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CELLULAR AND MOLECULAR

Collagenase-2 and -3 Mediate Epidermal Growth Factor Receptor Transactivation by Bradykinin B₂ Receptor in Kidney Cells

The Medical and Research Services of the Ralph H. Johnson Veterans Affairs Medical Center and Department of Medicine (Nephrology Division) of the Medical University of South Carolina, Charleston, South Carolina

Received March 3, 2006; accepted May 18, 2006.

	Abstract

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We have previously shown that stimulation of extracellular signal-regulated protein kinase (ERK) by bradykinin (BK) in murine inner medullary collecting duct (mIMCD)-3 cells is mediated by epidermal growth factor receptor (EGFR) transactivation. The mechanism of EGFR transactivation seemed to be novel, because it does not require phospholipase C, Ca²⁺, calmodulin, protein kinase C, G_i subunits, or EGFR-B₂ receptor heterodimerization. In this study, we demonstrated the involvement of matrix metalloproteinases (MMPs) in B₂ receptor-induced EGFR transactivation using their broad-spectrum inhibitors batimastat and N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (Galardin) (GM-6001). Selective inhibitors for collagenase-2 and -3 (MMP-8 and MMP-13, respectively) blocked BK-induced EGFR phosphorylation and ERK activation, whereas inhibitors for MMP-1, -2, -3, -7, or -9 were without effect. Transfection of mIMCD-3 cells with MMP-8 small interfering RNA (siRNA) resulted in 50% decrease of BK-induced ERK activation. A neutralizing antibody against MMP-13 as well as transfection with MMP-13 siRNA produced a similar effect. Inhibition of both collagenases resulted in 65% decrease of BK-induced ERK activation, supporting roles for both enzymes. Stimulation of mIMCD-3 cells with 10 nM BK increased the activity of collagenases in concentrated culture media within 10 min. Moreover, recombinant MMP-13 and MMP-8, when applied to mIMCD-3 cells for 10 min without BK, stimulated tyrosine phosphorylation of EGFR and caused 250% increase over basal ERK phosphorylation comparable with BK-induced ERK activation. Collagenases-induced ERK activation was inhibited by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478) and thus dependent on EGFR tyrosine kinase activity. This study demonstrates a novel role for collagenase-2 and -3 in signaling of the G_q-coupled BK B₂ receptor in mIMCD-3 cells.

In our previous work, we provided evidence that BK stimulates early mitogenic signals associated with activation of ERK1/2 in renal epithelia cell line derived from the inner medullary collecting duct of mice (mIMCD-3 cell line) (Mukhin et al., 2003). We showed that BK stimulated proliferation of mIMCD-3 cells that was dependent on activation of epidermal growth factor receptor (EGFR) tyrosine kinase and ERK1/2. Our results suggested that the bradykinin B₂ receptor stimulates Ras-dependent activation of ERK1/2 in mIMCD-3 cells via transactivation of the EGFR.

The mechanisms underlying GPCR-induced EGFR transactivation have not been clearly defined. One transactivation pathway recently proposed for the ₂-adrenergic receptor includes the isoproterenol-induced formation of a ₂-adrenergic receptor-EGFR complex (Maudsley et al., 2000). Multiple G protein-coupled receptors transactivate the EGFR through the intermediary actions of the calcium-dependent nonreceptor tyrosine kinase PYK-2 and of Src (a nonreceptor tyrosine kinase) kinase in various native and transfected cell types (Grosse et al., 2000; Keely et al., 2000). The thromboxane A₂ receptor transactivates the EGFR through G_q, phospholipase C, and protein kinase C-mediated phosphorylation of the thromboxane A₂ receptor, which results in coupling to G_i and Src-dependent activation of the EGFR (Gao et al., 2001). The angiotensin II AT_1A receptor transactivates the EGFR through activation of matrix metalloproteinases and release of tethered heparin-bound EGF (HB-EGF) in mesangial, vascular smooth muscle cells, and C9 hepatocytes (Eguchi et al., 2001; Uchiyama-Tanaka et al., 2001; Shah et al., 2004). The M₁ muscarinic and thrombin receptors use a similar pathway in Rat-1 cells and in rat smooth muscle cells, respectively (Prenzel et al., 1999; Kalmes et al., 2000). In contrast, carbachol-induced transactivation of the EGFR in colonic epithelial cells involves the matrix metalloproteinase (MMP)-dependent release of transforming growth factor (TGF)- (McCole et al., 2002). The gelatinases MMP-2 and MMP-9 have been implicated in transactivation of the EGFR by the G_q/11-coupled gonadotropin-releasing hormone receptor in gonadotropic cells (Roelle et al., 2003).

Bradykinin B₂ receptor-induced activation of the EGFR has been described in COS-7 cells transiently transfected with bradykinin B₂ receptors (Adomeit et al., 1999). In contrast, BK-induced activation of ERK1/2 in A431 epidermoid carcinoma cells is independent of the EGFR (Graness et al., 2000).

In our previous work, we suggested that the mechanism of B₂ receptor-induced transactivation of EGFR in mIMCD-3 cells is different from the mechanisms described in the literature, for it does not involve complex formation between the B₂ receptor and EGFR, and it does not require pertussis toxin-sensitive G proteins, Ca²⁺, calmodulin, phospholipase C, or protein kinase C activity (Mukhin et al., 2003). In the current study, we test the hypothesis that matrix metalloproteinases are involved in this signaling pathway.

