Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proliferation in mesangial cells

Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proliferation in mesangial cells

Immunopharmacology 45 Ž1999. 57–62 www.elsevier.comrlocaterimmpharm Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proli...

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Immunopharmacology 45 Ž1999. 57–62 www.elsevier.comrlocaterimmpharm

Effect of bradykinin on tyrosine kinase and phosphatase activities and cell proliferation in mesangial cells Celine Alric, Christiane Pecher, Jean-Loup Bascands, Jean-Pierre Girolami

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Institut National de la Sante´ et de la Recherche Medicale, INSERM U388, Institut Louis Bugnard, C.H.U. Rangueil, 31052 Toulouse, ´ France Accepted 3 June 1999

Abstract We investigated the relationship between protein tyrosine phosphorylation and bradykinin ŽBK. receptor activation in rat mesangial cells ŽMC.. Stimulation of the B2 receptor resulted in a dual effect consisting of an independent activation and inhibition of tyrosine kinase activity ŽTKA.. The activation was rapid and transient, reaching a peak value at 30 s whereas the inhibition was observed at 5 min and persisted up to 10 min. Treatments with pertussis-toxin and U73122 showed that only the BK-induced stimulation of TKA is dependent on phospholipase C activation via a pertussis-toxin sensitive G-protein. In addition, BK induced an increase in tyrosine phosphatase activity. Western-blot analysis demonstrated that the dual effect of BK on TKA was associated with both an increase and a decrease in tyrosine phosphorylation of the p125-focal adhesion kinase ŽFAK.. Moreover, BK was able to reduce the maximal stimulated MC cell proliferation induced by fetal calf serum. These data show that in rat MC, B2 receptor stimulation activates and inhibits two independent tyrosine kinase signaling pathways associated with tyrosine phosphorylation of p125-FAK that might be implicated in MC proliferation. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Bradykinin; Mesangial cells; Tyrosine kinase activity; Tyrosine phosphatase; Focal adhesion kinase

1. Introduction AbbreÕiations: Bradykinin B1 receptor, B1R; Bradykinin B2 receptor, B2R; Bradykinin, BK; Angiotensin II, AII; Prostaglandin E2, PGE 2 ; Insulin, Ins; Epidermal growth factor, EGF; Platelet derived growth factor, PDGF; Converting enzyme inhibitor, CEI; Calphostin C, Cal C; Thapsigargin, TG; Tyrosine kinase activity, TKA; Tyrosine phosphatase activity, TPA; PolyŽGlu80 , Tyr 20 ., pGT; Phospholipase C, PLC; Phospholipase A2, PLA2; Pertussistoxin, PTX; Fetal calf serum, FCS; Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; Focal adhesion kinase, FAK ) Corresponding author. Tel.: q33-05-61-32-26-21; fax: q3305-62-17-25-54; e-mail: [email protected]

In mesangial cells ŽMC., we previously demonstrated that both B1R and B2R are present ŽEmond et al., 1990; Bascands et al., 1991, 1993a, 1994. and are able to induce proliferation of quiescent cells which is potentiated by the presence of insulin ŽIns. ŽBascands et al., 1993b.. Recently, the effect of bradykinin ŽBK. on cell proliferation became controversial since antimitogenic properties of BK have been reported ŽDixon and Dennis, 1997., but no

0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 1 5 1 - 4

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clear hypotheses have been forwarded to explain these effects. Tyrosine phosphorylation of cellular proteins is a critical process associated with cell proliferation and differentiation. These processes are not only governed by growth factors receptors activation but can also be associated with the stimulation of G-protein coupled receptors. In response to BK, enhanced tyrosine phosphorylation of the focal adhesion kinase ŽFAK., paxillin, the tyrosine kinase p60-src, the p130-src substrate or mitogen-activated protein kinases ŽMAPK. has been demonstrated in different cell types ŽLeeb-Lundberg et al., 1994; Fleming et al., 1995; Tippmer et al., 1996.. MC proliferation is a hallmark of progressive glomerular disease. The proliferative and anti-proliferative effects of BK and its ability to induce tyrosine phosphorylation of cellular proteins in other cell-types suggest that BK receptors activation in MC may play a role in progressive glomerular disease. For these reasons, we investigated the effects of BK on tyrosine kinase activity ŽTKA. of MC in relationship with FAK phosphorylation and cell proliferation.

