Fig 1.
Investigation of the expression of genes involved in cell–cell and cell–matrix interactions using a PCR array.
The graph shows the fold changes of gene expression in IL-1Ra and control siRNA-transfected Ca9-22 cells. MMP-13 was up-regulated in IL-1Ra siRNA-transfected cells by 4.8-fold (n = 6). The red circle (most up-regulated gene) indicates MMP-13 expression in the graph. TIMP-1 and TIMP-2 expression levels are indicated by arrows.
Fig 2.
Suppression of IL-1Ra expression using siRNA.
(A) Determination of IL-1Ra mRNA levels using real-time PCR. At 48 hours after transfection, IL-1Ra siRNA-transfected cells showed clear knockdown of IL-1Ra mRNA compared with the control. Differences among groups were analyzed using the Student’s t-test. Data are expressed as the mean ± SD (n = 6). **p < 0.01 vs control. (B) Western blot of IL-1Ra (18 KDa) in cells transfected with either IL-1Ra or control siRNAs (n = 3). Data are representative of three independent experiments.
Fig 3.
Up-regulation of MMP-13 by IL-1Ra siRNA.
(A) MMP-13 mRNA expression in IL-1Ra and control siRNA-transfected cells. The cells were cultured for 1, 3, 6, 12, and 24 hours, and then mRNA levels were determined using real-time PCR. Values represent fold changes. Differences among groups were analyzed using the Student’s t-test. Data are expressed as the mean ± SD (n = 6). **p < 0.01 vs each time point control. (B) Western blot of MMP-13 (60 kDa) in cells transfected with either IL-1Ra or control siRNAs. Data are representative of three independent experiments. (C) Activity of the endogenous active form of MMP-13 was measured by a SensoLyte® Plus 520 MMP-13 assay. Differences among groups were analyzed using the Student’s t-test. Data are expressed as the mean ± SD (n = 3). **p < 0.01 vs control.
Fig 4.
TIMP expression in IL-1Ra siRNA-transfected cells.
(A) TIMP-1 and TIMP-2 mRNA expression in IL-1Ra and control siRNA-transfected cells. The cells were cultured for 1, 3, 6, 12, and 24 hours, and then mRNA levels were determined using real-time PCR. Values represent fold changes. Differences among groups were analyzed using the Student’s t-test. Data are expressed as the mean ± SD (n = 3). *p < 0.05 vs each time point control. (B) Western blot of TIMP-1 (23 kDa) and -2 (21 kDa) in cells transfected with either IL-1Ra or control siRNAs. Data are representative of three independent experiments.
Fig 5.
Down-regulation of MMP-13 expression by rhIL-1Ra.
MMP-13 mRNA expression in IL-1Ra and control siRNA-transfected cells treated with or without rIL-1Ra. The cells were cultured for 6 hours, and then MMP-13 mRNA levels were determined using real-time PCR. Values represent fold changes. Differences among groups were analyzed by one-factor ANOVA and Bonferroni’s multiple comparison test. Data are expressed as the mean ± SD (n = 3). **p < 0.01 vs control and IL-1Ra siRNA + rIL-1Ra 40 ng/ml.
Fig 6.
Neutralization of IL-1 does not affect MMP-13 up-regulation.
(A) IL-1α and (B) IL-1β mRNA (upper column) and protein expression (lower column) in IL-1Ra siRNA-transfected cells. The cells were cultured for 1, 3, 6, 12, and 24 hours, and then analyzed by real-time PCR. The protein levels in cells cultured for 24 hours were determined by an ELISA. Values represent fold changes (real-time PCR) or concentrations (ELISA). Differences among groups were analyzed using the Student’s t-test. Data are expressed as the mean ± SD (n = 3). *p < 0.05 vs each time point control. (C) MMP-13 mRNA expression in IL-1Ra and control siRNA-transfected cells treated with or without anti-IL-1α, anti-IL-1β, or isotype control antibodies (1 μg/ml). Cells were cultured for 6 hours, and then mRNA levels were determined using real-time PCR. Values represent fold changes. Differences among groups were analyzed by one-factor ANOVA and Bonferroni’s multiple comparison test. Data are expressed as the mean ± SD (n = 3). *p < 0.05.
Fig 7.
Expression and localization of MMP-13 in periodontal tissue of IL-1Ra KO and WT mice.
(A) At 4 weeks after infection, the mouse gingival epithelial layer was collected to examine MMP-13 levels. The levels of MMP-13 were measured by an ELISA. Differences among groups were analyzed by one-factor ANOVA and Bonferroni’s multiple comparison test. Data are expressed as the mean ± SD (n = 4). **p <0.01 vs IL-1Ra KO mice infected with A. actinomycetemcomitans. †p <0.05 vs IL-1Ra KO mice treated with PBS. (B) Localization of MMP-13 in periodontal tissue. Buccolingual sections of the second and third mandibular molars from A. actinomycetemcomitans-infected IL-1Ra KO and WT mice were stained immunohistochemically. In contrast to infected WT mice, immunohistochemical findings in periodontal tissues indicated considerable inflammatory reactions in IL-1Ra KO mice infected with A. actinomycetemcomitans.
Fig 8.
Localization of laminin-5 in periodontal tissue of IL-1Ra KO and WT mice.
Buccolingual sections of the second and third mandibular molars from A. actinomycetemcomitans-infected IL-1Ra KO and WT mice were stained immunohistochemically. In contrast to IL-1Ra KO mice, positive reactions were evident in the internal basal lamina of WT mice.