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Logo of annrheumdAnnals of the Rheumatic DiseasesCurrent TOCInstructions for authors
Ann Rheum Dis. Jan 1999; 58(1): 55–60.
PMCID: PMC1752754

Thrombin receptor mediated signals induce expressions of interleukin 6 and granulocyte colony stimulating factor via NF-κB 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 κ-chloramphenicol acetyltransferase (CAT) assay, and immunostaining for NF-κB subunit molecule was performed.
RESULTS—Thrombin stimulation activated the inducible transcription factor NF-κB, 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-κB activation.

Keywords: thrombin; NF-κB; cytokine; synovial cells

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Selected References

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Figures and Tables

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, ...
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 ...
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 ...
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 ...
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 ...

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