Focal adhesion kinase activation is required for TNF‐α‐induced production of matrix metalloproteinase‐2 and proinflammatory cytokines in cultured human periodontal ligament fibroblasts

Since focal adhesion kinase (FAK) was proposed as a mediator of the inflammatory response, we have investigated the role of this molecule in the release of inflammatory cytokines by cultured human periodontal ligament fibroblasts (HPDLFs), cells that are thought to be important in the patient's response to periodontal infection. Human periodontal ligament fibroblasts were stimulated by tumor necrosis factor alpha (TNF‐α) and its effects on interleukin (IL)‐6 and IL‐8 release were measured by ELISA. Expression of matrix metalloproteinase 2 (MMP‐2) protein was analysed by western blotting. The levels of IL6, IL8, and MMP2 mRNA were evaluated by real‐time PCR. Tumor necrosis factor alpha dose‐dependently induced the phosphorylation of FAK, whereas small interfering FAK (siFAK) inhibited TNF‐α‐induced FAK phosphorylation. Tumor necrosis factor alpha also stimulated the production of IL‐6, IL‐8, and MMP‐2 in a dose‐dependent manner. Knockdown of FAK significantly suppressed TNF‐α‐induced expression of IL6 and IL8 mRNA and release of IL‐6 and IL‐8 protein in HPDLFs. Similarly, MMP‐2 down‐regulation was significantly prevented by siFAK. Our results strongly suggest that knockdown of FAK can decrease the production of TNF‐α‐induced IL‐6, IL‐8, and MMP‐2 in HPDLFs. These effects may help in understanding the mechanisms that control expression of inflammatory cytokines in the pathogenesis of periodontitis.     

Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase‐3 in human gingival fibroblasts via extracellular signal‐regulated kinase

Zeldich E, Koren R, Dard M, Weinberg E, Weinreb M, Nemcovsky CE. Enamel matrix derivative induces the expression of tissue inhibitor of matrix metalloproteinase‐3 in human gingival fibroblasts via extracellular signal‐regulated kinase. J Periodont Res 2010; doi: 10.1111/j.1600‐0765.2009.01218.x © 2009 John Wiley & Sons A/S Background and Objective: Periodontal disease is characterized by increased expression and activity of matrix metalloproteinases (MMPs) and insufficient expression/activity of their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). This altered MMP–TIMP balance results in progressive destruction of gingival and periodontal extracellular matrix. Enamel matrix derivative (EMD), clinically used for periodontal regeneration in a device called Emdogain^®, has been suggested to enhance gingival healing following periodontal procedures in humans. We previously showed that EMD increases the proliferation of human and rat gingival fibroblasts and protects them from tumor necrosis factor‐induced apoptosis. In the present study, the modulation of MMP and TIMP expression by EMD was investigated. Material and Methods: Primary human gingival fibroblasts were treated in vitro with tumor necrosis factor, EMD or both in serum‐free conditions, and RNA was analyzed with an extracellular matrix‐focused microarray and quantitative real‐time polymerase chain reaction. Results: Microarray analysis showed detectable expression of MMP‐1, MMP‐2, MMP‐3, MMP‐7 and MMP‐13, as well as TIMP‐1 and TIMP‐3 in untreated cells. There was no apparent regulation of the expression of MMP‐2, MMP‐7, MMP‐13 and TIMP‐1 by either tumor necrosis factor or EMD. In contrast, tumor necrosis factor significantly increased MMP‐1 expression, and EMD reduced it when both agents were present. Also, EMD significantly induced TIMP‐3 expression, an effect which was dependent on activation of extracellular signal‐regulated kinase 1/2, since it was totally abolished by a selective extracellular signal‐regulated kinase pathway inhibitor. Conclusion: These data suggest that EMD may affect gingival health by ways other than cell proliferation/survival, i.e. by stimulation of TIMP‐3 production, which could improve the MMP–TIMP balance in gingival tissue and curb extracellular matrix destruction.     