2. Materials and methods 2.1. Rat MC cultures MC cultures were obtained as outgrowths of decapsulated collagenase-digested glomeruli obtained by graded sieving and characterized by morphological and functional criteria as described previously ŽEmond et al., 1990.. Primary cultures and passaged Žpassage 4 to 10. cells were used. 2.2. TKA and tyrosine phosphatase actiÕity (TPA) TKA was assayed by measuring the incorporation of 33 P from w g-33 Px ATP into the synthetic tyrosine kinase substrate polyŽGlu80 , Tyr 20 . ŽpGT.. TPA was assayed by measuring the release of 33 P from radiolabeled 33 P-polyŽGlu80 , Tyr 20 .. TKA and TPA were defined as the amount of 33 P incorporated or released, respectively and expressed as the percentage of value obtained in the absence of BK.

2.3. Western-blot analysis of p125-FAK phosphorylation For analysis of p125-FAK phosphorylation, the cell lysates were submitted to immunoprecipitation with anti-p125-FAK antibody, subjected to SDSPAGE, transferred to a nitrocellulose membrane and blotted either with anti-p125-FAK antibody or with the anti-phosphotyrosine antibody ŽPY20.. Proteins were visualized using an enhanced chemiluminescence kit ŽAMERSHAM.. 2.4. Cell proliferation Increasing concentrations of BK from 0.1 to 100 nM were tested in proliferating MC in the presence of FCS or MC rendered quiescent by 48 h deprivation in 0.5% FCS containing medium. Every 24 h, the cell number of triplicate wells was determined using a cell counter ŽCoulter counter ZM. as previously described ŽBascands et al., 1993b. and results were expressed as the percentage of BK-untreated cells.

3. Results 3.1. BK induced a biphasic effect on total TKA in MC MC lysates catalyzed the phosphorylation of pGT in a time-dependent manner up to 15 min at 258C and was linear with the protein concentration up to 50 mg per assay Ždata not shown.. MC thus have an intrinsic TKA under basal conditions Ždata not shown.. As shown in Fig. 1, BK induced a rapid Ž30 s. and transient activation of TKA reaching a maximum of 124.5 " 3.1%. When the effect of BK was compared with those of other ligands known to activate protein tyrosine phosphorylation, i.e., angiotensin II ŽAII., epidermal growth factor ŽEGF. and Ins, none of these three ligands induced inhibition. EGF, Ins and AII induced activation of TKA with a maximum of 150% at 5 min, 150% at 1 min and 135% at 5 min, respectively. The dose-dependent effect of BK on TKA ŽFig. 2. shows that a maximum of 90% increase in TKA was obtained

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inhibition of TKA were completely prevented by the B2 receptor antagonists Ždata not shown.. These findings strongly suggest that both activation and inhibition of TKA are mediated via the B2 receptor. No effect of the B1 antagonist Ždes-Arg 9 wLeu8 xBK. was observed Ždata not shown.. 3.2. BK-induced TKA inhibition does not require phospholipase C actiÕation and is pertussis-toxin (PTX) insensitiÕe

Fig. 1. Effect of BK, EGF, Ins and AII on TKA in rat MC stimulated at 378C with 100 nM BK, 300 ngrml EGF, 5 mgrml Ins or 100 nM AII. Results are the mean"S.E.M. of three experiments and are expressed as a percentage of the respective time untreated control. `: P - 0.05 and U : P - 0.01 when compared to values obtained in the absence of BK, AII, Ins and EGF.

with 1 nM BK after 2 min of stimulation that gradually decreased to stabilize at 127.9 " 6.3% up to 10 min. Incubation with higher concentrations Ž10 to 100 nM. resulted in a lower stimulation Ž20 to 40%., followed by a significant inhibition Žy30%. at 2 min that lasted up to 10 min. Activation and

Fig. 2. Concentration-dependent effect of BK on TKA in rat MC stimulated at 378C with increasing concentrations of BK. Results are the mean"S.E.M. of three experiments and are expressed as a percentage of the respective time untreated control. `: P - 0.05 and U : P - 0.01 when compared to the respective time values obtained in the absence of BK.

