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Am J Physiol Renal Physiol 294: F459-F468, 2008. First published January 2, 2008; doi:10.1152/ajprenal.00473.2007
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EDITORIAL FOCUS

Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells

Shougang Zhuang,1 Gilbert R. Kinsey,2,* Kyle Rasbach,2 and Rick G. Schnellmann2

1Department of Medicine, Brown University School of Medicine, Providence, Rhode Island; and 2Department of Pharmaceutical and Biomedical Sciences, South Carolina College of Pharmacy, Medical University of South Carolina, Charleston, South Carolina

Submitted 10 October 2007 ; accepted in final form 28 December 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our recent studies have shown that proliferation of renal proximal tubular cells (RPTC) in the absence of growth factors requires activation of the epidermal growth factor (EGF) receptor. We sought to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate in different models. RPTC expressed both pro- and cleaved forms of heparin-binding epidermal growth factor (HB-EGF) and several metalloproteinases (MMP-2, -3, -9, and ADAM10, ADAM17) that have been reported to cleave HB-EGF. Treatment of RPTC with CRM 197, an inhibitor of HB-EGF binding to the EGF receptor, or downregulation of HB-EGF with small interfering RNA inhibited RPTC proliferation following plating. Furthermore, GM6001 (pan-MMP inhibitor), tumor-necrosis factor protease inhibitor-1 (TAPI-1; MMP and ADAM17 inhibitor), and GW280264X (ADAM10 and -17 inhibitor), but not GI254023X (ADAM10 inhibitor), attenuated the proliferation after plating. Although EGF receptor activation is required for RPTC proliferation after oxidant injury, CRM197, GM6001, and TAPI-1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation after oxidant injury. In addition, PP1 treatment attenuated HB-EGF-enhanced RPTC proliferation. We suggest that RPTC proliferation after plating is mediated by HB-EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB-EGF.

renal proximal tubular cells; epidermal growth factor receptor; metalloproteinases

THE PROXIMAL TUBULE OF THE kidney is particularly susceptible to injury by ischemia-reperfusion (I/R) and toxicants. As a result of injury, renal epithelial cells undergo necrotic cell death or apoptosis and shed from the basement membrane. The remaining epithelial cells are thought to participate in regeneration of the tubular epithelium and the restoration of renal function through dedifferentiation, migration, proliferation, and redifferentiation (33).

Proliferation of renal epithelial cells is tightly regulated by multiple environmental influences, including soluble factors (e.g., polypeptide growth factors). The kidney is a site of synthesis of several growth factors, including EGF, insulin-like growth factor (IGF), hepatocyte growth factor (HGF), fibroblast growth factor 2, and transforming growth factor-{alpha}, and these growth factors promote renal proximal tubule cell (RPTC) proliferation to varying degrees (12, 32, 44, 55). For example, EGF is synthesized in the thick ascending limb and distal tubule of the kidney and is a strong mitogen (34, 42, 54). More recently, mRNA levels of heparin-binding (HB)-EGF, a member of the EGF family, increased in tubular epithelial cells of the inner cortex and outer medulla in rats subjected to I/R injury and mercuric chloride treatment (14, 15). Furthermore, exogenous HB-EGF stimulated proliferation in rabbit RPTC and NRK52E cells (15, 60). Although HB-EGF is mitogenic to RPTC, it is not known whether HB-EGF is the autocrine/paracrine stimulus that produces RPTC proliferation in vitro or in vivo.

HB-EGF is synthesized as a transmembrane protein (proHB-EGF) and is commonly cleaved at the apical plasma membrane to yield soluble HB-EGF (18, 19, 47). Ectodomain shedding, the proteolytic processing of the extracellular domain of proHB-EGF to form soluble HB-EGF, is observed in response to several stimuli, including 12-O-tetradecanoylphorbol-13-aceate, osmotic stress, and UV light (18, 49). Whereas initial studies of signaling and regulation of HB-EGF ectodomain shedding suggested that matrix metalloproteases -2, -3, and/or -9 were involved in this process (19, 38, 47), recent studies provide strong evidence that tumor necrosis factor-{alpha}-converting enzyme/a disintegrin and metalloprotease 17 protease (TACE/ADAM17) and ADAM10 are involved in HB-EGF shedding (10, 16, 41).

