Inhibition of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway in rheumatoid synovial fibroblasts using small molecule compounds

Abstract

Janus kinase (JAK) inhibitors have been developed as anti-inflammatory agents and have demonstrated clinical efficacy in rheumatoid arthritis (RA). We investigated if JAK-3-selective inhibition alone could disrupt cytokine signalling in rheumatoid synovial fibroblasts. In-vitro studies were performed using synovial fibroblasts isolated from patients with RA. Levels of activated JAK and signal transducer and activator of transcription (STAT) proteins were detected by immunoblot analysis. Target-gene expression levels were measured by reverse transcription-polymerase chain reaction (RT-PCR) or real-time PCR. The JAK inhibitors CP-690,550 and INCB028050 both suppressed activation of JAK-1/-2/-3 and downstream STAT-1/-3/-5, as well as the expression levels of target proinflammatory genes (MCP-I, SAA1/2) in oncostatin-M (OSM)-stimulated rheumatoid synovial fibroblasts. In contrast, the JAK-3-selective inhibitor, PF-956980, suppressed STAT-1/-5 activation but did not affect STAT-3 activation in OSM-stimulated rheumatoid synovial fibroblasts. In addition, PF-956980 significantly suppressed MCP-1 gene expression, but did not block SAA1/2 gene expression in OSM-stimulated rheumatoid synovial fibroblasts. These data suggest that JAK-3-selective inhibition alone is insufficient to control STAT-3-dependent signalling in rheumatoid synovial fibroblasts, and inhibition of JAKs, including JAK-1/-2, is needed to control the proinflammatory cascade in RA.

Keywords: CP-690,550; INCB020850; Janus kinase; rheumatoid arthritis; signal transducer and activator of transcription.

© 2013 British Society for Immunology.

Figures

Fig. 1

Phospho-Janus kinase (JAK)-3 expressions in synovial tissues. (a) Synovial tissue sections obtained from independent rheumatoid arthritis (RA) patients (n = 7) and osteoarthritis (OA) patients (n = 2) were stained using antibodies that were specific for phospho-JAK-3 protein. (original magnification ×200). (b) Staining synovial tissue sections with anti-CD3, anti-CD68 and anti-vimentin antibodies showed that phospho-JAK-3-expressing cells were CD3+ T cells and vimentin+ synovial fibroblasts. (original magnification ×200). A representative result of three independent experiments.

Fig. 2

Janus kinase (JAK) inhibitors suppressed oncostatin-M (OSM)-induced JAK-1/-2/-3 activations in rheumatoid synovial fibroblasts. Quiescent synovial fibroblasts were pretreated with various concentrations of CP-690,550 or INCB028050 or 2 h, then stimulated with OSM (20 ng/ml) for 20 min. Cellular lysates were subjected to Western blotting using phosphospecific antibodies against JAK-1, JAK-2, JAK-3, signal transducer and activator of transcription (STAT)-1, STAT-3 and STAT-5. Three experiments were performed using different rheumatoid synovial fibroblasts and a representative result is shown.

Fig. 3

PF956980 suppressed oncostatin-M (OSM)-induced Janus kinase (JAK)-3 activation in rheumatoid synovial fibroblasts. Quiescent synovial fibroblasts were pretreated with various concentrations of CP-690,550, 028050 or PF956980 for 2 h, then stimulated with OSM (20 ng/ml) for 20 min. Cellular lysates were subjected to Western blotting using phosphospecific antibodies against JAK-1, JAK-2, JAK-3, signal transducer and activator of transcription (STAT)-1, STAT-3 and STAT-5. Three experiments were performed using different rheumatoid synovial fibroblasts and a representative result is shown.

Fig. 4

PF956980 did not inhibit oncostatin-M (OSM)-induced A-SAA mRNA expression in synovial fibroblasts. (a) Quiescent synovial fibroblasts were pretreated with various concentrations of CP-690,550, INCB028050 or PF956980 for 2 h, then stimulated with OSM (20 ng/ml) for 6 h, after which, MCP-1 and GAPDH mRNA expression was determined by the real-time polymerase chain reaction (PCR) method. *P < 0·01 compared to OSM-stimulated synovial fibroblasts; **P < 0·0001 compared to OSM-stimulated synovial fibroblasts. Three experiments were performed using different rheumatoid synovial fibroblasts and a representative result is shown. (b) Quiescent synovial fibroblasts were pretreated with various concentrations of CP-690,550, INCB028050 or PF956980 for 2 h, then stimulated with OSM (20 ng/ml) for 6 h. A-SAA mRNA expression was analysed by PCR following reverse transcription. Three experiments were performed using different rheumatoid synovial fibroblasts and a representative result is shown.

Fig. 5

Phospho-Janus kinase (JAK)-1/-2/-3 expressions in synovial tissues. Synovial tissue sections obtained from rheumatoid arthritis (RA) (n = 1) or osteoarthritis (OA) patients (n = 1) were stained using antibodies that were specific for phospho-JAK-1, JAK-2 and JAK-3 protein (original magnification ×200).

Fig. 6

Janus kinase (JAK) inhibitors suppressed oncostatin-M (OSM)-induced JAK-1/-2/-3 activations in osteoarthritis (OA) synovial fibroblasts. Quiescent synovial fibroblasts isolated from OA patients were pretreated with various concentrations of CP-690,550 or INCB028050 for 2 h, then stimulated with OSM (20 ng/ml). Cellular lysates were subjected to Western blotting using phosphospecific antibodies against JAK-1, JAK-2, JAK-3, signal transducer and activator of transcription (STAT)-1, STAT-3 and STAT-5. Two experiments were performed using different OA synovial fibroblasts and a representative result is shown.