H2O2 activates matrix metalloproteinases through the nuclear factor kappa B pathway and Ca2+ signals in human periodontal fibroblasts

Abstract

BackgroundThe mechanisms involved in reactive oxygen species and matrix metalloproteinase (MMP)-mediated periodontal tissue breakdown are unknown.

ObjectiveTo determine the effect of H2O2 in MMP-2 and MMP-9 activity, and the involvement of nuclear factor kappa B (NFB) and Ca2+-mediated signals in human periodontal ligament fibroblasts.

Material and MethodsPrimary cultures were characterized for their phenotype and exposed for 24h to sublethal doses (2.5-10m) of H2O2 or control media. NFB involvement was evaluated through immunofluorescence of p65 subunit, using the NFB blocking peptide SN50 and catalase. Ca2+ 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 H2O2. 1,2-bis(o-aminophenoxy) ethane-N,N,N,N-tetraacetic acid-acetoxymethyl was used to chelate intracellular Ca2+. 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.

ResultsH(2)O(2) at concentrations of 2.5-5m induced Ca2+ signaling and NFB subunit p65 nuclear translocation, whereas catalase, SN50 and BAPTA-AM prevented p65 nuclear translocation. H2O2 at 2.5-5m significantly increased MMP-9 and MMP-2 activity, while SN50 resulted in lower MMP-2 and MMP-9 activity rates compared with controls.

ConclusionSublethal H2O2 induces Ca2+-dependent NFB signaling with an increase in MMP gelatinolytic activity in human periodontal ligament.