The biological actions of soluble HB-EGF are mediated through two members of the EGF receptor superfamily, EGF receptor/ErbB-1 and HER4/ErbB-4 (18, 37). The EGF receptor has been identified in RPTC in vitro and in vivo and has been reported to increase following folic acid or I/R injury in rats and rabbits, and in humans with acute renal failure (ARF) (3, 28, 40, 48). Using mice with a mutated EGF receptor that has minimal EGF receptor kinase activity and wild-type mice, Wang et al. (57) demonstrated that wild-type mice subjected to mercuric chloride-induced ARF experience subsequent RPTC proliferation and a return of renal function. In contrast, mice with the mutated EGF receptor had the same degree of ARF but neither underwent subsequent RPTC proliferation nor exhibited a return of renal function. Furthermore, Zhuang et al. (60) demonstrated that the EGF receptor is required for RPTC proliferation following plating, and after mechanical injury. These results demonstrate that the EGF receptor is a key regulator of RPTC proliferation and return of renal function following ARF.

The EGF receptor can be activated by different stimuli through diverse mechanisms. Ligand binding to the EGF receptor results in receptor dimerization, activation of tyrosine kinase domains, and autophosphorylation (37). Nonspecific stimuli, such as osmotic stress, UV light, oxidative stress, hypoxia-reoxygenation injury, and G protein-coupled receptor stimulation also trigger EGF receptor autophosphorylation through a process called transactivation (8, 22, 25, 39, 58). The release of a soluble EGF ligand such as HB-EGF from the cell membrane by an autocrine/paracrine mechanism and its subsequent binding and activation of the EGF receptor is an example of transactivation.

Non-receptor tyrosine kinases also contribute to EGF receptor activation. Three Src family members (Src, Fyn, and Yes) are expressed ubiquitously in mammalian cells (56, 62), and they have been shown to contribute to EGF receptor activation in several systems (45, 59, 61). For example, activation of Src family kinases is required for EGF receptor-mediated ERK phosphorylation in response to UV light, I/R, or H2O2 (20, 26, 61). The precise mechanism of Src family kinase-mediated EGF receptor activation is incompletely understood; however, Src can directly associate with and phosphorylate EGF receptor at tyrosine residues Tyr 845 and Tyr 1101 (4, 46). Mitogenic-promoting effects of Src through residue Tyr 845 of the EGF receptor has been reported in tumor cells (1). However, its role in renal regeneration has not been examined.

We used rabbit RPTC in our studies because they grow in the absence of exogenous growth factors, and the culture conditions have been improved such that the cells exhibit normal rates of aerobic metabolism, are gluconeogenic, and have a greater degree of differentiation that most other in vitro cellular models (35). Furthermore, RPTC undergo migration and proliferation following plating and mechanical injury in an EGF receptor-dependent manner (60). Thus this model is ideal to study autocrine/paracrine mechanisms of migration and proliferation in the kidney. Using RPTC undergoing proliferation following plating and following oxidant injury, we investigated the role of HB-EGF in autocrine/paracrine pathways of proliferation.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and reagents. Human recombinant HB-EGF was obtained from R&D Systems (Minneapolis, MN). AG1478 and PP1 were purchased from Biomol (Plymouth Meeting, PA). All other chemicals were purchased from Sigma (St. Louis, MO). GW280264X and GI254023X were generous gifts from Dr. Andreas Lugwig (Christian-Albrechts-University, Kiel, Germany). In all experiments using pharmacological inhibitors, control cells were treated with an equivalent amount of vehicle. Antibodies to phospho-EGFR and phospho-Src were obtained from Cell Signaling Technology. Antibodies to HB-EGF, EGF receptor, MMP-2, MMP-3, MMP-9, and ADAM17 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody to ADAM10 was obtained from Abcam (Cambridge, MA). Annealed HB-EGF (sense 5'-ggcagaucuggaccuuuugtt-3') and scrambled siRNA oligonucleotides were purchased from Ambion (Austin, TX).

Isolation and culture of renal proximal tubules. Female New Zealand White rabbits (2 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). The Medical University of South Carolina Institutional Animal Care and Use Committee reviewed and approved the animal protocol used in this study. RPTC were isolated using the iron oxide perfusion method and grown in six-well tissue culture dishes under improved conditions as previously described (36). 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.

Models. In model 1, RPTC were grown to 80–90% confluence and injured by exposure to 1 mM H2O2 for 5 h (61). Culture media were changed and RPTC were incubated for 24–48 h in the presence or absence of various pharmacological inhibitors. In model 2, RPTC were grown to 80–90% confluence, released from the plate by trypsin, and replated in culture dishes at 100,000 cells/dish. After the RPTC attached, they were incubated in the presence and absence of pharmacological inhibitors for 48 h. In model 3, RPTC were grown for 3 days and then incubated for 24 or 48 h in the presence or absence of various pharmacological agents.

