Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The vasoactive nonapeptide bradykinin (BK) has been implicated in the regulation of kidney function, particularly in electrolyte and water excretion (Vio et al., 1992; Mukai et al., 1996), but the cellular mechanisms by which BK alters functions of renal cells are not completely understood. In the inner medullary collecting duct (IMCD), BK regulates sodium excretion and, consequently, controls extracellular fluid volume (Tomita et al., 1985). We recently showed that BK stimulates Na⁺/H⁺ exchanger type 1, which is essential for regulation of cell growth, intracellular pH, and cellular volume, in a cultured murine cell model of IMCD (mIMCD-3 cells) (Mukhin et al., 2001). Because BK has been implicated in the regulation of mitogen-activated protein kinase (MAPK) in various cells (Liebmann, 2001), we proposed to study its possible role in the regulation of extracellular signal-regulated protein kinase (ERK1/2) in kidney cells. ERK1/2 belongs to the MAPK family of serine/threonine kinases that are rapidly activated in response to growth factor stimulation (Cobb and Goldsmith, 1995). ERK1/2 may be regulated by both receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). ERK1/2, after being activated through phosphorylation of threonine²⁰² and tyrosine²⁰⁴ by MEK1/2, translocates to the nucleus and activates transcription factors, thereby regulating cell proliferation (Widmann et al., 1999). The mechanism of ERK stimulation by GPCRs appears to be complex. Receptors coupled to G_i proteins usually cause ERK activation via a G subunit-mediated pathway, which involves activation of Ras and consecutive stimulation of Raf and MEK1/2. Other GPCRs mediate ERK activation via a G_q subunit pathway that is Ras-independent and involves protein kinase C (PKC) (Gutkind, 1998). In some systems Ca²⁺/calmodulin (CaM) has also been implicated in G_i- and G_q-coupled receptor-mediated ERK activation (Della Rocca et al., 1997). Another pathway used by GPCRs to activate ERK involves "transactivation" of growth factor receptors with intrinsic tyrosine kinase activity, such as the epidermal growth factor receptor (EGFR) (Daub et al., 1997; Carpenter, 1999). In this case, stimulation of G_i- or G_q-coupled receptors leads to activation of the EGFR tyrosine kinase, which in turn leads to the activation of Ras and initiates the phosphorylation cascade involving Raf, MEK1/2, and ERK.

The bradykinin B₂ receptor, a prototypical GPCR (McEachern et al., 1991) has been shown to activate ERK1/2 in vascular smooth muscle cells (VSMC) (Velarde et al., 1999), endothelial cells (Bernier et al., 2000), PC-12 cells (Dikic et al., 1996), and various tumor cell lines (Drube and Liebmann, 2000; Graness et al., 2000). There is contradictory evidence in the literature about the mechanisms through which BK induces ERK1/2 activation. In VSMC, BK stimulates ERK1/2 via the B₂ receptor, PKC, and the nonreceptor tyrosine kinase Src (Velarde et al., 1999). A role for Ca²⁺/CaM in BK-induced activation of ERK1/2 in VSMC has also been proposed (Naidu et al., 1999). In PC-12 cells, BK also uses a Ca²⁺-dependent pathway that involves the nonreceptor tyrosine kinases Pyk2 and Src to activate ERK1/2 (Dikic et al., 1996). In carcinoma cell lines SW-480 and A431, BK-induced ERK1/2 activation involves phosphatidylinositol 3-kinase (PI3K) and PKC (Graness et al., 1998, 2000). In other tumor cell lines, BK activates mitogenic pathways via pertussis toxin (PTX)-sensitive G_i/o proteins, PI3K, and PKC (Drube and Liebmann, 2000). Finally, the B₂ receptor transfected into COS-7 cells uses two different pathways to activate ERK1/2: a PKC-dependent pathway and EGFR transactivation (Adomeit et al., 1999).

Little is presently known about the role of BK in ERK1/2 signaling in kidney cells. However, it is extremely important because studies using in vivo renal model systems demonstrated connection of inflammatory or toxic renal injury with the activation of ERK1/2 in renal tissue (Tian et al., 2000). Activation of ERK1/2 by BK in kidney cells has been demonstrated for cultured mesangial cells, although its mechanism remains unclear (Jaffa et al., 1997; El-Dahr et al., 1998). Even less is known about ERK1/2 regulation by BK in renal tubular epithelial cells. The only paper published on this topic shows that BK activates ERK1/2 in rabbit cortical collecting duct cells via a PKC-dependent mechanism (Lal et al., 1998).

