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.     

Response of periodontitis and healthy patients in a Porphyromonas gingivalis‐stimulated whole‐blood model

Aim: To investigate the inflammatory responses of periodontitis patients and healthy patients in a whole‐blood model stimulated with Porphyromonas gingivalis. Methods: Whole blood collected from 17 periodontitis patients and six healthy patients was stimulated with Porphyromonas gingivalis cells. The secretion of cytokines and matrix metalloproteinases was quantified by enzyme‐linked immunosorbent assay. An analysis of covariance with the ancova model was used to evaluate the significance of differences in secreted host molecules by whole blood from the periodontitis and healthy groups. Results: Porphyromonas gingivalis induced the secretion of interleukin‐1β, interleukin‐6, interleukin‐8, tumor necrosis factor‐α, monocyte chemoattractant protein‐1, interferon inducible protein‐10 by whole blood from patients in the periodontitis and healthy groups. Matrix metalloproteinase‐8 and ‐9 levels secreted by whole blood also increased following stimulation. No significant differences (P < 0.05) in the amounts of secreted host molecules were observed between periodontitis and healthy patients. Conclusion: This study suggests that Porphyromonas gingivalis can provoke an inflammatory response and promote the progression of periodontitis by inducing the secretion of high levels of cytokines and matrix metalloproteinases by a mixed leukocyte population. However, the whole‐blood model did not reveal any significant differences in the inflammatory response between periodontitis patients (n = 17) and periodontally‐healthy patients (n = 6).     

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.     

Chemokine expression in Porphyromonas gingivalis–specific T‐cell lines

Autologous non‐T cells (monocytes and B cells) were added to Porphyromonas gingivalis–specific T cell lines established from 9 healthy adults together with P. gingivalis outer membrane antigens for 4–6, 16–18, 24 and 48 h. Flow cytometry was employed to analyze the CD4 and CD8 cells, monocytes and B cells for intracytoplasmic IP‐10 (interferon‐gamma inducible protein 10), MCP‐1 (monocyte chemoattractant protein 1), MIP‐1α (macrophage inflammatory protein 1α) and RANTES (regulated on activation normal T cell expressed and secreted) at the four time periods. All cell types were positive for each chemokine throughout the 48‐h time period. There were significantly fewer MCP‐1‐positive cells compared with the other 3 chemokines. However, the percentages of MCP‐1, MIP‐1α‐ and RANTES‐positive CD8 cells were significantly higher than the percentages of positive CD4 cells in all cultures. IP‐10‐positive CD4, CD14‐positive monocytes and CD19‐positive B cells were predominant compared with MIP‐1α‐ and RANTES‐positive cells at 24 h. In conclusion, the present study has shown that P. gingivalis–specific T cells, monocytes and B cells produce chemokines in response to P. gingivalis outer membrane antigens, IP‐10 being predominant, with MCP‐1 being significantly reduced in comparison with IP‐10, MIP‐1α and RANTES. Increased percentages of CD8 cells were induced to produce chemokines in comparison with CD4 cells, indicating a more preferential action on CD8 rather than CD4 cells.     

The proportion of interleukin-4, interferon-gamma and interleukin-10-positive cells in Porphyromonas gingivalis–specific T-cell lines established from P. gingivalis–positive subjects

T-cell cytokine profiles in ten adult periodontitis and seven age-matched healthy or gingivitis subjects were determined. Porphyromonas gingivalis–specific T-cell lines were established from the peripheral blood of these individuals all of whom had past or present evidence of P. gingivalis infection. FACS analysis was used to determine the percentage of CD4- and CD8-positive cells in each line staining for cytoplasmic interleukin (IL)-4, interferon-gamma and IL-10. There were no differences in the mean percentage of IL-4-, interferon-gamma- or IL-10-positive T cells between the two groups. However, the individual profiles showed that the CD4 cells in five of the seven healthy or gingivitis lines had a higher proportion of interferon-gamma-positive cells, with two lines demonstrating higher percentages of IL-10- and/or IL-4-positive CD4 cells. Five of the ten adult periodontitis lines demonstrated either equal or higher percentages of IL-4-positive and/or IL-10-positive CD4 cells. With respect to the CD8 cells, two of the seven lines established from the healthy or gingivitis subjects and six of the ten adult periodontitis lines showed profiles with a higher percentage IL-4- and/or IL-10-positive cells. When the total T-cell contribution (CD4 plus CD8) for each T-cell line was determined from the individual CD4:CD8 ratios, only one of the healthy or gingivitis lines showed a profile with a higher proportion of IL-10-positive cells, while the results for the adult periodontitis lines were the same as indicated for the CD4 cell profiles, with five lines showing a higher percentage of IL-4- and/or IL-10-positive cells. In conclusion, this study has shown that in P. gingivalis–responsive T-cell lines established from adult periodontitis and healthy or gingivitis subjects, there was a predominant trend towards a higher percentage of interferon-gamma positive cells than either IL-4- or IL-10-positive cells. However, there were variations from this trend, although whether these variations indicate true susceptibility to progressive disease has yet to be determined.     

