waved-2+) exhibited decreased cyst formation and improved kidney function relative to orpk+/+ mice, suggesting that a tyrosine kinase inhibitor (TKI) specific for EGFR may be an effective therapeutic strategy for PKD. Avner's group subsequently tested an EGFR TKI in rodent models of autosomal recessive and autosomal dominant PKD. The TKI effectively reduced cyst formation and improved renal function in the BPK mice and Han:SPRD rats, but it had no effect and in some cases worsened the pathogenesis of PKD in PCK rats (29, 34, 35). Unlike other rodent models of PKD, EGFR is expressed at normal levels and is localized in the basolateral membrane of PCK rats (35). Decreasing homeostatic EGFR activity with an EGFR TKI under these conditions was detrimental, aggravating instead of ameliorating renal pathology. Together, these studies suggest the efficacy of an EGFR-based therapeutic strategy may depend on abnormal abundance, activity, and/or localization of the EGFR in states of injury and disease.

Unilateral ureteral obstruction (UUO), similar to renal ablation and ischemia, induces extensive injury to renal tubules that culminates in apoptosis and necrosis (10). Activation of the EGFR with the administration of mitogenic EGF to animals with UUO should in theory promote tissue regeneration. In rats with chronic UUO, exogenous EGF significantly reduced tubule cell apoptosis by 80% in neonates (12) and by 50% in adults (17), relative to untreated animals. Using a cell culture model that simulates stretched cells lining dilated tubules in a hydronephrotic kidney, EGF reduced apoptosis in rat proximal tubule cells (PTC) by 50% relative to vehicle control, a response mediated by ERK1/2 signaling downstream of EGFR activation (18). However, when similar experiments were performed in a mouse model of UUO or with stretched mouse PTC in vitro, the response to EGF was very different. EGF administration to neonatal mice with UUO tended to increase both tubular apoptosis and atrophy relative to vehicle control (19). Addition of EGF to mouse PTC cultures undergoing stretch significantly increased apoptosis relative to vehicle control. Studies were also performed in mutant neonatal mice lacking endogenous EGF and mice with reduced EGFR tyrosine kinase activity (waved-2 mutant mice). Tubular apoptosis was 50% lower in the obstructed kidney of EGF knockout or waved-2 mice relative to wild-type and heterozygous animals. From these studies, we concluded that EGF is a survival factor in the neonatal rat, but potentiates tubule cell death in the neonatal mouse. Species differences maintained in cultured cells suggest that differences in EGFR signaling underlie these opposing effects. The present study investigates the molecular basis for species differences in EGF-stimulated EGFR activation.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 GRANTS
 REFERENCES
 
Cell culture, growth factors, and inhibitors. Temperature-sensitive SV-40 immortalized rat PTC (IRPTC) were grown in complete medium at a permissive temperature as described (30). Confluent cultures were shifted to nonpermissive temperature 39°C for 16 h to shut-off the transformed phenotype and induce normal epithelial cell differentiation before initiation of each experiment. Immortalized mouse PTC, PKSV-PR and -PCT, were derived and grown as described by Cartier et al. (11), except that EGF was omitted from normal growth conditions after initial passages. HK2 immortalized human PTC (American Type Culture Collection, Manassas, VA) and primary human renal PTC (Cambrex Bio Science, Walkersville, MD) were purchased and grown according to the manufacturers' recommendations. Confluent cultures were switched to low serum medium [≤1% fetal bovine serum (FBS) depending on cell line (Cambrex cell growth medium contains only 0.5% FBS)] at least 24 h before treatments were initiated. Where indicated, recombinant human EGF (Upstate Biotechnology, Lake Placid, NY) was added to cultures at a final concentration of 20 ng/ml. Normal mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA), neutralizing mouse anti-EGFR (clone LA1) monoclonal antibody (Upstate Biotechnology), and neutralizing mouse anti-TNF receptor I monoclonal antibody (R&D Systems, Minneapolis, MN) were added to cell cultures at the concentrations indicated 30 min before EGF addition. Src tyrosine kinase inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine] and EGFR tyrosine kinase inhibitor AG1478 [4-(3-chloroanilin)-6,7-dimethoxyquinazoline] were purchased from Calbiochem/EMD Biosciences (San Diego, CA) and added to cultures at concentrations indicated at least 4 h before addition of EGF.

Cell fixation and indirect TUNEL assay. Cells were fixed, permeabilized, and stained with an indirect TUNEL assay, ApopTag Red in situ (Chemicon International, Temecula, CA). TUNEL-positive cells were counted in 10 nonoverlapping fields and data are expressed as means ± SE apoptotic nuclei/field.

Cell lysate and tissue homogenate preparation. Cell lysates were prepared as described (18). Tissues were obtained from animals killed by lethal sodium pentabarbitol injection, according to a protocol approved by the University of Virginia Animal Care and Use Committee. For one set of immunoblots, left and right kidneys were harvested from six Sprague-Dawley rats (age: 3 mo) and from six C57/Blk6 mice (age: 6–10 mo). For another set of blots, kidney, liver, heart, brain, spleen, and lung tissues were harvested from 21-day-old Sprague-Dawley rats and C57/Blk6 mice (6 of each species). Tissues were rinsed in cold PBS, weighed, and flash-frozen in liquid nitrogen before storing in –80°C freezer. Flash-frozen paired specimens of normal human kidney tissue and renal clear cell carcinoma tumor were obtained through the University of Virginia Tissue Procurement Facility with University of Virginia IRB approval. Tissue homogenates were prepared on ice with a Polytron homogenizer, using 150–200 mg tissue/ml homogenization buffer, as previously described (18). Homogenate protein concentration was determined by the method of Bradford (9). Homogenates were normalized by protein concentration and solubilized with boiling Laemmli buffer (22).

