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Interleukin-1 Enhances Type I Collagen-induced Activation of Matrix Metalloproteinase-2 in Odontogenic Keratocyst Fibroblasts

Second Department of Oral and Maxillofacial Surgery, Faculty of Dentistry, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan;

View larger version (30K): [in a new window]   Figure 1. Effects of ECMs on gelatinases secretion in odontogenic keratocyst fibroblasts. Gelatin zymography of culture media. Fibroblasts were cultured on plastic plates (lanes 1 and 2), or 30 µg/cm2 fibronectin (lanes 3 and 4)-, 30 µg/cm2 laminin (lanes 5 and 6)-, or 30 µg/cm2 type I collagen (lanes 7, 8, and 9)-coated dishes in serum-free DMEM for 48 hrs at 37°C in the presence (lanes 2, 4, 6, 8, and 9) or absence (lanes 1, 3, 5, and 7) of 1 nM rhIL-1. Anti-hIL-1 antibody (Anti-IL-1) was added to the culture media prior to incubation with rhIL-1 (lane 9). The culture media (30 µL) were subjected to gelatin zymography. Similar results were seen in four independent experiments.

View larger version (19K): [in a new window]   Figure 2. Effects of IL-1 on the activation of proMMP-2 in odontogenic keratocyst fibroblasts cultured on type I collagen-coated dish. Fibroblasts were cultured in serum-free DMEM in the absence (0) or presence of various concentrations of rhIL-1 (0.01-10 nM) on plastic plates () or 30 µg/cm2 type I collagen-coated dishes () for 48 hrs at 37°C. Activity ratio of MMP-2 was calculated as described under "MATERIALS & METHODS". Vertical bars indicate mean ± SE (n = 4). *p < 0.05 compared with the value of the cells grown on type I collagen-coated dishes without rhIL-1.

View larger version (49K): [in a new window]   Figure 3. Effects of fibroblasts on proMMP-2 activation. Fibroblasts were cultured on 30 µg/cm2 type I collagen-coated dishes in serum-free DMEM for 48 hrs at 37°C. Then, the collected culture media were incubated with (lanes 1 and 2) or without (lane 3) the fibroblasts cultured on type I collagen-coated dishes for 18 hrs at 37°C in the absence (lane 1) or presence (lanes 2 and 3) of 1 nM rhIL-1. The culture media (30 µL) were subjected to gelatin zymography. Similar results were seen in four independent experiments.

View larger version (26K): [in a new window]   Figure 4. Effects of IL-1 and type I collagen on the expression of MT1-MMP mRNA and protein in odontogenic keratocyst fibroblasts. Agarose gel of the PCR products of MT1-MMP and ß-actin (A) and Western immunoblotting for MT1-MMP (B). Odontogenic keratocyst fibroblasts were incubated on plastic plates (lanes 1 and 2) or 30 µg/cm2 type I collagen-coated dishes (lanes 3 and 4) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 1 nM rhIL-1 for 48 hrs at 37°C. (A) Total cellular RNA was extracted, and PCR amplification was performed at 35 cycles. The PCR products of MT1-MMP and ß-actin were resolved by electrophoresis on 1.8% agarose gel. (B) The fibroblasts were lysed in SDS sample buffer, and the aliquots of cell lysates were subjected to Western immunoblotting for MT1-MMP as described under "MATERIALS & METHODS". Similar results were seen in six independent experiments.