Arthritis & Rheumatism, Volume 63,
November 2011 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Chicago, Illinois November 4-9, 2011.

Mechanism of Fractalkine/CX3CL1 Synthesis and Shedding in Rheumatoid Arthritis Synovial Fibroblasts.

Jones,  Brian, Beamer,  Maria, Rahman,  Ayesha, Aboualaiwi,  Wissam Ali, Ahmed,  Salahuddin


Tumor necrosis factor (TNF)-a and interferon (IFN)-g together induce the production of fractalkine/CX3CL1 (FKN), a well known chemoattractant and cell adhesion chemokine, in rheumatoid arthritis(RA) synovial fibroblasts by an unknown mechanism. The present study was undertaken to determine the cellular mechanisms governing RA synovial fibroblast FKN synthesis and shedding so that highly targeted therapeutic approaches against it may be developed for RA.


Effect of TNF-a and IFN-g, alone or in combination, on human RA synovial fibroblast FKN synthesis and shedding was determined over 6–72 hours by immunostaining, quantitative RT-PCR, and Western blotting methods. The role of enzymes ADAM17, ADAM10 and caspase-3, the signaling mediators such as mitogen-activated protein kinases (MAPK) and nuclear factor-kB (NF-kB) pathways were evaluated using signaling inhibitors. 20S Proteasome activity in the treated samples and the DNA-binding activity of NF-kB in the nuclear fractions were evaluated using the commercially available ELISA kits.


In RA synovial fibroblasts, the combination of TNF-a/IFN-g induced the cellular expression of FKN that peaked as early as 24 hours (P<0.05; n=3). Activation of ADAM17 expression, but not ADAM10, enhanced the proteolytic shedding of FKN, resulting in the release of soluble FKN (sFKN) that peaked around 48–72 hours of stimulation. TNF-a/IFN-g-induced FKN expression and sFKN release were markedly inhibited in RA synovial fibroblasts by the pretreatment of GM6001 (an inhibitor of ADAM17) or MG132 (a proteasome inhibitor), but not Z-DEVD-FMK (an inhibitor of caspase-3), suggesting an important role of ADAM17 in the proteolytic shedding of sFKN. This observation was further supported by an increase in the proteasome activity by 40% in the samples treated with TNF-a/IFN-g as compared to the untreated samples (P<0.001; n=3). Evaluation of the signaling pathways revealed that MG132 and SB203580 (a p38-MAPK inhibitor) selectively inhibited TNF-a/IFN-g-induced sFKN production in RA synovial fibroblasts (p<0.05; n=3), suggesting the clinical importance of these pathways in mediating the synthesis and proteolytic processing of FKN. Importantly, we also observed a rapid phosphorylation and proteasomal degradation of IkBa and an activation of phospho-p38 expression within 1–5 minutes of TNF-a/IFN-g stimulation, which further correlated with the increased DNA-binding activity of NF-kB in the nuclear fractions of the similarly treated RA synovial fibroblasts.


Our results provide the evidence of the role of ADAM17, p38-MAPK, and proteasome pathway in regulating TNF-a/IFN-g-induced synthesis and proteolytic shedding of FKN. Selective inhibition of these molecular targets may be one of the potential therapeutic approaches to limit the pathological role of FKN in RA synovial fibroblast mediated inflammation and tissue destruction.

To cite this abstract, please use the following information:
Jones, Brian, Beamer, Maria, Rahman, Ayesha, Aboualaiwi, Wissam Ali, Ahmed, Salahuddin; Mechanism of Fractalkine/CX3CL1 Synthesis and Shedding in Rheumatoid Arthritis Synovial Fibroblasts. [abstract]. Arthritis Rheum 2011;63 Suppl 10 :1813

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