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Role of NF-B in TNF--induced COX-2 Expression in Synovial Fibroblasts from Human TMJ

¹ Key Lab. for Oral Biomedical Engineering, Ministry of Education,
² Departments of Oral Maxillofacial Surgery and
⁴ Prosthodontics, School & Hospital of Stomatology,
³ Department of Radio-Chemotherapy of Zhongnan Hospital, Wuhan University, Wuhan 430079, PR China

View larger version (44K): [in this window] [in a new window]   Figure 1. Dose-dependent TNF--induced COX-2 mRNA and protein in synovial fibroblasts from TMJ. (A) Cells were incubated with various concentrations of TNF- for 2 hrs. Total RNA was isolated from cells and analyzed for COX-2 and GAPDH mRNA by RT-PCR. (B) Cells were incubated with various concentrations of TNF- for 6 hrs. Whole-cell lysates were prepared and subjected to Western blotting with antibody specific for COX-2 and actin. The data represent 1 of 3 separate experiments with similar results.

View larger version (39K): [in this window] [in a new window]   Figure 2. Time-dependent TNF--induced COX-2 mRNA and protein in synovial fibroblasts from TMJ. (A) Cells were incubated with 20 ng/mL of TNF- for various time intervals. Total RNA was then isolated from cells and analyzed for COX-2 and GAPDH mRNA by RT-PCR. (B) Cells were incubated with 20 ng/mL of TNF- for various time intervals, and the whole-cell lysates were prepared and subjected to Western blotting with antibody specific for COX-2 and actin. The data represent 1 of 3 separate experiments with similar results.

View larger version (42K): [in this window] [in a new window]   Figure 3. Time-course of TNF--induced NF-B-specific DNA-protein complex formation and NF-B translocation in synovial fibroblasts from TMJ. Cells were treated with 20 ng/mL TNF- for various time intervals, and then cytoplasmic and nuclear extracts were prepared. (A) NF-B-specific DNA-protein-binding activity in nuclear extracts was determined by EMSA. (B) Supershift assays were performed with specific p65 and p50 antibodies. Moreover, we added unlabeled NF-B probes in a 100-fold excess, to determine the specificity of NF-B-specific DNA protein. (C) Cytoplasmic and nuclear levels of p65 and p50 proteins were detected by Western blotting. The data represent 1 of 3 separate experiments with similar results.

View larger version (36K): [in this window] [in a new window]   Figure 4. Inhibitory effect of PDTC on NF-B activity and COX-2 protein expression induced by TNF- in synovial fibroblasts from the TMJ. (A) NF- B binding activity in the nuclear extracts from cells pre-treated with PDTC (50 µmol/L) 1 hr before 20 ng/mL of TNF- stimulation for 40 min was determined by EMSA. (B) COX-2 protein expression in the whole-cell lysates from cells pre-treated with PDTC (50 µmol/L) 1 hr before 20 ng/mL of TNF- stimulation for 6 hrs was assayed by Western blotting. The data represent 1 of 3 separate experiments with similar results.