Gel electrophoresis and immunoblotting. Cell or tissue proteins were separated on 7.5 or 10% acrylamide gels by SDS-PAGE and transferred to nitrocellulose membranes. Uniform protein loading and transfer were determined with Ponceau staining as previously described (18). Blots were blocked in 5% nonfat dry milk for 30 min, washed 3x in TBS (20 mM Tris·HCl, pH 7.4, and 0.5 M NaCl), and then incubated with agitation overnight at 4°C with one of the following antibodies diluted in TBS supplemented with 1% wt/vol bovine serum albumin and 0.02% wt/vol sodium azide: Cell Signaling Technology's rabbit anti-phospho-EGFR antibodies directed against EGFR tyrosines (Y)-845, -992 and -1068, rabbit anti-HER2-PY877, mouse anti-nonphospho Src-NPY416 antibody, and rabbit anti-phospho Src-PY416, all diluted 1:300; Lab Vision's rabbit anti-EGFR (Ab-17, 5 µg/ml), mouse anti-HER2/neu (Ab-17, 1 µg/ml), mouse anti-HER3 (Ab-7, 2 µg/ml), and rabbit anti-HER4 (2 µg/ml) antibodies. Immunoreactive bands were detected with ECL according to the manufacturer's recommendations.

Immunohistochemistry. Kidneys from healthy 14-day-old neonatal rats and mice were harvested, formalin-fixed, embedded in paraffin, and cut into 4-µm sections for staining. Kidney sections were prepared for immunohistochemical staining as previously described (19). Cell Signaling Technology (Beverly, MA) rabbit anti-Src-PY416 antibody no. 2101 was diluted 1:50 and rabbit anti-EGFR-PY845 antibody no. 2231 was diluted 1:500 in PBS with 5% goat serum and used according to the manufacturer's protocol. Slides were counterstained with methylene blue.

Stable expression of constitutively active Src in IRPTC. Dr. S. Parsons (University of Virginia, Charlottesville, VA) generously donated the cDNA construct of chicken pp60c-Src containing the Y527F activating mutation (21) cloned into the HindIII-EcoR1 site of the pcDNA3 vector (Invitrogen, Carlsbad, CA). Vector ± Y527F-pp60c-Src cDNA was transfected into IRPTC with Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. G418-resistant colonies were selected and four clonal cell lines were tested through passage 17. Cell lysates were screened by immunoblot using antibodies directed against Src-NPY416, Src-PY416, EGFR, and EGFR-PY845. Selected stable transfectant clones were maintained in normal growth medium containing 175 µg/ml G418.

Cell stretch. Mechanical stain was applied to confluent human PTC grown on collagen type I-coated Bioflex plates (Flexcell International, Hillsborough, NC) for 4 h as previously described (18). Cells were fixed, stained with a TUNEL assay, and TUNEL-positive cells were counted as described above.

Statistical analysis. Data are presented as means ± SE. Student's t-test for paired variables was used to compare vehicle vs. inhibitor and vehicle vs. EGF treatment groups. Student's t-test for unpaired variables was used to compare unstretched vs. stretched cells. Two-way ANOVA was also performed on cell stretch data in GoGoGoGoGoGoGoFig. 8. Statistical significance was defined as P < 0.01.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. Neutralizing anti-epidermal growth factor receptor (EGFR) antibody attenuates EGF-induced cell death of mouse PTC. A: indirect TUNEL staining of mouse PKSV-PR cells incubated with EGF for 4 h in the absence or presence of 10 µg antibody (IgG/Ab); control IgG (normal mouse), nTNFR IgG (neutralizing mouse anti-TNF Receptor Ab), and nEGFR IgG (neutralizing mouse anti-EGFR Ab). B: increasing concentrations of IgG were added to cell cultures before addition of vehicle (Veh) or EGF for 4 h: dashed line, nTNFR Ab; dash and dot line, control mouse Ab; solid line, nEGFR Ab. Mean TUNEL-positive cells counted shown and error bars indicate SE; *P < 0.001. Similar results were achieved in 3 independent experiments.

 

Figure 2
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 2. Species differences in EGFR and Src expression and regulation. A: differential phosphorylation and activation: immunoblots of cell lysate proteins (50 µg/lane) prepared from cell cultures ± EGF 20 min were probed with the anti-EGFR or EGFR-phosphotyrosine (PY) site-specific antibodies indicated on right. Cell lines: IRPTC, immortalized rat proximal tubule cells; PKSV-PCT and PKSV-PR are germline immortalized mouse PTC. Representative blots are shown from 1 of 3 experiments. B: differences in Src phosphorylation and HER2-4 expression: immunoblots of IRPTC (rat) and PKSV-PR (mouse) cell lysate proteins (50 µg/lane) were prepared from cell cultures treated ± EGF for 20 min (0.33 h) or 6 h. Blots were probed with antibodies directed against nonphosphorylated (NPY) or phosphorylated (PY) Src tyrosine-416, EGFR- or EGFR family members HER2-4 indicated on right. Blots are shown from a single experiment and similar results were achieved in 4 experiments.

 

Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		Fig. 3. Expression and differential activation of c-Src and EGFR in rat and mouse kidneys. A: kidney homogenates (60 µg/lane) were prepared from left and right kidneys of 3 adult rats and mice for immunoblot analysis with nonphospho (NP)- and phospho (P)-specific Src-Y416 and total EGFR and EGFR-PY845 antibodies indicated on right. Blots shown represent 1 of 2 sets of 6 kidneys per species. Similar results were obtained with both sets. B: representative photomicrographs of 14-day-old neonatal rat and mouse kidneys stained with anti-Src-PY416 or anti-EGFR-PY845 antibodies; scale bar, 100 µm. Kidney sections from at least 3 animals were examined with each antibody. C: tissue homogenates were prepared from weanling rat and mouse organs listed to the right, 3 animals/species. Immunoblots of homogenate proteins (60 µg/lane) were probed with Src-NPY416- or Src-PY416-specific antibodies. Representative blots are shown and similar results were achieved with 2 different sets of organ homogenates.

 

Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 4. Inhibition of Src-Y416 or EGFR-Y845 phosphorylation attenuates EGF-induced mouse PTC death. Mouse PKSV-PR cell cultures were pretreated overnight (~16 h) with increasing concentrations (0 to 1 µM) of Src kinase inhibitor PP2 (A and B) or EGFR tyrosine kinase inhibitor AG1478 (C and D), followed by ± 4-h EGF treatment. A and C: dose-dependent inhibition of EGFR phosphorylation: immunoblots of cell lysate proteins (50 µg/lane) from these cultures were probed with antibody specificities shown on right. B and D: dose-dependent inhibition of EGF-induced apoptosis: parallel cultures were fixed and stained using an indirect TUNEL technique to identify apoptotic cells; hatched bars, vehicle-treated; filled bars, EGF-treated. Mean TUNEL-positive cells per field are shown and error bars indicate SE; *P < 0.001 for EGF-treated, no inhibitor relative to inhibitor pretreatment. Representative data are shown from 1 of 3 experiments for each inhibitor.

 

Figure 5
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 5. Stable expression of a constitutively active c-Src construct renders rat PTC susceptible to EGF-induced cell death. A and C: cell lysates (50 µg/lane) were prepared from parental IRPTC (rat), mouse PKSV-PR (Ms), and successive passages of stably transfected IRPTC clones (*Src A5 and B1) cells for immunoblot analysis. Blots were probed with antibody specificities shown to the right. B: susceptibility to EGF-induced cell death increases as EGFR-PY845 increases. At passage 7, vector control (IRPTC) and stably transfected IRPTC*Src clones A5 and B1 were treated ± EGF for 6 h, then fixed and stained using an indirect TUNEL assay to identify apoptotic cells; hatched bar, vehicle-treated; solid bar, EGF-treated. Statistically significant comparisons are shown for vehicle- vs. EGF-treated cultures; *P < 0.001. D: susceptibility to EGF-induced death declines as EGFR-PY845 levels decrease with higher passage number. Successive passages (p8–17) of IRPTC *Src A5 and B1 cells were treated ± EGF for 6 h, fixed, and stained by TUNEL; filled bars, EGF-treated. Statistically significant comparisons are shown for vehicle- vs. EGF-treated cultures; *P < 0.001 and {dagger}P < 0.01. Representative data are shown from 1 of 3 experiments; i.e., stable transfectant clones were derived 3 times with similar results over the period of 1 yr.

 

Figure 6
View larger version (22K):
[in this window]
[in a new window]

  		Fig. 6. Differential expression of EGFR family members (A) and differential activation of Src (B) and EGFR/HER2 (C) in rat, mouse, and human PTC cultures. Immunoblots of cell lysate proteins (50 µg/lane) were prepared from immortalized rat, mouse (Ms), and human (HK2) PTC cultures and primary human PTC (1°Hu). Blots were probed with antibody specificities indicated on right. * On the left indicate Src-dependent tyrosine phosphorylations and the ratio of phosphorylated/activated-Src (Src-PY) to nonphosphorylated/inactive-Src (Src-NPY) is expressed as <1 or >1 for each cell line at the bottom. Results shown are from a single experiment and identical results were achieved using cell lysates prepared from different passages in a minimum of 3 experiments.

 

Figure 7
View larger version (17K):
[in this window]
[in a new window]

  		Fig. 7. Differential activation of c-Src and EGFR in rodent and human kidneys. Immunoblots of rat, mouse, and human kidney homogenate proteins (60 µg/lane) were prepared from frozen normal cortex (N) or tumor (T) and probed with antibodies shown on right. Tumors were graded G1 though G3 by a pathologist. Representative data are shown for 1 of 2 sets of tissues.

 

Figure 8
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 8. EGF attenuates stretch-induced death of primary human PTC. A: TUNEL staining of nonstretched control primary human PTC and PTC subjected to 20% axial stretch for 4 h ± EGF. B: TUNEL-positive cells were counted and statistically analyzed as previously described; hatched bars, vehicle-treated; filled bars, EGF-treated. Statistically significant comparisons are shown for vehicle vs. EGF (*P < 0.001) and control vs. stretch (#P < 0.001). Representative data are shown from 1 of 3 experiments.

 

   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 GRANTS
 REFERENCES
 
In previous studies, EGF was the only ligand added to resting mouse PTC cultures when cell death was induced, making alternative ligand (TGF-{alpha}, HB-EGF, amphiregulin, or betacellulin) binding to the EGFR an unlikely cause of cell death. To test the hypothesis that EGF binding to the EGFR triggers the cell death response, mouse PTC were preincubated with neutralizing anti-EGFR (blocks EGF binding site) or-TNFRI (unrelated receptor control) monoclonal antibodies, normal mouse IgG (nonimmune antibody control), or zero antibody before treatment with EGF. As shown in Fig. 1, A and B, only the neutralizing anti-EGFR antibody significantly attenuated EGF-induced cell death at effective concentrations 3–30 µg/ml relative to mouse IgG control (P < 0.001). These results indicate that EGF binding to the mouse kidney EGFR is key to the induction of tubule cell death.