As shown in Fig. 3, the activation of TKA induced by the low dose of BK was abolished by PTX pre-treatment. In contrast, in the presence of PTX, the inhibition of TKA was observed as soon as 2 min incubation indicating that TKA activation in the absence of PTX masks the TKA inhibition already present at 2 min. In addition, treatment with 0.3 mM U73122, an inhibitor of the phosphatidylinositolspecific PLC for 10 min, or inhibition of protein kinase C with Calphostin C ŽCal C., or chelation of free cytosolic Ca2q ŽBAPTA-AM. or depletion of inositol sensitive Ca2q stores Žthapsigargin, TG., abolished the BK-induced TKA activation, but did

Fig. 3. Effect of PTX Ž100 ngrml., 0.3 mM U73122, 100 nM Cal C, 20 mM BAPTA-AM, and 1.5 mM TG on BK-induced activation of TKA in rat MC stimulated for 2 min with 1 nM BK. Results are the mean"S.E.M. of three independent experiments and are expressed as a percentage of the respective time value obtained in the absence of any treatment. U : P - 0.01 when compared to the value obtained in the absence of BK. `: P - 0.01 when compared to the value obtained in the presence of BK.

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not block the BK-induced inhibition of TKA. However, the inhibitory effect of BK on TKA does not involve PLA 2 activation as indomethacin Ž1 mM. did not reverse the effects of BK on TKA and direct activation of adenylate cyclase by forskolin Ž1 mM., exogenous PGE 2 Ž1 mM. or treatment with the cAMP analogue dibutyryl cAMP Ž100 mM. has no effect on TKA Ždata not shown.. 3.3. Effect of BK on TPA As shown in Fig. 4, BK induced a 60% increase in TPA, which was completely inhibited in the presence of sodium orthovanadate. 3.4. BK induces tyrosine phosphorylation of p125FAK An increase in tyrosine phosphorylation of p125FAK ŽFig. 5A, blot P-Tyr and Fig. 5B. at 2 min with two doses of BK Žlanes 2 and 5. and at 10 min incubation with the low dose of BK Žlane 6. was observed compared to control lanes 1 and 4. When MC were incubated 10 min with a high dose of BK Žlane 3., the tyrosine phosphorylation of the p125FAK was significantly inhibited. When the nitrocellulose membrane was blotted with the anti-p125FAK antibody ŽFig. 5A, blot p125-FAK., no differ-

Fig. 5. Effect of BK on tyrosine phosphorylation of p125-FAK in rat MC stimulated at 378C with 1 nM and 100 nM BK for 2 and 10 min. The total cell lysate was immunoprecipitated with antip125-FAK antibody, subjected to a 10% SDS-PAGE and analysed by immunoblotting either with anti-phosphotyrosine antibody Ž blot P-Tyr ., or with anti-p125-FAK antibody Ž blot p125-FAK .. A representative experiment is shown in panel A. Molecular mass values are on the right in kDa. Control after 2 min Žlane 1., 100 nM BK for 2 min Žlane 2., 100 nM BK for 10 min Žlane 3., control after 10 min Žlane 4., 1 nM BK for 2 min Žlane 5., 1 nM BK for 10 min Žlane 6.. A mean"S.E.M. of the relative tyrosine phosphorylation of p125-FAK from three experiments is shown in panel B. U : P - 0.01 when compared to values obtained in the absence of BK.

ence between control and BK-stimulated cells was detected indicating no change in the p125-FAK protein level. 3.5. Effect of BK on cell proliferation

Fig. 4. Effect of BK on TPA of MC. MC were treated for 2 min with 100 nM BK in the absence or presence of 1 mM orthovanadate ŽV. added 4 h prior to stimulation with BK. Results are the mean"S.E.M. of three experiments and are expressed as a percentage of the TPA of untreated cells. U : P - 0.01 when compared to values of untreated cells.

