Tumor necrosis factor-α induces matrix metalloproteinases-3, -10, and -13 in human periodontal ligament cells

Abstract

Background: Various biologic mediators, including matrix metalloproteinases (MMPs), that are implicated in periodontal tissue breakdown can be induced by cytokines. MMPs are known to degrade periodontal ligament attachment, and bone matrix proteins and tissue inhibitors of metalloproteinase (TIMPs) inhibit the activity of MMPs. The aim of this study is to investigate the effect of tumor necrosis factor (TNF)-α on the expression of MMPs in human periodontal ligament (PDL) cells in vitro and establish which MMPs are expressed specifically in response to that stimulus.

Methods: Cultured PDL cells were stimulated with TNF-α and analyzed with an MMP antibody array. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and western blot with cell lysate and zymography were used to measure messenger RNA (mRNA) and protein levels of MMP-3, -10, and -13. To examine TNF receptor (TNFR) expression, PDL cells were examined by flow cytometry, and expression of MMP-3, -10, and -13 was observed after blocking the TNFR with an antagonist. Results from real-time PCR, ELISA, and western blot were analyzed by paired t test.

Results: The antibody array showed that the protein most strongly upregulated by TNF-α stimulation was MMP-3, followed by MMP-13 and MMP-10. The TNF-α receptor blocker specifically inhibited expression of MMP-3 and -13. In addition, TNF-α increased levels of MMP mRNAs in MMP-3, -13, and -10 (in decreasing order). However, ELISAs showed that MMP-13 was the most upregulated protein, followed by MMP-10 and MMP-3. Western blotting indicated that TNF-α increased MMP-3 and -13 levels but had no significant effect on the level of MMP-10, and zymography showed that TNF-α increased the activities of all forms of MMP-3 and -13, but MMP-10 was not detected. Flow cytometry demonstrated that the majority of PDL cells expressed TNFR1.

Conclusions: TNF-α (10 ng/mL) upregulates levels of MMP-3, -10, and -13 in human PDL cells. These results suggest that these proteins play an important role in the inflammation of PDLs.