Background/Purpose: Recently we described a hypersensitivity of rheumatoid arthritis synovial fibroblasts (RASF) compared to osteoarthritis synovial fibroblasts to autophagy under conditions of severe endoplasmic reticulum (ER) stress, leading to a massive cytoplasmic vacuolization, the formation of poly-ubiquitinated protein aggeregates and non-apoptotic cell death. Valosin containing protein (p97/VCP) is an ATPase implicated in the degradation of ubiquitin-labelled proteins through the proteasome. We hypothesized that the inhibition of p97 further sensitizes RASF to autophagy-associated cell death due to impaired proteasomal degradation and subsequent overloaded autophagy.

Methods: RASF were transfected with siRNA targeting p97 or treated with the selective p97 inhibitor DBeQ (5 μM). To induce ER stress, RASF were treated with thapsigargin (TG, 5 nM-5 μM). 3-methyladenine (5 mM) was used as an autophagy inhibitor. The distribution of poly-ubiquitinated proteins was evaluated by immunofluorescence microscopy. Cell death was evaluated by flow cytometry using annexin V/propidium iodide staining. Collagen-induced arthritis (CIA) was induced in Lewis rats. Scrambled or p97 siRNA-atelocollagen complexes were injected into ankle joints of rats. Three, seven and eleven days after the injection, CIA was scored from 0 to 4 according to paw thickness and ankle diameter.

Results: Both siRNA-mediated knockdown and inhibition of p97 in RASF boosted a cytoplasmic vacuolization, the formation of poly-ubiquitinated protein aggeregates and cell death under 5 μM TG treatment, and this 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 aggeregates in p97–inhibited RASF but not in control RASF. Intra-articular injection of p97 siRNA significantly suppressed CIA (Day 3, p = 0.002; Day 7, p = 0.002; Day 11, p = 0.04; n = 6) in rats.

Conclusion: Our data indicate that the inhibition of p97 promotes autophagy-associated cell death in RASF and suppresses CIA in vivo. p97 may be a new potential target in the treatment of arthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/aaa-atpase-p97-regulates-autophagy-associated-cell-death-in-arthritis/