MMPs, a large family of zinc-dependent, matrix-degrading enzymes, are thought to play a central role in degradation of extracellular matrix (ECM) (Nagase and Woessner, 1999). In the kidney, the accumulation of ECM molecules can lead to interstitial fibrosis and glomerulosclerosis (He et al., 1995; Norman and Lewis, 1996). MMPs are also involved in normal kidney development and potentially play roles in diabetic nephropathy and in inflammatory glomerulonephritis (Lenz et al., 2000). Despite the emerging roles of MMPs in the kidney, little is presently known about possible interactions between BK receptors and MMPs. The effect of BK on the expression of MMP-3, MMP-20, and membrane type-1-MMP has been described for cultured granulosa cells (Kimura et al., 2001). In vivo studies on the B₂ receptor knockout mice suggest that the B₂ receptor might play a protective role in renal tubulointerstitial fibrosis (Schanstra et al., 2002), but a possible connection between the B₂ receptor and MMPs in kidney cells remains unclear. Herein, we demonstrate novel roles for collagenase-2 and -3 (MMP-8 and MMP-13, respectively) in G_q-coupled B₂ receptor signaling in mIMCD-3 cells.

	Materials and Methods

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Materials. Cell culture supplies were obtained from Invitrogen (Grand Island, NY), or Corning Life Sciences (Cambridge, MA). Bradykinin and anti--actin antibody were from Sigma-Aldrich (St. Louis, MO). Phospho-ERK and phospho-MEK kits were obtained from Cell Signaling Technology Inc. (Beverly, MA). The Ras activation kit, EGFR polyclonal antibody, and anti-phospho-EGFR-(Tyr¹¹⁷³) monoclonal antibody were from Upstate Biotechnology (Lake Placid, NY). MMP inhibitors and neutralizing antibody against TGF- were from Calbiochem (San Diego, CA). Purified MMP-8 from human neutrophils, recombinant MMP-13, MMP-13 neutralizing antibodies, and type 1 collagenase activity assay kit were from Chemicon International (Temecula, CA). MMP-8 and MMP-13 antibodies, MMP-8 siRNA, MMP-13 siRNA, and control siRNA were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Neutralizing antibody against HB-EGF was from R&D Systems (Minneapolis, MN). The Mouse MMP MultiGene-12 RT-PCR Profiling kit was from SuperArray Bioscience Corporation (Frederick, MD).

Cell Culture. mIMCD-3 cells were obtained from American Type Culture Collection (Manassas, VA). mIMCD-3 cells were grown in equal mixtures of Dulbecco's modified Eagle's medium and Ham's F-12 medium, supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Transfections of mIMCD-3 Cells: MMP-8 and MMP-13 Silencing. Transfections of mIMCD-3 cells were achieved by nucleofection with an Amaxa Biosystems instrument (Giessen, Germany) in to transfer DNA or RNA directly into the nucleus of the cell. Cells (1 x 10⁶) were resuspended in 100 µl of Nucleofector Solution R (Amaxa Biosystems), and nucleofected with either 100 nM MMP-8 siRNA or MMP-13 siRNA or control siRNA, or with combination of MMP-8 and MMP-13 siRNAs (Santa Cruz Biotechnology, Inc.) using manufacturer's protocol T-16. Forty-eight hours postnucleofection, cells were stimulated with BK or vehicle, lysed, and analyzed for MMP-8 expression by Western blotting with an anti-MMP-8 goat polyclonal antibody, for MMP-13 expression by Western blotting with an anti-MMP-13 rabbit polyclonal antibody, and for ERK activation. Blots were reprobed with a mouse monoclonal anti--actin antibody to control for protein loading and for silencing specificity.

ERK Assay. ERK phosphorylation was assessed by immunoblot using phosphorylation state-specific antibodies as described previously (Mukhin et al., 2003) in mIMCD-3 cells treated for various times with varying concentrations of BK, EGF, MMPs, or vehicle.

Ras Assay. Ras activation was assessed by a nonradioactive Ras assay kit (Upstate Biotechnology) as described previously (Mukhin et al., 2003). Quiescent mIMCD-3 cell monolayers were pretreated with inhibitors or vehicle for 30 min, stimulated with 10 nM BK or vehicle for 5 min, and lysed in a 1 ml/100-mm dish of Mg²⁺ lysis buffer (150 mM NaCl, 25 mM HEPES, pH 7.5, 1 mM EDTA, 10 mM MgCl₂, 1% Igepal CA-630, 25 mM sodium fluoride, 1 mM Na₃VO₄, 10 µg/ml each of aprotinin and leupeptin, and 10% glycerol). Cell lysates were precleared by incubating with glutathione-agarose for 10 min at 4°C. Precleared lysates (1 µg/µl total cell protein) were incubated with 10 µg of Raf-1 Ras binding domain (glutathione S-transferase fusion protein, corresponding to the Ras binding domain of Raf-1) and bound to glutathione-agarose for 30 min at 4°C. The agarose beads were collected by centrifugation, washed three times with Mg²⁺ lysis buffer, resuspended in 2x Laemmli sample buffer, boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis and subsequent immunoblot analysis with monoclonal anti-Ras IgG.

EGF Receptor Phosphorylation Assay. The phosphorylation state of EGFR was assessed by immunoprecipitation/Western blotting studies as described previously (Mukhin et al., 2003). Quiescent mIMCD-3 cells, grown in 100-mm dishes, were pretreated with vehicle or inhibitors for 30 min. Cells were subsequently treated with 10 nM BK, 1 ng/ml EGF, or vehicle for 5 min and lysed in radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM sodium fluoride, 1 mM Na₃VO₄, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin). Cell lysates were precleared by incubating with protein A-agarose bead slurries for 30 min at 4°C. Precleared lysates (1 µg/µl total cell protein) were incubated with 4 µg of anti-EGFR polyclonal IgG (Upstate Biotechnology) overnight at 4°C. The immunocomplexes were captured by the addition of protein A-agarose bead slurries, with incubation for 2 h more at 4°C. The agarose beads were collected by centrifugation, washed three times with radioimmunoprecipitation assay buffer, resuspended in 2x Laemmli sample buffer, boiled for 5 min, and subjected to SDS-polyacrylamide gel electrophoresis. After semidry transfer to polyvinylidene difluoride membranes, the membranes were probed with monoclonal anti-phospho-EGFR(Tyr¹¹⁷³) antibody to assess the phosphorylation state of EGFR, or with EGFR antibody (Upstate Biotechnology).

