MMP-2 activation by Actinobacillus actinomycetemcomitans supernatant in human PDL cells was corresponded with reduction of TIMP-2

OBJECTIVE: Matrix metalloproteinase 2 (MMP-2) has been implicated to play a role in pathogenesis of periodontal disease. We recently reported that Porphyromonas gingivalis supernatant could activate MMP-2 in human periodontal ligament (HPDL) cells. In this study, activation of MMP-2 by Actinobacillus actinomycetemcomitans supernatant and the mechanism was investigated. METHODS: HPDL cells were treated with either A. actinomycetemcomitans or P. gingivalis supernatant for 48 h. To verify the mechanism, pretreated inhibitors were used. Gelatin zymography, RT-PCR and Western blot analysis were used to detect the activation of MMP-2, expression of MT1-MMP and TIMP-2 mRNA and the proteins, respectively. RESULTS: The supernatant from A. actinomycetemcomitans could activate MMP-2 in HPDL cells similar to that from P. gingivalis but by a different mechanism. Activation by A. actinomycetemcomitans supernatant was correlated with a reduction of TIMP-2 secretion without any alteration of MT1-MMP, while activation by P. gingivalis increased MT1-MMP but no change of TIMP-2 was found. CONCLUSION: The supernatant from A. actinomycetemcomitans and P. gingivalis could induce the activation of MMP-2 possibly through the imbalance of MT1-MMP and TIMP-2 in HPDL cells but by different mechanisms. The imbalance of MT1-MMP and TIMP-2 may be another factor that is involved in the severity of periodontal disease.     

Regulation of matrix metalloproteinase production by cytokines,pharmacological agents and periodontal pathogens in human periodontal ligament fibroblast cultures

Matrix metalloproteinases (MMPs). produced by both infiltrating and resident cells of the periodontium, play a role in physiologic and pathologic events. It is recognized that an imbalance between activated MMPs and their endogenous inhibitors leads to pathologic breakdown of the extracellular matrix during periodontitis. To date, little is known about the regulation of MMP synthesis and secretion in human periodontal ligament fibroblasts (PDLFs). The purpose of this study was to examine the effects of cytokines, pharmacological agents (protein synthesis inhibitor and protein kinase C inhibitors) and predominant periodontal pathogens (Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis) on MMP production in human PDLFs using gelatin zymography. The gelatin zymograms revealed that the main gelatinase secreted by human PDLFs migrated at 72 kDa and represents MMP-2. Minor gelatinolytic bands were also observed at 92 kDa regions that correspond to MMP-9. We found that A. actinomycetemcomitans, P. gingivalis and IL-1alpha can elevate MMP-2 secretion in human PDLFs. These results indicate that periodontal pathogens and inflammatory cytokines play an important role in tissue destruction and disintegration of extracellular matrix in periodontal diseases. Thus, activation of MMPs may be one of the distinct host degradative pathways in the pathogenesis of periodontitis. In addition, H7, staurosporine, cycloheximide and TGF-beta could suppress MMP-2 production. Agents that target protein synthesis or the protein kinase C pathway in human PDLFs inhibit MMP-2 production, and such inhibition may contribute to the pathogenesis of periodontal inflammation. Taken together, these findings suggest a possible new therapeutic approach, involving the use of drugs that modify host-response mechanisms to suppress or inhibit MMP-mediated tissue destruction.     

Effects of hyaluronan oligosaccharide on the expression of MMP-1 in periodontal ligament cells

It is well known that low-molecular weight hyaluronan (HA) is detected in human periodontal tissue under inflammatory conditions. HA oligosaccharide (HAoligo) has been demonstrated to induce matrix metalloproteinase (MMP)-1 expression in dendritic cells and chondrocytes, however, the bioactivities of HAoligo in periodontal ligament (PDL) cells remain unclear. In this study, we investigated the effect of HAoligo on MMP-1 expression in human PDL (HPDL) cells and the mechanisms in terms of the signal transmission. HAoligo was generated and purified from commercial human umbilical cord HA. HPDL cells were isolated from healthy ligaments, and cultured with HAoligo for 0-24 h. The expression of MMP-1, tissue inhibitor of MMPs (TIMP)-1 and TIMP-2 was analyzed by real-time PCR and Western blot analyses. Effects of specific inhibitors of p38 mitogen-activated protein kinase (MAPK) activity, MAPK kinase activity and NFkB activity on HAoligo-induced MMP-1 expression in HPDL cells were also investigated by real-time PCR and Western blot analyses. HAoligo remarkably enhanced MMP-1 expression in both mRNA and protein levels, but no effect was shown on the expression of TIMP-1 and TIMP-2 mRNAs. Inhibition of p38MAPK activity decreased the MMP-1 expression. Neither inhibition of NFkB nor MAPKK activity affected the MMP-1 expression. It was suggested that HAoligo induces MMP-1 expression in HPDL cells, and p38MAPK plays a crucial role in signal transduction for MMP-1 inducted by HAoligo.     

Membrane type (MT) 1-matrix metalloproteinase (MMP) and MMP-2 expression in ligature-induced periodontitis in the rat

BACKGROUND: Matrix metalloproteinases (MMPs) have been shown to be involved in the degradation of the extracellular matrix (ECM). In particular, MMP-2 and MMP-9 (gelatinase A and gelatinase B, respectively) have been identified as the predominant MMPs during periodontitis. Recent studies have indicated that a novel transmembrane MMP, mebrane-type 1 matrix metalloproteinase (MT1-MMP), can activate pro-MMP-2 in tumor metastasis. This study aims to elucidate the presence and localization of MT1-MMP and MMP-2 in periodontitis in a rat model. METHODS: In 2 groups of 40-day-old male Sprague-Dawley rats, periodontitis was initiated by ligating floss around maxillary second molars. A group of control animals were left untreated. Maxillary dentoalveolar segments were isolated after 7 and 21 days postinduction and were prepared for gross and radiographic analysis of bone loss and for histological analysis. Samples were also prepared for gel zymography to detect the presence of MMP-2, and for Northern blot analysis and in situ hybridization with MT1-MMP probes. RESULTS: MMP-2 expression increased at 21 days following ligature placement, in conjunction with MT1-MMP expression. MT1-MMP mRNA expression was observed in epithelial cells, fibroblasts, and in multinucleated cells in the periodontium. CONCLUSION: Our data suggest that MT1-MMP may play a role in extracellular matrix degradation during periodontitis, in concert with MMP-2 and other proteinases.     

Sonic extracts from a bacterium related to periapical disease activate gelatinase A and inactivate tissue inhibitor of metalloproteinases TIMP-1 and TIMP-2

Aim  To examine the effects of sonicated bacterial extracts (SBEs) from three related to periapical disease bacteria ( Porphyromonas gingivalis , P. endodontalis and F. nucleatum ) on the activation of matrix metalloproteinase (MMP-2) and the inactivation of tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2).
Methodology  Each SBE was added to cultures of human periodontal ligament (PL) cells or HT1080 cells and their supernatants were analysed by zymography for MMP-2. Each SBE was added to PL cell cultures, and the amount of TIMP-1 was determined by ELISA. P. gingivalis SBE was incubated with HT1080 cell culture supernatants, and the amounts of TIMP-1 and TIMP-2 were determined by ELISA. Statistical analysis was performed with the paired Student's t -test.
Results  In extracts of PL cells that had been incubated in the presence of P. gingivalis SBE, one representing pro-MMP-2 (72 kDa) and a band corresponding to the active MMP-2 (66 kDa) were observed; but in the other extracts it was not detected. When HT1080 cells were treated with P. gingivalis SBE, the pro-MMPs was processed into 86- and 66-kDa fragments, but in the other extracts, the processing did not occur when the other SBEs were used. When PL cells were incubated with the same SBEs, the amount of TIMP-1 was markedly decreased ( P  <   0.01), but in the other extracts, it was not. The amounts of both TIMP-1 and TIMP-2 were decreased in a dose-dependent manner when HT1080 cell culture supernatant was incubated with P. gingivalis SBE.
Conclusions  These findings suggest that P. gingivalis SBE may cause connective tissue to be destroyed, contributing to the process of periapical disease, by activating pro-MMP-2 as well as by inactivating TIMP-1 and TIMP-2.     

Examination of the signal transduction pathways leading to activation of gelatinolytic activity by interleukin-1alpha and Porphyromonas gingivalis in human osteosarcoma cells

BACKGROUND: Recently, evidence show that matrix metalloproteinases (MMP) play an important role in the pathogenesis of periodontal diseases. However, the mechanisms and signal transduction pathways involved in the production of MMPs in human osteosarcoma cells are not fully understood. OBJECTIVES: The purpose of this study was to investigate the gelatinolytic activity in human osteosarcoma cells stimulated with interleukin-1alpha (IL-1alpha) or Porphyromonas gingivalis in the absence or presence of SB203580 (p38 inhibitor), U0126 [mitogen-activated protein kinase kinase (MEK) inhibitor], and LY294002 [phosphatidylinositaol 3-kinase (PI3K) inhibitor]. METHODS: IL-1alpha and the supernatants of P. gingivalis were used to evaluate gelatinolytic activity in human osteosarcoma cells using gelatin zymography. Furthermore, to search possible signal transduction pathways, SB203580, U0126, and LY294002 were added to test how they modulated the gelatinolytic activity. Results: Gelatin zymography demonstrated that the latent proforms of gelatinases MMP-2 and MMP-9 were released by human osteosarcoma cells. Secretion of MMP-9 was time-dependent by stimulating with IL-1alpha or P. gingivalis. In addition, SB203580, U0126, and LY294002 significantly reduced the IL-1alpha or P. gingivalis-stimulated MMP-9 production, respectively (p < 0.05). However, none of the kinase inhibitors affected the MMP-2 level compared with the control during the 4-day culture period (p > 0.05). CONCLUSIONS: Our findings demonstrated that IL-1alpha and P. gingivalis enhance MMP-9 production in human osteosarcoma cells, and the signal transduction pathways p38, MEK, and PI3K are involved in the inhibition of MMP-9. SB203580, U0126, and LY294002 suppress MMP-9 production and/or activity and may therefore be valuable therapeutics in MMP-mediated periodontal destruction, and might be proved clinically useful agents, in combination with standard treatment modalities, in the treatment of periodontitis.     

