Porphyromonas gingivalis affects host collagen degradation by affecting expression, activation, and inhibition of matrix metalloproteinases.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Zhou
Jing
J
Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, IN 46202, USA. jzhou2@iupui.edu
Windsor
L Jack
LJ
D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D024042:Collagen Type I / Q000378:metabolism; D017077:Culture Media, Conditioned; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism; D005881:Gingiva / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000032:analysis; D020778:Matrix Metalloproteinase 2 / Q000032:analysis; D019278:Matrix Metalloproteinase 3 / Q000032:analysis; D020782:Matrix Metalloproteinases / Q000032:analysis / Q000502:physiology*; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000032:analysis; D019714:Tissue Inhibitor of Metalloproteinases / Q000032:analysis
D024042:Collagen Type I; D017077:Culture Media, Conditioned; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D019714:Tissue Inhibitor of Metalloproteinases; D003094:Collagen; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D020778:Matrix Metalloproteinase 2; D020781:Matrix Metalloproteinase 1