To determine whether differences in the regulation of rat vs. mouse EGFRs exist, cell lysates prepared from rat and mouse PTC were compared by immunoblot analysis for expression and phosphorylation of EGFR in the absence or presence of EGF. Mouse PTC express three- to fivefold more EGFR than rat PTC and expression level is unchanged by 20-min EGF treatment (Fig. 2A). Comparing basal (-EGF) phosphorylation of rat and mouse EGFRs at key tyrosine residues, additional differences are apparent. The rat EGFR is only phosphorylated after EGF treatment, whereas the mouse EGFR is highly phosphorylated in resting cell cultures and becomes hyperphosphorylated in response to EGF treatment (Fig. 2A). Tyrosine-845 is a Src-dependent phosphorylation site and critical for activation. To further explore species differences in EGFR expression and regulation, we compared immunoblots of rat and mouse PTC for differences in c-Src activity (activating Src phosphotyrosine-416 and inactive Src nonphosphorylated Y416) and for differences in receptor downmodulation. In Fig. 2B, top, blots indicate that rat and mouse PTC express comparable levels of nonphosphorylated Src-Y416 but differ in levels of Src phosphotyrosine (PY)-416. Mouse PTC express 2–3x more Src-PY416 than NonPhospho-Src-Y416 (P/NP-Src ratio >1), whereas rat cells express less Src-PY416 then Src-NPY416 (P/NP-Src ratio <1). Rat and mouse PTC express comparable levels of inactivating Src-PY527 phosphorylation (data not shown) and EGF treatment does not influence the expression or phosphorylation levels of Src-NPY416 in either species. Figure 2B immunoblots also demonstrate that the rat EGFR is downmodulated after 6-h EGF treatment, whereas mouse EGFR expression is unchanged. Immunoblots in Fig. 2B, bottom, compare the expression profiles of the alternate EGFR family members (HER2-4) in rat and mouse PTC. In addition to EGFR, rat cells express abundant HER2/neu, whereas mouse PTC express all three EGFR-related receptor proteins HER2-4. Thus mouse PTC can potentially form two EGFR heterodimer pairings (EGFR/HER3 and EGFR/HER4) for downstream signaling that rat PTC cannot form. Collectively, these results demonstrate distinct species differences in EGFR regulation and that a key regulator of EGFR kinase activity (Src) is constitutively active in mouse PTC.

In vitro studies point to a constitutive Src kinase activity in mouse PTC as a key difference in biological responses of rat and mouse cells to EGF. To look for evidence of constitutive Src activity in vivo, we compared immunoblots of normal adult rat and mouse kidney homogenates for phosphorylation and expression of c-Src and EGFR. As shown in Fig. 3A, rat and mouse kidneys express comparable levels of Src-NPY416 and total EGFR protein, but differ in levels of Src-PY416 and EGFR-PY845. Similar to findings with cultured PTC, mouse kidneys demonstrate 3–5x higher phosphorylation of Src-PY416 and EGFR-PY845 than detected in rat kidneys. Immunohistochemical staining of neonatal kidneys indicates that Src-PY416 and EGFR-PY845 are abundant in mouse tubules and concentrated in the basolateral region of tubule cells. These phosphorylated proteins were not detected in neonatal rat kidneys (Fig. 3B). Constitutive Src activity (P/NP-Src > 1) was also detected in the kidneys of weanling mice, but not weanling rats (Fig. 3C) indicating the age of the animals does not influence this species difference. Figure 3C also indicates this species difference may be restricted to the kidney. Evidence of constitutive Src-Y416 phosphorylation/activity was not detected in liver, heart, brain, spleen, or lung tissue of either rodent species.

Src kinase is a key regulator of EGFR signaling.

To determine whether Src kinase mediates EGF-induced mouse PTC death, we pretreated cell cultures with PP2, a selective inhibitor of Src kinase (37), before adding EGF to cultures and monitoring apoptosis. Pretreatment with PP2 decreased Src-PY416 and Src-dependent EGFR-PY845 in a dose-dependent manner without altering the total amount of Src-NPY416 or EGFR protein expressed in mouse PTC (Fig. 4A). Inhibition of EGF-induced cell death was also dependent on PP2 concentration used for cell pretreatment (Fig. 4B). Taken together, these results demonstrate that a reduction in Src kinase activity (Src-PY416) will attenuate EGF-induced death of mouse PTC. Data shown in Figs. 2A and 3 indicate mouse kidney EGFRs are constitutively phosphorylated on tyrosine-845 and suggest the receptor is constitutively active. To determine whether EGFR kinase activity is required for EGF-induced cell death, we pretreated cell cultures with EGFR-specific inhibitor AG1478 to knockdown constitutive EGFR tyrosine kinase activity and to block EGF-stimulated EGFR-tyrosine kinase activation. AG1478 pretreatment did not alter total Src-NPY416 (data not shown) or EGFR protein in mouse PTC lysates at any concentration tested (Fig. 4C); however, the highest concentration (1 µM) decreased phosphorylation of Src-dependent tyrosine-845 and autophosphorylation of tyrosine-992 and -1068. EGF-induced apoptosis declined in a dose-dependent manner (Fig. 4D), with AG1478 reducing apoptosis by 90% (P < 0.001) at the 1-µM dose, a concentration that corresponds to the greatest reduction in EGFR-tyrosine kinase activity (Fig. 4C). The data suggest that constitutive EGFR-tyrosine kinase activity together with constitutive Src activity play a critical role in mediating EGF-induced mouse PTC death.

