Background Valosin containing protein (p97/VCP) is an ATPase implicated in the degradation of ubiquitin-labelled proteins through the proteasome. We recently described a resistance to apoptotic cell death induced by proteasome inhibition and a hypersensitivity to autophagy-associated cell death under conditions of severe endoplasmic reticulum (ER) stress in rheumatoid arthritis synovial fibroblasts (RASF) compared to osteoarthritis synovial fibroblasts.
Objectives To investigate the role of p97 in apoptotic and autophagy-associated cell death in RASF and in in vivo arthritis model.
Methods RASF were transfected with siRNA targeting p97 or treated with the selective p97 inhibitor DBeQ (5 μM). To induce cell death, RASF were treated with TRAIL (100 ng/ml) for 24 hours or the ER stress inducer thapsigargin (TG, 5 nM-5 μM) for 72 hours. 3-methyladenine (5 mM) was used as an autophagy inhibitor. The distribution and amount of poly-ubiquitinated proteins were evaluated by immunofluorescence and immunoblotting. Cell death was evaluated by flow cytometry using annexin V/ propidium iodide staining and a caspase-3 activity assay (NucView 488, Biotium). Collagen-induced arthritis (CIA) was induced in Lewis rats. Scrambled or p97 siRNA-atelocollagen complexes were injected into ankle joints of rats. CIA was scored according to paw thickness and ankle diameter. Bone erosion was assessed by micro-CT. Proliferation of fibroblasts in rat synovial tissue was quantified by immunolabeling for Hsp47.
Results siRNA-mediated knockdown of p97 in RASF increased cell death induced by TRAIL (p=0.009, n=6), accompanied by caspase-3 activation. Both siRNA-mediated knockdown and inhibition of p97 in RASF boosted cell death induced by 5 μM TG, accompanied by a massive cytoplasmic vacuolization, the formation of poly-ubiquitinated protein aggregates and the accumulation of poly-ubiquitinated proteins, and cell death was inhibited by 3-methyladenine. Smaller amounts of TG (50 or 500 nM) induced a cytoplasmic vacuolization and the formation of poly-ubiquitinated protein aggregates in p97-inhibited RASF but not in control RASF. Intra-articular injection of p97 siRNA significantly suppressed CIA (p=0.002, n=6), bone erosion (p=0.02, n=6) and proliferation of synovial fibroblasts (p=0.004, n=6) in rats.
Conclusions Our data indicate that the inhibition of p97 promotes both apoptotic and autophagy-associated cell death in RASF and suppresses CIA, bone erosion and proliferation of synovial fibroblasts in vivo. p97 may be a new potential target in the treatment of arthritis.
Kato M, et al. Arthritis Rheum 2014 Jan;66(1):40-8.
Acknowledgements This work was supported by MHLW, MEXT, IMI-BT Cure, IAR Epalinges, euroTEAM.
Disclosure of Interest None declared
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