As shown in Fig. 6A, BK induces proliferation of quiescent MC. A BK dose–response of MC proliferation was observed with a maximum proliferation induced by 100 nM BK at 96 h. When experiments were repeated on MC in the presence of 15% FCS, BK induced a time- and dose-dependent inhibition of

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Fig. 6. Dose–response of BK on cell proliferation of quiescent MC Žpanel A. and on fetal calf serum ŽFCS.-stimulated MC Žpanel B.. Quiescent MC Ždeprived for 48 h in 0.5% FCS containing medium. or MC cultured in the presence of 15% FCS-containing medium were exposed to increasing concentrations of BK from 0.1 to 100 nM. Values Žmeans"S.E.M. of three cultures from three independent experiments. were expressed as a percentage of the cell number found in 0.5 or 15% FCS exposed MC. `: P - 0.05 and U : P - 0.01 when compared to the appropriate time control.

cell proliferation ŽFig. 6B.. A maximum 30% reduction in cell number was observed after 96 h and did not result from cell detachment as no cell ghosts were found in the culture supernatant.

4. Discussion The main result communicated in the present report is the coexistence of two opposite effects Žactivation and inhibition. of BK on the total TKA, the tyrosine phosphorylation of p125-FAK and cell proliferation of rat MC. Whereas, the stimulatory

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effect of BK on tyrosine phosphorylation is now well-documented in different cell types ŽLeeb-Lundberg et al., 1994; Tippmer et al., 1996; Fleming and Busse, 1997. the inhibition of TKA appears less ubiquitous and the mechanism is unknown. The data presented here suggest that the transient stimulation of TKA masked the concomitant inhibition. Very few reports describe decreases in TKA and only one group found an inhibitory effect of BK in human fibroblast cells ŽMcAllister et al., 1993.. This inhibitory effect of BK pass through a PGE 2-mediated cAMP-dependent pathway. A similar PGE 2-mediated cAMP-dependent pathway can be ruled out in our study since we report here that exogenous PGE 2 or stimulation of cAMP formation by forskolin, or dibutyryl cAMP did not change the basal TKA and thus, did not mimic BK-induced TKA inhibition. Although the type of the G-proteins coupled to the B2 receptor on MC is not known, a dual signaling pathway of BK involving the stimulation of different stimulated G-proteins, Ga q and Ga i has been previously reported in endothelial cells and in A431 epidermoid caranoma cells ŽLiao and Homcy, 1993; Liebmann et al., 1996.. The involvement of two distinct G-proteins in BK signaling in MC is consistent with the dissociation of activation and inhibition by PTX since the Ga q protein is insensitive to PTX. Another mechanism to explain the inhibition of tyrosine phosphorylation is a Gprotein-induced activation of a phosphatase activity as described for the AT2 receptor for AII and the SST2 somatostatin receptor. Such a link between B2 receptor activation and phosphatase activity has been recently proposed ŽFleming and Busse, 1997. and is consistent with the increase in TPA observed in MC exposed to BK. In the present study, we report for the first time the phosphorylation of p125-FAK by BK in MC. As the focal adhesion system, p125-FAK participates in the translocation of mechanical forces Ži.e., stretching., in biochemical events initiating cell proliferation, p125-FAK may be one of the important targets in BK receptor signaling in MC. The mitogenic effect of BK is well-documented and involved the Ca2q and the PLC pathway ŽBascands et al., 1993b; el-Dahr et al., 1996; Dixon and Dennis, 1997.. However, an inhibitory effect of BK on the cell proliferation stimulated by growth

