Crosstalk Between Human Monocytic U937 Cells and Gingival Fibroblasts in Coculturally Enhanced Matrix Metalloproteinase‐2 Expression

Background: Matrix metalloproteinases (MMPs) play a key role in inflammatory periodontal disease. Synergistically enhanced MMP‐2 expression in a coculture of human gingival fibroblasts (HGFs) and human monocytic U937 cells was observed. Crosstalk between these two cells via the extracellular matrix metalloproteinase inducer (EMMPRIN) was demonstrated. Methods: Enzyme levels of MMP‐2 in HGFs and direct coculture with U937 were examined by zymography. MMP‐2 and EMMPRIN expressions of HGFs and U937 were determined in coculture and conditioned cultures (using supernatants from HGF‐ or U937‐conditioned medium). The crosstalk was evaluated by EMMPRIN extrasupplement and EMMPRIN inhibition, through pretreatment of U937 with cyclosporine‐A. Results: Direct coculturing of HGFs and U937 enhanced MMP‐2 enzyme level and mRNA expression. Coculturing also increased membranous EMMPRIN expression of U937, but not from HGFs. In conditioned cultures, mRNA expression of MMP‐2 increased in HGFs which received U937‐conditioned medium. Increased MMP‐2 was not observed in U937 with HGF‐conditioned medium, although mRNA expression of EMMPRIN increased. Enhanced MMP‐2 was observed after administration of exogenous EMMPRIN in HGFs; however, reduced MMP‐2 enzyme level was noted if EMMPRIN of cocultured U937 was inhibited. Conclusions: In the coculture of HGFs and U937, upregulated EMMPRIN expression in U937, which may be triggered by HGFs, can enhance MMP‐2 expression in HGFs. Crosstalk between HGFs and U937 involving MMP‐2 from HGFs was proposed; EMMPRIN from U937 may play a particular role.     

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.     

Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E(2) by hypoxia-inducible factor-1α up-regulation in human periodontal ligament cells

Kim Y‐S, Shin S‐I, Kang K‐L, Herr Y, Bae W‐J, Kim E‐C. Nicotine and lipopolysaccharide stimulate the production of MMPs and prostaglandin E₂ by hypoxia‐inducible factor‐1α up‐regulation in human periodontal ligament cells. J Periodont Res 2012; 47: 719–728. © 2012 John Wiley & Sons A/S Background and Objective: Although hypoxia‐inducible factor 1α (HIF‐1α) is up‐regulated in the periodontal pockets of periodontitis patients, the expression and precise molecular mechanisms of HIF‐1α remain unknown in human periodontal ligament cells (PDLCs). The aim of this study was to explore the effects, as well as the signaling pathway, of nicotine and lipopolysaccharide (LPS) on the expression of HIF‐1α and on the production of its target genes, including cyclooxygenase‐2 (COX‐2)‐derived prostaglandin E₂ (PGE₂), MMP‐2 and MMP‐9 in PDLCs. Material and Methods: The expression of COX‐2 and HIF‐1α proteins was evaluated using western blotting. The production of PGE₂ and MMPs was evaluated using enzyme immunoassays and zymography, respectively. Results: LPS and nicotine synergistically induced the production of PGE₂, MMP‐2 and MMP‐9, and increased the expression of MMP‐2, MMP‐9, COX‐2 and HIF‐1α proteins. Inhibition of HIF‐1α activity by chetomin or knockdown of HIF1α gene expression by small interfering RNA markedly attenuated the production of LPS‐ and nicotine‐stimulated PGE₂ and MMPs, as well as the expression of COX‐2 and HIF‐1α. Furthermore, pretreatment with inhibitors of COX‐2, p38, extracellular signal‐regulated kinase, Jun N‐terminal kinase, protein kinase C, phosphatidylinositol 3‐kinase and nuclear factor‐kappaB decreased the expression of nicotine‐ and LPS‐induced HIF‐1α and COX‐2, as well as the activity of PGE₂ and MMPs. Conclusion: These data demonstrate novel mechanisms by which nicotine and LPS promote periodontal tissue destruction, and provide further evidence that HIF‐1α is a potential target in periodontal disease associated with smoking and dental plaque.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

The Upregulation of Transglutaminase‐2 by Cyclosporin A in Human Gingival Fibroblasts Is Augmented by Oxidative Stress