Here we used mIMCD-3 cells, a cell culture model of the terminal segment of the nephron, where the final urinary composition is made. mIMCD-3 cells are able to survive in high concentrations of NaCl and urea, and urea-inducible ERK1/2 activation has been shown in these cells (Yang et al., 1999). In the present study, we demonstrate for the first time that BK activates ERK1/2 in mIMCD-3 cells and describe the signal transduction pathway of this activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Culture. The mIMCD-3 cells (Rauchman et al., 1993) were obtained from American Type Culture Collection (Manassas, VA). mIMCD-3 cells were grown in an equal-part mixture of DMEM and Ham's F-12 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin in a cell culture incubator at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

ERK Assays. ERK activity was assessed using a phosphorylation state-specific ERK antibody (Cell Signaling Technology, Inc., Beverly, MA), which specifically recognizes threonine²⁰²- and tyrosine²⁰⁴-phosphorylated (but not nonphosphorylated) ERK-1 and ERK-2, and does not react with other members of the MAPK family, such as ERK-5, p38 MAPK, or stress-activated protein kinases.

Study of EGFR Phosphorylation and Association with B₂ Receptor and Grb2. The phosphorylation state of EGFR and its association with the adapter protein Grb2 were assessed by immunoprecipitation/Western blotting studies. Quiescent cells, grown in 100-mm dishes, were treated with 10 nM BK or with 10 ng/ml or 1 ng/ml EGF for 5 min and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM sodium fluoride, 1 mM Na₃VO₄, 1 µg/ml aprotinin, leupeptin, and pepstatin, each). Cell lysates were precleared by incubating with protein A-agarose bead slurry 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, Inc., Lake Placid, NY) overnight at 4°C. The immunocomplexes were captured by the addition of protein A-agarose bead slurry and incubation for 2 h more at 4°C. The agarose beads were collected by centrifugation, washed three times with RIPA buffer, resuspended in 2× Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE. After semidry transfer to polyvinylidine difluoride membranes, the membranes were probed with monoclonal anti-phospho-EGFR antibodies (Upstate Biotechnology, Inc.) to assess the phosphorylation state of EGFR or with monoclonal anti-Grb2 antibodies (Upstate Biotechnology, Inc.) to study the presence of Grb2 in EGFR immunoprecipitates. To examine the possibility that the B₂ receptor associates with EGFR to activate it, we also performed Western blotting with monoclonal anti-B₂ receptor antibody (Research Diagnostics, Inc., Flanders, NJ).

Association of Grb2 with Shc. Association of Grb2 with the adapter protein Shc was assessed by immunoprecipitation of cell lysates from mIMCD-3 cells treated with vehicle, 10 nM BK, or 1 ng/ml EGF for 5 min with polyclonal anti-Shc antibody (Upstate Biotechnology, Inc.) followed by Western blotting with monoclonal anti-Grb2 antibodies (Upstate Biotechnology, Inc.).

Ras Activation Assay. Ras activation was assessed by a nonradioactive Ras assay kit (Upstate Biotechnology, Inc.). Quiescent mIMCD-3 cell monolayers were pretreated with AG1478 or vehicle for 30 min, stimulated with 10 nM BK, 1 ng/ml EGF or vehicle for 5 min, and lysed in a 1 ml/100-mm dish of Mg²⁺ lysis buffer (MLB) (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 aprotinin and leupeptin each, 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 MLB buffer, resuspended in 2× Laemmli sample buffer, boiled for 5 min, and subjected to SDS-PAGE and subsequent immunoblot analysis with monoclonal anti-Ras IgG.

DNA Synthesis. DNA synthesis was assessed by measuring the incorporation of [³H]thymidine into DNA fragments. Quiescent mIMCD-3 cells grown in 24-well plates were stimulated for 18 h with 100 nM BK or with 10 ng/ml EGF in the presence and absence of MEK1 inhibitor PD98059 (50 µM) or EGFR tyrosine kinase inhibitor AG-1478 (100 nM). The cells were labeled for 6 h with 1 µCi/ml [³H]thymidine per well. Then, after two saline washes, 0.5 ml of 0.3 M perchloric acid was added for 30 s, followed by saline wash and solubilization in 0.1% SDS/0.1 N NaOH. Cells were scraped into vials, and incorporated radioactivity was quantified in a scintillation counter.

Data Analysis. ERK assays were performed in duplicate and repeated at least three times. EGFR assays and Ras activation assays were repeated three times for each condition. Thymidine incorporation assays were performed in triplicate and repeated at least four 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. Plots and graphs were prepared with SigmaPlot 4.0 for Windows (SPSS, Inc., San Rafael, CA). Nonlinear regression analysis was done using SigmaPlot 4.0 Regression Wizard with user-modified functions.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Activation of ERK by BK. Treatment of mIMCD-3 cells with 10 nM BK resulted in a time-dependent increase in tyrosine phosphorylation of ERK1/2 (Fig. 1). BK treatment produced a 4- to 5-fold increase in ERK phosphorylation over basal activity. ERK phosphorylation was detectable as early as 1 min, peaked at 3 to 5 min, and was sustained at least for 1 h. BK stimulated ERK activation in a concentration-dependent manner, with maximal activation at 10⁸ M (Fig. 2). In subsequent experiments ERK assays usually were carried out with stimulation by 10 nM BK for 5 min, unless otherwise mentioned.