Measurement of Intracellular Ca²⁺. We used a FLIPR (Molecular Devices, Sunnyvale, CA) fluorometric imaging plate reader system to measure intracellular Ca²⁺ in mIMCD-3 plated into 96-well microtiter plates as described previously (Mukhin et al., 2001). Cells were seeded (50,000 cells/well) in 96-well clear-bottomed black microplates (Corning Life Sciences) and left overnight in a CO₂ incubator at 37°C. On the day of the assay, cells were loaded with 4 µM Fluo-3 for 1 h in Hanks' balanced salt solution, pH 7.4, containing 20 mM HEPES and 2.5 mM probenecid. After loading, cells were washed four times with Hanks' balanced salt solution on an automated platewasher (Labsystems, Helsinki, Finland) and placed into the FLIPR. All 96 wells were simultaneously illuminated for 0.4 s by an argon laser (488 nm) set at 300 mW (Coherent Inc., Santa Clara, CA). Fluorescence emission readings were measured using a 540-nm bandpass filter at 1-s intervals until a baseline was obtained (about 10 readings). Cells were then exposed simultaneously to 10 nM BK, 1 µM ATP, or 1 µM A23187 [GenBank] (calcimycin; calcium ionophore), and fluorescence readings were obtained from each well every second for a total of 2 min and then every 6 s for 3 min. Replicate readings were averaged, and each tracing was normalized against background fluorescence from wells treated with buffer controls. Tracings were acquired, averaged, and normalized using the FLIPR Control software program (Molecular Devices).

Study of Expression Levels of MMPs in mIMCD-3 Cells. The expression levels of MMPs were monitored using the Mouse MMP MultiGene-12 RT-PCR Profiling kit (SuperArray Bioscience Corporation) according to the manufacturer's instructions by RT-PCR analysis. In brief, RNA from quiescent mIMCD-3 cells grown in six-well plates was isolated using TRIzol reagent (Invitrogen), and 5 µg of RNA was reverse-transcribed using SuperScript III First-Strand Synthesis System (Invitrogen). For polymerase chain reaction amplification of MMP-13, mRNA primer sets of the MMP-I kit (SuperArray Biosciences Corporation) were used. Polymerase chain reaction products were subjected to electrophoresis on 2% agarose gels, and DNA was visualized by ethidium bromide staining.

Collagenase Activity Assay. Quiescent mIMCD-3 cells grown in 100-mm dishes were treated with 10 nM BK for different periods. The cultured medium was collected and concentrated 100 times using Vivaspin concentrators (Vivascience, Stonehouse, UK). The activities of type 1 collagenases (MMP-1, -8, and -13) were assessed by assay kit (Chemicon International), which uses biotinylated collagen as a substrate for active collagenases. The biotinylated fragments were transferred to a biotin-binding 96-well plate and detected with streptavidin-enzyme complex. The addition of enzyme substrate resulted in a colored product, which was detected by its optical density at 450 nm using a Victor-2 1420 multilabel counter (PerkinElmer Life and Analytical Sciences, Boston, MA). Optical density values of the test samples were compared with the provided MMP-1 standards.

Data Analysis. ERK assays were performed in duplicate and repeated at least three times. EGFR assays and Ras activation assays were repeated 3 times for each condition. Collagenase activity assays were performed in triplicate and repeated at least two times. Data are presented as mean + S.E.M. and were analyzed for repeated measures by Student's t test for unpaired two-tailed analysis. Differences were considered significant at P < 0.05.

	Results

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MMPs Are Involved in Bradykinin-Induced ERK1/2 Activation and EGFR Transactivation in mIMCD-3 Cells. In the first set of experiments, we tested the involvement of MMPs in B₂ receptor-induced EGFR transactivation and ERK1/2 phosphorylation using synthetic hydroxamate-based broad-spectrum MMP inhibitors batimastat and GM-6001. Pretreatment of mIMCD-3 cells for 30 min with 5 µM batimastat or 5 µM GM-6001 completely blocked BK-induced activation of ERK without affecting EGF-induced ERK activation (Fig. 1A). In contrast, a negative control for GM-6001 was without effect. We also tested the ability of these inhibitors to affect phosphorylation of the EGFR. The phosphorylation state of the EGFR was assessed by immunoprecipitation/Western blotting studies. mIMCD-3 cells pretreated with inhibitors or vehicles were stimulated with 10 nM BK or with 1 ng/ml EGF for 5 min. GM-6001 abolished BK-induced, but not EGF-induced, phosphorylation of EGFR (n = 4), whereas a negative control for GM-6001 was without effect. Pretreatment with batimastat produced the same effect on BK-induced phosphorylation of EGFR (not shown). The inset in Fig. 1B shows a representative immunoblot.

View larger version (44K): [in this window] [in a new window]   Fig. 1. Metalloproteinases are involved in bradykinin-induced ERK1/2 activation and transactivation of EGFR in mIMCD-3 Cells. mIMCD-3 cells were pretreated for 30 min with 5 µM batimastat, 5 µM GM-6001, 5 µM negative GM-6001, or vehicle. Cells were then stimulated with 10 nM BK or with 1 ng/ml EGF for 5 min. A, ERK1/2 phosphorylation was measured as described under Materials and Methods. Experiments were performed at least four times in duplicates. Data are presented as mean ± S.E.M. (n = 4; *, P < 0.01 versus vehicle-treated cells; , P < 0.01 versus control). B, in the inset, a representative immunoblot of phospho-EGFR is shown. EGFR phosphorylation was measured as described under Materials and Methods. Experiments were performed at least four times. Data are presented as mean ± S.E.M. (n = 4; , P < 0.05 versus vehicle-treated cells; , P < 0.05 versus control).