Background: Transglutaminase‐2 (TGM‐2) has been implicated in several fibrotic disorders and can be induced by reactive oxygen species (ROS). Hence, the authors hypothesize that cyclosporin A (CsA) may regulate TGM‐2 via ROS, and this regulation may have a role in the pathogenesis of CsA‐induced gingival overgrowth. Methods: Cytotoxicity, 2′,7′‐dichlorodihydrofluorescein diacetate assay, and Western blot were used to investigate the effects of CsA in human gingival fibroblasts (HGFs). In addition, extracellular signal‐regulated kinase (ERK) inhibitor PD98059, phosphatidylinositol 3‐kinase inhibitor LY294002, glutathione precursor N‐acetyl‐L‐cysteine (NAC), curcumin, epigallocatechin‐3 gallate (EGCG), and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Concentrations of CsA >500 ng/mL demonstrated cytotoxicity to HGFs (P < 0.05). CsA enhanced the generation of intracellular ROS at concentrations >200 ng/mL (P <0.05). TGM‐2 protein induced by CsA was found in HGFs in a dose‐ and time‐dependent manner (P <0.05). The addition of PD98059, LY294002, NAC, curcumin, EGCG, and SB203580 markedly inhibited TGM‐2 expression induced by CsA (P <0.05). Conclusions: These results demonstrate that CsA significantly upregulates intracellular ROS generation and elevates TGM‐2 expression in HGFs. In addition, TGM‐2 induced by CsA is downregulated by PD98059, LY294002, NAC, curcumin, EGCG, and SB203580.     

Nicotine modulates gelatinase B (MMP‐9) and epilysin (MMP‐28) expression in reconstituted human oral epithelium

J Oral Pathol Med (2011) 40 : 33–36 Oral epithelial keratinocytes express nicotinic cholinergic receptors which activation modulates keratinocytes differentiation and migration through different metabolic pathways. Matrix metalloproteinases (MMPs) are Zn‐dependent enzyme involved in cell migration. Among them, gelatinase B (MMP‐9) and epilysin (MMP‐28) are two MMPs expressed by human keratinocytes during both wound healing and proliferation. Their expression has been investigated in a reconstituted human oral epithelium (HOE) exposed to nicotine (Nic, 1–50 μM) for 72 h both in the absence and presence of the nicotinic antagonist mecamylamine (Mec), H7, a PKC inhibitor and PD98059, a MAPK inhibitor (PD). At the end of treatment, MMP‐28 expression has been analyzed in epithelium sections using an anti‐MMP‐28 antibody, whereas MMP‐9 presence and activity has been measured in cell‐conditioned medium analyzed by gelatine zymography. The expression of MMP‐9 was reduced by Nic in a dose‐dependent fashion and this effect was antagonized by Mec, H7 and PD. On the other hand, Nic increased the expression of MMP‐28, and this effect was blocked both by H7 and PD, whereas Mec even enforced it. Nic effects on MMP‐9 and MMP‐28 expression by oral keratinocytes were not previously reported and these data suggest MMPs expression mediated by PKC and MAPK as a possible target for Nic toxicity in oral epithelium.     

Endoplasmic reticulum stress modulates nicotine-induced extracellular matrix degradation in human periodontal ligament cells

Lee S‐I, Kang K‐L, Shin S‐I, Herr Y, Lee Y‐M, Kim E‐C. Endoplasmic reticulum stress modulates nicotine‐induced extracellular matrix degradation in human periodontal ligament cells. J Periodont Res 2012; 47: 299–308. © 2012 John Wiley & Sons A/S Background and Objective: Tobacco smoking is considered to be one of the major risk factors for periodontitis. For example, about half the risk of periodontitis can be attributable to smoking in the USA. It is evident that smokers have greater bone loss, greater attachment loss and deeper periodontal pockets than nonsmoking patients. It has recently been reported that endoplasmic reticulum (ER) stress markers are upregulated in periodontitis patients; however, the direct effects of nicotine on ER stress in regard to extracellular matrix (ECM) degradation are unclear. The purpose of this study was to examine the effects of nicotine on cytotoxicity and expression of ER stress markers, selected ECM molecules and MMPs, and to identify the underlying mechanisms in human periodontal ligament cells. We also examined whether ER stress was responsible for the nicotine‐induced cytotoxicity and ECM degradation. Material and Methods: Cytotoxicity and cell death were measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyltetrazolium bromide assay and flow cytometric annexin V and propidium iodide staining. The mRNA and protein expressions of MMPs and ER markers were examined by RT‐PCR and western blot analysis. Results: Treatment with nicotine reduced cell viability and increased the proportion of annexin V‐negative, propidium iodide‐positive cells, an indication of cell death. Nicotine induced ER stress, as evidenced by survival molecules, such as phosphorylated protein kinase‐like ER‐resident kinase, phosphorylated eukaryotic initiation factor‐2α and glucose‐regulated protein‐78, and apoptotic molecules, such as CAAT/enhancer binding protein homologous protein (CHOP). Nicotine treatment led to the downregulation of ECM molecules, including collagen type I, elastin and fibronectin, and upregulation of MMPs (MMP‐1, MMP‐2, MMP‐8 and MMP‐9). Inhibition of ER stress by salubrinal and transfection of CHOP small interfering RNA attenuated the nicotine‐induced cell death, ECM degradation and production of MMPs. Salubrinal and CHOP small interfering RNA inhibited the effects of nicotine on the activation of Akt, JNK and nuclear factor‐κB. Conclusion: These results indicate that nicotine‐induced cell death is mediated by the ER stress pathway, involving ECM degradation by MMPs, in human periodontal ligament cells.     