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Bradykinin-induced ERK1/2 phosphorylation: time course. A, mIMCD-3 cells were stimulated with 10 nM BK for the indicated periods of time. ERK1/2 phosphorylation was measured by immunoblotting with anti-phospho-ERK1/2 antibodies, as described under Materials and Methods. This is a representative immunoblot (phospho-ERK). The same blot was stripped and reprobed with antibodies for total ERK1/2 that recognize ERK1/2 independently of the phosphorylation state to assure an equal loading of protein samples on a gel (total ERK). B, data points represent intensities of phospho-ERK1/2 bands relative to total-ERK expressed as percentage of control (samples without BK treatment). Experiments were performed at least three times. Data are shown as mean ± S.E.M.

View larger version (43K): [in this window] [in a new window]   Fig. 2.   Bradykinin-induced ERK1/2 phosphorylation: concentration response curve. A, mIMCD-3 cells were stimulated with the indicated concentrations of BK for 5 min. ERK1/2 phosphorylation was detected by immunoblotting with anti-phospho-ERK1/2 antibodies, as described under Materials and Methods. A representative phospho-ERK1/2 immunoblot is shown. The same blot was stripped and reprobed with antibodies for total ERK1/2 that recognize ERK1/2 independently of the phosphorylation state to assure an equal loading of a protein sample on a gel (total ERK). B, data points represent intensities of phospho-ERK1/2 bands relative to total ERK1/2 measured by densitometry and expressed as percentage of control (samples without BK treatment). Experiments were performed at least three times. Data are shown as mean ± S.E.M.

BK Stimulates ERK through a B₂ Receptor. mIMCD-3 cells were pretreated for 30 min with a B₁ receptor antagonist des-Arg¹⁰-HOE-140 (1 µM) or with a B₂ receptor antagonist HOE-140 (1 µM), then stimulated with 100 nM BK for 5 min. Figure 3 shows that pretreatment with HOE-140 prevented the BK-induced activation of ERK, whereas a B₁ receptor antagonist was without effect. Thus, BK stimulates ERK activity via the B₂ receptor.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Bradykinin-induced ERK1/2 phosphorylation is mediated by a B2 receptor. mIMCD-3 cells were pretreated for 30 min with the B1 receptor antagonist des-Arg10-HOE-140 (1 µM) or with the B2 receptor antagonist HOE-140 (1 µM), then stimulated with 100 nM BK for 5 min. ERK1/2 phosphorylation was detected by immunoblotting with anti-phospho-ERK1/2 antibodies, as described under Materials and Methods. Bars represent intensities of phospho-ERK1/2 relative to total ERK1/2 expressed as percentage of control (cells without BK treatment) phosphorylation. Experiments were performed three times in duplicate. Data are presented as mean ± S.E.M. The inset shows a representative phospho-ERK1/2 blot and the same blot, stripped and reprobed with antibody for total ERK1/2.

BK Receptors Do Not Couple to G_i/o Proteins in mIMCD-3 Cells. mIMCD-3 cells were preincubated with vehicle or PTX (200 ng/ml) overnight, then stimulated with 10 nM BK or 1 µM lysophosphatidic acid (LPA) for 5 min. Figure 4 shows that there was no difference in BK-induced ERK phosphorylation in PTX-treated and nontreated cells, whereas LPA-induced signal was inhibited by ~45%. LPA has been previously shown to use PTX-sensitive intracellular signaling pathways (Seewald et al., 1999; Goppelt-Struebe et al., 2000), so this result supports the fact that PTX-sensitive G_i/o  subunits were indeed inhibited under our experimental conditions.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   BK receptors do not couple to Gi/o proteins in mIMCD-3 cells. mIMCD-3 cells were preincubated with vehicle or PTX (200 ng/ml overnight), then stimulated with 10 nM BK or 1 µM LPA for 5 min. ERK1/2 phosphorylation was detected by immunoblotting with anti-phospho-ERK1/2 antibodies, as described under Materials and Methods. Bars represent intensities of phospho-ERK1/2 relative to total ERK expressed as percentage of control (cells without treatment) phosphorylation. Experiments were performed two times in duplicate. Data are presented as mean ± S.E.M. *, P < 0.05 versus control.

Effect of MEK Inhibitors on BK Stimulation of ERK. Next, the effect of two different inhibitors of ERK kinase (MEK) on BK stimulation of ERK tyrosine phosphorylation was investigated. Pretreatment of mIMCD-3 cells with 10 µM PD 098059 and with 10 µM U0126 for 30 min before stimulation with 10 nM BK for 5 min abolished BK-stimulated tyrosine phosphorylation of ERK1/2 (Fig. 5). These results confirm that in mIMCD-3 cells BK stimulation of ERK proceeds, as expected, through MEK.