View larger version (19K): [in this window] [in a new window]   Fig. 2. Bradykinin-induced activation of Ras in mIMCD-3 cells is metalloproteinase-dependent. mIMCD-3 cells were pretreated with vehicle or broad-spectrum inhibitors of MMPs: 5 µM batimastat or 5 µM GM-6001; or inactive form of GM-6001 (5 µM). Cells were then stimulated with 10 nM BK or with 1 ng/ml EGF for 5 min. Ras activity was measured using Ras activation assay kit as described under Materials and Methods. Experiments were performed at least three times in duplicates. Data are presented as mean ± S.E.M. *, P < 0.01 versus vehicle-treated cells; , P < 0.01 versus control.

MMP Inhibitors Do Not Affect Ca²⁺ Signaling in mIMCD3 Cells. We have shown previously that BK increases intracellular Ca²⁺ release in mIMCD-3 cells (Mukhin et al., 2001). In the next set of experiments, we tested whether MMP inhibitors are able to influence this effect. We used a FLIPR (Molecular Devices) fluorometric imaging platereader system to measure intracellular Ca²⁺ in mIMCD-3 cells. GM-6001 (5 µM), 5 µM GM-1489 (another broad-spectrum MMP inhibitor), and 10 µM MMP inhibitor III (selective for MMP-1, -2, -3, -7, and -13) had no effect on BK-induced intracellular Ca²⁺ release as measured by FLIPR (Fig. 3), suggesting that MMP inhibitors selectively affect BK-induced EGFR phosphorylation. This FLIPR experiment also shows that the effects of inhibitors on ERK and EGFR are not due to toxic effects of the inhibitors.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Inhibitors of MMPs do not affect Ca2+ signal in mIMCD-3 cells. mIMCD-3 cells were pretreated with vehicle (rows 2 and 3), 5 µM GM-1489 (rows 4 and 5), 5 µM inactive form of GM-6001 (rows 6 and 7), 5 µM GM-6001 (rows 8 and 9), or 10 µM MMP inhibitor III. Cells were then stimulated with 10 nM BK (B and C), 1 µM ATP (D and E), 1 µM Ca2+ ionophore A23187 (F and G), or vehicle (A and H). Intracellular Ca2+ release was measured in FLIPR experiments as described under Materials and Methods.

Characterization of Metalloproteinase Types Present in mIMCD-3 Cells. Because to date 28 different MMPs have been identified (Egeblad and Werb, 2002), we needed to determine which isoforms are present in mIMCD-3 cells to plan more specific experiments. We extracted RNA from the cells using the TRIzol reagent and used a mouse MMP MultiGene RT-PCR Profiling kit. Thus, we established that mIMCD-3 cells express mRNAs for the following MMPs: MMP-2, MMP-3, MMP-8, MMP-9, MMP-11, MMP-13, MMP-14, MMP-15, MMP-17, MMP-19, MMP-20, and MMP-23 as well as for all four tissue inhibitors of metalloproteinases (Table 1)

Inhibitor Data Suggest the Involvement of Collagenase-2 and -3 in the BK-Induced EGFR Transactivation and ERK1/2 Phosphorylation. In an attempt to identify the MMPs that mediate BK-induced EGFR transactivation, we used a number of commercially available broad-spectrum as well as more selective MMP inhibitors. Those studies showed that selective inhibitors for MMP-8 and MMP-13 were able to block BK-induced EGFR phosphorylation and ERK activation. Inhibitors for MMP-1, -2, -3, -7, or -9 were without effect (Table 2).

Neutralizing Antibody against MMP-13 Decreases BK-Induced ERK Activation. To support the involvement of MMP-13 in BK-induced ERK activation, we preincubated mIMCD-3 cells with 0.1 mg/ml neutralizing MMP-13 antibody for 2 h before stimulation with 10 nM BK for 5 min. Control samples were preincubated with 0.1 mg/ml normal goat IgG. Figure 4A shows that neutralizing MMP-13 antibody reduced BK-induced ERK phosphorylation by about 50%, supporting a role for MMP-13 in this process.

View larger version (23K): [in this window] [in a new window]   Fig. 4. MMP-8 and MMP-13 are involved in bradykinin-induced ERK1/2 activation. A, neutralizing antibody against MMP-13 decreases BK-induced ERK1/2 activation. mIMCD-3 cells were preincubated with 0.1 mg/ml neutralizing MMP-13 antibody for 2 h before stimulation with 10 nM BK for 5 min. Control samples were preincubated with 0.1 mg/ml normal goat IgG. Neutralizing MMP-13 antibody reduced BK-induced ERK phosphorylation by 50%. Experiments were performed three times in duplicates. Data are presented as mean ± S.E.M. *, P < 0.01 versus vehicle-treated cells; , P < 0.05 versus control. B, bradykinin-induced ERK phosphorylation in mIMCD-3 cells transfected with siRNA for MMP-8. mIMCD-3 cells were nucleofected with either MMP-8 siRNA or with control siRNA. Forty-eight hours postnucleofection, cells were stimulated with vehicle or 10 nM BK for 5 min, lysed, and analyzed for ERK phosphorylation. More than 50% reduction of BK-induced ERK activation was observed. Experiments were performed three times in duplicates. Data are presented as mean ± S.E.M. , P < 0.05 versus vehicle-treated cells; , P < 0.05 versus control. C, effective silencing of MMP-8 expression in mIMCD-3 cells transfected with MMP-8 siRNA was shown by immunoblotting with anti-MMP-8 antibody. Antibody against -actin was used to control the equal amounts of proteins in the lysates.

