title: 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(-1)), human, and frog PAR-1 agonist peptide (20-240 microM) 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 microM) 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 microM, a calcium/calmodulin (CaM) inhibitor), ML-7 (50 microM, myosin light chain kinase (MLCK) inhibitor), and HA1077 (100 microM, 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 microM) (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 thrombin-induced 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.
authors.affiliation: Laboratory of Dental Pharmacology and Toxicology, Department of Dentistry, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan.
authors.forename: Jiiang-Huei
authors.initials: JH
authors.lastname: Jeng
authors.forename: Wan-Hong
authors.initials: WH
authors.lastname: Lan
authors.forename: Juo-Song
authors.initials: JS
authors.lastname: Wang
authors.forename: Chiu-Po
authors.initials: CP
authors.lastname: Chan
authors.forename: Yuan-Soon
authors.initials: YS
authors.lastname: Ho
authors.forename: Po-Hsuen
authors.initials: PH
authors.lastname: Lee
authors.forename: Ying-Jen
authors.initials: YJ
authors.lastname: Wang
authors.forename: Tong-Mei
authors.initials: TM
authors.lastname: Wang
authors.forename: Yi-Jane
authors.initials: YJ
authors.lastname: Chen
authors.forename: Mei-Chi
authors.initials: MC
authors.lastname: Chang
mesh_terms: D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D048429:Cell Size; D002478:Cells, Cultured; D003094:Collagen / Q000502:physiology*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology*; D005782:Gels; D005881:Gingiva / Q000166:cytology*; D006801:Humans; D045726:Metalloproteases / Q000096:biosynthesis; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors; D010084:Oxidation-Reduction; D010455:Peptides / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D044462:Receptors, Proteinase-Activated / Q000819:agonists; D015398:Signal Transduction; D013917:Thrombin / Q000502:physiology*; D010738:Type C Phospholipases / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D005782:Gels; D010455:Peptides; D017382:Reactive Oxygen Species; D044462:Receptors, Proteinase-Activated; D003094:Collagen; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D048049:Extracellular Signal-Regulated MAP Kinases; D010738:Type C Phospholipases; D045726:Metalloproteases; D013917:Thrombin; D020741:rho GTP-Binding Proteins; D002118:Calcium