HB-EGF siRNA transfection. RPTC were transfected with small interfering RNA (siRNA) using an Amaxa Nucleofector (Gaithersburg, MD) and Basic Nucleofector Kit for Primary Mammalian Epithelial Cells (Amaxa) according to the manufacturer's protocol. Briefly, RPTC were cultured to 80–90% confluence, released from the plate by trypsinization, and suspended in nucleofection solution with the siRNA [either negative control (scrambled) or HB-EGF] and electroporated using the Amaxa nucleofection device. For each transfection, 1.5 x 106 cells and 2 µg negative control or HB-EGF siRNA were used. After transfection, RPTC were resuspended in RPMI media (GIBCO, Carlsbad, CA) and incubated at 37°C for 10 min, then plated in 35-mm culture dishes, and incubated in the presence and absence of AG1478 or diluent for an additional 48 h in normal culture medium described above.

Cell proliferation. Cell proliferation was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, by the number of cells in S phase of the cell cycle, and/or by the amount of cell protein in a dish. Cell protein was determined using the BCA assay. MTT was added (final concentration of 0.5 mg/ml) to each dish, incubated for an additional 30 min, and tetrazolium was released by dimethylsulfoxide. Optical density was determined with a spectrophotometer (570 nm). Data were normalized to solvent-treated cultures. The number of cells in the S phase of the cell cycle was determined using flow cytometry after the cells were stained with propidium iodide as previously described (61).

Immunoblot analysis. RPTC were 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), disrupted by sonication for 15 s, and lysates were stored at –20°C. Equal amounts of cellular protein lysate 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 after chemiluminescence detection on autoradiographic film.

Statistical analysis. Data are presented as means ± SE and were subjected to one-way ANOVA. Multiple means were compared using Tukey's test, and P < 0.05 was considered statistically different. RPTC isolated from an individual rabbit represents a single experiment (n = 1) consisting of data obtained from three dishes/well.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
RPTC express HB-EGF and effect of CRM 197 and AG1478 on EGF receptor and cell proliferation. Because the EGF receptor ligand HB-EGF has been identified in RPTC (14, 15), we investigated whether HB-EGF is expressed and cleaved in RPTC. RPTC were grown in the absence of exogenous growth factors until 40–50% confluence, harvested, and cell lysates were subjected to immunoblot analysis using an anti-HB-EGF COOH-terminal domain antibody. As reported previously, bands ranging from 20 to 30 kDa correspond to proHB-EGF and bands ranging from 6 to 17 kDa are proteolytic tail fragments, composed of cytoplasmic and transmembrane domains of proHB-EGF (49, 53). ProHB-EGF and proteolytic tail fragments were identified in RPTC lysates (Fig. 1A). Because the presence of proteolytic tail fragments corresponds to the release of soluble HB-EGF (49), these results suggest that soluble HB-EGF is produced by RPTC undergoing proliferation. Unfortunately, we failed to detect the soluble HB-EGF in the culture medium or in concentrated culture medium by immunoblot analysis (data not shown). This may be due to the low concentration of HB-EGF released from cells, its rapid binding to the EGF receptor once it is released, and/or its degradation.


Figure 1
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  		Fig. 1. Expression of heparin-binding epidermal growth factor (HB-EGF) in renal proximal tubular cells (RPTC) and the effect of CRM197 and AG1478 on RPTC proliferation and EGF receptor activation following plating. A: RPTC were grown to 40–50% confluence, harvested, and cell lysates were subjected to immunoblot analysis with anti-HB-EGF antibody. B: RPTC were grown to 40–50% confluence, incubated for 24 h in the presence or absence of 10 µg/ml CRM197 or 10 µM AG1478, stained with propidium iodide, and the cell cycle was determined by flow cytometry. Data are expressed as means ± SE; n = 5. Bars with different letters are significantly different from one another (P < 0.05). C: RPTC were treated as described in B, and cell lysates were prepared, subjected to immunoprecipitation using an anti-EGF receptor (EGFR) antibody, and then subjected to immunoblot analysis with an anti-phospho-EGFR (Tyr 1068) or total EGFR antibody. Representative immunoblots from 3 or more experiments are shown.

