jun-N-terminal kinase regulates thrombin-induced PAI-1 gene expression in proximal tubular epithelial cells

Abstract

Background. Interstitial activation of the coagulation cascade is a common finding in acute and chronic tubulointerstitial damage. We previously demonstrated that thrombin may induce proximal tubular epithelial cells (PTEC) proliferation and regulate, through plasminogen activator inhibitor (PAI)-1 and urokinase-type plasminogen activator (u-PA), their profibrotic activity. The signaling pathways leading to these effects are still unknown. The PAI-1 promoter contains several activator protein-1 (AP-1) consensus sequences. AP-1 activation is induced by different agonists through jun-N-terminal kinase (JNK). Thus, we investigated the role of the JNK-AP-1 axis on thrombin-induced PAI-1 and u-PA expression in immortalized PTEC and its modulation by PKC and src, two key signaling enzymes. Methods. JNK and src activation was investigated by Western blotting, PAR-1 cellular surface expression by flow cytometry, PAI-1 and u-PA gene expression by Northern blotting, AP-1 activation by transient transfection, and DNA synthesis by 3H-thymidine uptake. Results. Thrombin and PMA induced a time-dependent increase of JNK phosphorylation in immortalized PTEC that was inhibited by PKC down-regulation. Both thrombin and PMA caused AP-1 activation, significantly reduced by src inhibition. Phorbol 12-myristate 13-acetate (PMA), indeed, induced an increase in src phosphorylation. Both PMA- and thrombin-stimulated PAI-1 gene expression was abolished by JNK, protein kinase C (PKC), and src inhibition, and this effect was regulated at the trascriptional level. PKC, but not src or JNK inhibition, abolished thrombin-elicited u-PA expression. Finally, JNK inhibition did not influence thrombin-induced proliferation. Conclusion. Our data suggest that thrombin activates the JNK-AP-1 axis in a PKC- and src-dependent manner in PTEC. This axis, necessary for thrombin-stimulated PAI-1 expression, but not for its fibrinolytic and regenerative effect, may represent a therapeutic target in acute and chronic tubulointerstitial damage.