Thrombin receptor mediated signals induce expressions of interleukin 6 and granulocyte colony stimulating factor via NF-kappa B activation in synovial fibroblasts

Abstract

Objective: To clarify the mechanism of thrombin receptor mediated signal transduction and the induction of cytokines by thrombin stimulation in rheumatoid synovial fibroblasts.

Methods: Cytokines were measured by enzyme linked immunosorbent assay (ELISA) in the supernatants of cultured rheumatoid synovial fibroblasts stimulated by thrombin. To assess the mechanism of thrombin receptor mediated signal transduction in the rheumatoid synovial fibroblasts, electrophoretic mobility gel shift assay (EMSA), immunoglobulin kappa-chloramphenicol acetyltransferase (CAT) assay, and immunostaining for NF-kappa B subunit molecule was performed.

Results: Thrombin stimulation activated the inducible transcription factor NF-kappa B, and then induced subsequent expressions of interleukin 6 (IL6) and granulocyte colony stimulating factor (G-CSF) in the cells.

Conclusion: Thrombin receptor mediated signal transduction could induce the expressions of IL6 and G-CSF, and increase inflammatory events in the cavum articulare via NF-kappa B activation.

Figure 1

Induction of IL6 or G-CSF mRNA expressions by thrombin. Synovial fibroblasts (2 × 10⁵ cells) were starved for 48 hours in DMEM containing 0.5% FBS, then stimulated with thrombin (10 units/ml). At 30 minutes, and 1, 2, 4, 8, 12, and 24 hours after the stimulation, total RNA (2 µg) was isolated and converted to cDNA. RT-PCR analysis was performed as described in Methods. The RT-PCR products for β actin were used as an internal control. Results shown are obtained in a representative examination of two sets of measurements.

Figure 2

Activation of nuclear κB binding proteins by thrombin. Synovial cells were either untreated or stimulated with thrombin (10 units/ml) for the indicated times. Five micrograms of nuclear protein were incubated for 30 minutes at room temperature with ³²P-labelled probe. The mixtures were then run on a native 4% polyacrylamide gel. Nuclear protein from MT-2 cells was used as a positive control. Results shown are from two sets of experiments.

Figure 3

Identification of NF-kB protein in RA. Supershift assay was performed using antibodies against the NF-κB subunits, p50 (αp50), p65 (αp65), or c-Rel (αc-Rel). DNA binding mixture containing nuclear extracts from thrombin treated synovial cells and ³²P-labelled κB probe was treated with each antibody. The mixture was applied to a native 4% polyacrylamide gel. Competition assay was performed using a 25- (× 25) or 50- (× 50) fold molar excess of unlabelled probe. Results shown are obtained in a representative examination of three sets of measurements.

Figure 4

Nuclear translocation of NF-κB molecules on thrombin stimulation. Synovial cells were stimulated by thrombin (10 units/ml) and were fixed using acetone/methanol (1:1). The fixed cells were incubated with αp50, polyclonal anti-p50 antibody. The bound antibody was visualised with fluorescein isothiocyanate conjugated goat antirabbit IgG.

Figure 5

Effect of thrombin on immunoglobulin κ-CAT transactivation in synovial fibroblasts. Synovial fibroblasts were transfected with immunoglobulin κ (Igκ)-CAT plasmid by the DEAE dextran method. The cells were cotransfected with 2 µg of Igκ-CAT and 1 µg of SV-β gal control plasmid. The transfected cells were stimulated with vehicle (thrombin 0 units/ml), thrombin (5, 10, 50 units/ml) or TNFα (500 units/ml) for 48 hours. CAT activity was assayed by thin layer chromatography, and the relative CAT activities were quantified using Fuji computed radiographs. Results shown are obtained in a representative examination of three sets of measurements.