Combined Inhibition of MMP-8 and MMP-13 Further Decreases BK-Induced ERK Activation. Because inhibition of MMP-8 by transfection with MMP-8 siRNA and inhibition of MMP-13 neutralizing antibody resulted only in partial (50%) decrease in BK-induced ERK phosphorylation (Fig. 4), in the next set of experiments we used the combination of both treatments to block the activity of both collagenases and to clarify the role of two specific MMPs in BK-induced ERK activation. The results are presented in Fig. 5. Cells transfected with MMP-8 siRNA were further preincubated with 0.1 mg/ml normal goat IgG (MMP-8 siRNA) or with 0.1 mg/ml neutralizing MMP-13 antibody (MMP-13 Ab and MMP-8 siRNA). Control cells transfected with control siRNA were preincubated with 0.1 mg/ml normal goat IgG (control) or with neutralizing MMP-13 antibody (MMP-13 Ab). After 2-h preincubation with antibody or normal IgG, cells were stimulated with 10 nM BK or vehicle for 5 min and assessed for ERK activity.

View larger version (39K): [in this window] [in a new window]   Fig. 5. Combined inhibition of MMP-8 and MMP-13 further decreases bradykinin-induced ERK activation. A, mIMCD-3 cells were nucleofected with control siRNA or with either MMP-8 siRNA or MMP-13 siRNA alone or with both MMP-8 and MMP-13 siRNAs. Forty-eight hours postnucleofection, cells transfected with MMP-8 siRNA were further preincubated with 0.1 mg/ml normal goat IgG (MMP-8 siRNA) or with 0.1 mg/ml neutralizing MMP-13 antibody (MMP-13 Ab and MMP-8 siRNA). Control cells transfected with control siRNA were preincubated with 0.1 mg/ml normal goat IgG (control) or with neutralizing MMP-13 antibody (MMP-13 Ab). After 2-h preincubation with antibody or normal IgG, cells were stimulated with 10 nM BK or vehicle for 5 min and assessed for ERK activity. Cells nucleofected with MMP-13 siRNA alone (MMP-13 siRNA) or with both MMP-8 and MMP-13 siRNAs (MMP-8 and MMP-13 siRNA) were stimulated with vehicle or 10 nM BK for 5 min, lysed, and analyzed for ERK phosphorylation. Experiments were performed at least six times in duplicates. Data are presented as mean ± S.E.M. *, P < 0.01 versus cells transfected with both MMP-8 and MMP-13 siRNAs were inhibited; , P < 0.05 versus cells transfected with MMP-8 siRNA and treated with neutralizing MMP-13 antibody. B, effective silencing of MMP-13 expression in mIMCD-3 cells transfected with MMP-13 siRNA was shown by immunoblotting with anti-MMP-13 antibody. Antibody against -actin was used to control the equal amounts of proteins in the lysates and silencing specificity.

Blocking collagenase activity by silencing of MMP-8 and applying MMP-13 neutralizing antibody resulted in additional inhibition of BK-induced ERK activation (65%), supporting roles for both MMPs. Because neutralizing antibodies often cannot completely block protein function, we decided to support our data using an additional method of blocking MMP-13 activity by transfecting mIMCD-3 cells with MMP-13 siRNA. mIMCD-3 cells were nucleofected with either MMP-13 siRNA alone or with both MMP-8 and MMP-13 siRNAs. Forty-eight hours postnucleofection, cells were stimulated with vehicle or 10 nM BK for 5 min, lysed, and analyzed for ERK phosphorylation. mIMCD-3 cells transfected with MMP-13 siRNA (MMP-13 siRNA) demonstrated 50% less BK-induced ERK activation than cells transfected with control siRNA (Fig. 5A).

Cells transfected with both siRNAs (MMP-8 and MMP-13 siRNA) demonstrated 68% decrease in BK-induced ERK activation. Figure 5B demonstrates effective silencing of MMP-13 expression in mIMCD-3 cells transfected with MMP-13 siRNA. Immunoblot with antibody against -actin shows the equal amount of -actin in the lysates from cells transfected with MMP-13 siRNA and with control siRNA, supporting silencing specificity and serving as a control for protein loading. The residual (30–35%) BK-induced activation of ERK in mIMCD-3 cells that were treated to block both MMP-8 and MMP-13 is due most likely to the incomplete blockade of collagenases, although we cannot exclude possible involvement of other enzymes in this process.

BK Treatment Stimulates the Activity of Collagenases in mIMCD-3 Cells. The next set of experiments was aimed to determine whether BK stimulates the activity of MMPs in mIMCD-3 cells. To answer this question, we measured collagenase activity in concentrated conditioned media from mIMCD-3 cells that were treated with 10 nM BK for different periods. The activities of type 1 collagenases (MMP-1, -8, and -13) were assessed by assaying biotinylated collagen as a substrate for active collagenases. Experiments were performed at least four times in triplicate. The results presented on Fig. 6A demonstrated that BK treatment stimulated collagenase activity in mIMCD-3 cells within 10 min. We also assessed MMP gelatinase activity, but we were able to detect only basal activity of gelatinases, and not BK-dependent activity in the same samples (data not shown). This suggests that the most likely enzymes involved in BK-induced ERK activation are collagenase-2 and -3, rather than gelatinases MMP-2 or MMP-9.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Collagenase activity in concentrated conditioned media from mIMCD-3 cells. A, bradykinin treatment stimulates collagenase activity in mIMCD-3 cells. The activities of type 1 collagenases (MMP-1, -8, and -13) were assessed as described under Materials and Methods. Experiments were performed five times in triplicates. Data are presented as mean ± S.E.M. Highly uneven errors were due to a nonlinear calibration curves for the provided collagenase standards. , P < 0.05 versus control. B, silencing of MMP-8 and MMP-13 expression inhibits BK-induced activation of collagenases in mIMCD-3 cells. Cells were transfected with control siRNA, MMP-8 siRNA, MMP-13 siRNA, or with combination of both MMP-8 and MMP-13 siRNAs as described under Materials and Methods. Forty-eight hours postnucleofection, cells were stimulated with vehicle or 10 nM BK for 5 min, and collagenase activity in concentrated conditioned media was measured as described under Materials and Methods. Experiments were performed two times in triplicates. Data are presented as mean ± S.E.M. , P < 0.05 versus control.

