Signaling mechanism of thrombin‐induced gingival fibroblast‐populated collagen gel contraction

Abstract

1 Thrombin is activated during gingival tissue injury and inflammation. Thrombin (platelet)-rich plasma has been used for periodontal regeneration with success. Thrombin and other bacterial proteases also affect the functions of adjacent periodontal cells via stimulation of protease-activated receptors (PARs). 2 We noted that thrombin (0.1-2 U ml À1 ), human, and frog PAR-1 agonist peptide (20-240 mM) induced the gingival fibroblast (GF)-populated collagen gel contraction within 2 h of exposure. However, PAR-2, PAR-3, and PAR-4 agonist peptide (20-240 mM) showed little effect on collagen gel contraction. U73122 (phospholipase C inhibitor) and 2-APB (IP3 antagonist) were effective in inhibition of GF contraction. 3 Thrombin-induced GF contraction was inhibited by 5 mM EGTA (an extracellular calcium chelator) and verapamil (an L-type calcium channel blocker). In addition, W7 (10 and 25 mM, a calcium/calmodulin (CaM) inhibitor), ML-7 (50 mM, myosin light chain kinase (MLCK) inhibitor), and HA1077 (100 mM, Rho kinase inhibitor) completely inhibited the thrombin-induced collagen gel contraction. Thrombin also induced the phosphorylation of ERK1/ERK2 and elevated the Rho-GTP levels in GF. 4 However, U0126 only partially inhibited the thrombin-induced GF contraction. Similarly, wortmannin (100 nM), LY294002 (20 mM) (two PI3K inhibitor) and genistein also showed partial inhibition. Moreover, NAC was not able to suppress the GF contraction, as supported by the slight decrease in reactive oxygen species production in GF by thrombin. 5 Thrombin also stimulated metalloproteinase-2 (MMP-2) and MMP-3 production in GF. But addition of GM6001 or 1,10-phenanthroline, two MMP inhibitors, could not inhibit the thrombininduced GF contraction. 6 These results indicate that thrombin is crucial in the periodontal inflammation and wound healing by promoting GF contraction. This event is mainly mediated via PAR-1 activation, PLC activation, extracellular calcium influx via L-type calcium channel, and the calcium/CaM-MLCK and Rho kinase activation pathway.

The role of reactive oxygen species and hemeoxygenase-1 expression in the cytotoxicity, cell cycle alteration and apoptosis of dental pulp cells induced by BisGMA

“…Semi-quantitative RT-PCR e PCR was conducted by using specific primers for beta-actin (BAC), cdc2, cdc25C, cyclinB1 as described previously [27]. Briefly 3 mg of denatured total RNA was reverse transcribed in a total volume of 44.5 ml reaction mixture containing 4 ml of random primer (500 mg/ml), 8 ml of dNTP (2.5 mM), 4.5 ml of 10Â RT buffer, 1 ml of RNase inhibitor (40 U/ml) and 0.5 ml of RT (21 U/ml) at 42 C for 90 min.…”

Thrombin induces osteoprotegerin synthesis via phosphatidylinositol 3′‐kinase/mammalian target of rapamycin pathway in human periodontal ligament cells

“…Thrombin has been shown to stimulate proliferation and protein synthesis in human periodontal ligament (HPDL) cells (15). In addition, it could promote collagen gel contraction in gingival fibroblasts (16). Although there are few reports regarding the role of thrombin in periodontal tissue, the data suggest the possibility that thrombin may play a role in repair and healing of periodontal tissue.…”

A novel collagen gel-based measurement technique for quantitation of cell contraction force

“…Conventional techniques used to measure collagen gel contraction rely on imaging only the gel area from a top view. The estimation of contraction is often expressed by the area changes of the gels between the beginning and end of the treatment or cell contraction period [9,10]. To improve the measurement of cell contraction kinetics for time-dependent changes, timelapse-video-based imaging techniques have been developed to record changes in gel areas continuously while the cells contract [11].…”