View larger version (43K): [in this window] [in a new window]   Fig. 5.   Bradykinin-induced ERK1/2 phosphorylation is MEK1/2-dependent. mIMCD-3 cells were pretreated for 30 min with 10 µM PD 098059 and with 10 µM U 0126 for 30 min before stimulation with 10 nM BK for 5 min. ERK1/2 phosphorylation was detected by immunoblotting with anti-phospho-ERK1/2 antibodies, as described under Materials and Methods. Bars represent intensities of phospho-ERK1/2 relative to total ERK1/2 expressed as percentage of control (cells without treatment) phosphorylation. Experiments were performed three times in duplicate. Data are presented as mean ± S.E.M. The inset shows a representative phospho-ERK1/2 blot and the same blot, stripped and reprobed with antibody for total ERK1/2.

Lack of Involvement of PLC and PKC in BK-Induced Activation of ERK. Different chemical inhibitors of PLC and protein PKC were used to examine the role of these proteins in BK-induced activation of ERK in mIMCD-3 cells. The PKC inhibitor GF109203X (1 µM for 30 min) and PKC depletion by the prolonged (20 h) treatment of the cells with 160 nM phorbol 12-myristate 13-acetate (PMA) did not block BK-stimulated ERK activation. At the same time, both treatments inhibited ERK activation by PMA, showing that PKC was indeed inhibited under our experimental conditions (Fig. 6A). That result is consistent with the noninvolvement of PKC in BK-stimulated activation of ERK. Cells were also pretreated with vehicle or with three different PLC inhibitors [U-73122 (10 µM), D609 (50 µM), and ET-18-OCH₃ (50 µM)] for 30 min, and then stimulated with 10 nM BK for 5 min. Treatment with PLC inhibitors slightly elevated the basal ERK activity but did not impair the BK-stimulated activation of ERK, suggesting that PLC is not involved in this process (Fig. 6B).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Bradykinin-induced ERK1/2 phosphorylation is PKC- and PLC-independent. A, mIMCD-3 cells were preincubated with 1 µM GF109203X for 30 min or with 160 nM PMA for 20 h, followed by either BK (10 nM) or PMA (1 µM) stimulation for 5 min. ERK1/2 phosphorylation was detected by immunoblotting with anti-phospho-ERK1/2 antibodies, as described under Materials and Methods. Bars represent intensities of phospho-ERK1/2 relative to total ERK1/2 expressed as percentage of control (cells without treatment) phosphorylation. Experiments were performed three times in duplicate. Data are presented as mean ± S.E.M. **, P < 0.01 versus control. B, mIMCD-3 cells were preincubated with vehicle or with three different PLC inhibitors U-73122 (10 µM), D609 (50 µM), and ET-18-OCH3 (50 µM) for 30 min and then stimulated with 10 nM BK for 5 min. Experiments were performed at least four times in duplicate. Data are presented as mean ± S.E.M.

Lack of Involvement of Calcium and Calmodulin in BK-Induced ERK Activation. To examine the role of calcium in BK-induced ERK activation, cells were pretreated for 30 min with 10 µM BAPTA, a cell-permeable Ca²⁺ sequestrant. To determine whether CaM activity is required for BK-mediated ERK activation, cells were pretreated for 30 min with a CaM antagonist, fluphenazine (50 µM). As in the case of PLC inhibitors, both treatments caused a slight increase in a basal ERK activity. At the same time, neither of these treatments impaired BK-stimulated ERK activation, indicating that Ca²⁺ and CaM are not important for BK stimulation of ERK in mIMCD-3 cells (Fig. 7).

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Bradykinin-induced ERK1/2 phosphorylation is Ca2+- and CaM-independent. mIMCD-3 cells were pretreated for 30 min with vehicle, 10 µM BAPTA, or 50 µM fluphenazine and then stimulated with 10 nM BK for 5 min. ERK1/2 phosphorylation was detected by immunoblotting with anti-phospho-ERK1/2 antibodies, as described under Materials and Methods. Bars represent intensities of phospho-ERK1/2 relative to total ERK1/2 expressed as percentage of control (cells without BK treatment) phosphorylation. Experiments were performed at least three times in duplicate. Data are presented as mean ± S.E.M.

Role of PI3 Kinase and Tyrosine Kinases. Because activation of ERK typically requires activation of phosphorylation cascades, the roles of PI3K and different tyrosine kinases in the effect of BK was examined. Two different specific inhibitors of PI3K (50 nM wortmannin and 50 µM LY294002) were used to pretreat mIMCD-3 cells for 30 min before stimulation with 10 nM BK for 5 min. Neither pretreatment altered the ability of BK to stimulate ERK activity (Fig. 8), suggesting the noninvolvement of PI3K in BK-induced ERK activation in mIMCD-3 cells.

View larger version (34K): [in this window] [in a new window]   Fig. 8.   Role of PI3K and tyrosine kinases in BK-induced ERK1/2 phosphorylation. mIMCD-3 cells were pretreated for 30 min with vehicle or with two different specific inhibitors of PI3K (50 nM wortmannin and 50 µM LY294002) or with tyrosine kinase inhibitors (50 µM genistein or 50 µM of its negative control daidzein, 50 µM AG-490, or 100 nM AG1478) before stimulation with 10 nM BK for 5 min. ERK1/2 phosphorylation was detected by immunoblotting with anti-phospho-ERK1/2 antibodies, as described under Materials and Methods. Bars represent intensities of phospho-ERK1/2 relative to total ERK1/2 expressed as percentage of control (cells without BK treatment) phosphorylation. Experiments were performed at least three times in duplicate. Data are presented as mean ± S.E.M. **, P < 0.01 versus control.