BK-Induced ERK Phosphorylation Does Not Require the Release of HB-EGF or TGF-. Because most of the published mechanisms of MMP-dependent EGFR activation by GPCRs involve extracellular release of EGF-like growth factors such as HB-EGF and/or TGF- (Prenzel et al., 1999; Kalmes et al., 2000; Uchiyama-Tanaka et al., 2001; McCole et al., 2002; Shah et al., 2004), we next decided to test whether EGF-like ligands are involved as extracellular soluble factors in BK-induced transactivation of the EGFR in mIMCD-3 cells. In the first set of experiments, we used a glycosaminoglycan heparin, which is structurally similar to the heparan sulfate proteoglycan (HSPG) that is present on the cell surface and serves as coreceptor for many growth factors. Heparin acts as a competitor for HSPG and can adsorb the HB-EGF as well as other HSPG-tethered molecules (e.g., amphiregulin or betacellulin). mIMCD-3 cells were pretreated with 0.1 mg/ml heparin for 30 min before stimulation with 10 nM BK or with 1 ng/ml EGF for 5 min. We also pretreated cells with 0.1 mg/ml neutralizing antibody against HB-EGF for 1 h and measured phosphorylation of ERK after 5 min of stimulation with BK or EGF. We have previously used this antibody to successfully inhibit HB-EGF- and serotonin-induced ERK activation in renal mesangial cells (M. Gooz, submitted for publication; data not shown). In another set of experiments, we pretreated mIMCD-3 cells with 50 µg/ml neutralizing antibody against TGF-, another potential EGFR ligand, before stimulation with 10 nM BK or 10 ng/ml TGF-. TGF- neutralizing antibody blocked TGF- -induced ERK activation, supporting that this antibody is functional under our experimental conditions. We found that BK-induced MMP-dependent phosphorylation of EGFR does not require extracellular release of TGF- and/or HB-EGF, in that neutralizing TGF- and HB-EGF antibodies (as well as heparin) failed to prevent ERK activation by BK. Results are shown in Fig. 7, A and B.

View larger version (30K): [in this window] [in a new window]   Fig. 7. HB-EGF and TGF- are not involved in BK-induced ERK phosphorylation. A, mIMCD-3 cells were pretreated for 30 min with vehicle, 100 µg/ml heparin, or with 100 µg/ml HB-EGF neutralizing antibody (R&D Systems). Phosphorylation of ERK was measured after 5-min treatments with vehicle, 10 nM BK, or 1 ng/ml EGF. B, mIMCD-3 cells were pretreated for 30 min with vehicle or with 50 µg/ml TGF- neutralizing antibody (Calbiochem). Phosphorylation of ERK was measured after 5-min stimulation with vehicle, 10 nM BK, or 10 ng/ml TGF-. Experiments were performed at least three times in duplicates. Data are presented as mean ± S.E.M. *, P < 0.01 versus vehicle-treated cells; , P < 0.01 versus control.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Matrix metalloproteinases stimulate ERK1/2 activity in mIMCD-3 cells. mIMCD-3 cells were preincubated with different inhibitors and then stimulated with vehicle, BK, active MMP-8, or active MMP-13. ERK1/2 phosphorylation was assessed. Experiments were performed at least three times in duplicates. Data are presented as mean ± S.E.M. *, P < 0.01 versus vehicle-treated cells; , P < 0.01 versus control.

View larger version (33K): [in this window] [in a new window]   Fig. 9. Matrix metalloproteinases stimulate EGFR phosphorylation in mIMCD-3 cells. mIMCD-3 cells were stimulated with vehicle, active MMP-8, or active MMP-13. EGF receptor phosphorylation was assessed as described under Materials and Methods. Experiments were performed at least four times in duplicates. Data are presented as mean ± S.E.M. *, P < 0.01 versus vehicle-treated cells.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The current work describes a novel mechanism of EGFR transactivation by the G_q-coupled bradykinin B₂ receptor that involves activation of collagenase-2 and -3. In addition, we demonstrate for the first time that 1) collagenases (rather than gelatinases) are activated by the B₂ receptor in a cultured murine cell model of the IMCD, 2) collagenases are involved in a cross-talk between the B₂ receptor and EGFR, and 3) EGFR activation does not require the release of HB-EGF or TGF-.

Several mechanisms of EGFR transactivation by G_q-coupled receptors have been demonstrated. A protein kinase C-dependent mechanism of EGFR transactivation has been shown for the receptor of gonadotropin-releasing hormone in pituitary T3-1 gonadotropes and in transfected COS-7 cells (Grosse et al., 2000). Metalloproteinases-dependent EGFR activation by HB-EGF was described for angiotensin II AT₁ receptors in vascular smooth muscle cells (Eguchi et al., 2001), glomerular mesangial cells (Uchiyama-Tanaka et al., 2001), C9 hepatocytes (Shah et al., 2004), and for muscarinic M₁ and thrombin receptors in other cell types (Prenzel et al., 1999; Kalmes et al., 2000). Carbachol-stimulated transactivation of the EGFR by M₃ receptors in T84 cells is mediated by metalloproteinase-dependent extracellular release of TGF- and intracellular Src activation (McCole et al., 2002).