Effects of relaxin on collagen type I released by stretched human periodontal ligament cells

Authors – Takano M, Yamaguchi M, Nakajima R, Fujita S, Kojima T, Kasai K Objectives – Relapse of teeth that have moved during orthodontic treatment is a major clinical issue with respect to the goals of successful treatment. Such relapse is a physiologic response of the supporting tissues to application of force, and is mainly attributed to occlusal instability and increased mechanical tension exerted by the periodontal ligament (PDL). Relaxin, a member of the insulin/relaxin family of structurally related hormones, has an influence on many physiologic processes, such as collagen turnover, angiogenesis, and antifibrosis. Therefore, relaxin may also affect orthodontic tooth movement through alterations of the PDL, though little is known regarding the relationship between relaxin and stretched human PDL (hPDL) cells. In the present study, we investigated the effects of relaxin on the expression of collagen type I (Col‐I) and matrix metalloproteinase 1 (MMP‐1) in stretched hPDL cells in vitro. Materials and Methods – The release and gene expression of Col‐I, as well as those of MMP‐1 in stretched hPDL cells treated with relaxin were investigated using enzyme‐linked immunosorbent assay and real‐time PCR methods. Results – Relaxin decreased the release and gene expression of Col‐I, and increased those of MMP‐1 by stretched hPDL cells in a magnitude‐dependent manner. Conclusion – Our results indicate that relaxin modulates collagen metabolism in stretched hPDL cells via the release and expression of Col‐I and MMP‐1. This hormone may be useful to prevent orthodontic relapse following orthodontic treatment.     

Alterations of collagen-I, MMP-1 and TIMP-1 in the periodontal ligament of diabetic rats under mechanical stress

Zhang L, Li X, Bi L‐J. Alterations of collagen‐I, MMP‐1 and TIMP‐1 in the periodontal ligament of diabetic rats under mechanical stress. J Periodont Res 2011; 46: 448–455. ©2011 John Wiley & Sons A/S Background and Objective: The present study assessed effects of hyperglycemia on production of proteins involved in remodeling of the periodontal ligament under mechanical stress. Material and Methods: Forty‐eight Sprague–Dawley rats were randomly divided into the following two groups: nondiabetic (ND) and diabetes induced (DI; n = 24 each group). Diabetes was induced in the DI group by a single dose of streptozotocin, and saline solution was injected in the ND group. Rats were killed 1–14 d after induction of mechanical pressure (50 g) on the first upper left molar. Alterations of collagen type I (Col‐I), MMP‐1 and TIMP‐1 in the upper left periodontal ligament of these rats were measured immunohistochemically and compared with those on the contralateral side of the same rat (control; no force induction). Results: The DI group showed a decrease in Col‐I and an increase in MMP‐1 compared with the ND group. Both Col‐I and MMP‐1 increased in both groups, whereas TIMP‐1 was decreased following mechanical pressure. The DI group exhibited a longer duration of increased MMP‐1 and MMP/TIMP ratio compared with the ND group. Conclusion: Diabetes affects proteins involved in remodeling of periodontal ligament during mechanical pressure. This may delay the reconstruction and remodeling of periodontal ligament in diabetic individuals.     

Down‐regulation of interleukin‐1α‐induced matrix metalloproteinase‐13 expression via EP1 receptors by prostaglandin E2 in human periodontal ligament cells