In the mouse kidney, constitutive phosphorylation of Src on tyrosine-416 and the consequent activation of Src kinase appear to be driving the constitutive and dysregulated activity of the EGFR in mouse tubule cells. To test this hypothesis, we stably expressed a mutant Src (Y527F) cDNA in rat PTC for which EGF is a survival factor (18). Phosphorylation of Src-Y527 in the COOH terminus occurs in vivo to maintain Src in an inactive state (21). Dephosphorylation of Src-PY527 results in a conformational change that permits autophosphorylation of Src-Y416 in the activation loop rendering Src kinase fully active. Mutating tyrosine-527 to phenylalanine mimics dephosphorylation at that site, resulting in increased phosphorylation of Src-Y416 and a constitutively active kinase (21). Stable expression of Src (Y527F) did not alter expression of Src-NPY416 or total EGFR protein expression, but did increase the level of Src-PY416 to levels detected in mouse PTC cultures as expected (Fig. 5A). EGFR-Y845 phosphorylation increased with successive passages of activated (*) Src-rat PTC clones A5 and B1. Susceptibility of *Src-rat PTC to EGF-induced cell death was first recorded at passage 5 and results of a passage 7 TUNEL assay are shown in Fig. 5B. These data indicate that susceptibility to EGF-induced cell death is most closely correlated with high levels of Src-dependent phosphorylation of EGFR-Y845. As EGFR-PY845 levels waned in successive passages of *Src-rat PTC (Fig. 5C), susceptibility to EGF-induced apoptosis also declined and was eventually lost when EGFR-PY845 levels matched levels detected in vector control PTC (Fig. 5D). These results demonstrate that expression of an activated Src construct in differentiated proximal tubule epithelial cells leads to constitutive activation and dysregulation of the EGFR. This single modification completely reversed the biological response of rat PTC to EGF.

To determine the expression and phosphorylation profiles of Src, EGFR and HERs 2-4 in human kidney tubule cells, we compared cell lysates of primary human PTC (1°Hu) and immortalized human PTC line (HK2) with our immortalized rat and mouse PTC by immunoblot analysis. Blots probed with antibodies specific for EGFR and HERs 2-4 demonstrate that only mouse PTC express all four HERs (Fig. 6A). All four cell lines express EGFR and HER2/neu. Primary human PTC also express HER3, although it is much less abundant than EGFR or HER2. Blots probed with antibodies directed against Src-NPY416 and PY416 indicate that primary human PTC express more Src-NPY416, than PY416 (P/NP-Src < 1), similar to rat PTC (Fig. 6B). In contrast, HK2 levels of Src-PY416 are greater than levels of Src-NPY416 (P/NP-Src > 1), similar to mouse PTC. The EGFR in HK2 cells was highly abundant and heavily phosphorylated on tyrosines-845, -992 and -1068, whereas phosphorylation of the 1°Hu EGFR was barely detectable (Fig. 6C). Similar results were found for the Src-dependent phosphorylation site of HER2/neu. HER2 phosphotyrosine-877 was only detected in mouse PTC and HK2 cells. Of note, the level of Src-dependent phosphorylations (EGFR-PY845 and HER2-PY877) are proportional to limiting HER2/neu content in HK2 cells and to limiting EGFR content in mouse PTC. This suggests that EGFR may be held in a constitutive state in part through heterodimerization with HER2/neu, in addition to Src-dependent phosphorylations. The EGFR/HER2 heterodimer is highly correlated with dysregulated cell growth in cancer. Phenotypically, HK2 cells are transformed and exhibit characteristics of tumor cells (spindle shape and loss of contact inhibition) unlike the other cell lines shown.

To determine whether basal phorphorylation levels of c-Src and EGFR in vivo were similar to levels detected in cultured cells, homogenates were prepared from "normal human renal cortex" and clear cell carcinoma tumor for comparison by immunoblot analysis. Rat and mouse kidney homogenates were included as negative and positive controls, respectively. In Fig. 7, Src-Y416 is largely unphosphorylated in "normal renal cortex" homogenates (P/NP-Src < 1), although levels vary with the grade of associated tumor (G1 vs. G2). In contrast, Src is highly phosphorylated on Y416 in human renal tumor and therefore constitutively active (P/NP > 1), similar to findings in mouse kidney. The level of EGFR-Y845 phosphorylation is directly proportional to the level of Src-Y416 phosphorylation for each specimen analyzed. These results further demonstrate that Src activity in primary human PTC is comparable to that detected in normal renal cortex, whereas HK2 Src activity is comparable to that detected in renal tumors. Thus correlation between constitutively active Src (ratio > 1) and a constitutively active EGFR was consistent in rodent and human kidneys, as well as cultured cell lines. From these results, we predict that EGF will be a survival factor for human PTC.

To test this prediction, primary human PTC were subjected to mechanical stretch in vitro to approximate axial strain injury induced in vivo by ureteral obstruction. Axial strain applied to human PTC cultures increased apoptosis 545% (P < 0.001) above control (Fig. 8). Addition of EGF to culture medium during the mechanical stretch procedure reduced apoptosis by 60% (P < 0.001) relative to vehicle/stretch. Addition of EGF to unstretched control cultures decreased background apoptosis by 40% (P < 0.001) compared with vehicle/control. Two-way ANOVA indicates a statistically significant interaction (P < 0.001) between phenotype (control vs. stretch) and drug (vehicle vs. EGF). Thus our prediction was correct; EGF is a survival factor for primary human PTC in vitro.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 GRANTS
 REFERENCES
 
The goal of this study was to identify species differences in the regulation and/or activation of the EGFR that might explain the opposing life and death responses of rat and mouse tubule cells to EGF. Our results identify four distinct species differences in renal EGFR regulation. First, EGFR phosphorylation and activation are transient in rat PTC, but prolonged in mouse PTC (Fig. 2). Second, EGF induces downregulation of the rat EGFR, but not the mouse EGFR (Fig. 2B). Third, rat EGFR can dimerize with itself and HER2/neu, but mouse EGFR can potentially form dimers with all four receptor family members (HER1-4) and activate additional signaling pathways (Fig. 2B). Fourth, the constitutive Src kinase activity in mouse cells phosphorylates and activates EGFR, rendering it constitutively active (Figs. 4 and 5). Sustained Src activity in mouse kidney appears to be the underlying cause of EGFR dysregulation and susceptibility to EGF-induced cell death.

