Bradykinin B₂ Receptor Activates Extracellular Signal-Regulated Protein Kinase in mIMCD-3 Cells via Epidermal Growth Factor Receptor Transactivation

Materials and Methods

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.

Cells were grown in 12-well plastic culture plates to 80% confluence and serum-starved for 48 h in DMEM/F-12 medium with 0.5% bovine serum albumin, 100 U/ml penicillin, and 100 μg/ml streptomycin. Treatments with different inhibitors were carried out for 30 min before stimulation with BK for 5 min. After the treatments, cells were scraped into Laemmli buffer and subjected to SDS-PAGE under reducing conditions using 4 to 20% precast gels (Novex, San Diego, CA). After semidry transfer to polyvinylidine difluoride membranes, the membranes were blocked with a BLOTTO (bovine lacto transfer optimizer) buffer and incubated with the phospho-ERK antibody (at 1:1000 dilution). After incubation with goat anti-rabbit alkaline phosphatase-conjugated IgG, immunoreactive bands were visualized by a chemiluminescent method (CDP Star; New England Biolabs, Beverly, MA) using Kodak X-AR film, and quantified using a GS-670 densitometer and Molecular Analyst software (Bio-Rad, Hercules, CA).

The same membranes were stripped using a Re-Blot Plus Western blot recycling kit (Chemicon International, Temecula, CA) and re-probed with the control ERK antibody, which recognizes equally well the phosphorylated and nonphosphorylated ERK to quantify total ERK1/2. Results were presented as intensities of phospho-ERK1/2 bands relative to total ERK1/2 and expressed as percentage of control (cells without BK treatment) phosphorylation.

Study of EGFR Phosphorylation and Association with B₂Receptor 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 (glutathioneS-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 atP < 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

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 10⁻⁸ M (Fig. 2). In subsequent experiments ERK assays usually were carried out with stimulation by 10 nM BK for 5 min, unless otherwise mentioned.

BK Stimulates ERK through a B₂ Receptor.

mIMCD-3 cells were pretreated for 30 min with a B₁receptor 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. Figure3 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 B₂receptor.

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. Figure4 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 α subunits were indeed inhibited under our experimental conditions.

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.

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).

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).

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.

Genistein (50 μM), a broad-spectrum tyrosine kinase inhibitor, markedly attenuated the ability of BK to activate ERK, whereas the structurally similar but inactive compound, daidzein, had no effect (Fig. 8). Another inactive analog of genistein, genistin (50 μM), was also without effect (data not shown). These results suggest the involvement of a tyrosine kinase in the pathway of ERK activation by BK. Next, several tyrosine kinase inhibitors selective for different tyrosine kinases were used to find out which of them is involved in the BK-induced activation of ERK. The specific inhibitor of nonreceptor Janus kinase 2 (AG-490, 50 μM) had no effect on the BK-induced activation of ERK. At the same time, AG1478 (100 nM), a selective inhibitor of EGFR tyrosine kinase, effectively blocked the BK-induced activation of ERK, suggesting that activation of EGFR is involved in the BK-induced ERK activation in mIMCD-3 cells.

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).

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 β₂-adrenergic receptor involves the complex formed between EGFR and the β₂-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).

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).

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.

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. Figure13 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.

Discussion

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 and2).

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 B₂receptor to ERK1/2 in other cells also were shown to involve PI3K (Graness et al., 1998), we thought that the B₂receptor 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 B₂receptor 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 β₂-adrenergic receptor signaling includes the isoproterenol-induced formation of a β₂-adrenergic receptor-EGFR complex (Maudsley et al., 2000). We considered the possibility that BK stimulation induces a complex formation between the B₂receptor 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 T₈₄intestinal 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 αT3-1 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_qcoupling 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_βγ-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 B₂receptor. 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 B₂receptor-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.

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.