In the present study, we investigated the effect of prostaglandin (PG) E₂ on matrix metalloproteinase (MMP)‐13 production in human periodontal ligament cells stimulated with interleukin (IL)‐1α. IL‐1α enhanced both MMP‐13 and PGE₂ production. Indomethacin, a nonselective cyclooxygenase inhibitor, and NS‐398, a specific cyclooxygenase‐2 (COX‐2) inhibitor, significantly enhanced IL‐1α‐induced MMP‐13 production in periodontal ligament cells, although both the agents completely inhibited IL‐1α‐induced PGE₂ production. Exogenous PGE₂ reduced IL‐1α‐induced MMP‐13 mRNA and protein production in a dose‐dependent manner. 17‐phenyl‐ω‐trinor PGE₂, a selective EP₁ receptor agonist, mimicked the inhibitory effect of PGE₂ on IL‐1α‐induced MMP‐13 mRNA and protein production. On the basis of these data, we suggest that COX‐2‐dependent PGE₂ down‐regulates IL‐1α‐elicited MMP‐13 production via EP₁ receptors in human periodontal ligament cells. PGE₂ may be involved in the regulation of destruction of extracellular matrix components in periodontal lesions.     

Analysis of matrix metalloproteinase (MMP‐8 and MMP‐2) activity in gingival crevicular fluid from children with Down’s syndrome

Yamazaki‐Kubota T, Miyamoto M, Sano Y, Kusumoto M, Yonezu T, Sugita K, Okuda K, Yakushiji M, Ishihara K. Analysis of matrix metalloproteinase (MMP‐8 and MMP‐2) activity in gingival crevicular fluid from children with Down’s syndrome. J Periodont Res 2010; doi: 10.1111/j.1600‐0765.2009.01214.x. © 2009 John Wiley & Sons A/S Background and Objective: High levels of colonization by periodontopathic bacteria and a high prevalence of chronic inflammatory periodontal disease have been reported in children with Down’s syndrome. Matrix metalloproteinases (MMPs) are mediators of extracellular matrix degradation and remodelling, and are deeply involved in the course of periodontal disease. To clarify the relationship between Down’s syndrome and periodontitis, we investigated levels of MMP‐2 and MMP‐8 in gingival crevicular fluid (GCF) and detection of periodontopathic bacteria from subgingival plaque. Material and Methods: Samples of GCF and plaque were isolated from central incisors. Levels of MMPs were evaluated by enzyme‐linked immunosorbent assay, and periodontopathic bacteria were detected by polymerase chain reaction. Results: Levels of MMP‐2 and MMP‐8 in Down’s syndrome patients were higher than those in healthy control subjects. In the Down’s syndrome group, increases in these MMPs were observed in GCF from patients with an oral hygiene index score of < 2 and in GCF from sites that were negative for bleeding on probing. The detection rate of periodontopathic bacteria in Down’s syndrome patients was higher than that in the control subjects. Matrix metalloproteinase‐2 levels in sites harbouring Porphyromonas gingivalis or Aggregatibacter (Actinobacillus) actinomycetemcomitans were lower than in those without these microorganisms. Conclusion: These results suggest an increase in MMP‐2 and MMP‐8 in Down’s syndrome patients, regardless of whether inflammation of periodontal tissue is present or not.     

The effect of centrifugal force on mRNA levels of collagenase, collagen type-I, tissue inhibitors of metalloproteinases and beta-actin in cultured human periodontal ligament fibroblasts

BACKGROUND: The aim of orthodontic treatment is to relocate teeth abnormally positioned in the jaws. This is achieved by application of continuous force on the tooth, which is immediately being sensed by the periodontal ligament (PDL), bone and the gingiva. Since the bony response is mediated by the PDL, tooth movement is primarily a PDL phenomenon. OBJECTIVES: Thus, the purpose of the present study was to evaluate the direct effect of force (excluding the in vivo tissue response) on the molecular level of matrix metalloproteinase-1 (MMP-1) and collagen type-I (Col-I) in human PDL fibroblasts. METHODS: PDL cell culture flasks were centrifuged for 10, 20, 30, 60, 90 and 120 min by horizontal microplate rotor. The effect of force on mRNA levels of beta-actin, MMP-1, Col-I, tissue inhibitors-1 and -2 (TIMPs) genes was analyzed by RT-PCR. RESULTS: The results showed that force had no effect on the mRNA levels of beta-actin during the first 90 min of application of force, indicating for the first time the use of beta-actin gene as an internal invariant control. It increased the mRNA levels of MMP-1 while almost no effect on Col-I and TIMPs was observed. CONCLUSIONS: The results indicate that PDL remodeling following application of orthodontic force could be partly attributed to the direct effect of the force on MMP-1 gene expression in fibroblasts.     