Ligand binding induces rapid phosphorylation and activation of the EGFR. In healthy tissue and nontransformed cells, activation is followed by gradual dephosphorylation that inactivates the receptor. Inactivated EGFRs are then internalized via endocytosis and targeted for degradation/downmodulation (36). In tumor-derived cells, Src and EGFR are frequently overexpressed and/or constitutively active (dysregulated) as a consequence of mutation (24). Src-dependent phosphorylation of EGFR-Y845 is particularly critical for sustaining EGFR activation (6, 32). In transformed cells, Src overexpression or sustained activation alters c-Cbl regulation of receptor ubiquitylation and prevents EGFR endocytosis (3). Thus the absence of EGF-induced receptor downmodulation in mouse cells is consistent with cells that either overexpress Src or have constitutive Src activity (3). Based on these criteria, rat and human renal EGFRs are transiently activated and downmodulated similar to nontransformed cells, and mouse renal EGFRs are continuously activated and resistant to downmodulation similar to transformed cells.

Mouse PTC express all four EGFR-related receptors (Her1-4) and rat PTC express only two (HER1-2). This means that mouse PTC can potentially form two heterodimer pairings (EGFR/HER3 and EGFR/HER4) that rat PTC cannot form. Species differences in the expression of alternate EGFR family members can potentially alter downstream signaling and affect different biological outcomes (23, 25, 26). However, in coimmunoprecipitation experiments EGF alone did not induce formation of EGFR/HER3 or EGFR/HER4 heterodimers in mouse PTC, although these heterodimer pairings did form if cells were first treated with heregulin (a HER3/Her4-specific ligand) and subsequently treated with EGF (data not shown). Serial sections of developing mouse kidneys were stained with lotus lectin (to identify proximal tubules), heregulin, EGFR, and HER3-specific antibodies to determine whether all were expressed in or basolateral to PTC at the time EGF was administered to neonatal mice in a previous study (19). All three proteins were abundant in the proximal tubules of mice at birth (day 0). However, by day 3, when exogenous EGF was administered to mice, heregulin and HER3 were primarily vascular (data not shown) and only EGFR remained in the proximal tubule (19). These findings, along with results from the in vitro coimmunoprecipitation studies, indicate it is unlikely that alternate EGF family members, HER3 and/or HER4, contribute to EGF-induced tubule cell death in the mouse.

Several reports described mechanisms of EGF-induced cell death. In fibroblasts and breast cancer cell lines expressing high levels of EGFR and HER2, EGF induces p38 kinase-dependent apoptosis (33). Pretreatment of mouse PTC with p38 kinase inhibitor (SB203580) before EGF addition did not inhibit EGF-induced cell death (data not shown). The HER2/HER3 heterodimer is a low-affinity receptor for EGF (25) and can induce cell death through prolonged p38 kinase activation (13). Neither neutralizing anti-HER3 antibodies nor SB203580 inhibited EGF-induced cell death in mouse PTC relative to vehicle control nor did we detect prolonged activation of p38 kinase after EGF treatment in immunoblot studies of cell lysates (data not shown). Extracellular signal-regulated kinase (ERK) lies downstream of EGFR and is generally associated with cell growth and survival. A number of recent studies link ERK activation to cell death (reviewed in Ref. 38). Pretreatment of mouse PTC with ERK inhibitor (PD98059) did not attenuate EGF-induced cell death in our cell culture model (data not shown). Indeed, EGF-induced cell death of mouse PTC does not proceed by any EGF-induced death pathway described to date.

The identification of constitutive Src and EGFR tyrosine kinase activities in C57/Bl6 mouse kidneys and mouse PTC is a novel finding. Sustained tyrosine kinase activities have not been previously reported in normal tissue or nontransformed cell lines. Overexpression and/or mutational activation of Src and EGFR occur frequently in carcinomas of the lung, breast, and colon and cell lines derived from these tumors retain Src and EGFR overexpression/activity (3,4,6). Studies in breast cancer cells demonstrated that Src-dependent EGFR-Y845 phosphorylation is required for most biological activities mediated by the receptor including cell proliferation via STAT3/5b activation (20) and binding EGFR to the mitochondrial cytochrome c oxidase subunit II to inhibit drug-induced apoptosis and promote cancer cell survival (7). Our studies indicate that the Src-dependent phosphorylation of EGFR-Y845 correlates with susceptibility to EGF-induced apoptosis. Inhibition of Src or EGFR tyrosine kinase activity in mouse PTC significantly reduced EGFR-Y845 phosphorylation and EGF-induced cell death (P < 0.001; Fig. 4). Conversely, expression of an "activated" Src transgene in rat PTC reversed the survival response to EGF and rendered cells susceptible to EGF-induced cell death (Fig. 5). Taken together, these results indicate the novel constitutive Src and EGFR activities in nontransformed mouse kidney cells mediate EGF intracellular signaling that culminates in cell death.

Constitutive tyrosine kinase activity, as opposed to overexpression is gaining importance in cell biology as TKIs have entered clinical trials for the treatment of human cancers. Mutations in the tyrosine kinase domain that render EGFR constitutively active in nonsmall cell lung tumors also render tumors sensitive to TKIs, gefitinib and erlotinib (1, 15). Gifitinib reduced PY845 levels in sensitive tumor cell lines, thereby blocking downstream STAT3 activation for cell proliferation (1) and the anti-apoptotic effects of EGFR binding to cytochrome c oxidase subunit II (7). The type of mutation and activation status of EGFR in the tumor can be more important than EGFR expression level for tumor sensitivity to therapy. Similar findings have been reported for imatinib treatment for gastrointestinal stromal tumors expressing mutant c-kit (16). In renal carcinoma, EGFR is activated not only in the transformed epithelial cells, but also in tumor-associated endothelium responding to high levels of TGF-{alpha} produced by metastatic tumor cells (2). EGFR TKIs have been used to treat renal carcinoma with limited success; however, little if any information is available on EGFR mutations or the activation state of Src or EGFR in renal carcinoma (5). TKIs could be effective therapies for renal carcinoma if targeted to tumors with sensitizing mutations (2).


