Protease nexin-1: a cellular serpin down-regulated by thrombin in rat aortic smooth muscle cells.
Protease nexin-1 (PN-1), a potent inhibitor of serine proteases, is present in vascular cells and forms complexes with thrombin, plasminogen activators, and plasmin. We examined the effect of thrombin on PN-1 expression by rat aortic smooth muscle cells (RASMCs). PN-1 expression was determined by measuring protein and mRNA levels, using respectively immunoblotting and semi-quantitative reverse transcriptase polymerase chain reaction (PCR). Thrombin down-regulated PN-1 expression in a dose- and time-dependent manner. This effect was mediated via the interaction of thrombin with its receptor protease activated receptor (PAR-1) since the peptide thrombin receptor activating peptide (TRAP) reduced PN-1 expression. PN-1 secreted by smooth muscle cells remained essentially associated to cell-surface glycosaminoglycans and was released from the cell surface by heparin. A lower amount of PN-1 was released by heparin from TRAP-stimulated versus unstimulated cells and correlated with a decreased capacity to inhibit thrombin. In addition, the ability to generate peri-cellular plasmin was increased in cells with a low PN-1 expression. Pre-treatment of smooth muscle cells with cycloheximide abolished the reduction of PN-1 expression by thrombin. Furthermore, conditioned media from thrombin-treated cells reproduced the effect of thrombin, suggesting that thrombin acted via the induction of auto/paracrine mediator(s). We observed that fibroblast growth factor-2 (FGF-2)-neutralizing antibodies abolished thrombin effect whereas FGF-2 reproduced it, indicating that FGF-2 is one of the involved mediator. Together, these results indicate that (i) PN-1 modulates the activity of endogenous and exogenous serine proteases in RASMCs, (ii) thrombin down-regulates PN-1 expression and thus may increase its own activity on cells.
Richard
Benjamin
B
Equipe INSERM E0348, Faculté Xavier Bichat, Paris Cedex, France.
Arocas
Véronique
V
Guillin
Marie-Claude
MC
Michel
Jean-Baptiste
JB
Jandrot-Perrus
Martine
M
Bouton
Marie-Christine
MC
D016564:Amyloid beta-Protein Precursor; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001011:Aorta / Q000166:cytology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation; D016222:Fibroblast Growth Factor 2 / Q000276:immunology / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006490:Hemostatics / Q000494:pharmacology*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000502:physiology*; D058255:Protease Nexins; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D011956:Receptors, Cell Surface; D013917:Thrombin / Q000494:pharmacology*
D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D006490:Hemostatics; D058255:Protease Nexins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D016222:Fibroblast Growth Factor 2; D003513:Cycloheximide; D013917:Thrombin