Extracellular Matrix Metalloproteinase Inducer And Periodontitis

Periodontitis is a kind of infectious diseases, characterized by the epithelial cells proliferation and migration, degradation of Sharpy’s fibre, and alveolar bone loss at last. Its histological change is the breakdown of periodontal extracellular matrix (ECM), including collagen, fibronectin, laminin and proteglycan. Matrix metalloproteinases (MMPs) have been proved to play a vital role in the process of ECM breakdown. Studies both in vivo and in vitro indicated that MMPs are involved in the initiation and progression of periodontitis. Expression and activation of MMPs are regulated by a complicated mechanism, such as bacteria and its virulence, cytokines, growth factors and hormones. Extracellular matrix metalloproteinase inducer (EMMPRIN) is a newly-purified protein which shows potent regulatory effect on the production of MMPs.EMMPRIN was first purified from LX-1, a human lung carcinoma cell line. It was named as tumor cell-derived collagenase stimulatory factor (TCSF) because of its capacity of collagenase induction. Latter, it was found to be existed in numerous normal human tissues and possess an ability to stimulate the production of MMPs, so renamed as EMMPRIN. EMMPRIN was broadly distributed in normal human cells, including peripheral haemocytes, endothelial cells, cultured haemocytes and other tissues. Till now, the exact function of EMMPRIN is unclear, yet, it was convinced that EMMPRIN, through stimulating the production of MMPs in near cells as a signaling molecular or a regulator of cell-cell attachment, plays a role in physiological and pathological processes, including embryo development, tissue remodeling, wound repairment, tumor invasion and metastasis, rheumatoid arthritis, vascular diseases and diabetes, and so on.Since the ECM degradation by MMPs is the similar pathological process among periondontitis, rheumatoid arthritis and tumor invasion, it is reasonable to hypothesize that EMMPRIN might be expressed in the clinical healthy periodontal tissues and participate in the periodontal connective tissue destruction. Emingil et al have detected the soluble EMMPRIN in human gingival crevicular fluid (GCF) from both clinical healthy individuals and periodontitis-affected patients, and the results suggested that the level of EMMPRIN in GCF was correlated with the severity of periodontal inflammation. However, few studies were performed to detect the expression of EMMPRIN in human gingival tissues up to date.The present study will investigate the expression of EMMPRIN in clinical healthy and inflamed human gingival tissues, and analyze its possible role in different periodontal status according to experiments in vivo and in vitro.Part I Expression of EMMPRIN in periodontal tissuesObjective:To investigate the expression of EMMPRIN in periodontal tissues and determine its potential role during the progression of periodontitis.Materials and methods:The EMMPRIN protein level was detected with immunohistochemistry and western blot in gingival tissues from clinical healthy individuals and chronic periodontitis-affected patients. The human gingival epithelial cells (HGEC) were cultured from different objects in different periodontal status. Then, reverse transcription-polymerase chain reaction (RT-PCR) was used to measure the mRNA level of EMMPRIN, MMP-1, MMP-2 and MMP-3 in HGEC after treatment with Porphyromonas gingivalis Lipopolysaccharides (Pg LPS).Results:EMMPRIN was stained weakly to moderately in gingival epithelia of clinical healthy individuals, with more intensive in basal layer. In inflamed gingival tissues, EMMPRIN reaction was stronger and broader, and mainly located in epithelial cells, and broadly existed in subepithelial connective tissues. Results of western blot also demonstrated the positive expression of EMMPRIN protein in healthy tissues, and the level was significantly increased in inflamed ones. EMMPRIN mRNA, together with MMP-1, MMP-2 and MMP-3 was expressed in untreated HGEC. Interestingly, their mRNA expression showed different reaction to Pg LPS. The mRNA of these four genes had no significant change after treatment in HGEC from clinical healthy individuals or moderate periodontitis-affected patients, but it was upregulated significantly in HGEC from patients with severe periodontitis.Conclusion:EMMPRIN was expressed in normal human gingival tissues and might play a role in the physiological remodeling of periodontal tissues. Its increased level in inflamed status suggested that EMMPRIN might participate in periodontal tissue destruction as an MMPs stimulator. Experiment in vitro indicated that EMMPRIN might be a susceptible gene for severe periodontitis.Par II Possible function of EMMPRIN in pathological process of periodontitsObjective:To determine whether EMMPRIN was involved in regulation of MMPs by cytokines in periodontal fibroblasts as a signal molecule and to determine the role of EMMPRIN in pathological periodontal stastus.Materials and methods:Cultured human periodontal ligament cells (PDLC) and gingival fibroblasts (HGF) were stimulated with recombinant human IL-1βor TNF-αrespectively. The protein and mRNA levels of EMMPRIN, MMP-1, MMP-2 and MMP-3 were measured with RT-PCR, western blot and gelatin zymography. The levels of EMMPRIN and MMPs were also detected in a coculture model between HaCat and HGF, which was added with EMMPRIN antibody to block the signaling of soluble EMMPRIN.Results:Both IL-1βand TNF-a could upregulate the expression of MMPs in PDLC and HGF except for MMP-2 in HGF. However, this regulation process was not followed by changes of EMMPRIN expression level. Soluble EMMPRIN from HaCat could stimulate the production of MMPs in HGF, and this effect was inhibited by EMMPRIN antibody.Conclusion:MMPs in periodontal fibroblasts could be regulated by cytokines and EMMPRIN independently. EMMPRIN derived from epithelial cells could stimulate the expression of MMPs in fibroblasts and therefore play a role in the breakdown of periodontal connective tissues during progression of periodontitis.