T.denticola protease mediates MMP-2-dependent fibronectin degradation in PDL cells

Objectives: Periodontal disease is a bacterially-mediated chronic inflammatory disease that results in destruction of the periodontal ligament (PDL) and alveolar bone that surround and support the dentition. Periodontal pathogens, including Treponema denticola, are believed to initiate the destructive inflammatory responses and dysregulation of tissue homeostasis that characterize the disease. These responses are believed to result both from proinflammatory effects of acylated bacterial membrane components (lipopolysaccharides and lipoproteins) and degradative effects of secreted bacterial proteases. Host-derived matrix metalloproteases (MMPs) are key enzymes in both tissue homeostasis and tissue destruction. MMP expression is modulated in part by specific proteolytic fragments of fibronectin (FN), which are associated with periodontal disease. In addition, FN fragments induce apoptosis and suppress osteoblast differentiation of PDL cells. The purpose of this study is to investigate the ability of T. denticola and its acylated serine outer membrane protease complex, PrtP (dentilisin/CTLP), to induce both activation of MMP-2 and generation of FN fragments in cultured human PDL cells. Methods: PDL cells were challenged with T. denticola parent and isogenic mutant strains for 2 hours, washed, then replenished with fresh medium. Protein expression, MMP activity, and FN fragmentation were measured by Western immunoblotting and gelatin zymography in 24 h post challenged cell culture supernatants. The activation of MMP-2 mediated by T. denticola and its purified PrtP protease complex were both evaluated. Also the role of MMP-2 in mediating FN fragmentation was examined using MMP-2 siRNA and specific chemical inhibitors of MMP-2 Results: T. denticola and its purified protease induced activation of MMP-2, and active MMP-2 is required for FN fragmentation. Conclusions: These results delineate a potential new mechanism by which the T. denticola protease may disrupt homeostatic processes required for maintenance of periodontal health. (Supported by R01 DE013725 to YLK, R01 DE018221 to JCF, China Scholarship Council to DM).