Signaling mechanism of thrombin-induced gingival fibroblast-populated collagen gel contraction.

Laboratory of Dental Pharmacology and Toxicology, Department of Dentistry, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan.
British Journal of Pharmacology (Impact Factor: 4.99). 02/2006; 147(2):188-98. DOI: 10.1038/sj.bjp.0706462
Source: PubMed

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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Background/purpose Various toxic products are generated by periodontal pathogens. Porphyromonas gingivalis has been found to generate lipopolysaccharide (LPS) that may potentially affect periodontal health. However, the precise effects of P. gingivalis LPS on human gingival fibroblasts (GFs) await further investigation. Materials and methods Human GFs were cultured and exposed to different concentrations of P. gingivalis LPS (0.1–10 μg/mL) for 24 hours. Cytotoxicity was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Total ribonucleic acid (RNA) was isolated and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primer sets. Culture medium was collected for determination of prostaglandin E2 (PGE2), PGF2α, and interleukin-8 (IL-8) production by enzyme-linked immunosorbent assay. ERK1/2 phosphorylation in GFs was evaluated by Western blotting. In some experiments, U0126 (a MEK/ERK inhibitor) was added to the GFs culture 30 minutes before LPS and culture medium was also collected for analysis. Results P. gingivalis LPS (0.1–10 μg/mL) showed little cytotoxicity and morphologic changes of GFs. P. gingivalis LPS obviously stimulated the cyclooxygenase-2 (COX-2) and IL-8 messenger RNA expression of GFs after 24 hours of exposure. Moreover, P. gingivalis LPS induced PGE2, PGF2α, and IL-8 production in GFs. P. gingivalis LPS also induced ERK1/2 phosphorylation in GFs. Stimulation of PGE2 production by P. gingivalis LPS was completely attenuated by U0126, whereas U0126 only partially inhibited the LPS-induced IL-8 production in the same condition. Conclusion Our data indicate that P. gingivalis LPS stimulates gene expression of differential inflammatory mediators (COX-2 and IL-8) as well as prostanoids and IL-8 production in GFs. These events are associated with MEK/ERK signaling and crucial in the pathogenesis of inflammatory periodontal diseases.
    Journal of dental sciences 03/2014; 9(1):78–84. · 0.47 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Understanding the mechanisms that regulate mechanosensitivity in osteoblasts is important for controlling bone homeostasis and the development of new drugs to combat bone loss. It is believed that prestress or force generation (the tensile stress within the cell body) plays an important role in regulating cellular mechanosensitivity. In the present study, a three-dimensional (3D) collagen culture was used to monitor the change in prestress of the osteoblast-like cells. Collagen hydrogel compaction has been used as an indicator of the change in the degree of cell prestress. Previous results in this model demonstrated that extracellular ATP reduced the mechanosensitivity of osteoblasts by reducing cellular prestress. To elucidate the potential mechanisms involved in this process, the signaling pathways downstream of P2 purinoceptors involved in regulating the compaction of type I collagen gels were investigated. By using specific inhibitors to these signaling pathways, we found that ATP-induced reduction in collagen gel compaction rate is dependent on mitogen-activated protein kinase (MAKP) and NF-kappaB pathways. However, blocking protein kinase C with GF109203X did not change the compaction kinetics in the presence of ATPgammaS. Moreover, blocking cyclic AMP (cAMP), phosphatidylinositol-3 kinase (PI3K), calmodulin (CaM) or L-type voltage sensitive calcium channels did not affect ATP's ability to reduce collagen gel compaction. The results from the present and previous studies indicate that extracellular ATP may act as a negative feedback modulator in the mechanotransduction system since mechanical stimuli increase ATP release from stimulated cells.
    Experimental Cell Research 07/2009; 315(11):1990-2000. · 3.37 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Gingival enlargement is a fibrotic condition that can arise from systemic administration of the dihydropyridine calcium channel blocker nifedipine. Periostin, a transforming growth factor-beta (TGF-β)-inducible matricellular protein, has been associated with fibrosis in numerous tissues, but its expression has never been examined in nifedipine-influenced gingival enlargement (NIGE). The objective of this study was to assess if periostin up-regulation is associated with NIGE and whether nifedipine induces periostin expression in gingival fibroblasts. In NIGE tissue (n = 6), periostin is overexpressed in the gingival connective tissue compared with healthy control tissue (n = 6). The transcription factor p-SMAD2/3, which is associated with canonical TGF-β signaling, localizes to the nuclei in both HGFs and oral epithelial cells in NIGE tissues, but not in control healthy tissue. In vitro culture of HGFs with 30 and 100 ng/mL of nifedipine significantly increased periostin mRNA and protein levels, which correlated with increased levels of active TGF-β and increased phosphorylation and nuclear localization of SMAD3. Blocking of canonical TGF-β signaling through inhibition of the TGF-β receptor I with SB431542 significantly reduced nifedipine-induced SMAD3 phosphorylation and periostin expression. Our results demonstrate that nifedipine up-regulates periostin in HGFs in a TGF-β-dependent manner.
    Journal of dental research 09/2013; · 4.14 Impact Factor

Full-text (2 Sources)

Available from
May 31, 2014