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SAT0063 Functional analysis of the primary cilium in rheumatoid arthritis synovial fibroblasts
  1. K. Klein1,2,
  2. B. Michel1,2,
  3. R.E. Gay1,2,
  4. S. Gay1,2,
  5. C. Ospelt1,2
  1. 1Center of Experimental Rheumatology, University Hospital Zurich
  2. 2Zurich Center of Integrative Human Physiology (ZIHP), Zurich, Switzerland

Abstract

Background The primary cilium is a microtubule-based, polarized, antenna structure that emanates from the cell surface of most mammalian cell types. It serves as a sensor that mediates reactions to mechanical and chemical signals from the environment and is therefore, a crucial factor in the communication with neighbouring cells and the environment.

Objectives To investigate the functional role of the primary cilium expressed on synovial fibroblasts (SF) in the pathogenesis of rheumatoid arthritis (RA).

Methods The expression of the primary cilium was verified in serum- starved RASF by immunofluorescence microscopy using acetylated tubulin as a marker. Ciliogenesis of RASF was disrupted by transfection of siRNA targeting the ciliary component kinesin family member 3a (KIF3a) before stimulation with TNF-α (10ng/ml) and IL-1β (1ng/ml). Knockdown of KIF3a was verified by Western blotting. Migration and adhesion properties of transfected RASF were analysed by scratch assay (n=2) and a fibronectin-based adhesion assay (n=7), respectively. Expression levels of integrin beta 1 (ITGB1), integrin beta 3 (ITGB3), and integrin alpha V (ITGAV) were measured by SYBR green Real-time PCR and compared to expression levels of RASF that were transfected with scrambled siRNA (n=6).

Results The primary cilium was detected on the surface of RASF. Migration of RASF with blocked formation of the primary cilium by transfection with siRNA targeting KIF3a was reduced compared to RASF transfected with scrambled siRNA. RASF adhesion to fibronectin was induced by TNF-α (2.1 fold ±0.9, n.s.) and IL-1β (2.3 fold ±1.3, p<0.05), and was reduced in KIF3a siRNA transfected RASF (TNF-α: 1.2 fold ±0.7, p<0.05; IL-1β: 1.3 fold ±0.7, p=0.1142). While IL-1β significantly induced mRNA expression of ITGB1 (1.7 fold ±0.3, p<0.001), ITGB3 (1.8 fold ±0.6, p<0.01) and ITGAV (1.7 fold ±0.7, p<0.01) in control transfected RASF, it had no effect on KIF3a siRNA transfected RASF in which the levels of the analysed integrins after IL-1β stimulation were significantly lower compared to control transfected RASF (ITGB1: 0.9 fold ±0.2, p<0.001; ITGB3: 1.1 fold ±0.3, p<0.001; ITGAV: 1.1 fold ±0.1, p<0.01).

Conclusions Our data show a functional role of the primary cilium in the migratory and adhesive properties of RASF. Since disruption of ciliogenesis in RASF altered their response to stimulation with TNF-α and IL-1β, we hypothesise that the primary cilium also plays a key role in the transmission of pro-inflammatory signals in RASF.

Acknowledgements IAR Epalinges, FP7 Masterswitch, IMI-BT Cure, EMDO-Stiftung

Disclosure of Interest None Declared

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