Background/Purpose: The AAA-ATPase valosin containing protein (p97) and histone deacetylase 6 (HDAC6) interact with each other and are implicated in the degradation of ubiquitin-labelled proteins. 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 (OASF). The objective of this study was to investigate the role of p97-HDAC6 controlled poly-ubiquitin turnover in apoptotic and autophagy-associated cell death in RASF and in an in vivo arthritis model.

Methods: p97, HDAC6 and poly-ubiquitinated proteins were evaluated by immunohistochemistry, Real-time PCR, immunoblotting and proximal ligation assay. RASF were transfected with siRNA targeting p97 or HDAC6 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. Cell death was evaluated by flow cytometry using annexin V/ propidium iodide staining and a caspase-3 activity assay. 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: The expression of p97 and the expression of HDAC6 were restricted to the lining layer of synovial tissues and were similarly detected in RA and OA patients. The expression of p97 in RASF and OASF was positively related with the expression of HDAC6 at both mRNA (R² = 0.77, n = 31) and protein (R² = 0.77, n = 14) level. Interactions of p97-ubiquitin, HDAC6-ubiquitin and p97-HDAC6 were detected in RASF. The siRNA-mediated knockdown of p97 in RASF decreased the expression of HDAC6 but not vice versa. Knockdown of p97 in RASF increased cell death induced by TRAIL (p = 0.009, n = 6), accompanied by caspase-3 activation. Both knockdown and inhibition of p97 in RASF boosted cell death induced by 5 μM TG, accompanied by a massive cytoplasmic vacuolization and the formation of poly-ubiquitinated protein aggregates, 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), cartilage destruction (p = 0.03) and proliferation of synovial fibroblasts (p = 0.004) in rats.

Conclusion: Our data indicate that the inhibition of the ATPase p97 promotes both apoptotic and autophagy-associated cell death in RASF and suppresses CIA and proliferation of synovial fibroblasts in vivo. The p97-HDAC6 controlled poly-ubiquitin turnover 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-hdac6-controlled-poly-ubiquitin-turnover-regulates-apoptotic-and-autophagy-associated-cell-death-in-arthritis/