BK Induces Phosphorylation of the EGF Receptor. Because GPCR-induced tyrosine phosphorylation (transactivation) of EGFR has been recently described (Daub et al., 1997; Carpenter, 1999), we next studied the possibility that the B₂ receptor induces phosphorylation of the EGFR in mIMCD-3 cells. We performed immunoprecipitation/Western blotting studies to assess the phosphorylation state of EGFR and demonstrated that BK induced a time-dependent ~2-fold increase in EGFR phosphorylation that started as early as 1 min after BK application. Pretreatment of mIMCD-3 cells with 100 nM AG1478 (a selective inhibitor of EGFR tyrosine kinase) completely blocked BK-induced phosphorylation of EGFR (Fig. 9).

View larger version (33K): [in this window] [in a new window]   Fig. 9.   Bradykinin induces phosphorylation of EGF receptors. Inset: the time course for EGFR phosphorylation. Cells were stimulated with 10 nM BK for the indicated periods of time. EGFR phosphorylation was measured as described under Materials and Methods. Data points represent intensities of phosphorylated EGFR bands measured by densitometry and expressed as fold increase over control (nonstimulated cells). A representative phospho-EGFR immunoblot is shown at the top part of the inset. The same blots were stripped and reprobed with antibodies for total EGFR that recognize EGFR independently of the phosphorylation state to assure an equal loading of a protein sample on a gel (data not shown). Experiments were performed two times in duplicate. Data are presented as mean ± S.E.M. BK-induced EGFR phosphorylation depends on EGFR tyrosine kinase activity. mIMCD-3 cells were preincubated for 30 min with vehicle or with 100 nM AG1478, and stimulated for 5 min with 10 nM BK or 10 ng/ml EGF. EGFR phosphorylation was measured as described under Materials and Methods. A, a representative phospho-EGFR immunoblot; B, bars represent intensities of phospho-EGFR bands measured by densitometry and expressed as fold increase over control (without BK treatment) phosphorylation. Experiments were performed at least three times in duplicate. Data are presented as mean ± S.E.M. **, P < 0.01 versus control.

BK Does Not Stimulate Complex Formation of B₂ Receptor with EGFR. One of the mechanisms of GPCR-induced transactivation of EGFR that has been recently described for the ₂-adrenergic receptor involves the complex formed between EGFR and the ₂-adrenergic receptor (Maudsley et al., 2000). We considered the possibility that the same mechanism exists in mIMCD-3 cells and used immunoprecipitation/Western blotting studies to test it. However, there was no B₂ receptor in EGFR immunoprecipitates from mIMCD-3 cells either nonstimulated or stimulated with 10 nM BK (Fig. 10).

View larger version (48K): [in this window] [in a new window]   Fig. 10.   Immunoprecipitation/Western blotting studies do not support complex formation of B2 receptor with EGFR. Cells were stimulated for 5 min with 10 nM BK or 1 ng/ml EGF. Immunoprecipitation of cell lysates with anti-EGFR antibody followed by immunoblotting with anti-B2 receptor and/or with anti-phospho-EGFR antibody was performed as described under Materials and Methods. A, a representative phospho-EGFR immunoblot. The arrow shows a band corresponding to phosphorylated EGFR. The same blot was stripped and reprobed with anti-EGFR antibody to assure an equal loading of a protein sample on a gel (data not shown). B, a representative B2 receptor immunoblot. The arrow points to a band corresponding to B2 receptors in positive control lanes. As a positive control we used 20 µg of total protein of nonstimulated mIMCD-3 cell lysates per lane. The same blot was stripped and reprobed with anti-EGFR antibody to assure that EGFR was indeed immunoprecipitated (data not shown). Experiments were performed three times. IP, immunoprecipitation; IB, immunoblot.

BK Stimulates Complex Formation of Grb2 with EGFR and Association of Grb2 with Shc in mIMCD-3 Cells. Tyrosine phosphorylation of EGFR usually leads to activation of ERK1/2 via consecutive recruitment or activation of the adapter proteins Grb2 and/or Shc, the small GTP-binding protein Ras, and a cascade of protein kinases consisting of Raf, MEK, and ERK. Figure 10 demonstrates that treatment of mIMCD-3 cells with 10 nM BK for 5 min stimulates complex formation of EGFR with Grb2 (Fig. 11A) and also increased the amount of the adapter protein Shc associated with Grb2 (Fig. 11B).