   CONCLUSIONS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
GRANTS
 REFERENCES
 
Predicting outcomes for EGFR-based therapies increasingly depends on prior knowledge of receptor activation in the diseased tissue. The same can be said of EGF-mediated responses in normal kidney tissue and cells. Differences in basal EGFR phosphorylation and activation among species will alter downstream responses to EGF and limit interspecies comparisons. We urge caution in choosing human renal cell culture models (e.g., tumor-like HK2 cells) and in comparing mouse and human EGFR-mediated physiology or pathology.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 GRANTS
REFERENCES
 
This research was supported in part by the UVA Children's Medical Center (S. C. Kiley), the University of Virginia School of Medicine Research and Development Committee (S. C. Kiley), National Institutes of Health Center of Excellence in Pediatric Nephrology and Urology, DK-45179 and DK-52612 (R. L. Chevalier), and National Institutes of Health Child Health Research Center, HD-01421 (R. L. Chevalier).


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge the contributions of B. A. Thornhill (rodent kidney tissue), K. Neale (immunohistochemistry), M. S. Forbes (images of immunohistology), and A. Vandewalle (mouse PTC lines).


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. C. Kiley, Dept. of Pediatrics, Univ. of Virginia, Box 801334, 409 Lane Road, Charlottesville, VA 22908 (e-mail: sck3k{at}virginia.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 GRANTS
 REFERENCES
 