On the role of MT1-MMP,a matrix metalloproteinase essential to collagen remodeling,in murine molar eruption and root growth

Although the connective tissues of the periodontium are subject to a high turnover rate, no conclusive evidence has yet emerged that periodontal collagen turnover is essential for the eruption of teeth or for root elongation. These processes were studied in mice deficient in MT1-MMP, a membrane type matrix metalloproteinase essential for remodeling of soft tissue-hard tissue interfaces. Mandibular first molars of deficient mice and their wild-type littermates were subjected to stereological analysis in order to assess root length, eruption and the volume density of phagocytosed collagen in periodontal ligament fibroblasts. The data showed that both eruption and root elongation were severely inhibited in animals lacking the enzyme. We also found, in periodontal ligament fibroblasts from MT1-MMP-deficient mice, a massive age-related accumulation (up to 60-fold over controls) of collagen fibril-containing phagosomes. Phagolysosomes, which represent the next downstream step in collagen fibril degradation by the lysosomal pathway, did not accumulate. These observations indicate that MT1-MMP plays a central role in periodontal remodeling. The stunted root growth and the failure to erupt indicate the important role of the enzyme in tooth development.     

Type VI collagen is associated with microfibrils and oxytalan fibers in the extracellular matrix of periodontium,mesenterium and periosteum

Type VI collagen was immunolocalized in several soft connective tissues at the light and electron microscopic level. Positive labeling was found in all tissues examined, periodontal ligament, gingiva, mesenterium and periosteum. The labeled structures could be divided into 2 categories: microfibrils intermingling with collagen fibrils, and those that formed bundles (oxytalan fibres and elastin-associated microfibrils). Control sections incubated with antibody pre-absorbed to purified type VI collagen, or with non-immune antibody, proved to be negative. Our observations indicate that the structural organization of type VI collagen varies from small microfibrillar structures associated with the collagen and elastin fibre systems to highly ordered parallel arrays of oxytalan bundles.     

Stimulation of epithelial cell matrix metalloproteinase (MMP‐2, ‐9, ‐13) and interleukin‐8 secretion by fusobacteria

Background/aims: Bacterial pathogens involved in periodontal diseases exert their destructive effects primarily by stimulating the host cells to increase their secretion of proinflammatory cytokines and matrix metalloproteinases (MMPs). This study aimed to determine the epithelial cell matrix metalloproteinase and interleukin‐8 (IL‐8) secretion upon exposure to fusobacteria. Methods: Eight different oral and non‐oral Fusobacterium strains were incubated with HaCaT epithelial cells. Gelatin zymography and Western blot analysis were performed to detect collagenase 3 (MMP‐13), gelatinase A (MMP‐2), gelatinase B (MMP‐9), and IL‐8 secretion by epithelial cells. Results: All Fusobacterium strains, especially Fusobacterium necrophorum ATCC 25286, Fusobacterium nucleatum ATCC 25586, and Fusobacterium varium ATCC 51644, increased MMP‐9 and MMP‐13 secretion. Fusobacterium simiae ATCC 33568, and to a lesser extent F. nucleatum and F. necrophorum, increased epithelial MMP‐2 secretion. F. nucleatum and F. necrophorum also increased IL‐8 secretion. F. varium ATCC 27725, a strain that only weakly stimulated MMP production, strongly increased the IL‐8 production, suggesting that their expression is differently regulated. Conclusion: We conclude that the pathogenic potential of fusobacteria may partly result from their ability to stimulate secretion of MMP‐9, MMP‐13, and IL‐8 from epithelial cells.