Porphyromonas gingivalis affects host collagen degradation by affecting expression, activation, and inhibition of matrix metalloproteinases.
ABSTRACT Studies have shown that Porphyromonas gingivalis and host matrix metalloproteinases (MMPs) play important roles in the tissue destruction associated with periodontal disease. It is still unclear which MMPs or their inhibitors are regulated by P. gingivalis at the transcriptional and/or at the protein levels. Therefore, this study was conducted to determine what effects P. gingivalis supernatant has on the collagen degrading ability of human gingival fibroblasts (HGFs) and how it regulates the activation, mRNA expression, and inhibition of MMPs.
Culture supernatant from P. gingivalis ATCC 33277 was added to HGFs cultured in six-well plates coated with Type I collagen. At certain time intervals, the cell conditioned media was collected for zymography and/or western blot analyses to determine the MMP and tissue inhibitor of MMPs (TIMP) protein levels. The cells were then removed and the collagen cleavage visualized by Coomassie blue staining. The mRNA expression of multiple MMPs and TIMPs by the treated and untreated HGFs was determined by reverse transcription-polymerase chain reaction.
The collagen in the six-well plates was degraded more rapidly by the HGFs treated with 10% v/v P. gingivalis supernatant. More active MMP-1, MMP-2, MMP-3, and MMP-14 were detected in the conditioned media from the HGFs treated with the P. gingivalis supernatant. TIMP-1, but not TIMP-2, was decreased in the presence of the P. gingivalis supernatant. MMP-1 mRNA expression by the treated HGFs increased more than two-fold over the untreated HGFs. MMP-3 mRNA was unchanged, MMP-2 mRNA had a slight increase, MMP-14 mRNA decreased, and MMP-15 increased. MMP-12 mRNA was induced in the P. gingivalis treated HGFs. TIMP-1 and TIMP-2 mRNA had a slight increase with P. gingivalis treatment.
Porphyromonas gingivalis increased the collagen degrading ability of HGFs, in part, by increasing MMP activation and by lowering the TIMP-1 protein level, as well as by affecting the mRNA expression of multiple MMPs and TIMPs.
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ABSTRACT: Tissue destruction in periodontitis is initiated by bacteria, such as Porphyromonas gingivalis (P. gingivalis), and is caused largely by host responses. Resolvins protect the host against acute inflammation by blocking the migration of polymorphonuclear neutrophils (PMNs) to initiate resolution. The effects of Resolvins on human gingival fibroblasts (HGFs) are unknown. The effects of Resolvin D1 on HGF survival and cytokine expression when treated with/without P. gingivalis supernatant were examined. Methods: Cytotoxicity of Resolvin D1 on HGFs with/without a toxic level of P. gingivalis supernatant was measured with Lactate dehydrogenase (LDH) assays. Cytokine arrays were performed on HGFs conditioned media treated with/without Resolvin D1, and with/without P. gingivalis supernatant. Results: Resolvin D1 had no cytotoxic effects on the HGFs at concentrations between 1-1000 nM (all p values > 0.05). Resolvin D1 (1000 nM) significantly inhibited the toxic effects of 13.5% (v/v) P. gingivalis supernatant (p= 0.002) on HGFs. Resolvin D1 significantly reduced the expression of Interleukin (IL)-6 (p= 0.010) and Monocyte Chemoattractant Protein (MCP)-1 (p= 0.04) from untreated fibroblasts. P. gingivalis (10%) supernatant significantly increased the levels of expression of Granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, Growth Regulated Oncogene (GRO), IL-5, IL-6, IL-7, IL-8, IL-10, MCP-1, MCP-2, MCP-3 and Monokine induced by Gamma Interferon (MIG). Resolvin D1 significantly reduced the expression of GRO (p= 0.04) from P. gingivalis supernatant treated HGFs, marginally reduced the levels of MCP-1(p= 0.10) and marginally increased the levels of Transforming Growth Factor (TGF)-β1 (p= 0.07). Conclusion: Resolvin D1 altered the cytotoxicity of P. gingivalis supernatant on HGFs. Resolvin D1 significantly reduced GRO, marginally reduced MCP-1 and marginally increased TGF-β1 from P. gingivalis treated HGFs, which could alter the ability of P. gingivalis to induce inflammation.Journal of Periodontology 02/2013; · 2.40 Impact Factor