Bradykinin has been shown to induce tyrosine phosphorylation of the EGFR in COS-7 cells where protein kinase C activation and EGFR transactivation independently activate ERK (Adomeit et al., 1999). Previously, we identified a novel mechanism of BK-induced transactivation of EGFR in mIMCD-3 cells that does not involve phospholipase C, elevations of Ca²⁺, calmodulin, protein kinase C, G_i/o subunits, or B₂ receptor heterodimerization with EGFR (Mukhin et al., 2003). In the current study, we looked further into this mechanism testing the hypothesis that B₂ receptor-induced transactivation of the EGFR in mIMCD-3 cells involves matrix metalloproteinases.

MMPs, a large family of zinc-dependent, matrix-degrading enzymes, play a central role in degradation of ECM (Nagase and Woessner, 1999). To date, 28 secreted or transmembrane MMPs have been identified and divided into collagenases, gelatinases, stromelysins, and matrilysins, based on their preference for ECM components (Egeblad and Werb, 2002). In the kidney, the accumulation of ECM molecules can lead to interstitial fibrosis and glomerulosclerosis (He et al., 1995; Norman and Lewis, 1996). MMPs are also involved in normal kidney development, and they potentially play roles in diabetic nephropathy and in inflammatory glomerulonephritis (Lenz et al., 2000). In vivo experiments using transgenic mice have established a role for gelatinase MMP-9 in nephron mass formation and preservation of renal function, and they have revealed a protective effect of MMP-9 on the development of fibrin-induced glomerular lesions (Lelongt and Ronco, 2002). It has been suggested also that MMP-2, MMP-9, and MMP-14 play roles in tubular segmentation (Kanwar et al., 1999).

Despite the emerging roles of MMPs in the kidney, little is presently known about possible interactions between BK receptors and MMPs. The effect of BK on the expression of MMP-3, MMP-20, and membrane type-1-MMP has been described for cultured granulosa cells (Kimura et al., 2001). In vivo studies on the B₂ receptor knockout mice suggest that the B₂ receptor plays a protective role in renal tubulointerstitial fibrosis, probably by increasing the activity of a plasminogen activator/MMP-2 cascade (Schanstra et al., 2002), but it does not mediate the antifibrotic effect of angiotensin-converting enzyme inhibitors (Schanstra et al., 2003). Thus, a possible connection between the B₂ receptor and MMPs in kidney cells remains unclear.

Our data support the involvement of MMPs in a BK-induced signaling pathway that leads to the activation of ERK1/2 in mIMCD-3 cells. Using broad-spectrum inhibitors of MMPs, batimastat, and GM-6001, we demonstrated that MMPs are involved in BK-induced ERK activation and EGFR phosphorylation (Fig. 1, A and B), and in Ras activation (Fig. 2). To plan more specific experiments, we first established that mIMCD-3 cells express mRNAs for the MMP-2, MMP-3, MMP-8, MMP-9, MMP-11, MMP-13, MMP-14, MMP-15, MMP-17, MMP-19, MMP-20, and MMP-23 (Table 1). Next, we used a panel of commercially available chemical inhibitors for different MMPs present in mIMCD-3 cells that allowed us to propose involvement of MMP-8 (collagenase-2) and/or MMP-13 (collagenase-3) in B₂ receptor-induced EGFR transactivation (Table 2). Because we questioned the specificity of MMP inhibitors, the next series of experiments were aimed to inhibit MMP-8 and MMP-13 with other methods to support the inhibitor data. Figure 4A shows that neutralizing MMP-13 antibody reduced BK-induced ERK phosphorylation by about 50%, supporting a role for MMP-13 in this process. The same level of decrease in BK-induced ERK phosphorylation was achieved by transfection of mIMCD-3 cells with MMP-8 siRNA (Fig. 4B) or with MMP-13 siRNA (Fig. 5). The simultaneous blockade of MMP-8 and MMP-13 resulted in a further decrease in BK-induced ERK phosphorylation, supporting roles for both collagenases. Having established that collagenases are involved in BK-induced ERK stimulation in mIMCD-3 cells, we hypothesized that BK stimulates the activity of collagenases. Figure 6A demonstrates that BK treatment stimulates collagenase activity in concentrated conditioned media from mIMCD-3 cells.

To further assess possible involvement of MMPs in ERK signaling in mIMCD-3 cells we studied the ability of exogenous activated collagenases to stimulate ERK phosphorylation. Interestingly, exogenous activated collagenase-2 and -3 induced ERK activation (Fig. 8) and EGFR phosphorylation (Fig. 9). MMP-8 and MMP-13-induced ERK activation was MEK-dependent, did not require B₂ receptor, was not due to release of HB-EGF, and required EGFR kinase activity (Fig. 8).

Thus, our data support specific roles for MMP-8 and MMP-13 in BK-induced signaling based on 1) inhibitor studies, 2) neutralizing antibody against MMP-13, 3) siRNA studies, 4) stimulation of collagenase activity by BK, and 5) phosphorylation of EGFR and ERK by exogenously applied activated MMP-8 and MMP-13.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Proposed pathway of BK-induced activation of ERK1/2 in mIMCD-3 cells. This scheme is described under Discussion.