Kaempferol Inhibits P. intermedia Lipopolysaccharide‐Induced Production of Nitric Oxide Through Translational Regulation in Murine Macrophages: Critical Role of Heme Oxygenase‐1‐Mediated ROS Reduction

Background: Nitric oxide (NO) could be a potential target for the development of new therapeutic approaches to the treatment of periodontal disease because this molecule plays a significant role in the tissue destruction observed in periodontitis. In this study, the authors investigate the effect of kaempferol on the production of NO by murine macrophage‐like RAW264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen implicated in periodontal disease, and try to determine the underlying mechanisms of action. Methods: NO production was assayed by measuring the accumulation of nitrite in culture supernatants. Real‐time polymerase chain reaction was performed to quantify inducible NO synthase (iNOS) and heme oxygenase‐1 (HO‐1) mRNA expression. iNOS and HO‐1 protein expression and phosphorylation of c‐Jun N‐terminal kinase and p38 were characterized via immunoblot analysis. Reactive oxygen species (ROS) production was measured using the redox‐sensitive fluorescent probe 2′,7′‐dichlorodihydrofluorescein diacetate. Results: Kaempferol significantly inhibited NO production and expression of iNOS protein in P. intermedia LPS‐stimulated RAW246.7 cells without affecting iNOS mRNA expression. Kaempferol upregulated HO‐1 expression in LPS‐activated cells. Inhibition of HO‐1 activity by tin protoporphyrin IX (SnPP) abolished the suppressive effect of kaempferol on NO production. In addition, kaempferol significantly attenuated P. intermedia LPS‐induced increase of intracellular ROS, and SnPP blocked this reduction. Treatment with antioxidants downregulated the production of LPS‐induced NO. Conclusions: Kaempferol inhibits NO production and iNOS protein expression in P. intermedia LPS‐stimulated RAW264.7 cells at the translational level via HO‐1‐mediated ROS reduction and could be an efficient modulator of host response in the treatment of periodontal disease.     

l‐arginine‐dependent nitric oxide production of a murine macrophage‐like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg‐LPS). RAW264.7 cells were incubated with i) various concentrations of Pg‐LPS or Salmonella typhosa LPS (St‐LPS), ii) Pg‐LPS with or without l ‐arginine and/or N^G‐monomethyl‐l ‐arginine (NMMA), an arginine analog or iii) Pg‐LPS and interferon‐γ (IFN‐γ) with or without anti‐IFN‐γ antibodies or interleukin‐10 (IL‐10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg‐LPS, but was observed after stimulation with St‐LPS. Exogenous l ‐arginine restored the ability of Pg‐LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg‐LPS with exogenous l ‐arginine was abolished by NMMA. IFN‐γ induced independent NO production by Pg‐LPS‐stimulated macrophages and this stimulatory effect of IFN‐γ could be completely suppressed by anti‐IFN‐γ antibodies and IL‐10. These results suggest that Pg‐LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an l ‐arginine‐dependent mechanism which is itself independent of the action of IFN‐γ.     