  1. Alvarez JV, Greulich H, Sellers WR, Meyerson M, Frank DA. Signal transducer and activator of transcription 3 is required for the oncogenic effects of nonsmall-cell lung cancer-associated mutations of the epidermal growth factor receptor. Cancer Res 66: 3162–3168, 2006.[Abstract/Free Full Text]
  2. Baker CH, Pino MS, Fidler IJ. Phosphorylated epidermal growth factor receptor on tumor-associated endothelial cells in human renal carcinoma is a primary target for therapy by tyrosine kinase inhibitors. Neoplasia8: 470–476, 2006.[CrossRef][Web of Science][Medline]
  3. Bao J, Gur G, Yarden Y. Src promotes destruction of c-Cbl: implications for oncogenic synergy between Src and growth factor receptors. Proc Natl Acad Sci USA 100: 2438–2443, 2003.[Abstract/Free Full Text]
  4. Baselga J. Targeting tyrosine kinases in cancer: the second wave. Science 312: 1175–1178, 2006.[Abstract/Free Full Text]
  5. Bellmunt J, Montagut C, Abiol S, Carles J, Maroto P, Orsola A. Present strategies in the treatment of metastatic renal cell carcinoma: an update on molecular targeting agents. BJU Int 99: 274–280, 2006.[Web of Science][Medline]
  6. Biscardi JS, Maa MC, Tice DA, Cox ME, Leu TH, Parsons SJ. c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J Biol Chem 274: 8335–8343, 1999.[Abstract/Free Full Text]
  7. Boerner JL, Demory ML, Silva C, Parsons SJ. Phosphorylation of Y845 on the epidermal growth factor receptor mediates binding to the mitochondrial protein cytochrome c oxidase subunit II. Mol Cell Biol 24: 7059–7071, 2004.[Abstract/Free Full Text]
  8. Boerner JL, Biscardi JS, Silva CM, Parsons SJ. Transactivating agonists of the EGF receptor require Tyr 845 phosphorylation for induction of DNA synthesis. Mol Carcinog 44: 262–273, 2005.[CrossRef][Web of Science][Medline]
  9. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][Web of Science][Medline]
  10. Cachat F, Lange-Sperandio B, Chang AY, Kiley SC, Thornhill BA, Forbes MS, Chevalier RL. Ureteral obstruction in neonatal mice elicits segment-specific tubular cell responses leading to nephron loss. Kidney Int 63: 564–575, 2002.[Web of Science]
  11. Cartier N, Lacave R, Vallet V, Hagege R, Hellio J, Robine S, Pringault E, Cluzeaud F, Briand P, Kahn A, Vandewalle A. Establishment of proximal tubule cell lines by targeted oncogenesis in transgenic mice using the L-pyruvate kinase-SV40 (T) antigen hybrid gene. J Cell Sci 104: 695–704, 1993.[Abstract]
  12. Chevalier RL, Goyal S, Wolstenholme JT, Thornhill BA. Obstructive nephropathy in the neonatal rat is attenuated by epidermal growth factor. Kidney Int 54: 38–47, 1998.[CrossRef][Web of Science][Medline]
  13. Daly JM, Olayioye MA, Wong AML, Neve R, Lane HA, Maurer FG, Hynes NA. NDF/heregulin-induced cell cycle changes and apoptosis in breast cancer cells: role of PI3 kinase and p38 MAP kinase pathways. Oncogene 18: 3440–3451, 1999.[CrossRef][Web of Science][Medline]
  14. Emlet DR, Moscatello DK, Ludlow LB, Wong AJ. Subsets of epidermal growth factor receptors during activation and endocytosis. J Biol Chem 272: 4079–4086, 1997.[Abstract/Free Full Text]
  15. Guillermo Paez J, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, Herman P, Kaye FJ, Lindeman N, Boggon TJ, Naoki K, Sasaki H, Fujii Y, Eck MJ, Sellers WR, Johnson BE, Meyerson M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304: 1497–1500, 2004.[Abstract/Free Full Text]
  16. Heinrich MC, Corless CL, Demetri GD, Blanke CD, von Mehren M, Joensuu H, McGreevey LS, Chen CJ, Van den Abbeele AD, Druker BJ, Kiese B, Eisenberg B, Roberts PJ, Singer S, Fletcher CDM, Silberman S, Dimitrijevic S, Fletcher JA. Kinase mutations and imatinib response in patients with metastatic gastrointestinal stromal tumor. J Clin Oncol 21: 4342–4349, 2003.[Abstract/Free Full Text]
  17. Kennedy WA, Buttyan R, Garcia-Montes E, D'Agati V, Olsson CA, Sawczuk IS. Epidermal growth factor suppresses renal tubular apoptosis following ureteral obstruction. Urology 49: 973–980, 1997.[CrossRef][Web of Science][Medline]
  18. Kiley SC, Thornhill BA, Tang SS, Ingelfinger JR, Chevalier RL. Growth factor-mediated phosphorylation of proapoptotic BAD reduces tubule cell death in vitro and in vivo. Kidney Int 63: 33–42, 2003.[Web of Science][Medline]
  19. Kiley SC, Thornhill BA, Belyea BC, Neale K, Forbes MS, Luetteke NC, Lee DC, Chevalier RL. Epidermal growth factor potentiates renal cell death in hydronephrotic neonatal mice, but cell survival in rats. Kidney Int 68: 504–514, 2005.[CrossRef][Web of Science][Medline]
  20. Kloth MT, Laughlin KK, Biscardi JS, Boerner JL, Parsons SJ, Silva CM. STAT5b, a mediator of synergism between c-Src and the epidermal growth factor receptor. J Biol Chem 278: 1671–1679, 2003.[Abstract/Free Full Text]
  21. Kmiecik TE, Shalloway D. Activation and suppression of pp60c-Src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell 49: 65–73, 1987.[CrossRef][Web of Science][Medline]
  22. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.[CrossRef][Medline]
  23. Muthuswamy SK, Gilman M, Brugge JS. Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo- and hetrodimers. Mol Cell Biol 19: 6845–6857, 1999.[Abstract/Free Full Text]
  24. Ono M, Kuwano M. Molecular mechanisms of epidermal growth factor receptor activation and response to gefitinib and other EGFR-targeting drugs. Clin Cancer Res 12: 7242–7251, 2006.[Abstract/Free Full Text]
  25. Pinkas-Kramarski R, Lenferink AEG, Bacus SS, Lyass L, van de Poll MLM, Klapper LN, Sela M, van Zoelen EJJ, Yarden Y. The oncogenic ErbB2/ErbB3 heterodimer is a surrogate receptor of the epidermal growth factor and betacellulin. Oncogene 16: 1249–1258, 1998.[CrossRef][Web of Science][Medline]
  26. Ram TG, Hosick HL, Ethier SP. Heregulin-beta is especially potent in activating phosphatidylinositol 3-kinase in nontransformed human mammary epithelial cells. J Cell Physiol 18: 301–313, 2000.
  27. Richards WG, Sweeney WE, Yoder BK, Wilkinson JE, Woychik RP, Avner ED. Epidermal growth factor receptor activity mediates renal cyst formation in polycystic kidney disease. J Clin Invest 101: 935–939, 1998.[Web of Science][Medline]
  28. Rojas M, Yao S, Lin YZ. Controlling epidermal growth factor (EGF)-stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor. J Biol Chem 271: 27456–27461, 1996.[Abstract/Free Full Text]
  29. Sweeney WE, Chen Y, Nakanishi K, Frost P, Avner ED. Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor. Kidney Int 57: 33–40, 2000.[CrossRef][Web of Science][Medline]
  30. Tang SS, Jung F, Diamant D, Brown D, Bachinsky D, Hellman P, Ingelfinger JR. Temperature-sensitive SV40 immortalized rat proximal tubule cell line has functional renin-angiotensin system. Am J Physiol Renal Fluid Electrolyte Physiol 268: F435–F446, 1995.[Abstract/Free Full Text]
  31. Terzi F, Burtin M, Hekmati M, Federici P, Grimber G, Briand P, Friedlander G. Targeted expression of a dominant-negative EGF-R in the kidney reduces tubulo-interstital lesions after renal injury. J Clin Invest 106: 225–234, 2000.[Web of Science][Medline]
  32. Tice DA, Biscardi JS, Nickles AL, Parsons SJ. Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc Natl Acad Sci USA 96: 1415–1420, 1999.[Abstract/Free Full Text]
  33. Tikhomirov O, Carpenter G. Ligand-induced, p38-dependent apoptosis in cells expressing high levels of epidermal growth factor receptor and ErbB-2. J Biol Chem 279: 12988–12996, 2004.[Abstract/Free Full Text]
  34. Torres VE, Sweeney WE, Wang X, Qian Q, Harris PC, Frost P, Avner ED. EGF receptor tyrosine kinase inhibition attenuates the development of PKD in Han:SPRD rats. Kidney Int 64: 1573–1579, 2003.[CrossRef][Web of Science][Medline]
  35. Torres VE, Sweeney WE, Wang X, Qian Q, Harris PC, Frost P, Avner ED. Epidermal growth factor receptor tyrosine kinase inhibition is not protective in PCK rats. Kidney Int 66: 1766–1773, 2004.[CrossRef][Web of Science][Medline]
  36. Warren CM, Landgraf R. Signaling through ErbB receptors: multiple layers of diversity and control. Cell Signal 18: 923–933, 2006.[CrossRef][Web of Science][Medline]
  37. Zhu X, Kim JL, Newcomb JR, Rose PE, Stover DR, Toledo LM, Zhao H, Morgenstern KA. Structure analysis of the lymphocyte-specific kinase Lck in complex with nonselective and Src family selective kinase inhibitors. Structure 7: 651–661, 1999.[Medline]
  38. Zhuang S, Schnellmann RG. A death-promoting role for extracellular signal-regulated kinase. J Pharmacol Exp Ther 319: 991–997, 2006.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/3/F895    most recent
00227.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kiley, S. C.
Right arrow Articles by Chevalier, R. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kiley, S. C.
Right arrow Articles by Chevalier, R. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.