 
To determine whether autocrine/paracrine-produced HB-EGF mediates proliferation of RPTC. RPTC were incubated with diluent or 10 µg/ml CRM197 for 24 h, and cell proliferation was determined by measuring the number of cells in the S phase of the cell cycle. CRM197 is a nontoxic and catalytically inactive (Glu-52) mutant of diphtheria toxin that binds specifically to the extracellular HB-EGF domain, thereby inhibiting HB-EGF mitogenic activity (30). Approximately 22% of RPTC were in the S phase 4 days following plating (Fig. 1B). CRM197 decreased the number of cells in the S phase to 12%. As a control, the EGF receptor inhibitor AG1478 decreased the number of cells in the S phase to the same degree as CRM197 (Fig. 1B).

EGF receptor phosphorylation was detected in RPTC 4 days after plating using immunoprecipitation with anti-EGF receptor antibody and immunoblot analysis with anti-phospho-EGF receptor antibody (Tyr 1068). Treatment with either CRM197 or AG1478 decreased EGF receptor phosphorylation (Fig. 1C). These data reveal that HB-EGF is produced and cleaved during RPTC proliferation following plating, that RPTC proliferation under these conditions is through the EGF receptor, and that CRM197 blocked RPTC proliferation. We suggest that HB-EGF may be the autocrine/paracrine factor responsible for RPTC proliferation under these conditions.

Effect of EGF receptor inhibition and HB-EGF siRNA on RPTC morphology and proliferation. To confirm the functional role of endogenous HB-EGF in RPTC proliferation, RPTC were transfected with HB-EGF siRNA or scrambled siRNA and replated. siRNA decreased HB-EGF levels 48 h later, as demonstrated by immunoblot analysis. HB-EGF protein (22 kDa) in RPTC transfected with HB-EGF siRNA decreased ~55% compared with RPTC transfected with scrambled siRNA (Fig. 2). No change in HB-EGF protein expression was observed in scrambled siRNA-transfected cells compared with control cells (data not shown).


Figure 2
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  		Fig. 2. Effect of HB-EGF siRNA on RPTC HB-EGF levels. RPTC were transfected with scrambled small interfering RNA (siRNA) or HB-EGF siRNA and then cultured for 48 h. Cells were harvested, and immunoblot analysis was carried out using antibodies against HB-EGF or GAPDH. A: representative immunoblot from 4 experiments is shown. B: soluble HB-EGF (22 kDa) was quantified by densitometry and is expressed as the percentage of HB-EGF levels in RPTC treated with the scrambled siRNA.

 
RPTC morphology and proliferation were determined 48 h after transfection using light microscopy, an MTT assay, and dish protein content. As a control, RPTC transfected with scrambled siRNA were incubated in the presence and absence of AG1478. siRNA HB-EGF decreased the formation of cell islands and decreased the proliferation of RPTC compared with RPTC expressing scrambled siRNA alone (Fig. 3). Treatment of RPTC transfected with scrambled siRNA and AG1478 resulted in further inhibition of RPTC proliferation compared with RPTC expressing HB-EGF siRNA (Fig. 3). These data confirm that RPTC proliferation following plating is the result of autocrine/paracrine-produced HB-EGF. The partial decrease in HB-EGF is consistent with the partial decrease in RPTC proliferation.


Figure 3
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  		Fig. 3. Effect of EGF receptor inhibition and HB-EGF siRNA on RPTC morphology and proliferation. RPTC were transfected with scrambled siRNA or HB-EGF siRNA and then cultured in the presence or absence of 10 µM AG1478 for 48 h. A: photomicrographs (x10) of RPTC 4 h (a–c) and 48 h (d–f) after transfection with scrambled siRNA or HB-EGF siRNA and cultured in the presence (b) or absence (e) of the EGF receptor inhibitor AG1478 (10 µM). Results are representative of 4 separate RPTC preparations. Cell numbers were estimated using an MTT assay (B) and measuring total cellular protein (C). Data are presented as means ± SE of the percentage of scrambled siRNA control (n = 4–5). Means with different superscripts are significantly different from each other (P < 0.05).

 
Expression of MMP-2, -3, -9, and ADAM10 and ADAM17 in RPTC and effects of MMP and ADAM inhibition on EGF receptor phosphorylation and RPTC proliferation. proHB-EGF is cleaved at the apical plasma membrane to yield soluble HB-EGF (19, 47). The proteolytic processing of the extracellular domain of proHB-EGF has been reported to be mediated by MMP -2, -3, -9, and/or ADAM10 and ADAM17 in a variety of cell types (2, 13, 19, 38, 43, 47). To determine whether these metalloproteases are expressed in RPTC and involved in RPTC proliferation, immunoblot analysis was performed using specific antibodies against each of them. All five of these proteases were detected in RPTC lysates, suggesting that all of them are expressed in RPTC (Fig. 4A).


