H 2 O 2 activates matrix metalloproteinases through the nuclear factor kappa B pathway and C a 2+ signals in human periodontal fibroblasts

Background The mechanisms involved in reactive oxygen species and matrix metalloproteinase ( MMP )‐mediated periodontal tissue breakdown are unknown. Objective To determine the effect of H 2 O 2 in MMP ‐2 and MMP ‐9 activity, and the involvement of nuclear factor kappa B ( NF κB) and Ca 2+ ‐mediated signals in human periodontal ligament fibroblasts. Material and Methods Primary cultures were characterized for their phenotype and exposed for 24 h to sublethal doses (2.5–10 μ m ) of H 2 O 2 or control media. NF κB involvement was evaluated through immunofluorescence of p65 subunit, using the NF κB blocking peptide SN 50 and catalase. Ca 2+ signals were analyzed by loading the cells with Fluo4‐ AM and recording the fluorescence changes in a confocal microscope before and after the addition of H 2 O 2 . 1,2‐bis(o‐aminophenoxy) ethane‐ N , N , N ′, N ′‐tetraacetic acid‐acetoxymethyl was used to chelate intracellular Ca 2+ . The activity and levels of MMP ‐2 and MMP ‐9 were analyzed by gelatin zymogram and densitometric scanning, and enzyme‐linked immunosorbent assay , respectively. Statistical analysis was performed with stata V11.1 software using the ANOVA test. Results H 2 O 2 at concentrations of 2.5–5 μ m induced Ca 2+ signaling and NF κB subunit p65 nuclear translocation, whereas catalase, SN 50 and BAPTA ‐ AM prevented p65 nuclear translocation. H 2 O 2 at 2.5–5 μ m significantly increased MMP ‐9 and MMP ‐2 activity, while SN 50 resulted in lower MMP ‐2 and MMP ‐9 activity rates compared with controls. Conclusion Sublethal H 2 O 2 induces Ca 2+ ‐dependent NF κB signaling with an increase in MMP gelatinolytic activity in human periodontal ligament .