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factor ŽEGF, PDGF. has been recently reported ŽMcAllister et al., 1993; Dixon and Dennis, 1997.. The inhibition pathway does not involve PLC and PLA 2 but the involvement of the TK was neither tested nor evoked ŽDixon and Dennis, 1997.. In conclusion, the dual effect of BK on TKA and on tyrosine phosphorylation of p125-FAK might be related to the mitogenic and antimitogenic effect of BK on MC. Depending on cell culture conditions, BK can either induce or reduce MC proliferation. The confirmation of the antimitogenic effect of BK on MC provides a very interesting basis for therapeutic applications especially with respect to converting enzyme inhibitor ŽCEI. treatments which are known to favor the generation of kinin and demonstrate very efficient protective effects against the development of glomerulosclerosis.

Acknowledgements This work was partly funded by a grant from region Midi-Pyrenees ´ ´ no. RECHr9407562. Ms. C. Alric is supported by a grant from MENESRIP. We thank Dr. J.P Schanstra for help during the preparation of the manuscript.

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Evidence for existence of two distinct bradykinin receptors on rat mesangial cells. Am. J. Physiol. 264, F548–F556. Bascands, J.L., Pecher, C., Bompart, G., Rakotoarivony, J., Tack, J.L., Girolami, J.P., 1994. Bradykinin-induced in vitro contraction of rat mesangial cells via a B2 receptor type. Am. J. Physiol. 267, F871–F878. Dixon, B.S., Dennis, M.J., 1997. Regulation of mitogenesis by kinins in arterial smooth muscle cells. Am. J. Physiol. 273, C7–C20. el-Dahr, S.S., Dipp, S., Yosipiv, I.V., Baricos, W.H., 1996. Bradykinin stimulates c-fos expression, AP-1-DNA binding activity and proliferation of rat glomerular mesangial cells. Kidney Int. 50, 1850–1855. Emond, C., Bascands, J.L., Pecher, C., Cabos-Boutot, G., Pradelles, P., Regoli, D., Girolami, J.P., 1990. Characterization of a B2-bradykinin receptor in rat renal mesangial cells. Eur. J. Pharmacol. 190, 381–392. Fleming, I., Busse, R., 1997. Tyrosine phosphorylation and bradykinin-induced signaling in endothelial cells. Am. J. Cardiol. 4, 102A–109A. Fleming, I., Fisslthaler, B., Busse, R., 1995. Calcium signaling in endothelial cells involves activation of tyrosine kinases and leads to activation of mitogen-activated protein kinases. Circ. Res. 76, 522–529. Leeb-Lundberg, L.M., Song, X.H., Mathis, S.A., 1994. Focal adhesion-associated proteins p125FAK and paxillin are substrates for bradykinin-stimulated tyrosine phosphorylation in Swiss 3T3 cells. J. Biol. Chem. 269, 24328–24334. Liao, J.K., Homcy, C.J., 1993. The G proteins of the G alpha i and G alpha q family couple the bradykinin receptor to the release of endothelium-derived relaxing factor. J. Clin. Invest. 92, 2168–2172. Liebmann, C., Graness, A., Ludwig, B., Adomeit, A., Boehmer, A., Boehmer, F.D., Nurnberg, B., Wetzker, R., 1996. Dual ¨ bradykinin B2 receptor signalling in A431 human epidermoid carcinoma cells: activation of protein kinase C is counteracted by a GS-mediated stimulation of the cyclic AMP pathway. Biochem. J. 313, 109–118. McAllister, B.S., Leeb-Lundberg, F., Olson, M.S., 1993. Bradykinin inhibition of EGF- and PDGF-induced DNA synthesis in human fibroblasts. Am. J. Physiol. 265, C477–C484. Tippmer, S., Bossenmaier, B., Haring, H., 1996. Bradykinin in¨ duces tyrosine phosphorylation in human foreskin fibroblasts and 293 cells transfected with rat B2 kinin receptor. Eur. J. Biochem. 236, 953–959.