Figure 4
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  		Fig. 4. Expression of MMP-2, MMP-3, MMP-9, ADAM10, and ADAM17 and the effect of GM6001 and TAPI-1 on RPTC proliferation and EGF receptor activation following plating. A: RPTC were grown to 40–50% confluence, and cell lysates were subjected to immunoblot analysis with antibodies to MMP-2, MMP-3, MMP-9, MMP-10, and ADAM17. B: RPTC were grown to 40–50% confluence, incubated for 24 h in the presence or absence of 10 µM GM6001 (GM) or 50 µM TAPI-1, stained with propidium iodide, and the cell cycle was determined by flow cytometry. Data are expressed as means ± SE; n = 3. Bars with different letters are significantly different from one another (P < 0.05). C: RPTC were treated as described in B. Cell lysates were prepared, subjected to immunoprecipitation using an anti-EGF receptor (EGFR) antibody, and subjected to immunoblot analysis with an anti-phospho-EGFR (Tyr 1068) or total EGFR antibody. Representative immunoblots from 3 or more experiments are shown. D: RPTC were grown to 40–50% confluence, incubated with HB-EGF (10 ng/ml) for 24 h in the presence or absence of 10 µM GM6001 (GM) or 50 µM TAPI-1 and then analyzed as described in B. Data are expressed as means + SE; n = 3. Bars with different letters are significantly different from one another (P < 0.05).

 
The role of metalloproteases in RPTC proliferation was examined using GM6001, a pan-MMP inhibitor, and tumor-necrosis factor protease inhibitor-1 (TAPI-1), a MMP and ADAM17 inhibitor (5, 23). Treatment of RPTC on day 3 after plating with either GM 6001(10 µM) or TAPI-1 (50 µM) equally decreased the number of cells in the S phase from 22 to 14% 24 h later (Fig. 4B). These two inhibitors also decreased EGF receptor phosphorylation 24 h after plating (Fig. 4C). However, these two inhibitors did not affect RPTC proliferation in the presence of HB-EGF (Fig. 4D).

To further explore the role of these metalloproteases in RPTC proliferation, RPTC were replated and allowed to proliferate for 48 h in the presence and absence of the ADAM10 inhibitor GI254023X or the ADAM10/17 inhibitor GW280264X (17). Whereas GW280264X inhibited RPTC proliferation, GI254023X had no effect on RPTC proliferation as measured by the MTT assay (Fig. 5). The EGF receptor inhibitor AG1478 was used as a positive control and inhibited RPTC proliferation. These results reveal that ADAM17 is responsible for RPTC proliferation following plating. We suggest that HB-EGF shedding and subsequent EGF receptor activation and RPTC proliferation are mediated by ADAM17.


Figure 5
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  		Fig. 5. Effect of ADAM10, ADAM10/17 and EGF receptor inhibition on RPTC proliferation. RPTC were grown to 80–90% confluence, released from the plate, and reseeded at 100,000 cells/dish. RPTC were treated with diluent, 3 µM GW280264X (GW), 10 µM GI1254023X (GI), or 10 µM AG1478 for 24 h, and cell number was determined by an MTT assay. Data are expressed as means ± SE; n = 3–4. Bars with different superscripts are significantly different from one another (P < 0.05).

 
EGF receptor activity is required for RPTC proliferation following oxidant injury. To investigate whether EGF receptor activation is required for RPTC proliferation following oxidant injury, cells were subjected to 1 mM H2O2 for 5 h and then incubated in the presence or absence of AG1478. RPTC proliferation was measured by the MTT assay and by the number of cells in the S phase. H2O2 exposure led to ~40% cell death 5 h after H2O2 addition (Fig. 6A), and the cell monolayer recovered 48 h after H2O2 removal after injury (Fig. 6B). The presence of AG1478 blocked the monolayer recovery and RPTC proliferation (Fig. 6, A and B).


