jun-N-terminal kinase regulates thrombin-induced PAI-1 gene expression in proximal tubular epithelial cells.
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 (3)H-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.


CONCLUSIONS
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.
Pontrelli
Paola
P
Division of Nephrology, Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy.
Ranieri
Elena
E
Ursi
Michele
M
Ghosh-Choudhury
Goutham
G
Gesualdo
Loreto
L
Paolo Schena
Francesco
F
Grandaliano
Giuseppe
G
D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000378:metabolism*; D008954:Models, Biological; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics; D019061:src-Family Kinases / Q000378:metabolism
D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D004247:DNA; D019061:src-Family Kinases; D011493:Protein Kinase C; D048031:JNK Mitogen-Activated Protein Kinases; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate