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Signaling Pathways Regulating IL-1-induced COX-2 Expression

¹ Department of Oral and Maxillofacial Surgery, Graduate School of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; and
² Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science and Station for Collaborative Research, Kyushu University, Fukuoka, Japan

View larger version (20K): [in this window] [in a new window]   Figure 1. Effect of IL-1 on the expression of COX-2 in odontogenic keratocyst fibroblasts. (A) A representative immunohistochemical staining for COX-2 in a section of an odontogenic keratocyst. Subepithelial layer fibroblasts were positively stained with anti-COX-2 antibody (arrowheads). Scale bar: 12 µm. (B,C) Fibroblasts (4 x 104 cells/cm2) were incubated in the absence (Co) or presence of various concentrations of rhIL-1 for 6 hrs (B), or were incubated with 0.1 nM rhIL-1 for various times (C). The expression of COX-2 mRNA was measured as described in "MATERIALS & METHODS". The amounts of COX-2 mRNA in the control were normalized as 1.0. Vertical bars indicate mean ± SD (n = 3). * Significant difference at p < 0.05. (D) Fibroblasts were incubated in the absence or presence of 0.1 nM rhIL-1 for 12 hrs, and the aliquots of cell lysates were subjected to Western immunoblotting for COX-2, as described in "MATERIALS & METHODS".

View larger version (32K): [in this window] [in a new window]   Figure 2. Effects of IL-1 on the phosphorylation of ERK1/2, p38, and JNK in odontogenic keratocyst fibroblasts. Fibroblasts (4 x 104 cells/cm2) were incubated with 0.1 nM rhIL-1 for various intervals (A,B,C), or with various concentrations of rhIL-1 for 10 min (D,E) or 20 min (F) at 37°C. Western immunoblotting for phospho-ERK1/2 (A,D), phospho-p38 (B,E), and phospho-JNK (C,F) was performed as described in "MATERIALS & METHODS". The values were normalized to the amounts of the control. Vertical bars indicate mean ± SD (n = 3). * Significant difference at p < 0.05.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of inhibitors for MAPKs and NF-B on IL-1-induced expression of COX-2 mRNA and secretion of PGE2 in odontogenic keratocyst fibroblasts. Fibroblasts (4 x 104 cells/cm2) were incubated with 20 µM PD-98059, 20 µM SB-203580, 40 µM SP-600125, and 20 µM PDTC for 1 hr, and then incubated with 0.1 nM rhIL-1 for 6 hrs (A) or 24 hrs (B) at 37°C. (A) The expression of COX-2 mRNA was measured as described in "MATERIALS & METHODS". (B) The concentration of PGE2 in the culture media was measured as described in "MATERIALS & METHODS". The values were normalized to the concentration of the control. Vertical bars indicate mean ± SD (n = 3). * Significant difference at p < 0.05.

View larger version (28K): [in this window] [in a new window]   Figure 4. PKC-dependent COX-2 mRNA expression and MAPKs phosphorylation in odontogenic keratocyst fibroblasts. (A) Fibroblasts (1.5 x 106 cells) were incubated without (Co) or with 0.1 nM rhIL-1 (IL-1) for 30 sec at 37°C. The amounts of Ins(1,4,5)P3 in the cells (n = 4) were measured as described in "MATERIALS & METHODS". (B) Fibroblasts (4 x 104 cells/cm2) were stimulated with 0.1 nM rhIL-1 for 10, 30, and 60 min. Then, the activity of PKC for the cells (n = 3) was measured as described in "MATERIALS & METHODS". Vertical bars indicate mean ± SD. * Significant difference at p < 0.05. (C) Fibroblasts (4 x 104 cells/cm2) were stimulated with 0.1 nM rhIL-1 (lanes 2,3), 0.01% DMSO (lane 4), or 1 µM PMA (lane 5,6) for 6 hrs after pre-incubation without (lanes 1,2,4,5) or with (lanes 3,6) 2 µM staurosporine for 1 hr at 37°C. Then, the expression of COX-2 mRNA was measured as described in "MATERIALS & METHODS". (D) Fibroblasts (4 x 104 cells/cm2) were pre-incubated in the absence (lanes 1,2) or presence (lane 3) of 2 µM staurosporine for 1 hr at 37°C. The cells were then stimulated with 0.1 nM rhIL-1 (lanes 2,3) for 10 min for measurement of the phosphorylation of ERK1/2 and p38, or for 20 min for measurement of the phosphorylation of JNK. The aliquots of cell lysates were subjected to Western immunoblotting as described in "MATERIALS & METHODS".