Figure 6
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  		Fig. 6. EGF receptor mediates RPTC proliferation following oxidant injury. RPTC were grown to confluence and then exposed to 1 mM H2O2 for 5 h. After being washed with culture medium, RPTC were then incubated with 10 µM AG1478 for 24 (A) or 48 h (B). A: RPTC were photographed under various conditions as shown. B: MTT activity was determined immediately after injury and 48 h postinjury. Data are expressed as means ± SE of the percentage of MTT activity in controls at 48 h; n = 4–5. After H2O2 injury, RPTC were cultured with HB-EGF in the presence and absence of AG1478 (10 µM) for an additional 24 (C) or 1 h (D). C: cells were stained with propidium iodide, and the cell cycle was determined by flow cytometry. Data are expressed as means ± SE; n = 4–5. D: RPTC were treated as described in B. Cell lysates were prepared, subjected to immunoprecipitation using an anti-EGF receptor (EGFR) antibody, and subjected to immunoblot analysis with an anti-phospho-EGFR (Tyr 1068) or total EGFR. Representative immunoblots from 3 or more experiments are shown. Bars with different letters are significantly different from one another (P < 0.05).

 
We previously reported that exogenous HB-EGF further stimulated RPTC proliferation following plating in an EGF receptor-dependent manner (60). To determine whether this occurred after oxidant injury, exogenous HB-EGF was added at 5 h after H2O2 exposure and the number of cells in the S phase were determined 24 h later in the presence and absence of AG1478. HB-EGF further stimulated RPTC proliferation, and EGF receptor inhibition blocked basal and HB-EGF-mediated proliferation (Fig. 6C).

We previously reported that the EGF receptor is activated following oxidant injury (61). To examine whether exogenous HB-EGF further activates the EGF receptor following oxidant injury, RPTC were exposed to 1 mM H2O2 for 5 h and then incubated with HB-EGF for an additional hour. Cells were harvested and immunoblot analysis was performed using an anti-phospho-EGF receptor (Tyr 1068) antibody. EGF receptor phosphorylation was detected after oxidant injury and was further activated by exogenous HB-EGF (Fig. 6D). AG1478 inhibited EGF receptor phosphorylation following oxidant injury in the absence and presence of HB-EGF. Therefore, RPTC proliferation following oxidant injury is dependent on EGF receptor activation and exogenous HB-EGF potentiates cell proliferation under this condition.

Effect of CRM197, GM6001, and TAPI-1 on RPTC proliferation following oxidant injury. To determine whether oxidant-induced RPTC proliferation is mediated by HB-EGF or MMP/ADAM, RPTC were incubated with CRM197, GM6001, or TAPI-1 and the number of cells in the S phase were determined 24 h later. Following oxidant injury, none of these compounds inhibited RPTC proliferation (Fig. 7). These data reveal that EGF receptor-dependent RPTC proliferation following oxidant injury is not mediated by the release of an EGF receptor ligand such as HB-EGF.


Figure 7
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  		Fig. 7. Effect of CRM197, GM6001 and TAPI-1 on RPTC proliferation following oxidant injury. Confluent RPTC were exposed to 1 mM H2O2 for 5 h. RPTC were washed with culture medium and incubated with vehicle, 10 µg/ml CRM197, 10 µM GM6001(GM), or 50 µM TAPI-1 for an additional 24 h. After being stained with propidium iodide, the cell cycle was determined by flow cytometry. Data are expressed as means ± SE; n = 3. No significant differences were noted (P > 0.05).

 
Inhibition of Src family kinases blocks RPTC proliferation following oxidant injury. Our previous studies indicated that H2O2 induced EGF receptor activation by a Src-dependent mechanism (61). We hypothesized that Src may be mediating EGF receptor-dependent RPTC proliferation following oxidant injury. RPTC were exposed to H2O2 for 5 h and then incubated with PP1 (10 µM), a selective Src family kinase inhibitor, for an additional 24 h. Oxidant injury resulted in cell loss, and the remaining islands of RPTC grew out from the edges 24 h after the injury (Fig. 8A). The addition of exogenous HB-EGF promoted outgrowth of RPTC, whereas PP1 blocked these responses. PP1 treatment decreased the number of RPTC in the S phase following oxidant injury in the absence and presence of exogenous HB-EGF (Fig. 8B).


