Effects Of IL-17on MMPs Expression And Cell Migration In Human Periodontal Ligament Fibroblasts

Periodontal ligament fibroblasts are a prominent cell type in the periodontal ligament, which play an important role in periodontal regeneration, integrity maintenance and modulating homeostasis. Interleukin-17(IL-17) has gained significant research attention by the recognition of a third effector CD4T cell population dubbed "Th17," that produces IL-17as a signature cytokine. IL-17has pleiotropic activities, one of which is to induce the expression of various cytokines on fibroblasts, epithelial, endothelial and stromal cells. IL-17was recently shown to mediate tissue inflammation and remodeling by targeting metalloproteinases (MMPs).MMPs are a group of structurally and functionally related proteinases that are responsible for degradation of extracellular matrix (ECM) components. MMP activity is regulated in part by its interaction with the tissue inhibitor of matrix metalloproteinase (TIMP). Increasing evidence shows that MMPs promote ECM remodeling, thereby facilitating cell migration, and are implicated in the wound-healing process. In particular, MMP-1is suggested to mediate cell migration and collagen remodeling in various cell types. Actin stress fibers and focal adhesions are all prerequisite processes required for ECM degradation and cell migration to occur. However, the mechanism underlying the regulation of IL-17on MMP-1expression as well as cell migration has yet to be thoroughly explored. We speculated that IL-17might regulate migration of periodontal ligament fibroblasts by modifying the expression of MMP-1.The present study was divided into three parts for investigating the effects of11-17in MMP-1expression and cell migration and to analyze the correlation between IL-17and MMP-1expression within human and rat periapical lesions.Part one: Effects of IL-17on MMPs expression in human periodontal fibroblasts and signaling pathway involvedExperiment one: Expression of MMPs, TIMP-1and EMMPRIN by IL-17in human periodontal fibroblastsObjective: The aim of present study was to investigate the effects of IL-17on the mRNA expression and protein production of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and EMMPRIN through human periodontal ligament cells.Methods: The cells were stimulated with IL-17for different time periods. The mRNA levels of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and EMMPRIN were evaluated via reverse transcription-polymerase chain reaction(RT-PCR) and quantitative real-time RT-PCR, whereas the protein secretion into the culture medium was assessed via enzyme-linked immunosorbentassay(ELISA) and zymography analysis.Results: IL-17significantly up-regulated MMP-1and MMP-13mRNA expression but down-regulated MMP-2, MMP-9, and TIMP-1mRNA expression. Furthermore, IL-17increased the secreted protein level of MMP-1and MMP-13and conversely reduced the level of MMP-2, MMP-9, and TIMP-1. However, IL-17exerted no effect on EMMPRIN mRNA or protein secretion.Conclusion:This study reported the ability of IL-17to regulate MMP and TIMP-1production through human periodontal ligament cells, a phenomenon that may contribute to periapical tissue remodeling.Experiment two: Effects of IL-17on MMP-1expression and signaling pathway involved.Objective: To investigate the signaling pathways mediating the upregulation of MMP-1expression by IL-17.Methods: Cells were treated with IL-17. MMP-1expression was analyzed by quantitative real-time RT-PCR, western blot and ELISA; localization of MMP-1by immunofluorescence analysis; transcriptional activation by Western blotting. Signal transduction pathways were investigated using pharmacological inhibitors, small interfering RNA (siRNA); transcription factor binding by EMSA.Results: IL-17can increase the expression of MMP-1in human periodontal ligament fibroblasts. IL-17-induced MMP-1expression was attenuated by pre-treatment with IL-17receptor neutralizing antibody and TIMP-1. IL-17stimulation resulted in rapid activation of Akt, p38mitogen-activated protein kinase (MAPK), extracellular signalregulated kinase (ERK)1/2, c-Jun-N-terminal kinase (JNK) and nuclear factor-kappaB (NF-κB) in periodontal ligament fibroblasts. In addition, a p38-MAPK inhibitor (SB203580) inhibited IL-17A-induced MMP-1upregulation of periodontal ligament fibroblasts. The involvement of p38-MAPK in IL-17Ainduced MMP-1expression was further confirmed by transfection of p38a siRNA. A NF-κB inhibitor (pyrrolidine dithiocarbamate) also suppressed MMP-1expression enhanced by IL-17A. Moreover, transfection with p38a siRNA inhibited IL-17A-induced NF-κB nuclear translocation as well as NF-κB binding activity.Conclusion:IL-17increased MMP-1expression through the IL-17receptor, p38-MAPK, and NF-κB signal transduction