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ABSTRACT: Elderly people exhibit increased susceptibility to a number of autoimmune and infectious diseases, such as periodontitis. Although aging is reportedly associated with a decline in immune function, age-related alterations in periodontal tissue have remained elusive. In the present study, we comprehensively analyzed the effect of aging on the expression of selected genes using mouse gingival fibroblasts. Gingival fibroblasts derived from young (8 wk of age) and old (≥ 24 mo of age) C57BL/6 mice were stimulated with Porphyromonas gingivalis lipopolysaccharide or live P. gingivalis strain W83. Expression of cytokines/chemokines, innate immune receptors, growth factors, matrix metalloproteinases, tissue inhibitors of metalloproteinases and osteoclastogenesis-related molecules were evaluated using real-time polymerase chain reaction and ELISA for interleukin-6 and transforming growth factor-β1. Gingival fibroblasts derived from old mice exhibited decreased gene expression of Il-6, Cxcl1, Tlr2, Tlr4, Irak3 (IRAK-M), Kgf, Timp1, Timp3 and Rankl under resting conditions, whereas the expression levels of Tgfβ1, Mmp3, Mmp13 and Opg were increased. Age-related differences were also detected at the protein level. Although P. gingivalis W83 upregulated Vegf, Fgf-2 and Bmp2 expression in both young and old gingival fibroblasts, the stimulatory effect on these genes was significantly lower in old gingival fibroblasts. Our findings demonstrated that aging altered the expression of a number of genes in gingival fibroblasts. Thus, alterations in the balance of these molecules could play a critical role in periodontitis progression in the elderly.Journal of Periodontal Research 10/2013; · 1.99 Impact Factor
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ABSTRACT: BACKGROUND: Porphyromonas gingivalis lipopolysaccharide (LPS) is a crucial virulence factor strongly associated with chronic periodontitis which is the primary cause of tooth loss in adults. It exhibits remarkable heterogeneity containing tetra-(LPS1435/1449) and penta-(LPS1690) acylated lipid A structures. Human gingival fibroblasts (HGFs) as the main resident cells of human gingiva play a key role in regulating matrix metalloproteinases (MMPs) and contribute to periodontal homeostasis. This study investigated the expression and regulation of MMPs1-3 and tissue inhibitors of MMP-1 (TIMP-1) in HGFs in response to P. gingivalis LPS1435/1449 and LPS1690 and hexa-acylated E. coli LPS as a reference. The expression of MMPs 1--3 and TIMP-1 was evaluated by real-time PCR and ELISA. RESULTS: The MMP-3 mRNA and protein were highly upregulated in P. gingivalis LPS1690- and E. coli LPS-treated cells, whereas no induction was observed in P. gingivalis LPS1435/1449-treated cells. On the contrary, the expression of MMP-1 and -2 was not significantly affected by P. gingivalis LPS lipid A heterogeneity. The TIMP-1 mRNA was upregulated in P. gingivalis LPS1435/1449- and E. coli LPS-treated cells. Next, signal transduction pathways involved in P. gingivalis LPS-induced expression of MMP-3 were examined by blocking assays. Blockage of p38 MAPK and ERK significantly inhibited P. gingivalis LPS1690-induced MMP-3 expression in HGFs. CONCLUSION: The present findings suggest that the heterogeneous lipid A structures of P. gingivalis LPS differentially modulate the expression of MMP-3 in HGFs, which may play a role in periodontal pathogenesis.BMC Microbiology 03/2013; 13(1):73. · 3.10 Impact Factor