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α2B-Adrenergic Receptor Activates MAPK via a Pathway Involving Arachidonic Acid Metabolism, Matrix Metalloproteinases, and Epidermal Growth Factor Receptor Transactivation

2002; Elsevier BV; Volume: 277; Issue: 22 Linguagem: Inglês

10.1074/jbc.m110142200

1083-351X

Daniel Cussac, Stéphane Schaak, Colette Denis, Hervé Paris,

Pharmacogenetics and Drug Metabolism

We have investigated the mechanisms whereby α2B-adrenergic receptor (α2B-AR) promotes MAPK activation in a clone of the renal tubular cell line, LLC-PK1, transfected with the rat nonglycosylated α2-AR gene. Treatment of LLC-PK1-α2Bwith UK14304 or dexmedetomidine caused arachidonic acid (AA) release and ERK2 phosphorylation. AA release was abolished by prior treatment of the cells with pertussis toxin, quinacrine, or methyl arachidonyl fluorophosphonate but not by the addition of the MEK inhibitor U0126. The effects of α2-agonists on MAPK phosphorylation were mimicked by cell exposure to exogenous AA. On the other hand, quinacrine abolished the effects of UK14304, but not of AA, suggesting that AA released through PLA2 is responsible for MAPK activation by α2B-AR. The effects of α2-agonists or AA were PKC-independent and were attenuated by indomethacin and nordihydroguaiaretic acid. Treatment with batimastat, CRM 197, or tyrphostin AG1478 suppressed MAPK phosphorylation promoted by α2-agonist or AA. Furthermore, conditioned culture medium from UK14304-treated LLC-PK1-α2B induced MAPK phosphorylation in wild-type LLC-PK1. Based on these data, we propose a model whereby activation of MAPK by α2B-AR is mediated through stimulation of PLA2, AA release, generation of AA derivatives, activation of matrix metalloproteinases, release of heparin-binding EGF-like growth factor, transactivation of epidermal growth factor receptor, and recruitment of Shc. Whether this pathway is particular to α2B-AR and LLC-PK1 or whether it can be extended to other cell types and/or other G-protein-coupled receptors remains to be established. We have investigated the mechanisms whereby α2B-adrenergic receptor (α2B-AR) promotes MAPK activation in a clone of the renal tubular cell line, LLC-PK1, transfected with the rat nonglycosylated α2-AR gene. Treatment of LLC-PK1-α2Bwith UK14304 or dexmedetomidine caused arachidonic acid (AA) release and ERK2 phosphorylation. AA release was abolished by prior treatment of the cells with pertussis toxin, quinacrine, or methyl arachidonyl fluorophosphonate but not by the addition of the MEK inhibitor U0126. The effects of α2-agonists on MAPK phosphorylation were mimicked by cell exposure to exogenous AA. On the other hand, quinacrine abolished the effects of UK14304, but not of AA, suggesting that AA released through PLA2 is responsible for MAPK activation by α2B-AR. The effects of α2-agonists or AA were PKC-independent and were attenuated by indomethacin and nordihydroguaiaretic acid. Treatment with batimastat, CRM 197, or tyrphostin AG1478 suppressed MAPK phosphorylation promoted by α2-agonist or AA. Furthermore, conditioned culture medium from UK14304-treated LLC-PK1-α2B induced MAPK phosphorylation in wild-type LLC-PK1. Based on these data, we propose a model whereby activation of MAPK by α2B-AR is mediated through stimulation of PLA2, AA release, generation of AA derivatives, activation of matrix metalloproteinases, release of heparin-binding EGF-like growth factor, transactivation of epidermal growth factor receptor, and recruitment of Shc. Whether this pathway is particular to α2B-AR and LLC-PK1 or whether it can be extended to other cell types and/or other G-protein-coupled receptors remains to be established. The α2-adrenergic receptors (α2-ARs) are members of the G-protein-coupled receptor superfamily that mediate physiological responses to the endogenous catecholamines, such as reduction of blood pressure, sedation, platelet aggregation, and inhibition of renin release or insulin secretion. Three subtypes of α2-ARs (namely α2A, α2B, and α2C) have been identified (1Bylund D.B. Eikenberg D.C. Hieble J.P. Langer S.Z. Lefkowitz R.J. Minneman K.P. Molinoff P.B. Ruffolo R.R. Trendelenburg U. Pharmacol. Rev. 1994; 46: 121-136PubMed Google Scholar). Although recent studies, conducted on mice with genetic alterations of α2-AR expression, have clarified the respective roles of α2A- and α2B-ARs in the mediation of the cardiovascular and sedative effects of α2-agonists, the precise functions of each subtype are far from being definitively elucidated (2Kable J.W. Murrin L.C. Bylund D.B. J. Pharmacol. Exp. Ther. 2000; 293: 1-7PubMed Google Scholar). Until recently, the effects of α2-ARs were generally considered as exclusively due to the modulation of effectors such as adenylyl cyclase or phospholipase Cβ. There is now accumulating evidence that, in addition to these pathways, α2-ARs are also involved in the regulation of cell growth via stimulation of mitogen-activated protein kinases (MAPKs). The phosphorylation of MAPKs has been observed in transfected cells (3Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 4Schaak S. Cussac D. Cayla C. Devedjian J.C. Guyot R. Paris H. Denis C. Gut. 2000; 47: 242-250Crossref PubMed Scopus (38) Google Scholar) as well as in various types of cells spontaneously expressing α2-ARs (5Bouloumie A. Planat V. Devedjian J.C. Valet P. Saulnier-Blache J.S. Record M. Lafontan M. J. Biol. Chem. 1994; 269: 30254-30259Abstract Full Text PDF PubMed Google Scholar, 6Kribben A. Herget-Rosenthal S. Lange B. Erdbrugger W. Philipp T. Michel M.C. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 225-232Crossref PubMed Scopus (29) Google Scholar). The three receptor subtypes promoted phosphorylation of ERK1 and ERK2 in Chinese hamster ovary cells (3Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). According to results obtained in HEK 293 and COS cells (7Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), this effect is independent of receptor internalization via clathrin-coated pits.Like for other ARs (9Pierce K.L. Luttrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (360) Google Scholar), it is probable that the mechanisms whereby α2-ARs promote MAPK activation are highly dependent upon the subtype considered and the particular cell type it is expressed in. So far, the precise pathways of the mitogenic signal transmission were exclusively examined for α2A-AR. In HEK 293 cells (10Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), activation of ERK1/2 by α2A-AR is primarily triggered through release of βγ subunits from pertussis toxin-sensitive G proteins, stimulation of phospholipase Cβ, phosphoinositide hydrolysis, increase of intracellular Ca2+, and successive activation of Pyk2 and Src. Activation of Src causes the formation of Shc-Grb2-Sos complex, which leads to ERK phosphorylation via the Ras/Raf/MEK cascade. In COS cells (11Pierce K.L. Tohgo A. Ahn S. Field M.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 23155-23160Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), α2A-AR-induced phosphorylation of ERK2 proceeds via two distinct pathways, which are dependent ("transactivation pathway") or not ("direct pathway") on the tyrosine kinase activity of the EGF receptor (EGF-R). The early steps of both pathways involve the release of βγ subunits from Gi proteins and the activation of Src by an unknown process that is independent of inositol 1,4,5-trisphosphate production (12Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Then, the phosphorylation of MAPKs occurs either directly through recruitment of the MEK cascade via phosphorylation of the adapter protein Shc or indirectly through activation of unidentified matrix metalloproteinases, release of heparin-binding EGF-like growth factor (HB-EGF), and subsequent transactivation of EGF-R.Recent experiments carried out on rat proximal tubule cells in primary culture and on LLC-PK1 cells transfected with the rat nonglycosylated α2-AR (RNG) gene (LLC-PK1-α2B) have shown that α2B-ARs promote MAPK activation and arachidonic acid (AA) release (13Cussac D. Schaak S. Gales C. Flordellis C. Denis C. Paris H. Am. J. Physiol. Renal Physiol. 2002; 282: F943-F952Crossref PubMed Scopus (27) Google Scholar). The sequential relationship between PLA2 and MAPK activation was not investigated. As demonstrated in eosinophils during the process of adhesion to fibronectin (14Sano H. Zhu X. Sano A. Boetticher E.E. Shioya T. Jacobs B. Munoz N.M. Leff A.R. J. Immunol. 2001; 166: 3515-3521Crossref PubMed Scopus (33) Google Scholar), PLA2 activation may result from its phosphorylation by MAPKs. Conversely, as shown in rabbit renal epithelial cells for angiotensin II receptor, the activation of MAPK could be the consequence of AA release (15Jiao H. Cui X.L. Torti M. Chang C.H. Alexander L.D. Lapetina E.G. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7417-7421Crossref PubMed Scopus (43) Google Scholar). Based on the use of different inhibitors, the present work demonstrates that activation of MAPK by α2B-AR is, in LLC-PK1-α2B, primarily mediated by a pathway involving stimulation of PLA2, generation of AA derivatives, activation of matrix metalloproteinases, release of HB-EGF, and transactivation of EGF-R.DISCUSSIONThe phosphorylation of MAPKs (ERK1/2) by α2-agonists has been reported in a variety of cells, including Chinese hamster ovary cells transfected with the RNG gene (3Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and COS or HEK 293 cells transfected with the α2C2 gene (7Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), which encode the rat and human α2B-AR subtypes, respectively. More recently, this effect has also been observed in rat proximal tubule cells in primary culture as well as in LLC-PK1-α2B (13Cussac D. Schaak S. Gales C. Flordellis C. Denis C. Paris H. Am. J. Physiol. Renal Physiol. 2002; 282: F943-F952Crossref PubMed Scopus (27) Google Scholar). In these two models, receptor stimulation resulted in an acceleration of cell proliferation, suggesting that the action of catecholamines on α2B-AR may play, in rat, a role in the adaptive response to acute renal tissue injury. Additionally, α2B-AR is known to enhance Na+ reabsorption as a consequence of increased activity of the Na+/H+ exchanger, NHE3 (20Nord E.P. Howard M.J. Hafezi A. Moradeshagi P. Vaystub S. Insel P.A. J. Clin. Invest. 1987; 80: 1755-1762Crossref PubMed Scopus (105) Google Scholar). Since NHE3 was recently found to be controlled by MAPK in mouse proximal tubule cells (21Liu F. Gesek F.A. Am. J. Physiol. 