pathways.Part two: Effects of IL-17on migration of human periodontal ligament fibroblastsExperiment three: Effects of IL-17on migration of human periodontal ligament fibroblastsObjective: The purpose of the present study was to investigate the effects of IL-17on cell migration and proliferation of human periodontal ligament fibroblasts.Methods:Cell proliferation was examined by CKK-8; cell migration by wound healing assay and transwell chamber-based migration assay; location of the actin cytoskeleton as well as paxillin were visualized by immunofluorescence analysis; transcriptional activation by Western blotting. Signal transduction pathways were investigated using pharmacological inhibitors, small interfering RNA (siRNA).Results: IL-17can increase the migration in human periodontal ligament fibroblasts but has no effect on cell proliferation. IL-17A-induced MMP-1expression led to cell migration, which was attenuated by pre-treatment with IL-17receptor neutralizing antibody or TIMP-1. The IL-17-induced cell migration was also attenuated by. small interfering RNA (siRNA) for MMP-1or p38-MAPK. Specifically, IL-17-induced remodeling of the cell cytoskeleton led to the rapid formation of actin stress fibers and required the catalytic activity of MMP-1, which was suppressed by MMP-1siRNA transfection and p38-MAPK inhibitor. IL-17-induced redistribution of paxillin at the focal adhesions in an p38-MAPK-dependent manner. IL-17could also induced tyrosine phosphorylation of focal adhesion kinase (FAK), which was independent of p38-MAPK and MMP-1activation.Conclusion:Our results demonstrated the IL-17induced migration and morphological changes of periodontal ligament fibroblasts in vitro require the corporation of MMP-1with p38-MAPK and FAK mediated signaling pathways.Part three: The potential role and mechanism of IL-17and MMP-1in human chronic periapical lesions and experimental apical periodontitis in ratsExperiment four: Expression of IL-17and MMP-1in human chronic periapical lesionsObjective: The purpose of the present study was to investigate the expression of IL-17and MMP-1in the human periapical lesions and study the relationship between them.Methods:Sixteen human periapical lesion samples (ten granulomas and five cysts) as well as healthy periapical samples were obtained in the clinic. Then seven periapical lesion samples were selected for immunohistochemical and double immunofluoresc-ence analysis to determine the presence of IL-17and MMP-1. Another eight periapical lesion samples and healthy samples were analyzed using RT-PCR to examine the mRNA expression of IL-17and MMP-1. The correlation between the expression of IL-17and MMP-1was also analyzed.Results: The data demonstrated the large amount of IL-17and MMP-1was present in periapical lesions. Immunofluorescence analysis revealed different localization of IL-17-positive and RANKL-positive cells. The expression of IL-17was a little higher in periapical granulomas than cysts, but MMP-1expression was less in granulomas. Compared with healthy control, the mRNA expression of both IL-17and MMP-1was remarkable elevated in periapical lesions. In addition, there was a positive correlation between the mRNA levels of IL-17and MMP-1.Conclusion: The results indicated the differential expression of IL-17and MMP-1in the chronic periapical lesions.Experiment five: Expression of IL-17and MMP-1in experimental apical periodontitis in ratsObjective: The purpose of the present study was to determine immunohistochemical localization of IL-17and MMP-1during the development of periapical lesions in rats.Methods: Periapical lesions developed within35days following mandibular first molar pulp exposure in Sprague-Dawley rats. The animals were randomly sacrificed at0,7,14,21,28and35days after pulpal exposure. The jaws which contained the first molar were obtained and routinely prepared for histological and immunohistochemical examination. In addition, collagen fibers were staining by picro-sirius red staining according to the previous method.Results: From day0to day21, the number of IL-17-positive and MMP-1-positive cells and neutrophils ascended andpeaked on day21, and the collagen bundles became severely broken at day21. From day28to day35, the number of IL-17-positive cells decreased, while MMP-1-positive cells remained high, and there were new collagen formation. There was a remarkable positive correlation between the number of IL-17positive cells and MMP-1positive cells and there was also a remarkable positive correlation between the number of IL-17positive cell and inflammatory cells.Conclusion:These findings demonstrated that IL-17and MMP-1might possibly be involved during the acute and chronic inflammatory response and associated with development and outcome process of periapical lesion.