UBC Theses and Dissertations
The role of granzyme B in the degradation of periodontal connective tissues Ahmadi, Amir Reza
Objective: Periodontitis (gum disease) is characterized by the degradation of tooth- supporting connective tissues, largely due to matrix metalloproteinase (MMP) release by fibroblasts residing in the gingival (gum) tissue. Granzyme B (GzmB) is a serine protease whose role in periodontitis is undefined. Here we studied the role of GzmB in the context of periodontitis. Methods: Gingival fibroblasts were cultured in the presence or absence of recombinant GzmB. Culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA) to quantify GzmB-induced release of collagenase (MMP-1). MMP-1 expression was quantified by qPCR. In some experiments, cells were pre-treated with PD98059 to block MEK/ERK signaling. The protease-activated receptor-1 (PAR-1) was blocked with ATAP-2 neutralizing antibody prior to GzmB stimulation. Human samples of gingival crevicular fluid (GCF), an inflammatory transudate released at the dento-gingival (tooth- gum) interface, were obtained from teeth with periodontal disease and healthy control teeth. GzmB was quantified in the GCF ([GzmB]GCF) by ELISA. Results: Recombinant GzmB induced a ~4-5-fold increase in MMP-1 secretion by cultured fibroblasts. GzmB induced phosphorylation of Erk1/2 which was abrogated by PD98059. GzmB-induced upregulation of MMP-1 gene expression/secretion were also reduced by PD98059. Blockade of PAR-1 function by ATAP-2 abrogated the GzmB- induced increase in MMP-1 secretion by GF. The [GzmB]GCF was ~4-5 fold higher at sites of periodontal disease (gingivitis/periodontitis) compared to healthy control sites, suggesting an association between GzmB and matrix degradation. Conclusions: These data point to a novel GzmB-driven signaling pathway in fibroblasts in which MMP-1 expression and secretion are upregulated in a PAR1- and Erk1/2- dependent manner.
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