H2 O2 activates matrix metalloproteinases through the nuclear factor kappa B pathway and Ca(2+) signals in human periodontal fibroblasts.

Abstract

The mechanisms involved in reactive oxygen species and matrix metalloproteinase (MMP)-mediated periodontal tissue breakdown are unknown. To determine the effect of H2 O2 in MMP-2 and MMP-9 activity, and the involvement of nuclear factor kappa B (NFκB) and Ca(2+) -mediated signals in human periodontal ligament fibroblasts. Primary cultures were characterized for their phenotype and exposed for 24 h to sublethal doses (2.5-10 μm) of H2 O2 or control media. NFκB involvement was evaluated through immunofluorescence of p65 subunit, using the NFκB blocking peptide SN50 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 H2 O2 . 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. H2 O2 at concentrations of 2.5-5 μm induced Ca(2+) signaling and NFκB subunit p65 nuclear translocation, whereas catalase, SN50 and BAPTA-AM prevented p65 nuclear translocation. H2 O2 at 2.5-5 μm significantly increased MMP-9 and MMP-2 activity, while SN50 resulted in lower MMP-2 and MMP-9 activity rates compared with controls. Sublethal H2 O2 induces Ca(2+) -dependent NFκB signaling with an increase in MMP gelatinolytic activity in human periodontal ligament.