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: Members of the matrix metalloproteinase (MMP) family have been shown to be involved in periodontal disease. Risk factors for periodontal disease include tobacco smoking. Cigarette smoke condensate (CSC) is comprised of thousands of chemicals. Nicotine is one of the active components in tobacco. This study compares the effects of CSC and nicotine at the level in CSC on the collagen-degrading ability of human gingival fibroblasts (HGFs) and the expression of selected MMPs and tissue inhibitors of metalloproteinases (TIMPs). HGFs were seeded in six-well collagen-coated plates, exposed to 100 μg/mL (2.4 μg/mL nicotine) of CSC or 2.4 μg/mL nicotine for 3 days, and then collagen degradation was analyzed. After 3 days exposure to CSC or nicotine, the conditioned media from HGFs was collected and the membrane proteins were extracted for gelatin zymography and Western blot analyses. The mRNA levels of MMP-2, MMP-14, and TIMP-2 were measured by reverse transcription-polymerase chain reaction. The CSC increased collagen degradation, and increased the levels of TIMP-2, MMP-14, and the active MMP-2 in the membrane extracts, and their mRNA levels. CSC also increased the level of active MMP-2 in the conditioned media. Nicotine at the level in CSC (2.4 μg/mL) had little influence on collagen degradation, as well as on the protein and mRNA levels of MMP-2, MMP-14, and TIMP-2. CSC may increase HGF-mediated collagen degradation by affecting membrane-associated MMPs and TIMPs.Journal of Periodontology 12/2010; 82(7):1071-9. · 2.40 Impact Factor