Thrombin-stimulated proliferation of cultured human synovial fibroblasts through proteolytic activation of proteinase-activated receptor-1.
We examined the mechanism of thrombin on proliferation of synovial fibroblasts obtained from rheumatoid arthritis (RA). Thrombin concentration-dependently induced proliferation of synovial fibroblasts. Proliferation in response to thrombin (10 U/ml) was completely blocked by hirudin. TP367 and TP508, peptides corresponding to 2 noncatalytic regions of thrombin, failed to induce cell proliferation. Thrombin did not induce the production of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and epidermal growth factor (EGF) in synovial fibroblasts. Expression of proteinase-activated receptor (PAR)-1 and PAR-3 mRNAs was observed in synovial fibroblasts. Thrombin and PAR-1 agonist peptide (AP), but not PAR-3 AP, induced intracellular calcium mobilization. PAR-1 AP induced cell proliferation whereas PAR-3 AP and PAR-4 AP had no effect on proliferation. Pertussis toxin (PTX), a Gialpha protein inhibitor; wortmannin, a PI (phosphatidylinositol) 3-kinase inhibitor; and PD98059, a specific MEK [mitogen-activated protein (MAK) kinase kinase] inhibitor, inhibited the thrombin-induced cell proliferation. Furthermore, the proliferation of synovial fibroblasts was suppressed by U-73122, a PLC (phospholipase C) inhibitor; 2-APB, an antagonist of InsP3 (inositol 1,4,5-triphosphate) receptor; and GF-109203X, a PKC (protein kinase C) inhibitor. These results suggest that thrombin induces the proliferation of RA synovial fibroblasts through the activation of PAR-1, leading to the PTX-sensitive G proteins - PI3 kinase pathway and PTX-insensitive G proteins - PLC (InsP3 receptor) Ca(2+)-PKC branch.
Furuhashi
Ikue
I
Pharmacological Research Department, Research Laboratory, Minophagen Pharmaceutical Co. Ltd., 2-2-3 Komatsubara, Zama, Kanagawa, Japan.
Abe
Kazuki
K
Sato
Toshitsugu
T
Inoue
Hideo
H
D002118:Calcium / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000187:drug effects*; D006490:Hemostatics / Q000494:pharmacology*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000096:biosynthesis / Q000502:physiology*; D018179:Receptors, Thrombin / Q000096:biosynthesis / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D013268:Stimulation, Chemical; D013582:Synovial Fluid / Q000166:cytology*; D013917:Thrombin / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D006490:Hemostatics; D036341:Intercellular Signaling Peptides and Proteins; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C105295:protease-activated receptor 3; D013917:Thrombin; C112908:protease-activated receptor 4; D002118:Calcium