View larger version (35K): [in this window] [in a new window]   Fig. 11.   BK stimulates complex formation of Grb2 with EGFR and association of Grb2 with Shc in mIMCD-3 cells. A, BK stimulates complex formation between EGFR and Grb2. Cells were stimulated for 5 min with 10 nM BK or 1 ng/ml EGF. Immunoprecipitations of cell lysates with anti-EGFR antibody followed by immunoblotting with anti-Grb2 antibody were performed as described under Materials and Methods. Bars represent intensities of Grb2 bands measured by densitometry and expressed as percentage of control (nontreated samples). Experiments were performed three times. Data are presented as mean ± S.E.M. *, P < 0.05 versus vehicle. B, BK induces association of Grb2 with Shc. Cells were stimulated for 5 min with 10 nM BK or 1 ng/ml EGF. Immunoprecipitations of cell lysates with anti-Shc antibody followed by immunoblotting with anti-Grb2 antibody were performed as described under Materials and Methods. Bars represent intensities of Grb2 bands measured by densitometry and expressed as percentage of control (nontreated samples). Experiments were performed three times. Data are presented as mean ± S.E.M. *, P < 0.05 versus vehicle. IP, immunoprecipitation; IB, immunoblot.

BK Activates Ras in mIMCD-3 Cells in an EGFR-Dependent Manner. Using a direct measurement of Ras activation in mIMCD-3 cells treated with BK, we found that 10 nM BK caused time-dependent activation of Ras. This activation occurs as early as 1 min after BK application and peaks at 2.5 min (Fig. 12A). Pretreatment of the cells with 100 nM AG1478, which blocks EGFR tyrosine kinase, completely prevented Ras activation by BK and/or EGF (Fig. 12B), supporting an important role for EGFR tyrosine kinase activity in this process.

View larger version (19K): [in this window] [in a new window]   Fig. 12.   BK activates Ras in mIMCD-3 cells. A, time course for BK-induced Ras activation. mIMCD-3 cells were stimulated with 10 nM BK for indicated periods of time. Ras activity was measured using a Ras activation kit, as described under Materials and Methods. Data points represent intensities of Ras immunoreactive bands measured by densitometry and expressed as percentage of control (samples without BK treatment). Experiments were performed two times in duplicate. Data are shown as mean ± S.E.M. B, mIMCD-3 cells were pretreated with vehicle or 100 nM AG1478 for 30 min and then stimulated with 10 nM BK or 1 ng/ml EGF for 5 min. Ras activity was measured using a Ras activation kit as described under Materials and Methods. Bars represent intensities of Ras immunoreactive bands measured by densitometry and expressed as percentage of control (nontreated samples). Experiments were performed at least three times in duplicate. Data are shown as mean ± S.E.M. *, P < 0.05 versus control; **, P < 0.01 versus control.

BK-Induced DNA Synthesis in mIMCD-3 Cells Depends on EGFR and Activation of ERK. The mitogenic effect of BK in mIMCD-3 cells was evaluated by measuring DNA synthesis. There was a significant increase in [³H]thymidine incorporation in BK-treated cells (87 ± 19% over control values; P < 0.01, n = 6), which was comparable to EGF-induced DNA synthesis (102 ± 32% over control; P < 0.05, n = 6). To assess a role for EGFR tyrosine kinase and ERK in the mitogenic actions of BK, cells were pretreated with 100 nM AG1478 (EGFR tyrosine kinase inhibitor) or with 50 µM PD98059 (MEK1 inhibitor) for 1 h before stimulation with 100 nM BK or 10 ng/ml EGF for 18 h in the continuous presence of the inhibitors. Figure 13 shows that each inhibitor completely blocked the increase in [³H]thymidine incorporation induced by BK or EGF. AG1478 and PD98059 alone had no significant effect on the basal rate of DNA synthesis. These findings directly demonstrate that BK stimulates proliferation of mIMCD-3 cells and further implicates a role for activation of EGFR tyrosine kinase and ERK1/2 in the BK mitogenic activity.

View larger version (30K): [in this window] [in a new window]   Fig. 13.   BK-induced DNA synthesis in mIMCD-3 cells depends on EGFR and ERK1/2 activity. Serum-deprived mIMCD-3 cells grown in 24-well plates were preincubated for 1 h with 50 µM PD98059, 100 nM AG1478, or vehicle before addition of 100 nM BK or 10 ng/ml EGF for 24 h. [3H]Thymidine was included in the final 6 h of incubation, and [3H]thymidine incorporation into DNA was measured as described under Materials and Methods. Experiments were performed at least four times in triplicate. Data are shown as mean ± S.E.M. *, P < 0.05 versus vehicle-treated cells; **, P < 0.01 versus vehicle-treated cells; , P < 0.05 versus control; , P < 0.01 versus control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Despite an obviously important role of BK in the kidney, the processes by which BK alters functions of renal cells are not completely understood. Because BK acts as a mitogenic factor in different cell types (Liebmann, 2001) and because aberrant ERK1/2 regulation is implicated in several models of proliferative or toxic renal injury (Tian et al., 2000), we studied a role of BK in the regulation of ERK1/2 in kidney cells. Using a cultured murine cell model of the IMCD (mIMCD-3 cells), we showed that BK induces time- and concentration-dependent tyrosine phosphorylation of ERK1/2 (Figs. 1 and 2).

