It has been reported that matrix metalloproteinase (MMP) produced by host cells plays a major role in periodontal tissue destruction. In addition, secreted virulence factors from Porphyromonas gingivalis can alter MMP secretion and cause activation in host cells that lead to the tissue degradation. In this study, we examine the effects of P. gingivalis supernatant on matrix metalloproteinase-2 (MMP-2) activation in human periodontal ligament (HPDL) cells. Cultures of HPDL cells were treated with P. gingivalis supernatant for 48 h and the level of MMP-2 activation was monitored by gelatin zymography. The profound activation of MMP-2 was seen only in the treated group. The activation of MMP-2 was inhibited by MMP inhibitors phenanthroline and EDTA, but not serine protease or cysteine protease inhibitors. To study the correlation between the expression of membrane-type-1 matrix metalloproteinase (MT1-MMP) and the activation of MMP-2, the level of MT1-MMP was analyzed. The results from reverse-transcription polymerase chain reaction (RT-PCR) and Western analysis indicated that P. gingivalis supernatant up-regulated the expression of MT1-MMP in both transcription and translation levels within 48 h. These results suggest that P. gingivalis supernatant can activate MMP-2 in HPDL cells and the mechanism of activation may involve the increased amount of MT1-MMP. It is possible that the activation of MMP-2 by P. gingivalis plays a role in the process of chronic periodontitis.

Our previous study showed that mechanical stress induced the expression of osteopontin (OPN) in human periodontal ligament (HPDL) cells through the Rho kinase pathway. The increase of OPN expression via Rho kinase has been demonstrated to be triggered by nucleotide. Therefore, we hypothesized that nucleotides, particularly adenosine triphosphate (ATP), participated in the stress-induced OPN expression in HPDL cells. In the present study, the roles of ATP and P2Y1 purinoceptor were examined. Reverse-transcription polymerase chain-reaction and Western blot analysis revealed that the stress-induced ATP exerted its stimulatory effect on OPN expression. The inductive effect was attenuated by apyrase and completely inhibited by the Rho kinase inhibitor, as well as by the P2Y1 antagonist. We here propose that stress induces release of ATP, which in turn mediates Rho kinase activation through the P2Y1 receptor, resulting in the up-regulation of OPN. Stress-induced ATP could play a significant role in alveolar bone resorption.

The effect of the LPS of A. actinomycetemcomitans on HPDL cells is serum-independent and the induction of the activation of MMP-2 and the expression of RANKL are serine protease-dependent pathways. The results suggest the role of HPDL cells in the pathogenesis of periodontitis.

Extracellular ATP stimulates RANKL expression in human periodontal ligament cells through a pathway dependent on the P2Y(1) receptor, cAMP-dependent protein kinase, NF kappaB and COX. Our results suggest that, among the molecules responsible for the effect of mechanical stress, ATP participates in bone resorption or bone homeostasis by mediating its signal through the P2Y(1) receptor and the NF kappaB-COX-RANKL axis in periodontal tissue.

Mechanical stress affects OPN expression in HPDL cells through the Rho kinase pathway. Because OPN participates in bone resorption and remodeling induced by mechanical and biologic signals, these results suggest the significance of stress-induced OPN in HPDL cells in alveolar bone resorption and remodeling.