Matrix metalloproteinase inhibitors reduce collagen gel contraction and alpha-smooth muscle actin expression by periodontal ligament cells

Background and Objective:  Orthodontic tooth movement requires remodeling of the periodontal tissues. The matrix metalloproteinases (MMPs) degrade the extracellular matrix components of the periodontal ligament, while the tissue inhibitors of metalloproteinases (TIMPs) control their activity. Synthetic MMP inhibitors have been developed to inhibit MMP activity. In this study, periodontal ligament cells in contracting collagen gels served as a model for enhanced periodontal remodeling. The effect of MMP inhibitors on gel contraction and on MMP and TIMP expression was analyzed.
Material and Methods:  Human periodontal ligament cells were cultured in three-dimensional collagen gels and incubated with the MMP inhibitors BB94, CMT-3, doxycycline and Ilomastat. Gel contraction was determined using consecutive photographs. The relative amounts of MMPs and TIMPs were analyzed using substrate zymography and mRNA expression using quantitative polyermase chain reaction.
Results:  All MMP inhibitors reduced MMP activity to about 20% of the control activity. They all reduced contraction, but CMT-3 and doxycycline had the strongest effect. These inhibitors also reduced MMP-2, MMP-3 and α-smooth muscle actin mRNA expression. The expression of MMP-1 mRNA seemed to be increased by CMT-3. No effects were found on the amounts of MMPs and TIMPs.
Conclusion:  Synthetic MMP inhibitors strongly reduced gel contraction by periodontal ligament cells. This was primarily caused by an inhibitory effect on MMP activity, which reduces matrix remodeling. In addition, α-smooth muscle actin expression was reduced by CMT-3 and doxycycline, which limits the contractile activity of the fibroblasts.     

Effect of hydroxamic acid-based matrix metalloproteinase inhibitors on human gingival cells and Porphyromonas gingivalis

BACKGROUND: Matrix metalloproteinases (MMPs) are considered to play key roles in tissue destruction during periodontitis. In this study, we evaluated the cytotoxicity of hydroxamic acid-based MMP inhibitors (ONO-4817, ONO-MI1-514, and ONO-MI1-570), and their inhibitory effects on MMP-2 and -9 activities and growth of Porphyromonas gingivalis. METHODS: Human gingival fibroblasts (HGF) and human gingival epithelial cells (HGE) were incubated with test inhibitors prior to investigating cell viability, cell proliferation, and mRNA expression for MMP-2 and -9. Gelatin zymography and a colorimetric MMP assay were performed to study the inhibitory effects on MMP-2 and -9 activities derived from HGF and HGE, respectively. The effect of MMP inhibitors on keratinocyte migration and P. gingivalis growth was also tested. RESULTS: Cell viability was not affected by any of the inhibitors at a final concentration of 50 microM, nor was cell proliferation at 20 microM. All inhibitors clearly inhibited MMP-2 produced by HGF and MMP-9 produced by HGE in a dose-dependent manner. No change was found in mRNA expression of MMPs by gingival cells treated with the inhibitors. ONO-4817 and ONO-MI1-514 inhibited keratinocyte migration. ONO-4817 showed a slightly inhibitory effect on the growth of P. gingivalis. CONCLUSION: Data obtained in this study support the potential use of the three MMP inhibitors for the prevention and treatment of periodontal disease.     