Figure 8
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  		Fig. 8. Effect of PP1 on RPTC proliferation and EGF receptor activation following oxidant injury in the presence or absence of exogenous HB-EGF. RPTC were grown to confluence and then exposed to 1 mM H2O2 for 5 h. RPTC were washed with culture medium and exposed to 10 µM PP1 for 24 h in the presence and absence of exogenous HB-EGF. A: RPTC were photographed under various conditions as shown. B: after being stained with propidium iodide, the cell cycle was determined by flow cytometry. Data are expressed as means ± SE; n = 5. Bars with different letters are significantly different from one another (P < 0.05). C: cell lysates were prepared, subjected to immunoprecipitation using an anti-EGF receptor (EGFR) antibody, and subjected to immunoblot analysis with an anti-phospho-EGFR (Tyr 1068) or total EGFR. D: cell lysates were prepared and subjected to immunoblot analysis using antibodies against phospho-Src and Src. Representative immunoblots from 3 or more experiments are shown.

 
It has been shown that the EGF receptor at Tyr 845 is a specific phosphorylation site of Src and is able to initiate mitogenic signaling in a variety of cell types (21). We therefore examined the effect of Src inhibition on Tyr 845 phosphorylation in the absence or presence of HB-EGF following oxidant injury. Phosphorylation of the EGF receptor at Tyr 845 was detected after oxidant injury and further increased in the presence of HB-EGF (Fig. 8C). PP1 blocked H2O2-induced phosphorylation of the EGF receptor at Tyr 845 and blocked HB-EGF-enhanced EGF receptor phosphorylation.

Src activity is regulated primarily by phosphorylation of different tyrosine sites with phosphorylation at Tyr 416, in the catalytic domain, being an activating signal. Therefore, we also examined the effect of PP1 on Src phosphorylation using anti-phospho (Tyr 416) antibody. H2O2-induced Src phosphorylation was increased in the presence of HB-EGF and blocked by PP1 treatment (Fig. 8D). These data suggest that Src is critically involved in regulating RPTC proliferation following oxidant injury and is also required for HB-EGF potentiation of oxidant-induced RPTC proliferation.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we provide evidence that HB-EGF acts as an autocrine/paracine EGF receptor ligand that mediates RPTC proliferation following plating. In contrast, Src, but not HB-EGF, is involved in the activation of EGF receptor and RPTC proliferation following oxidant injury. Furthermore, Src is implicated in the activation of the EGF receptor by exogenous HB-EGF following oxidant injury. This study reveals that two activation mechanisms of the EGF receptor are present in the same cell type and operate under different conditions.

Ligand-dependent transactivation has been reported to be involved in EGF receptor activation in response to a variety of nonspecific stimuli. Among several members of the EGF receptor family of ligands, HB-EGF has been recognized to be a common EGF receptor ligand subjected to ectodomain shedding, leading to the transactivation of the EGF receptor in a variety of cells (9, 31). The results from our studies also support the idea that metalloprotease-dependent ectodomain shedding of HB-EGF is necessary for autocrine/paracrine proliferation of RPTC following plating. First, proHB-EGF and cleaved forms are present in cell lysates of proliferating RPTC. Second, inhibition of HB-EGF binding to the EGF receptor with CRM197 and depletion of HB-EGF with RNA interference inhibited RPTC proliferation. Third, treatment with GM6001, a general metalloproteases inhibitor, and TAPI-1, an ADAM17 and MMP inhibitor, decreased RPTC proliferation. Fourth, MMP-2, MMP-3, MMP-9, ADAM10, and ADAM17 are expressed in RPTC. Finally, the ADAM10/17 inhibitor GW280264X, but not the ADAM10 inhibitor GI254023X, inhibited RPTC proliferation. Based on these data, we suggest that HB-EGF shedding and subsequent EGF receptor activation and RPTC proliferation are mediated by ADAM17 following plating.

Despite many reports in the literature pointing to HB-EGF ectodomain shedding as a major pathway mediating EGF receptor transactivation, only a few studies directly demonstrated the release of HB-EGF from cells containing high levels of proHB-EGF (7, 49). Using immunoblot analysis of concentrated medium, we also were unable to detect soluble HB-EGF in the cultured medium as reported by other laboratories (27, 52). This may be due to the low concentration of HB-EGF released from cells, its rapid binding to the EGF receptor once it is released, and/or it degradation. In addition, proHB-EGF may also directly activate the EGF receptor on neighboring cells. It has been reported that the membrane-bound forms of some EGF receptor ligands may possess juxtacrine activity (11).

