Resumen de Tumor Necrosis Factor-a Induces Matrix Metalloproteinases-3, -10, and -13 in Human Periodontal Ligament Cells
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)-a 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-a 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-a stimulation was MMP-3, followed by MMP-13 and MMP-10. The TNF-a receptor blocker specifically inhibited expression of MMP-3 and -13. In addition, TNF-a 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-a increased MMP-3 and -13 levels but had no significant effect on the level of MMP-10, and zymography showed that TNF-a 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-a (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.
Breakdown of the periodontium results from a complex interaction among microorganisms and host immune and inflammatory responses1,2 and involves the local production of proinflammatory cytokines and growth factors. Cytokines are believed to be important in periodontal disease;3 release of cytokines such as interleukin (IL)-1a, IL-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF)-a is stimulated by bacterial lipopolysaccharides, and these cytokines are strongly associated with periodontal disease.4 Of the cytokines, TNF-a plays the most important role in periodontal inflammation and induces the expression of matrix metalloproteinases (MMPs).5,6 TNF-a is a proinflammatory cytokine with neutrophilotactic activity and may trigger distinct chemotactic responses in neutrophils.7,8 MMPs have gained considerable attention, especially in periodontal research.9,10 They can degrade all the components of the extracellular matrix (ECM) and regulate turnover of the ECM in both physiologic and pathologic conditions. MMPs are usually divided into five groups according to their substrate specificity: collagenases, gelatinases, stromelysins, membrane-type MMPs, and others.11 Of the collagenases (MMP-1, -8, -13, and -18), MMP-1 is the key enzyme responsible for breakdown of collagen networks. Whereas the main substrates of the gelatinases (MMP-2 and -9) are denatured collagens and gelatins, the stromelysins (MMP-3, -10, and -11) have substantial activity on the ECM and can activate proMMPs.12 The production of MMPs by periodontal ligament (PDL) cells is stimulated by bacterial extracts, and activated MMPs break down the ECM in the periodontium. Furthermore, individual MMPs have been found to play critical roles in specific conditions. For example, MMP-13 is highly expressed in destructive periodontal disease,13 and MMP-3 is involved in collagen lysis by activating proMMP-1 and degrades the ECM.14 MMP-10 is important because of its proteolytic activity against ECM components in wound repair15 and tumor invasion,16 but its function in periodontal inflammation is unclear. Tissue inhibitors of metalloproteinases (TIMPs), endogenous MMP inhibitors, can inhibit the activity of MMPs. The balance between MMPs and TIMPs is important for the maintenance of structural homeostasis in periodontal tissue. In healthy periodontium, the level of TIMPs is higher than that of MMPs, whereas in regions of periodontal disease, MMP levels exceed those of TIMPs.17 This study investigates the effect of TNF-a, a proinflammatory cytokine, on the expression of MMPs derived from human PDL cells in vitro, to determine which MMPs are expressed specifically in response to that stimulus. Antibody arrays, real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assays (ELISAs), western blot analysis, zymography, and flow cytometry analyses were used to observe the individual steps of transcription, secretion, and activation of MMPs in PDL cells.
