Integrin alpha(v)beta(3), metalloproteinases, and sphingomyelinase-2 mediate urokinase mitogenic effect.
Plasminogen activators are implicated in the pathogenesis of several diseases such as inflammatory diseases and cancer. Beside their serine-protease activity, these agents trigger signaling pathways involved in cell migration, adhesion and proliferation. We previously reported a role for the sphingolipid pathway in the mitogenic effect of plasminogen activators, but the signaling mechanisms involved in neutral sphingomyelinase-2 (NSMase-2) activation (the first step of the sphingolipid pathway) are poorly known. This study was carried out to investigate how urokinase plasminogen activator (uPA) activates NSMase-2. We report that uPA, as well as its catalytically inactive N-amino fragment ATF, triggers the sequential activation of MMP-2, NSMase-2 and ERK1/2 in ECV304 cells that are required for uPA-induced ECV304 proliferation, as assessed by the inhibitory effect of Marimastat (a MMP inhibitor), MMP-2-specific siRNA, MMP-2 defect, and NSMase-specific siRNA. Moreover, upon uPA stimulation, uPAR, MT1-MMP, MMP-2 and NSMase-2 interacted with integrin alpha(v)beta(3), evidenced by co-immunoprecipitation and immunocytochemistry experiments. Moreover, the alpha(v)beta(3) blocking antibody inhibited the uPA-triggered MMPs/uPAR/integrin alpha(v)beta(3) interaction, NSMase-2 activation, Ki67 expression and DNA synthesis in ECV304. In conclusion, uPA triggers interaction between integrin alpha(v)beta(3), uPAR and MMPs that leads to NSMase-2 and ERK1/2 activation and cell proliferation. These findings highlight a new signaling mechanism for uPA, and suggest that, upon uPA stimulation, uPAR, MMPs, integrin alpha(v)beta(3) and NSMase-2 form a signaling complex that take part in mitogenic signaling in ECV304 cells.
Maupas-Schwalm
Françoise
F
Inserm U858 Team 10, Dept of Biochemistry and Molecular Biology, Faculty of Medicine-Rangueil, University Paul Sabatier Toulouse-3, IFR-150, Toulouse, France. francoise.schwalm@inserm.fr
Bedel
Aurélie
A
Augé
Nathalie
N
Grazide
Marie-Hélène
MH
Mucher
Elodie
E
Thiers
Jean-Claude
JC
Salvayre
Robert
R
Nègre-Salvayre
Anne
A
D045744:Cell Line, Tumor; D004247:DNA / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D008934:Mitogens / Q000378:metabolism*; D008938:Mitosis; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D039302:Integrin alphaVbeta3; D061965:Matrix Metalloproteinase Inhibitors; D008934:Mitogens; D055293:Receptors, Urokinase Plasminogen Activator; D004247:DNA; C100342:marimastat; C491484:SMPD3 protein, human; D013108:Sphingomyelin Phosphodiesterase; D014568:Urokinase-Type Plasminogen Activator; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14