Levels of matrix metalloproteinases-8 and -9 with simultaneous presence of periodontal pathogens in gingival crevicular fluid as well as matrix metalloproteinase-9 and cholesterol in blood

BACKGROUND AND OBJECTIVES: To investigate the levels of matrix metalloproteinase (MMP) -8 and -9 with the simultaneous presence of periodontal pathogens in gingival crevicular fluid (GCF) as well as MMP-9 and cholesterol in blood. Although bacterial pathogens are required to initiate the periodontal disease process, in some individuals the reaction to bacteria may lead to an excessive host response, resulting in a general inflammatory response. METHODS: MMP-9 and lipids were analyzed from the blood samples of 33 subjects with a 16-year history and oral health records of periodontal disease as well as from 31 periodontally healthy controls. Information was obtained on education, body mass index, and family history of atherosclerosis. GCF was taken to determine MMP-8 and MMP-9 levels, and bacterial samples were simultaneously collected for polymerase chain reaction assessment of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Prevotella nigrescens, Tannerella forsythia, and Treponema denticola. Analysis of variance, chi-squared test, and multiple logistic regression analysis were used to analyze the results. RESULTS: Demographic data showed significant differences between patients and controls in smoking (P < 0.01), body mass index (P < 0.05), family history of atherosclerotic disease (P < 0.01), and education (P < 0.01). Significant differences were also observed in oral health data, in the detection of P. gingivalis (P < 0.001), P. intermedia (P < 0.01), P. nigrescens (P < 0.001), and T. forsythia (P < 0.001) and in the levels of MMP-8 and MMP-9 in GCF between patients and controls. T. forsythia[odds ratio(OR) 10.1; P = 0.001] and age (OR 5.54; P = 0.008) appeared to be the main independent predictors for high MMP-8 in GCF. Patients had significantly higher total cholesterol (P < 0.01), low-density lipoprotein cholesterol (P = 0.05), and triglycerides (P < = 0.01) than controls. Plasma levels of MMP-9 were significantly higher in patients than in controls (P = 0.001). CONCLUSIONS: Specific periodontal microorganisms appeared to induce host response, with increased release of MMP-8 and MMP-9 in gingival pockets as well as of MMP-9 in plasma, possibly triggering its up-regulation in blood.     

Expression of MMP-7 and MT1-MMP in oral squamous cell carcinoma as predictive indicator for tumor invasion and prognosis

BACKGROUND: Squamous cell carcinoma of the oral cavity is a highly invasive neoplasm that spreads locally and metastasizes to regional lymph nodes. This process involves multiple proteolytic enzymes including matrilysin (MMP-7) and membrane type I-matrix metalloproteinase (MT1-MMP). This study was designed to explore the association between MMP-7 and MT1-MMP in the invasiveness and prognosis of oral squamous cell carcinoma (OSCC). METHODS: About 4-microM, formalin-fixed, paraffin-embedded tissue sections from 69 patients with OSCC were immunohistochemically studied using specific antibodies against MMP-7 and MT1-MMP proteins. Immunostaining was semiquantitatively scored, and results were correlated with histologic and clinical variables including clinical behavior and survival. RESULTS: MMP-7 was observed only in cancer cells, and MT1-MMP in both tumoral tissue and stroma. MMP-7 expression was significantly correlated with lymph node metastasis (P = 0.03; RR = 3.2). MT1-MMP showed a significant association with TIMP-2 (in N+ cases) and p53 expression (P = 0.01). MMP-7 and MT1-MMP displayed a survival relevance, and in multivariate analysis they were independent prognostic indicators, particularly in neck node-positive cases.     

Proteinase-activated receptor-2 (PAR2) agonist causes periodontitis in rats

Proteinase-activated receptor-2 (PAR2) is a G-protein-coupled receptor that mediates cellular responses to extracellular proteinases. Since PAR2 is expressed by oral epithelial cells, osteoblasts, and gingival fibroblasts, where its activation releases interleukin-8, we hypothesized that PAR2 activation may participate in periodontal disease in vivo. We investigated the role of PAR2 activation in periodontal disease in rats. Radiographic and enzymatic (myeloperoxidase) analysis revealed that topical application of PAR2 agonist causes periodontitis but also exacerbates existing periodontitis, leading to significant alveolar bone loss and gingival granulocyte infiltration. Inhibition of matrix metalloproteinase (MMP) and cyclo-oxygenase (COX) decreased PAR2 agonist-induced periodontitis. More specifically, the overexpression of COX-1, COX-2, MMP-2, and MMP-9 in gingival tissues suggests that they are involved in PAR2-induced periodontitis. In conclusion, PAR2 agonist causes periodontitis in rats through a mechanism involving prostaglandin release and MMP activation. Inhibition of PAR2 may represent a novel approach to modulate host response in periodontitis.     

Down-regulation of interleukin-1alpha-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)-1alpha. IL-1alpha 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-1alpha-induced MMP-13 production in periodontal ligament cells, although both the agents completely inhibited IL-1alpha-induced PGE2 production. Exogenous PGE2 reduced IL-1alpha-induced MMP-13 mRNA and protein production in a dose-dependent manner. 17-phenyl-omega-trinor PGE2, a selective EP1 receptor agonist, mimicked the inhibitory effect of PGE2 on IL-1alpha-induced MMP-13 mRNA and protein production. On the basis of these data, we suggest that COX-2-dependent PGE2 down-regulates IL-1alpha-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.