Parathyroid hormone administration may modulate periodontal tissue levels of interleukin-6, matrix metalloproteinase-2 and matrix metalloproteinase-9 in experimental periodontitis

Background and Objective: Intermittent administration of the parathyroid hormone (1–34) has an anabolic effect on bone and it has been shown to reduce alveolar bone loss in experimental periodontitis models. The aim of the present study was to investigate the effect of parathyroid hormone on tissue degradation‐related factors in an experimental periodontitis model in rats. Material and Methods: Periodontitis was induced in seventy‐six male Wistar rats using ligature around the lower right first molars. The animals were then treated with parathyroid hormone (1–34) (T‐group) or vehicle (C‐group), three times a week for 15 d (C15, T15) or 30 d (C30, T30). At each experimental time‐point, the 19 rats were killed in each group and the gingival tissue around the first lower molar was removed and prepared for the following analyses: mRNA expression of interleukin‐1beta, interleukin‐6, matrix metalloproteinase (MMP)‐2 and MMP‐9, and gelatinolytic activity of MMP‐2 and MMP‐9. Hemimandibles were decalcified, and serial sections were processed and analyzed for interleukin‐6 immohistochemistry. Samples were also histochemically stained by tartrate‐resistant acid phosphatase (TRAP) to evaluate the number of osteoclasts present. Results: Parathyroid hormone‐treated samples showed decreased of levels of mRNA for interleukin‐6 in the T30 group (p < 0.01) and of MMP‐2 in the T15 and T30 groups (p < 0.05). Zymography assays demonstrated that treatment with parathyroid hormone led to a decrease in MMP‐9 activity (p < 0.01). TRAP staining of alveolar bone revealed that osteoclasts were present in higher numbers (p < 0.05) in the groups not treated with parathyroid hormone. Conclusion: These data suggest that intermittent administration of parathyroid hormone can down‐regulate the expression of biomarkers responsible for connective tissue breakdown and bone resorption, and potentially affect alveolar bone resorption activity.     

Peroxisome proliferator‐activated receptor δ inhibits Porphyromonas gingivalis lipopolysaccharide‐induced activation of matrix metalloproteinase‐2 by downregulating NADPH oxidase 4 in human gingival fibroblasts

We investigated the roles of peroxisome proliferator‐activated receptor δ (PPARδ) in Porphyromonas gingivalis‐derived lipopolysaccharide (Pg‐LPS)‐induced activation of matrix metalloproteinase 2 (MMP‐2). In human gingival fibroblasts (HGFs), activation of PPARδ by GW501516, a specific ligand of PPARδ, inhibited Pg‐LPS‐induced activation of MMP‐2 and generation of reactive oxygen species (ROS), which was associated with reduced expression of NADPH oxidase 4 (Nox4). These effects were significantly smaller in the presence of small interfering RNA targeting PPARδ or the specific PPARδ inhibitor GSK0660, indicating that PPARδ is involved in these events. In addition, modulation of Nox4 expression by small interfering RNA influenced the effect of PPARδ on MMP‐2 activity, suggesting a mechanism in which Nox4‐derived ROS modulates MMP‐2 activity. Furthermore, c‐Jun N‐terminal kinase and p38, but not extracellular signal‐regulated kinase, mediated PPARδ‐dependent inhibition of MMP‐2 activity in HGFs treated with Pg‐LPS. Concomitantly, PPARδ‐mediated inhibition of MMP‐2 activity was associated with the restoration of types I and III collagen to levels approaching those in HGFs not treated with Pg‐LPS. These results indicate that PPARδ‐mediated downregulation of Nox4 modulates cellular redox status, which in turn plays a critical role in extracellular matrix homeostasis through ROS‐dependent regulation of MMP‐2 activity.     

Observations on experimental marginal periodontitis in rats

OBJECTIVE: The purpose of this study was to assess periodontal destruction following experimentally induced marginal periodontitis in rats by ligatures over a 60-day observation period. The extent to which the physiological movement of teeth influenced the effect of the ligatures was also examined. In addition, two methods for measuring bone loss in the defleshed jaw were compared. METHODS: Thirty-five male Sprague-Dawley rats (SD) were divided into five groups. Marginal periodontitis was induced by ligatures on the second maxillary molars. Rats were killed after 15, 30, and 60 days. Rats in the control group were killed on day 1 and day 60. Bone loss was determined with two different methods on the buccal and palatinal surfaces of the defleshed jaw. In the first method, the distance of the cementoenamel junction (CEJ) from the alveolar bone crest (ABC) was measured at different sites; in the second method, the area of the exposed root surface of the molars was measured. RESULTS: Comparison of the control groups from day 1 and day 60 using both measuring methods showed significant differences in bone loss. In the area where the ligature was located, test rats exhibited significantly greater bone loss than control rats. Comparison of control rats from day 1 with test rats from day 15 showed that the increase in bone loss between the groups within the area of the ligature was significantly greater than outside it. The age-dependent bone loss increases over the entire observation period of 60 days. The ligature-induced bone loss increased most from day 1 to day 15; on days 30 and 60, slighter increases in bone loss were observed. CONCLUSIONS: The application of this model can only be recommended for short (相似文献   

The role of cytokines in a Porphyromonas gingivalis‐induced murine abscess model

Introduction: Porphyromonas gingivalis is an important periodontopathic bacterium that is strongly associated with periodontal disease and is part of human dental plaque. Periodontal disease results from the interaction of the host with bacterial products, and T‐cell‐derived cytokines remain critical in the immunoregulation of periodontal disease. Methods: The aim of this study was to examine the role of T helper type 1 [interleukin‐12p40 (IL‐12p40), interferon‐γ, tumour necrosis factor (TNF)] and type 2 (IL‐4, IL‐10) cytokines in the immune response to a subcutaneous challenge with P. gingivalis using a well‐established murine abscess model, in genetically modified cytokine‐specific knockout mice. Results: IL‐12p40^−/− mice exhibited more advanced tissue destruction and a reduced inflammatory cell infiltrate after subcutaneous P. gingivalis challenge. Deficiency of IL‐4 or IL‐10 did not result in increased susceptibility to P. gingivalis‐mediated tissue destruction. Furthermore, TNF deficiency appeared to reduce local tissue destruction. Interestingly, serum‐specific antibodies suggested a strong T helper type 2 response. Conclusion: The results of our study indicate an important role for IL‐12 in a primary P. gingivalis subcutaneous challenge.     

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.