Latent TNFa-mediated expression of MMPs by pulp versus PDL cells

Objective: To determine the effects of the pro-inflammatory mediator TNF-a on the secretion of matrix metalloproteinases (MMPs) by primary human pulp and periodontal ligament (PDL) cells. Methods: In dose-response experiments, cells were treated with increasing concentrations of TNF-a (0.01ng/ml, 0.1ng/ml, 1ng/ml, 10ng/ml, 50ng/ml) or with serum-free medium, and the conditioned medium was collected after 24 hours. In time-dependent experiments, cells were treated with 10ng/ml of TNF-a or serum-free medium, and the medium was collected after 3, 6, 12, 24, 48 and 96 hours. The results were evaluated by Gelatin Zymography and confirmed with Western Blotting. The intensity of the bands was analyzed by densitometry. Quantitative densitometric data were derived as fold change relative to baseline control levels. The effects of TNF-a on MMP expression were analyzed by one way ANOVA and post test comparisons by Tukey (a=0.05). Results: The zymograms demonstrated that pulp and PDL cells secreted MMPs that migrated at 72kDa, 60kDa and 55kDa, corresponding to the latent forms of MMP-2, 13 and 1 respectively. The secretion of MMP-2 increased in a dose-dependent manner with the maximum amount of MMP-2 at 24 hours for both pulp and PDL cells. The same dose-response pattern was observed for MMP-1 and MMP-13. However pulp cells exhibited a more latent response to TNF-a, with the maximum amount of MMP-1 at 96 hours and the appearance of active MMP-13 at 48 and 96 hours. Conclusion: Our results show that pulp and PDL cells can secrete MMP-1, 2 and 13 under the inflammatory stimulus of TNF-a. Furthermore, compared to PDL cells, pulp cells exhibit a latent response to TNF-a with regards to secretion of MMP-1 and MMP-13 thereby suggesting a possible mechanism by which these cells respond differentially to inflammatory stimuli. (Supported by Grant NIH grant R01-DE013725 to YLK; CAPES Foundation Fellowship to FWGPS 0668/07-9).