2001; 280: F415-F425Crossref PubMed Google Scholar), it is possible that MAPK activation is also involved in the regulation of NHE3 by α2B-AR.The mechanisms whereby G-protein-coupled receptors activate the MAPK cascade are highly dependent upon the receptor considered and the cell type it is expressed in. Although previous studies have shown that the action of the α2B-AR is independent of receptor internalization (7Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), the signaling pathway(s) accounting for the phosphorylation of MAPK by this receptor subtype remains poorly defined. The results obtained in this study provide substantial evidence that, in LLC-PK1-α2B, the activation of ERK by α2-agonists is triggered via a mechanism comprising the activation of matrix metalloproteinases, the release of HB-EGF, and the subsequent activation of the EGF-R. This cascade was demonstrated by the following observations. First, UK14304-induced phosphorylation of MAPK is totally abrogated in the presence of the matrix metalloproteinase inhibitors (batimastat or 1,10-phenanthroline) and by prior treatment of the cells with CRM 197. Second, conditioned medium from LLC-PK1-α2B cells treated with UK14304 causes activation of MAPK in wild-type LLC-PK1, even in the presence of batimastat. Third, the consequences of LLC-PK1-α2B exposure to α2-agonists or of wild-type LLC-PK1 exposure to conditioned medium are abolished by prior treatment of the cells with the inhibitor of EGF-R tyrosine kinase activity, tyrphostin AG1478. Previous studies of lysophosphatidic acid receptor or α2A-AR have demonstrated that the contribution of the EGF-R transactivation pathway is largely dependent on the cell type (10Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 22Della Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). In HEK 293, the major pathway of MAPK activation by α2A-AR is via the activation of Pyk2, a calcium-dependent tyrosine kinase of the focal adhesion kinase family (10Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). On the other hand, the effects of α2A-AR in COS cells are mediated by both EGF-R transactivation and direct recruitment of the MEK cascade by Src (9Pierce K.L. Luttrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (360) Google Scholar,23Pierce K.L. Maudsley S. Daaka Y. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1489-1494Crossref PubMed Scopus (196) Google Scholar). According to our results, the transactivation pathway is predominant in LLC-PK1-α2B; whether it is exclusive awaits definitive demonstration.An other major effort of the present work was to define the pathway leading from stimulated α2B-AR to transactivation of EGF-R. Because α2-agonists activate AA release in LLC-PK1-α2B and because AA is responsible for MAPK phosphorylation following angiotensin II treatment in rabbit proximal tubule cells (16Dulin N.O. Alexander L.D. Harwalkar S. Falck J.R. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8098-8102Crossref PubMed Scopus (77) Google Scholar), we sought to evaluate its implication. Consistent with a crucial role of the lipid second messenger, exposure of LLC-PK1-α2B to exogenous AA resulted in tyrosine phosphorylation of p52 Shc and in activation of MAPK with time courses that show a striking parallel to those observed with α2-agonists. Like for α2-agonists, the action of AA was prevented by batimastat, 1,10-phenanthroline, CRM 197, or tyrphostin AG1478. However, a major divergence was that, unlike the effects of α2-agonists, those of AA are resistant to pretreatment with pertussis toxin or to the addition of quinacrine. Additional support for implication of AA in the mediation of α2B-AR signal came from the study of the effects of inhibitors of AA metabolism. According to these experiments, phosphorylation of MAPK by UK14304 was strongly inhibited by NDGA and indomethacin. Ketoconazole was by contrast ineffective, indicating that AA products generated by lipoxygenase and/or cyclooxygenase, but not epoxygenase, are involved in α2-agonist effect. The observation that AA causes MAPK phosphorylation in our model is in opposition with results obtained on LLC-PK1/C14 (24Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. J. Biol. Chem. 1999; 274: 4764-4769Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In this clone, ERK phosphorylation was observed in response to epoxyeicosatrienoic acids but not to AA. Moreover, AA became efficient after cell transfection with an active form of cytochrome P450 epoxygenase of bacterial origin, indicating eicosanoid-dependent activation of MAPK. The reason for these discrepancies is enigmatic. However, activation of MAPK by AA was repeatedly reported in rabbit proximal tubules as well as in various cell types, including vascular smooth muscle cells and neutrophils. In vascular smooth muscle cells, efficacy of AA was dependent on its conversion into 15-hydroxyeicosatetraenoic acid and on PKC activation (25Rao G.N. Baas A.S. Glasgow W.C. Eling T.E. Runge M.S. Alexander R.W. J. Biol. Chem. 1994; 269: 32586-32591Abstract Full Text PDF PubMed Google Scholar). Dependence on lipoxygenase and PKC activity was also found in human neutrophils (26Hii C.S. Huang Z.H. Bilney A. Costabile M. Murray A.W. Rathjen D.A. Der C.J. Ferrante A. J. Biol. Chem. 1998; 273: 19277-19282Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In this cell type, the effects of AA engaged a membrane receptor linked to Gi/o proteins (27Capodici C. Pillinger M.H. Han G. Philips M.R. Weissmann G. J. Clin. Invest. 1998; 102: 165-175Crossref PubMed Scopus (62) Google Scholar). This is not the case in LLC-PK1-α2B, since neither staurosporine nor pertussis toxin treatment abolished ERK phosphorylation caused by AA. Regarding these points, LLC-PK1-α2B resembles rabbit renal epithelial cells. However, it is epoxy derivatives that mediate the effects of AA on MAPK phosphorylation in these cells (16Dulin N.O. Alexander L.D. Harwalkar S. Falck J.R. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8098-8102Crossref PubMed Scopus (77) Google Scholar). Whereas involvement of the cytochrome P450 pathway can be excluded in our model, the respective contribution of COX and LOX is still unclear, since it is difficult to reconcile why products from either pathway could function similarly. The implication of COX activity is beyond doubt, because the effects of α2-agonists and AA were also blocked by aspirin (not shown). By contrast, that of LOX is more questionable, since NDGA can also interfere with COX activity and act as an antioxidant. Alternatively, the possibility that prostaglandins and leukotrienes act in concert cannot be definitively ruled out. Indeed, the combined action of COX and LOX was already demonstrated to be necessary for some of the effects of angiotensin II in rat kidney and bovine bronchi (28Oyekan A. Balazy M. McGiff J.C. Am. J. Physiol. 1997; 273: R293-R300PubMed Google Scholar, 29Nally J.E. Bunton D.C. Martin D. Thomson N.C. Pulm. Pharmacol. 1996; 9: 211-217Crossref PubMed Scopus (5) Google Scholar). It is therefore clear that the identification of the AA metabolites responsible for MAPK activation in LLC-PK1-α2B will require future study. In addition, the mechanism whereby these products may affect matrix metalloproteinase activity has yet to be defined. In line with the existence of a relationship between the two phenomena, constitutive expression of cyclooxygenase-2 in human colon cancer cells results in increased activation of MMP-2 (30Tsujii M. Kawano S. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3336-3340Crossref PubMed Scopus (1325) Google Scholar), whereas inhibitors of PLA2 and cyclooxygenase-2 reduce the release of matrix metalloproteinases in prostate tumor cells (31Attiga F.A. Fernandez P.M. Weeraratna A.T. Manyak M.J. Patierno S.R. Cancer Res. 2000; 60: 4629-4637PubMed Google Scholar).In conclusion, our results provide evidence for a pathway by which α2B-AR activates MAPK through stimulation of PLA2, generation of AA metabolites by cyclooxygenase and/or lipoxygenase, stimulation of matrix metalloproteinases, release of HB-EGF, and transactivation of the EGF-R (Fig. 9). Whether this scenario is particular to α2B-AR in LLC-PK1 or whether it can be extended to other cell types and/or other G-protein-coupled receptors remains to be established. The α2-adrenergic receptors (α2-ARs) are members of the G-protein-coupled receptor superfamily that mediate physiological responses to the endogenous catecholamines, such as reduction of blood pressure, sedation, platelet aggregation, and inhibition of renin release or insulin secretion. Three subtypes of α2-ARs (namely α2A, α2B, and α2C) have been identified (1Bylund D.B. Eikenberg D.C. Hieble J.P. Langer S.Z. Lefkowitz R.J. Minneman K.P. Molinoff P.B. Ruffolo R.R. Trendelenburg U. Pharmacol. Rev. 1994; 46: 121-136PubMed Google Scholar). Although recent studies, conducted on mice with genetic alterations of α2-AR expression, have clarified the respective roles of α2A- and α2B-ARs in the mediation of the cardiovascular and sedative effects of α2-agonists, the precise functions of each subtype are far from being definitively elucidated (2Kable J.W. Murrin L.C. Bylund D.B. J. Pharmacol. Exp. Ther. 2000; 293: 1-7PubMed Google Scholar). Until recently, the effects of α2-ARs were generally considered as exclusively due to the modulation of effectors such as adenylyl cyclase or phospholipase Cβ. There is now accumulating evidence that, in addition to these pathways, α2-ARs are also involved in the regulation of cell growth via stimulation of mitogen-activated protein kinases (MAPKs). The phosphorylation of MAPKs has been observed in transfected cells (3Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 4Schaak S. Cussac D. Cayla C. Devedjian J.C. Guyot R. Paris H. Denis C. Gut. 2000; 47: 242-250Crossref PubMed Scopus (38) Google Scholar) as well as in various types of cells spontaneously expressing α2-ARs (5Bouloumie A. Planat V. Devedjian J.C. Valet P. Saulnier-Blache J.S. Record M. Lafontan M. J. Biol. Chem. 1994; 269: 30254-30259Abstract Full Text PDF PubMed Google Scholar, 6Kribben A. Herget-Rosenthal S. Lange B. Erdbrugger W. Philipp T. Michel M.C. Naunyn-Schmiedeberg's Arch. Pharmacol. 1997; 356: 225-232Crossref PubMed Scopus (29) Google Scholar). The three receptor subtypes promoted phosphorylation of ERK1 and ERK2 in Chinese hamster ovary cells (3Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). According to results obtained in HEK 293 and COS cells (7Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), this effect is independent of receptor internalization via clathrin-coated pits. Like for other ARs (9Pierce K.L. Luttrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (360) Google Scholar), it is probable that the mechanisms whereby α2-ARs promote MAPK activation are highly dependent upon the subtype considered and the particular cell type it is expressed in. So far, the precise pathways of the mitogenic signal transmission were exclusively examined for α2A-AR. In HEK 293 cells (10Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), activation of ERK1/2 by α2A-AR is primarily triggered through release of βγ subunits from pertussis toxin-sensitive G proteins, stimulation of phospholipase Cβ, phosphoinositide hydrolysis, increase of intracellular Ca2+, and successive activation of Pyk2 and Src. Activation of Src causes the formation of Shc-Grb2-Sos complex, which leads to ERK phosphorylation via the Ras/Raf/MEK cascade. In COS cells (11Pierce K.L. Tohgo A. Ahn S. Field M.E. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 2001; 276: 23155-23160Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar), α2A-AR-induced phosphorylation of ERK2 proceeds via two distinct pathways, which are dependent ("transactivation pathway") or not ("direct pathway") on the tyrosine kinase activity of the EGF receptor (EGF-R). The early steps of both pathways involve the release of βγ subunits from Gi proteins and the activation of Src by an unknown process that is independent of inositol 1,4,5-trisphosphate production (12Hawes B.E. van Biesen T. Koch W.J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17148-17153Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Then, the phosphorylation of MAPKs occurs either directly through recruitment of the MEK cascade via phosphorylation of the adapter protein Shc or indirectly through activation of unidentified matrix metalloproteinases, release of heparin-binding EGF-like growth factor (HB-EGF), and subsequent transactivation of EGF-R. Recent experiments carried out on rat proximal tubule cells in primary culture and on LLC-PK1 cells transfected with the rat nonglycosylated α2-AR (RNG) gene (LLC-PK1-α2B) have shown that α2B-ARs promote MAPK activation and arachidonic acid (AA) release (13Cussac D. Schaak S. Gales C. Flordellis C. Denis C. Paris H. Am. J. Physiol. Renal Physiol. 2002; 282: F943-F952Crossref PubMed Scopus (27) Google Scholar). The sequential relationship between PLA2 and MAPK activation was not investigated. As demonstrated in eosinophils during the process of adhesion to fibronectin (14Sano H. Zhu X. Sano A. Boetticher E.E. Shioya T. Jacobs B. Munoz N.M. Leff A.R. J. Immunol. 2001; 166: 3515-3521Crossref PubMed Scopus (33) Google Scholar), PLA2 activation may result from its phosphorylation by MAPKs. Conversely, as shown in rabbit renal epithelial cells for angiotensin II receptor, the activation of MAPK could be the consequence of AA release (15Jiao H. Cui X.L. Torti M. Chang C.H. Alexander L.D. Lapetina E.G. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7417-7421Crossref PubMed Scopus (43) Google Scholar). Based on the use of different inhibitors, the present work demonstrates that activation of MAPK by α2B-AR is, in LLC-PK1-α2B, primarily mediated by a pathway involving stimulation of PLA2, generation of AA derivatives, activation of matrix metalloproteinases, release of HB-EGF, and transactivation of EGF-R. DISCUSSIONThe phosphorylation of MAPKs (ERK1/2) by α2-agonists has been reported in a variety of cells, including Chinese hamster ovary cells transfected with the RNG gene (3Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and COS or HEK 293 cells transfected with the α2C2 gene (7Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), which encode the rat and human α2B-AR subtypes, respectively. More recently, this effect has also been observed in rat proximal tubule cells in primary culture as well as in LLC-PK1-α2B (13Cussac D. Schaak S. Gales C. Flordellis C. Denis C. Paris H. Am. J. Physiol. Renal Physiol. 2002; 282: F943-F952Crossref PubMed Scopus (27) Google Scholar). In these two models, receptor stimulation resulted in an acceleration of cell proliferation, suggesting that the action of catecholamines on α2B-AR may play, in rat, a role in the adaptive response to acute renal tissue injury. Additionally, α2B-AR is known to enhance Na+ reabsorption as a consequence of increased activity of the Na+/H+ exchanger, NHE3 (20Nord E.P. Howard M.J. Hafezi A. Moradeshagi P. Vaystub S. Insel P.A. J. Clin. Invest. 1987; 80: 1755-1762Crossref PubMed Scopus (105) Google Scholar). Since NHE3 was recently found to be controlled by MAPK in mouse proximal tubule cells (21Liu F. Gesek F.A. Am. J. Physiol. 2001; 280: F415-F425Crossref PubMed Google Scholar), it is possible that MAPK activation is also involved in the regulation of NHE3 by α2B-AR.The mechanisms whereby G-protein-coupled receptors activate the MAPK cascade are highly dependent upon the receptor considered and the cell type it is expressed in. Although previous studies have shown that the action of the α2B-AR is independent of receptor internalization (7Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), the signaling pathway(s) accounting for the phosphorylation of MAPK by this receptor subtype remains poorly defined. The results obtained in this study provide substantial evidence that, in LLC-PK1-α2B, the activation of ERK by α2-agonists is triggered via a mechanism comprising the activation of matrix metalloproteinases, the release of HB-EGF, and the subsequent activation of the EGF-R. This cascade was demonstrated by the following observations. First, UK14304-induced phosphorylation of MAPK is totally abrogated in the presence of the matrix metalloproteinase inhibitors (batimastat or 1,10-phenanthroline) and by prior treatment of the cells with CRM 197. Second, conditioned medium from LLC-PK1-α2B cells treated with UK14304 causes activation of MAPK in wild-type LLC-PK1, even in the presence of batimastat. Third, the consequences of LLC-PK1-α2B exposure to α2-agonists or of wild-type LLC-PK1 exposure to conditioned medium are abolished by prior treatment of the cells with the inhibitor of EGF-R tyrosine kinase activity, tyrphostin AG1478. Previous studies of lysophosphatidic acid receptor or α2A-AR have demonstrated that the contribution of the EGF-R transactivation pathway is largely dependent on the cell type (10Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 22Della Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). In HEK 293, the major pathway of MAPK activation by α2A-AR is via the activation of Pyk2, a calcium-dependent tyrosine kinase of the focal adhesion kinase family (10Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). On the other hand, the effects of α2A-AR in COS cells are mediated by both EGF-R transactivation and direct recruitment of the MEK cascade by Src (9Pierce K.L. Luttrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (360) Google Scholar,23Pierce K.L. Maudsley S. Daaka Y. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1489-1494Crossref PubMed Scopus (196) Google Scholar). According to our results, the transactivation pathway is predominant in LLC-PK1-α2B; whether it is exclusive awaits definitive demonstration.An other major effort of the present work was to define the pathway leading from stimulated α2B-AR to transactivation of EGF-R. Because α2-agonists activate AA release in LLC-PK1-α2B and because AA is responsible for MAPK phosphorylation following angiotensin II treatment in rabbit proximal tubule cells (16Dulin N.O. Alexander L.D. Harwalkar S. Falck J.R. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8098-8102Crossref PubMed Scopus (77) Google Scholar), we sought to evaluate its implication. Consistent with a crucial role of the lipid second messenger, exposure of LLC-PK1-α2B to exogenous AA resulted in tyrosine phosphorylation of p52 Shc and in activation of MAPK with time courses that show a striking parallel to those observed with α2-agonists. Like for α2-agonists, the action of AA was prevented by batimastat, 1,10-phenanthroline, CRM 197, or tyrphostin AG1478. However, a major divergence was that, unlike the effects of α2-agonists, those of AA are resistant to pretreatment with pertussis toxin or to the addition of quinacrine. Additional support for implication of AA in the mediation of α2B-AR signal came from the study of the effects of inhibitors of AA metabolism. According to these experiments, phosphorylation of MAPK by UK14304 was strongly inhibited by NDGA and indomethacin. Ketoconazole was by contrast ineffective, indicating that AA products generated by lipoxygenase and/or cyclooxygenase, but not epoxygenase, are involved in α2-agonist effect. The observation that AA causes MAPK phosphorylation in our model is in opposition with results obtained on LLC-PK1/C14 (24Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. J. Biol. Chem. 1999; 274: 4764-4769Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In this clone, ERK phosphorylation was observed in response to epoxyeicosatrienoic acids but not to AA. Moreover, AA became efficient after cell transfection with an active form of cytochrome P450 epoxygenase of bacterial origin, indicating eicosanoid-dependent activation of MAPK. The reason for these discrepancies is enigmatic. However, activation of MAPK by AA was repeatedly reported in rabbit proximal tubules as well as in various cell types, including vascular smooth muscle cells and neutrophils. In vascular smooth muscle cells, efficacy of AA was dependent on its conversion into 15-hydroxyeicosatetraenoic acid and on PKC activation (25Rao G.N. Baas A.S. Glasgow W.C. Eling T.E. Runge M.S. Alexander R.W. J. Biol. Chem. 1994; 269: 32586-32591Abstract Full Text PDF PubMed Google Scholar). Dependence on lipoxygenase and PKC activity was also found in human neutrophils (26Hii C.S. Huang Z.H. Bilney A. Costabile M. Murray A.W. Rathjen D.A. Der C.J. Ferrante A. J. Biol. Chem. 1998; 273: 19277-19282Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In this cell type, the effects of AA engaged a membrane receptor linked to Gi/o proteins (27Capodici C. Pillinger M.H. Han G. Philips M.R. Weissmann G. J. Clin. Invest. 1998; 102: 165-175Crossref PubMed Scopus (62) Google Scholar). This is not the case in LLC-PK1-α2B, since neither staurosporine nor pertussis toxin treatment abolished ERK phosphorylation caused by AA. Regarding these points, LLC-PK1-α2B resembles rabbit renal epithelial cells. However, it is epoxy derivatives that mediate the effects of AA on MAPK phosphorylation in these cells (16Dulin N.O. Alexander L.D. Harwalkar S. Falck J.R. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8098-8102Crossref PubMed Scopus (77) Google Scholar). Whereas involvement of the cytochrome P450 pathway can be excluded in our model, the respective contribution of COX and LOX is still unclear, since it is difficult to reconcile why products from either pathway could function similarly. The implication of COX activity is beyond doubt, because the effects of α2-agonists and AA were also blocked by aspirin (not shown). By contrast, that of LOX is more questionable, since NDGA can also interfere with COX activity and act as an antioxidant. Alternatively, the possibility that prostaglandins and leukotrienes act in concert cannot be definitively ruled out. Indeed, the combined action of COX and LOX was already demonstrated to be necessary for some of the effects of angiotensin II in rat kidney and bovine bronchi (28Oyekan A. Balazy M. McGiff J.C. Am. J. Physiol. 1997; 273: R293-R300PubMed Google Scholar, 29Nally J.E. Bunton D.C. Martin D. Thomson N.C. Pulm. Pharmacol. 1996; 9: 211-217Crossref PubMed Scopus (5) Google Scholar). It is therefore clear that the identification of the AA metabolites responsible for MAPK activation in LLC-PK1-α2B will require future study. In addition, the mechanism whereby these products may affect matrix metalloproteinase activity has yet to be defined. In line with the existence of a relationship between the two phenomena, constitutive expression of cyclooxygenase-2 in human colon cancer cells results in increased activation of MMP-2 (30Tsujii M. Kawano S. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3336-3340Crossref PubMed Scopus (1325) Google Scholar), whereas inhibitors of PLA2 and cyclooxygenase-2 reduce the release of matrix metalloproteinases in prostate tumor cells (31Attiga F.A. Fernandez P.M. Weeraratna A.T. Manyak M.J. Patierno S.R. Cancer Res. 2000; 60: 4629-4637PubMed Google Scholar).In conclusion, our results provide evidence for a pathway by which α2B-AR activates MAPK through stimulation of PLA2, generation of AA metabolites by cyclooxygenase and/or lipoxygenase, stimulation of matrix metalloproteinases, release of HB-EGF, and transactivation of the EGF-R (Fig. 9). Whether this scenario is particular to α2B-AR in LLC-PK1 or whether it can be extended to other cell types and/or other G-protein-coupled receptors remains to be established. The phosphorylation of MAPKs (ERK1/2) by α2-agonists has been reported in a variety of cells, including Chinese hamster ovary cells transfected with the RNG gene (3Flordellis C.S. Berguerand M. Gouache P. Barbu V. Gavras H. Handy D.E. Bereziat G. Masliah J. J. Biol. Chem. 1995; 270: 3491-3494Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and COS or HEK 293 cells transfected with the α2C2 gene (7Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), which encode the rat and human α2B-AR subtypes, respectively. More recently, this effect has also been observed in rat proximal tubule cells in primary culture as well as in LLC-PK1-α2B (13Cussac D. Schaak S. Gales C. Flordellis C. Denis C. Paris H. Am. J. Physiol. Renal Physiol. 2002; 282: F943-F952Crossref PubMed Scopus (27) Google Scholar). In these two models, receptor stimulation resulted in an acceleration of cell proliferation, suggesting that the action of catecholamines on α2B-AR may play, in rat, a role in the adaptive response to acute renal tissue injury. Additionally, α2B-AR is known to enhance Na+ reabsorption as a consequence of increased activity of the Na+/H+ exchanger, NHE3 (20Nord E.P. Howard M.J. Hafezi A. Moradeshagi P. Vaystub S. Insel P.A. J. Clin. Invest. 1987; 80: 1755-1762Crossref PubMed Scopus (105) Google Scholar). Since NHE3 was recently found to be controlled by MAPK in mouse proximal tubule cells (21Liu F. Gesek F.A. Am. J. Physiol. 2001; 280: F415-F425Crossref PubMed Google Scholar), it is possible that MAPK activation is also involved in the regulation of NHE3 by α2B-AR. The mechanisms whereby G-protein-coupled receptors activate the MAPK cascade are highly dependent upon the receptor considered and the cell type it is expressed in. Although previous studies have shown that the action of the α2B-AR is independent of receptor internalization (7Schramm N.L. Limbird L.E. J. Biol. Chem. 1999; 274: 24935-24940Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 8DeGraff J.L. Gagnon A.W. Benovic J.L. Orsini M.J. J. Biol. Chem. 1999; 274: 11253-11259Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), the signaling pathway(s) accounting for the phosphorylation of MAPK by this receptor subtype remains poorly defined. The results obtained in this study provide substantial evidence that, in LLC-PK1-α2B, the activation of ERK by α2-agonists is triggered via a mechanism comprising the activation of matrix metalloproteinases, the release of HB-EGF, and the subsequent activation of the EGF-R. This cascade was demonstrated by the following observations. First, UK14304-induced phosphorylation of MAPK is totally abrogated in the presence of the matrix metalloproteinase inhibitors (batimastat or 1,10-phenanthroline) and by prior treatment of the cells with CRM 197. Second, conditioned medium from LLC-PK1-α2B cells treated with UK14304 causes activation of MAPK in wild-type LLC-PK1, even in the presence of batimastat. Third, the consequences of LLC-PK1-α2B exposure to α2-agonists or of wild-type LLC-PK1 exposure to conditioned medium are abolished by prior treatment of the cells with the inhibitor of EGF-R tyrosine kinase activity, tyrphostin AG1478. Previous studies of lysophosphatidic acid receptor or α2A-AR have demonstrated that the contribution of the EGF-R transactivation pathway is largely dependent on the cell type (10Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 22Della Rocca G.J. Maudsley S. Daaka Y. Lefkowitz R.J. Luttrell L.M. J. Biol. Chem. 1999; 274: 13978-13984Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). In HEK 293, the major pathway of MAPK activation by α2A-AR is via the activation of Pyk2, a calcium-dependent tyrosine kinase of the focal adhesion kinase family (10Della Rocca G.J. van Biesen T. Daaka Y. Luttrell D.K. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1997; 272: 19125-19132Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). On the other hand, the effects of α2A-AR in COS cells are mediated by both EGF-R transactivation and direct recruitment of the MEK cascade by Src (9Pierce K.L. Luttrell L.M. Lefkowitz R.J. Oncogene. 2001; 20: 1532-1539Crossref PubMed Scopus (360) Google Scholar,23Pierce K.L. Maudsley S. Daaka Y. Luttrell L.M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1489-1494Crossref PubMed Scopus (196) Google Scholar). According to our results, the transactivation pathway is predominant in LLC-PK1-α2B; whether it is exclusive awaits definitive demonstration. An other major effort of the present work was to define the pathway leading from stimulated α2B-AR to transactivation of EGF-R. Because α2-agonists activate AA release in LLC-PK1-α2B and because AA is responsible for MAPK phosphorylation following angiotensin II treatment in rabbit proximal tubule cells (16Dulin N.O. Alexander L.D. Harwalkar S. Falck J.R. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8098-8102Crossref PubMed Scopus (77) Google Scholar), we sought to evaluate its implication. Consistent with a crucial role of the lipid second messenger, exposure of LLC-PK1-α2B to exogenous AA resulted in tyrosine phosphorylation of p52 Shc and in activation of MAPK with time courses that show a striking parallel to those observed with α2-agonists. Like for α2-agonists, the action of AA was prevented by batimastat, 1,10-phenanthroline, CRM 197, or tyrphostin AG1478. However, a major divergence was that, unlike the effects of α2-agonists, those of AA are resistant to pretreatment with pertussis toxin or to the addition of quinacrine. Additional support for implication of AA in the mediation of α2B-AR signal came from the study of the effects of inhibitors of AA metabolism. According to these experiments, phosphorylation of MAPK by UK14304 was strongly inhibited by NDGA and indomethacin. Ketoconazole was by contrast ineffective, indicating that AA products generated by lipoxygenase and/or cyclooxygenase, but not epoxygenase, are involved in α2-agonist effect. The observation that AA causes MAPK phosphorylation in our model is in opposition with results obtained on LLC-PK1/C14 (24Chen J.K. Wang D.W. Falck J.R. Capdevila J. Harris R.C. J. Biol. Chem. 1999; 274: 4764-4769Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). In this clone, ERK phosphorylation was observed in response to epoxyeicosatrienoic acids but not to AA. Moreover, AA became efficient after cell transfection with an active form of cytochrome P450 epoxygenase of bacterial origin, indicating eicosanoid-dependent activation of MAPK. The reason for these discrepancies is enigmatic. However, activation of MAPK by AA was repeatedly reported in rabbit proximal tubules as well as in various cell types, including vascular smooth muscle cells and neutrophils. In vascular smooth muscle cells, efficacy of AA was dependent on its conversion into 15-hydroxyeicosatetraenoic acid and on PKC activation (25Rao G.N. Baas A.S. Glasgow W.C. Eling T.E. Runge M.S. Alexander R.W. J. Biol. Chem. 1994; 269: 32586-32591Abstract Full Text PDF PubMed Google Scholar). Dependence on lipoxygenase and PKC activity was also found in human neutrophils (26Hii C.S. Huang Z.H. Bilney A. Costabile M. Murray A.W. Rathjen D.A. Der C.J. Ferrante A. J. Biol. Chem. 1998; 273: 19277-19282Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In this cell type, the effects of AA engaged a membrane receptor linked to Gi/o proteins (27Capodici C. Pillinger M.H. Han G. Philips M.R. Weissmann G. J. Clin. Invest. 1998; 102: 165-175Crossref PubMed Scopus (62) Google Scholar). This is not the case in LLC-PK1-α2B, since neither staurosporine nor pertussis toxin treatment abolished ERK phosphorylation caused by AA. Regarding these points, LLC-PK1-α2B resembles rabbit renal epithelial cells. However, it is epoxy derivatives that mediate the effects of AA on MAPK phosphorylation in these cells (16Dulin N.O. Alexander L.D. Harwalkar S. Falck J.R. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8098-8102Crossref PubMed Scopus (77) Google Scholar). Whereas involvement of the cytochrome P450 pathway can be excluded in our model, the respective contribution of COX and LOX is still unclear, since it is difficult to reconcile why products from either pathway could function similarly. The implication of COX activity is beyond doubt, because the effects of α2-agonists and AA were also blocked by aspirin (not shown). By contrast, that of LOX is more questionable, since NDGA can also interfere with COX activity and act as an antioxidant. Alternatively, the possibility that prostaglandins and leukotrienes act in concert cannot be definitively ruled out. Indeed, the combined action of COX and LOX was already demonstrated to be necessary for some of the effects of angiotensin II in rat kidney and bovine bronchi (28Oyekan A. Balazy M. McGiff J.C. Am. J. Physiol. 1997; 273: R293-R300PubMed Google Scholar, 29Nally J.E. Bunton D.C. Martin D. Thomson N.C. Pulm. Pharmacol. 1996; 9: 211-217Crossref PubMed Scopus (5) Google Scholar). It is therefore clear that the identification of the AA metabolites responsible for MAPK activation in LLC-PK1-α2B will require future study. In addition, the mechanism whereby these products may affect matrix metalloproteinase activity has yet to be defined. In line with the existence of a relationship between the two phenomena, constitutive expression of cyclooxygenase-2 in human colon cancer cells results in increased activation of MMP-2 (30Tsujii M. Kawano S. DuBois R.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3336-3340Crossref PubMed Scopus (1325) Google Scholar), whereas inhibitors of PLA2 and cyclooxygenase-2 reduce the release of matrix metalloproteinases in prostate tumor cells (31Attiga F.A. Fernandez P.M. Weeraratna A.T. Manyak M.J. Patierno S.R. Cancer Res. 2000; 60: 4629-4637PubMed Google Scholar). In conclusion, our results provide evidence for a pathway by which α2B-AR activates MAPK through stimulation of PLA2, generation of AA metabolites by cyclooxygenase and/or lipoxygenase, stimulation of matrix metalloproteinases, release of HB-EGF, and transactivation of the EGF-R (Fig. 9). Whether this scenario is particular to α2B-AR in LLC-PK1 or whether it can be extended to other cell types and/or other G-protein-coupled receptors remains to be established. We thank Dr. C. Flordellis for valuable discussion and F. Quinchon for excellent technical assistance.