To test the mechanism of MMP-dependent ERK activation in mIMCD-3 cells, we tried to block BK-induced activation of ERK1/2 by heparin or neutralizing HB-EGF antibody (Fig. 7A). Neither treatment changed the ability of BK to activate ERK1/2, suggesting that B₂ receptor does not use HB-EGF-like growth factor to activate EGFR. To test the possibility that MMP-dependent extracellular release of TGF- is involved in BK-induced signaling, we used neutralizing antibody against TGF- (Fig. 7B). Treatment with neutralizing TGF- antibody did not change the ability of BK to activate ERK1/2, but it blocked TGF--induced ERK1/2 activation, suggesting that B₂ receptor does not use TGF-.

Thus, we demonstrated that B₂ receptor-induced ERK phosphorylation in mIMCD-3 cells does not require the release of HB-EGF or TGF-. Inability of heparin to inhibit BK-induced signaling also does not suggest the importance of amphiregulin and betacellulin, other heparin-binding members of EGF family. How exactly collagenases mediate BK-induced transactivation of EGFR is not clear at the moment (Fig. 10). One possibility is that MMP-8 and/or MMP-13 upon stimulation with BK cause shedding of other nonheparin-sensitive EGFR ligands (e.g., epiregulin). Another possibility would be that in this case collagenases activate EGFR without releasing EGFR ligand, probably by proteolytic activation of EGFR molecule. Regulation of EGFR activity during apoptosis by proteolytic cleavage in a caspase-dependent manner has been described in A431 cells, although in this case protease inactivated EGFR (Bae et al., 2001). MMP-mediated ectodomain shedding has been shown for ErbB2 and ErbB4 receptors, the members of the EGFR family (Codony-Servat et al., 1999; Junttila et al., 2000). Although the shedding of ErbB4 molecule is activated by protein kinase C stimulation and most likely is mediated by tumor necrosis factor--converting enzyme (Junttila et al., 2000), the cleavage of ErbB2 does not require protein kinase C activation and involves metalloprotease different from tumor necrosis factor--converting enzyme, which can be inhibited by tissue inhibitor of metalloproteinases-1 (Codony-Servat et al., 1999). Finally, BK-induced stimulation of collagenases may cause the recruitment of other mediators of EGFR activation such as nonreceptor tyrosine kinases Src that has been shown to mediate EGFR proligand release in response to GPCR ligands in head and neck cancer cells (Zhang et al., 2004).

In conclusion, this study demonstrates a novel mechanism of EGFR transactivation by the G_q-coupled bradykinin B₂ receptor that involves activation of collagenase-2 and -3 but does not require the release of HB-EGF or TGF-. Although the exact mechanism of BK-induced MMP-dependent transactivation of EGFR is still to be defined, these findings reveal a novel functional link between collagenases and EGFR, suggesting that besides their matrix-degrading abilities, MMP-8 and MMP-13 are essential for BK-induced proliferation of kidney cells.

	Footnotes

 
This work was supported by grants from the Department of Veterans Affairs (Merit Awards and a REAP Award to M.N.G. and J.R.R.), the National Institutes of Health (DK52448-02 to J.R.R. and DK02694 to Y.V.M.), and a laboratory endowment jointly supported by the Medical University of South Carolina Division of Nephrology and Dialysis Clinics, Incorporated (to J.R.R.). The FLIPR is a shared Medical University of South Carolina resource obtained with Grant S10RR013005 from the Public Health Service.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.104000.

ABBREVIATIONS: BK, bradykinin; GPCR, G protein-coupled receptor; ERK, extracellular signal-regulated protein kinase; mIMCD, cultured murine cell model of inner medullary collecting duct; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; HB-EGF, heparin-binding EGF-like growth factor; MMP, matrix metalloproteinase; TGF, transforming growth factor; ECM, extracellular matrix; MEK, mitogen and extracellular signal-regulated kinases kinase; siRNA, small interfering RNA; RT-PCR, reverse transcription-polymerase chain reaction; GM-1489, N-[(2R)-2-(carboxymethyl)-4-methylpentanoyl]-L-tryptophan-(S)-methyl-benzylamide; GM-6001, N-[(2R)-2-(hydroxamidocarbonyl-methyl)-4-methylpentanoyl]-L-tryptophan methylamide (Galardin); GM-6001 inactive form, N-t-butoxycarbonyl-L-leucyl-L-tryptophan methylamide; AB, antibody; HSPG, heparan sulfate proteoglycan; PD98059, 2'-amino-3'-methoxyflavone; AG-1478, 4-(3-chloroanilino)-6,7-dimethoxyquinazoline; MMPs inhibitor II, N-hydroxy-1,3-di-(4-methoxybenzenesulfonyl)-5,5-dimethyl-[1,3]-piperazine-2-carboxamide; MMPs inhibitor III, a homophenylalanine-hydroxamic acid-based broad-spectrum inhibitor; MMP-2 inhibitor I, cis-9-octadecenoyl-N-hydroxylamide; MMP-2/MMP-9 inhibitor II, (2R)-[(4-biphenylylsulfonyl) amino]-N-hydroxy-3-phenylpropionamide; MMP-3 inhibitor II, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid; MMP-3 inhibitor III, N-[[(4,5-dihydro-5-thioxo-1,3,4-thiadiazol-2-yl)amino]carbonyl]-L-phenylalanine; MMP-8 inhibitor I, (3R)-(+)-[2-(4-methoxybenzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-hydroxamate]; MMP-9/MMP-13 inhibitor I, N-hydroxy-1-(4-methoxyphenyl)sulfonyl-4-(4-biphenylcarbonyl)piperazine-2-carboxamide.

Address correspondence to: Dr. Maria N. Garnovskaya, Medical University of South Carolina, 96 Jonathan Lucas St., Room 829 CSB, P.O. Box 250623, Charleston, SC 29425-2227. E-mail: garnovsk{at}musc.edu

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