Next, the signal transduction pathway that leads to ERK1/2 activation by BK was studied. BK-induced ERK1/2 activation was blocked by a B₂ bradykinin receptor antagonist, indicating that BK activates ERK1/2 via a B₂ receptor, a member of the seven-transmembrane GPCR superfamily (McEachern et al., 1991). Our data suggest that PTX-sensitive GTP-binding proteins (G_i/o) are not involved in BK-induced ERK1/2 activation, which is different from the pathway described in human carcinoma cell lines, where BK uses G_i/o proteins to activate ERK1/2 (Drube and Liebmann, 2000). The B₂ receptor usually couples to the GTP-binding protein G_q, and stimulates PLC activity, leading to the generation of inositol 1,4,5-triphosphate and diacylglycerol, the latter of which is involved in activation of PKC (Bascands et al., 1991). Activation of PLC also leads to an increase in intracellular calcium, which in turn can stimulate different signaling targets inside the cell (Dixon et al., 1994). It has been previously published that BK stimulates ERK1/2 activation in VSMC via PKC-dependent and Ca²⁺/CaM-dependent mechanism (Naidu et al., 1999; Velarde et al., 1999). A PKC-dependent pathway of BK-induced ERK1/2 activation has also been described in transfected COS cells and multiple tumor cell lines (Liebmann, 2001). It seemed reasonable to expect that BK would use a similar pathway to activate ERK1/2 in mIMCD-3 cells. It appears, however, that inhibition of PLC or PKC did not alter ERK1/2 activation by BK, suggesting the noninvolvement of these proteins in the signal transduction pathway (Fig. 6). We also demonstrated that Ca²⁺ and CaM are not important for BK stimulation of ERK1/2 in mIMCD-3 cells (Fig. 7). Thus, in mIMCD-3 cells, in contrast to other cell lines, BK activates ERK1/2 via PKC- and Ca²⁺/CaM-independent pathways, indicating that in different types of cells BK can induce different signaling pathways that result in ERK1/2 activation.

Because the signaling pathways that link the B₂ receptor to ERK1/2 in other cells also were shown to involve PI3K (Graness et al., 1998), we thought that the B₂ receptor might stimulate ERK1/2 via activation of PI3K. However, two different inhibitors of PI3K had no effect on the BK-induced activation of ERK1/2, suggesting a lack of PI3K involvement in the signaling pathway (Fig. 8). Next, we assumed that the B₂ receptor would use tyrosine kinase-dependent pathways to stimulate ERK. Genistein, an isoflavonoid that is commonly used as a broad-specificity tyrosine kinase inhibitor, significantly suppressed the increase in ERK1/2 phosphorylation in response to BK stimulation. Because genistein has been reported to have a wide range of biological activities, we also used its inactive analogs daidzein and genistin, neither of which had any effect on BK-induced ERK1/2 activation. Furthermore, the effects of more specific cell-permeable inhibitors of nonreceptor Janus kinase (Jak2) and EGFR tyrosine kinase were examined. Although it has been shown recently that BK activates Jak2 in mIMCD-3 cells (Mukhin et al., 2001), our data did not support the involvement of Jak2 in BK-induced ERK signaling (Fig. 8). However, the EGFR kinase inhibitor AG1478 eliminated the increase in ERK1/2 phosphorylation induced by BK. These findings support a role of EGFR kinase in the signal transduction pathway leading to ERK1/2 activation by BK. This phenomenon has been recently described for many GPCRs (Daub et al., 1997; Carpenter, 1999). EGFR is a growth factor receptor with intrinsic tyrosine kinase activity. EGF binds to its receptor at the plasma membrane and activates it through a mechanism that involves dimerization, activation of the receptor tyrosine kinase, and autophosphorylation of the receptor. Activated EGFR then stimulates ERK1/2 through a chain of events that includes binding to the adapter protein Grb2, tyrosine phosphorylation of the adaptor protein Shc, and subsequent formation of an Shc-Grb2-Sos1 complex. The Ras guanine nucleotide exchange factor Sos1 stimulates small G protein Ras, which in turn initiates the phosphorylation cascade consisting of protein kinases Raf, MEK, and ERK.

We used immunoprecipitation/Western blot studies and showed that in mIMCD-3 cells BK indeed stimulated time-dependent tyrosine phosphorylation of EGFR that was dependent on the kinase activity of EGFR (Fig. 9), induced complex formation between EGFR and Grb2, and stimulated association of Grb2 with the adaptor protein Shc (Fig. 11, A and B). Finally, we showed that stimulation of mIMCD-3 cells with BK caused time-dependent activation of Ras, which occurred as early as 1 min after BK application and peaked at 2.5 min (Fig. 12A), slightly preceding the peak activation of ERK, consistent with a potential role for Ras upstream of ERK. This BK-stimulated Ras activation was comparable with Ras activation induced by EGF and was also dependent on EGFR tyrosine kinase activity (Fig. 12B). Thus, our results provide evidence that the B₂ receptor activates ERK1/2 via a pathway that shares mediators of growth signaling initiated by EGF, including activation of EGFR, Grb2, Shc, Ras, and MEK1/2, similar to a pathway described for other GPCRs (Carpenter, 1999).