Unlike RPTC proliferation that occurs after plating, our data reveal that HB-EGF is not involved in EGF receptor activation and RPTC proliferation following oxidant injury. Previously, we showed that H2O2-induced EGF receptor activation was not inhibited by CRM197 and GM6001 (61). In this study, we further show that although EGF receptor activation is critically involved in regulation of RPTC proliferation following oxidant injury, inhibition of either HB-EGF or MMP/ADAM failed to block cell cycle progression into the S phase. However, exogenous HB-EGF was still able to stimulate proliferation of RPTC after oxidant injury (see below). One possibility is that oxidant injury results in suppression of MMP/ADAM synthesis and/or activation and subsequently decreased production of soluble HB-EGF. In this context, a recent study showed that oxidant injury to retinal pigment epithelium decreased activity of MMP-2 (29).

Our data provide evidence that Src plays a critical role in regulating RPTC proliferation after oxidant injury. Using a common inhibitor of Src family kinases, PP1, we previously demonstrated that Src mediates EGF receptor phosphorylation at Tyr 845 and Tyr 1068 in response to H2O2 (61). In this study, we show that inactivation of Src inhibited RPTC proliferation after oxidant injury. Phosphorylation of Tyr 845, a specific Src phosphorylation site on the EGF receptor, has been reported to be involved in the modulation of EGF receptor function and cell cycle progression (21). Recently, Stat 5b, a transcription factor, has been identified as a substrate of Tyr 845 and mediates proliferation of tumor cells (1, 21). In addition, this pathway also was implicated in DNA synthesis induced by the G protein-coupled agonists endothelin and lysophosphatidic acid and cytokine and growth hormones in tumor cells (6). Thus RPTC proliferation after oxidant injury may also occur through a Tyr 845-mediated signaling pathway. Experiments are underway to examine whether Stat5b and additional signaling intermediates downstream of the EGF receptor mediate proliferation of RPTC after oxidant injury.

In addition, Src also may be involved in the regulation of HB-EGF-enhanced RPTC proliferation after oxidant injury. HB-EGF potentiated RPTC proliferation after oxidant injury, which was inhibited by PP1. Enhancement of proliferation by HB-EGF in RPTC exposed to H2O2 is consistent with the idea that the EGF receptor is partially activated by the oxidant and is still able to respond to stimulation to exogenous EGF receptor ligands. Indeed, addition of HB-EGF led to the phosphorylation of EGF receptor Tyr 845 to a greater degree than cells treated with H2O2 alone. HB-EGF treatment also enhanced phosphorylation of Src in RPTC that were oxidant injured (Fig. 8D). Thus activation of the EGF receptor by Src may represent a convergent signaling pathway accessible to both EGF receptor ligands and oxidant stress. In this context, Src also has been reported to be activated in response to EGF (24) and Src-mediated phosphorylation of Y845 is required for the EGF-induced mitogenic function of the EGF receptor in COS-7 cells (51).

Src is one of three Src family kinases (Src, Fyn, Yes) that are ubiquitously expressed in mammalian cells. In renal epithelial cells, it remains unclear which member(s) of Src is responsible for activation of the EGF receptor. Recent studies have shown that active Src is highly expressed in regenerating tubular cells in a rat model with ischemia/reperfusion injury (50), suggesting that Src may be an important mediator of renal regeneration. Further studies are required to explore the role of individual members as mediators of EGF receptor activation following injury in RPTC.

In conclusion, our results provide strong evidence that HB-EGF is an autocrine/paracrine factor that mediates RPTC proliferation. In addition, our results demonstrate that HB-EGF and Src mediate EGF receptor transactivation and RPTC proliferation under different conditions. Whereas EGF receptor activation and proliferation following RPTC plating requires ADAM17-dependent production of HB-EGF, Src activation contributes to EGF receptor activation and RPTC proliferation following oxidant injury and treatment with exogenous HB-EGF.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health (NIH) Grant DK-071997 to S. Zhuang. K. Rasbach was supported by NIH Training Grant T32-HL-007260, and G. R. Kinsey was supported by NIH Training Grant T32-ES-012878.


   FOOTNOTES
 

Addresses for reprint requests and other correspondence: R. G. Schnellmann, Dept. of Pharmaceutical and Biomedical Sciences, Medical Univ. of South Carolina, 280 Calhoun St., POB 250140, Charleston, SC 29425 (e-mail: schnell{at}musc.edu) or S. Zhuang, Dept. of Medicine, Brown Univ. School of Medicine, Rhode Island Hospital-Middle House 301, 593 Eddy St., Providence, RI 02903 (e-mail: szhuang{at}lifespan.org)

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.

* Present address of G. R. Kinsey: University of Virginia, Charlottesville, VA 22904 (e:mail: grk4n{at}Virginia.edu). Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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