Proteolytic roles of matrix metalloproteinase (MMP)‐13 during progression of chronic periodontitis: initial evidence for MMP‐13/MMP‐9 activation cascade

Aim: Matrix metalloproteinases (MMP)‐13 can initiate bone resorption and activate proMMP‐9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP‐13 activity and TIMP‐1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy‐terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP‐13 could potentiate gelatinase activation in diseased gingival tissue. Materials and Methods: In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP‐13 activity and TIMP‐1. Diseased gingival explants were cultured, treated or not with MMP‐13 with or without adding CL‐82198, a synthetic MMP‐13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis. Results: Active sites demonstrated increased ICTP levels and MMP‐13 activity (p<0.05) in progression subjects. The MMP‐9 activation rate was elevated in MMP‐13‐treated explants (p<0.05) and MMP‐13 inhibitor prevented MMP‐9 activation. Conclusions: MMP‐13 could be implicated in the degradation of soft and hard supporting tissues and proMMP‐9 activation during progression of chronic periodontitis. MMP‐13 and ‐9 can potentially form an activation cascade overcoming the protective TIMP‐1 shield, which may become useful for diagnostic aims and a target for drug development.     

Differentiated expression of membrane type metalloproteinases (MMP‐14, MMP‐15) and pro‐MMP2 in laryngeal squamous cell carcinoma. A novel mechanism

Cancer progression involves multiple proteolytic interactions, with metalloproteinases (MMPs) performing a crucial role. MMP‐2, a major MMP, plays a key role in the degradation of basement membranes. Mechanisms underlying MMP‐2 activation had to be investigated. Membrane‐type matrix metalloproteinases are not only responsible for the regulation of extracellular matrix remodeling, but also involved in the activation of several inactive MMPs. The aim of this study was to evaluate the expression of pro‐MMP2, MMP‐14, and MMP‐15 in tumor cells and tumor stroma. Immunohistochemical studies were performed on paraffin‐embedded tissue sections including laryngeal squamous cell carcinoma (SCC). We found the expression of pro‐MMP2 in 58% of cases, MMP‐14 in 78%, and MMP‐15 in 98% of cases of SCC. In all tumor cases, we revealed a higher expression of pro‐MMP2 in tumor stoma than in tumor cells. The expression of MMP‐14 and MMP‐15 was higher in tumor cells than in the stroma. Moreover, we found a statistically significant difference between the expression of MMP‐14 and MMP‐15 in the tumor in comparison with the surrounding stroma (P < 0.05). An analysis of expression levels of MT‐MMPs by classification trees showed that the probability of metastases was related to decreased expression of MMP‐14 and increased expression of MMP‐15. Our results may suggest that tumor cells with low MMP‐14 expression invade tumor stroma and form metastases. Probably, in such cases, tumor progression is stimulated by MMP‐15 in an MMP‐14 independent pathway, a novel (alternative) mechanism.     

A Sialidase‐Deficient Porphyromonas gingivalis Mutant Strain Induces Less Interleukin‐1β and Tumor Necrosis Factor‐α in Epi4 Cells Than W83 Strain Through Regulation of c‐Jun N‐Terminal Kinase Pathway

Background: Porphyromonas gingivalis is one of the major periodontal pathogens. In a previous study, a mouse abscess model showed that sialidase deficiency of P. gingivalis weakened its virulence, but the mechanism behind this observation remains unknown. Methods: A sialidase‐deficient mutant strain (△PG0352) and a complemented strain (com△PG0352) were constructed. Epi4 cells were stimulated by wild‐type strain P. gingivalis W83, △PG0352, or com△PG0352. Real‐time polymerase chain reaction was carried out to detect expression of virulent genes in P. gingivalis and interleukin (IL)‐1β, IL‐6, IL‐8, and tumor necrosis factor (TNF)‐α in epi4 cells. Activities of sialidase, gingipains, and lipopolysaccharide (LPS) were compared among the different P. gingivalis strains. Levels of IL‐1β and TNF‐α in the epi4 cells supernatant were detected by enzyme‐linked immunosorbent assay and levels of p38, extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase (JNK), and phospho‐c‐Jun were detected by western blotting. Results: Compared with P. gingivalis W83 and com△PG0352, activities of Kgp and Rgp gingipains and amount of LPS decreased in △PG0352, whereas there were no differences in LPS activity among these three strains. Level of phospho‐JNK was lower in epi4 cells stimulated by △PG0352. △pG0352 induced less IL‐1β and TNF‐α and more IL‐8 in epi4 cells; differences in IL‐1β and TNF‐α could not be detected after JNK blocking. Conclusion: A sialidase‐deficient P. gingivalis mutant strain induces less IL‐1β and TNF‐α in epi4 cells than W83 strain through regulation of JNK pathway.