The mechanisms underlying GPCR-induced EGFR transactivation have not been clearly defined. One transactivation pathway recently proposed for the ₂-adrenergic receptor signaling includes the isoproterenol-induced formation of a ₂-adrenergic receptor-EGFR complex (Maudsley et al., 2000). We considered the possibility that BK stimulation induces a complex formation between the B₂ receptor and EGFR in mIMCD-3 cells. However, our experimental data do not support this possibility (Fig. 10B).

Several mechanisms of EGFR transactivation by G_q-coupled receptors have been demonstrated. A metalloprotease-dependent EGFR activation by heparin-binding EGF-like growth factor was described for the angiotensin II AT₁ receptor in vascular smooth muscle cells (Eguchi et al., 2001) and glomerular mesangial cells (Uchiyama-Tanaka et al., 2001), and for the muscarinic M₁ receptor and thrombin receptor (Prenzel et al., 1999; Kalmes et al., 2000). Another published mechanism of EGFR transactivation that involves Ca⁺²-dependent tyrosine kinase PYK-2 and Src kinase has been demonstrated for AT₁ receptor in VSMC (Eguchi et al., 1999) and for the muscarinic M₃ receptor in T₈₄ intestinal epithelial cells (Keely et al., 2000). A third PKC-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) and for M₁ muscarinic acetylcholine receptors transfected into HEK-293 cells (Tsai et al., 1997). Finally, the last mechanism of G_q-coupled receptor-dependent EGFR transactivation described for the thromboxane A2 (TxA2) receptor involves stimulation of PKC through receptor-G_q coupling and PLC activation. Activated PKC then phosphorylates the TxA2 receptor directly or a mediator, facilitating TxA2 receptor coupling to G_i, which in turn leads to a G-Src-dependent phosphorylation of EGFR, in this case PTX-dependent (Gao et al., 2001).

The ability of bradykinin B₂ receptors to induce tyrosine phosphorylation of EGFR has been described in COS-7 cells, transiently transfected with bradykinin B₂ receptor. In these cells, BK used two independent pathways to activate ERK1/2: a PKC-dependent pathway and EGFR transactivation, although the mechanism of the latter was not elucidated (Adomeit et al., 1999). BK-induced trans-inactivation of EGFR by stimulation of protein-tyrosine phosphatase was shown in epidermoid carcinoma A431 cells. Interestingly, in these cells, BK simultaneously activated ERK1/2 independent of EGFR (Graness et al., 2000).

Our results suggest that the mechanism of the B₂ receptor-induced transactivation of EGFR in mIMCD-3 cells is different from those described in the literature, for it does not involve a complex formation between the B₂ receptor and EGFR and does not require PTX-sensitive G proteins, Ca²⁺, CaM, PLC, or PKC activity (Fig. 14). The precise mechanism of BK-induced activation of EGFR in mIMCD-3 cells remains unclear. This novel mechanism may involve Ca⁺²-independent activation of nonreceptor tyrosine kinase Src or the release of EGFR ligand. Future studies are required to examine these possibilities. In summary, a cultured murine cell model (mIMCD-3) of the inner medullary collecting duct was used to examine the regulation of ERK1/2 by BK.

View larger version (16K): [in this window] [in a new window]   Fig. 14.   Proposed model for BK-induced ERK1/2 activation. BK stimulates EGFR transactivation through the B2 receptor-Gq/11 pathway and subsequent Ras-dependent activation of ERK1/2. The mechanism of BK-induced transactivation of EGFR is different from published mechanisms of Gq/11-coupled receptor-induced EGFR transactivation in that it does not require PLC, PKC, Ca2+/calmodulin, and direct association of the B2 receptor with EGFR. We speculate that this novel mechanism may involve Ca2+-independent activation of nonreceptor tyrosine kinase Src or the release of EGFR ligand.

In this study for the first time, we provide evidence that BK stimulates early mitogenic signals associated with activation of ERK1/2 in mIMCD cells. First, we demonstrated that bradykinin B₂ receptor stimulates Ras-dependent activation of ERK1/2 in mIMCD-3 cells via a novel mechanism of EGFR transactivation. Second, we showed that BK-stimulated proliferation of mIMCD-3 depends on activation of EGFR tyrosine kinase and ERK1/2 (Fig. 13). Although the exact nuclear events linking BK-induced ERK1/2 activation with DNA synthesis in mIMCD-3 cells are still to be defined, the findings of the present study contribute to better understanding of the molecular and cellular mechanisms by which BK stimulates proliferation of kidney cells, which could lead to the development of new strategies for treatment of kidney diseases.