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Fibrillin-2 Degradation by Matrix Metalloproteinase-2 in Periodontium

Department of Oral Anatomy, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Tobetsu, Hokkaido 061-0293, Japan

View larger version (31K): [in this window] [in a new window]   Figure 1. Active MMP-2 is present only in human periodontal ligament (PDL) fibroblast cell layers. (A) Immunoblotting of MMP-2, membrane-type matrix metalloproteinases-1 (MT1-MMP), and tissue inhibitor of matrix metalloproteinsase-2 (TIMP-2). Gingival and PDL fibroblasts were cultured for 6 wks. A 10-µg quantity of cell lysate underwent electrophoresis on 10% polyacrylamide gels and was immunoblotted for MMP-2, MT1-MMP, and TIMP-2 analyses. Equivalent protein loading was confirmed by ß-actin. (B) MMP immunoreactivity in biotinylated and intracellular fractions of PDL fibroblasts. PDL fibroblasts were cultured for 6 wks. Cell-surface and extracellular proteins were then biotinylated with non-cell-permeable biotin, and the biotinylated and intracellular fractions were immunoblotted with MMP-2 (upper panel) and actin (lower panel). (C) MMP-2 in the culture media. Serum-free culture media were collected from gingival and PDL fibroblasts at 6 wks. The harvested media (10 µg) were subjected to gelatin zymography.

View larger version (34K): [in this window] [in a new window]   Figure 2. MMPI or TIMP-2 inhibits active MMP-2. Gingival and PDL fibroblasts were cultured for 6 wks, and then the medium was changed to MEM containing DMSO or various concentrations of MMPI (0, 5, 50 µM) for an additional 2 wks. TIMP-2 (0, 50, 100 ng/mL) was also added to the media through the same experimental periods. At 8 wks, the gingival and PDL fibroblast cell layers were harvested, and the cell lysates were analyzed by gelatin zymography.

View larger version (41K): [in this window] [in a new window]   Figure 3. MMP-2 degrades fibrillin-2, not fibrillin-1, in PDL fibroblast cell layers. (A) Gingival fibroblasts (G) and PDL fibroblasts (P) were cultured for 6 wks, after which MMPI (5 µM) or TIMP-2 (50 ng/mL) was added to the media for 2 wks. Microfibril-containing fractions (10 µg) were analyzed by Western blotting. (B) Densitometric scanning of the bands was performed with National Institute of Health imaging software, as shown in (A). The values for each six-week culture sample were arbitrarily assigned a value of 100. The results represent the means ± SD (standard deviation) of 3 independent experimental determinations (*p < 0.05).

View larger version (37K): [in this window] [in a new window]   Figure 4. MMPs degrade fibrillin-2 and produce a fibrillin-2 fragment in PDL fibroblast cell layers. The microfibril-containing fraction of the PDL fibroblast cell layer was subjected to fibrillin-2 immunoprecipitation. In control samples at 6 wks, a single 350-kDa protein (fibrillin-2) was detectable (arrowhead). From 6 to 8 wks of culture, a 180-kDa band (asterisk), as well as the 350-kDa band, was detectable. Upon treatment with MMPI, the 180-kDa band decreased in parallel with an increase of the 350-kDa band.