J Korean Acad Conserv Dent.  2005 Sep;30(5):372-384.

MMP and TIMP production in periodontal ligament fibroblasts stimulated by Prevotella nigrescens lipopolysaccharide

• Yang WK
• Lee W
• Kim MR
• Son HH

Abstract

The purpose of this study was to monitor the secretion of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) by human periodontal ligament (PDL) fibroblasts stimulated with Prevotella nigrescens lipopolysaccharide (LPS), and to examine the effect of calcium hydroxide treatment on P. nigrescens LPS. LPS was extracted and purified from anaerobically cultured P. nigrescens. PDL fibroblasts were stimulated by the LPS (0, 0.1, 1, 10 ug/ml) or LPS (10 ug/ml) pretreated with 12.5 mg/ml of Ca(OH)2 for 3 days, for various periods of time (12, 24, 48 h). Immunoprecipitation were performed for protein level analysis of MMP-1, MMP-2 and TIMP-1. Total RNA was isolated and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 mRNA. According to this study, the results were as follows: 1. The production of MMP-1 by stimulation with P. nigrescens LPS increased in time-dependent manner, and showed maximum value at 48 h in both protein and mRNA level. But there was no dose-dependent increase. 2. MMP-2 production time-dependently increased when stimulated with 1 and 10 ug/ml LPS, but there was no dose-dependent increase. 3. TIMP-1 production increased to 24 h, but decreased at 48 h. It increased when stimulated with 0.1 and 1 ug/ml LPS, but suppressed at 10 ug/ml. 4. P. nigrescens LPS pretreated with Ca(OH)2 markedly downregulated MMP-1 gene expression.

Keyword

MMP-1; MMP-2; TIMP-1; P. nigrescens; LPS; Ca(OH)2; PDL fibroblast

MeSH Terms

Calcium Hydroxide
Fibroblasts*
Gene Expression
Humans
Immunoprecipitation
Periodontal Ligament*
Polymerase Chain Reaction
Prevotella nigrescens*
Prevotella*
RNA
RNA, Messenger
Tissue Inhibitor of Metalloproteinase-1
Calcium Hydroxide
RNA
RNA, Messenger
Tissue Inhibitor of Metalloproteinase-1

Figure

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  Figure 1 E. coli (left) and P. nigrescens (Right) LPS confirmed by SDS-PAGE and silver staining.

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  Figure 2 Immunohistochemical staining of human PDL cells for the confirmation of cytoplasmic marker (β-subunit of proly-4-hydroxlyase and disulfideisomerase) of fibroblasts.

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  Figure 3 Effects of LPS on MMP-1 protein production in PDL fibroblasts. upper. immunoprecipitation analysis, lower. quantification of band intensity relative to negative control (0 µg/ml, 12 h).

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  Figure 4 Effects of LPS on MMP-2 protein production in PDL fibroblasts. upper. immunoprecipitation analysis, lower. quantification of band intensity relative to negative control (0 µg/ml, 12 h).

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  Figure 5 Effects of LPS on TIMP-1 protein production in PDL fibroblasts. upper. immunoprecipitation analysis, lower. quantification of band intensity relative to negative control (0 µg/ml, 12 h).

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  Figure 6 Effects of LPS on MMP-1 mRNA expression in PDL fibroblasts analyzed by real-time PCR and normalized with GAPDH mRNA level, relative to negative control (0 µg/ml, 12 h).

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  Figure 7 Effects of LPS pretreated with Ca(OH)2 on MMP-1 mRNA expression in PDL fibroblasts analyzed by real-time PCR and normalized with GAPDH mRNA level, relative to negative control (0 µg/ml, 12 h). *C-H; calcium hydroxide treated LPS

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