Background Sequestosome 1 (p62/SQSTM1) is a multifunctional ubiquitin-binding protein implicated in selective autophagy, cell signaling pathways and regulation of cell death. Recently, we described a functional role of p62 in the formation of poly-ubiquitinated protein aggregates and non-apoptotic cell death associated with autophagy in rheumatoid arthritis synovial fibroblasts (RASF).
Objectives To investigate the expression of p62 and its function in shuttling of poly-ubiquitinated proteins and apoptosis in RASF.
Methods p62 and poly-ubiquitinated proteins were evaluated by quantitative Real-time PCR, immunoblotting, immunofluorescence and immunohistochemistry. RASF were incubated with TNF-a (10 ng/ml), IL-1b (1 ng/ml) and IL-6 (50 ng/ml) in presence or absence of soluble IL-6 receptor (30 ng/ml) for 24 hours. To inhibit proteasome and lysosome functions, RASF were treated with MG132 (5 nM) and bafilomycin A1 (100 nM) for 4 hours, respectively. RASF were transfected with siRNA targeting p62. To induce cell death, RASF were incubated with TRAIL (100 ng/ml) in presence of a pan-caspase inhibitor (20 mM Z-VAD), a necroptosis inhibitor (30 mM necrostatin-1) or an autophagy inhibitor (5 mM 3-methyladenine) for 24 hours. Cell death was evaluated by flow cytometry using annexin V/propidium iodide staining and a caspase-3 activity assay (NucView 488, Biotium).
Results The expression of p62 was restricted to the lining layer of synovial tissues and was increased in synovial tissues from RA patients (n=30) compared to osteoarthritis (OA) patients (n=9, p=0.01). Interestingly, the expression of p62 in RA patients treated with anti-TNF agents (n=14) was similar to the p62 expression observed in OA patients and was decreased compared to patients treated with non-biologics (n=9, p=0.006), tocilizumab (n=4, p=0.008) or abatacept (n=3, p=0.02). In RASF, p62 mRNA (n=4, p=0.02) and protein (n=5, p=0.002) were induced by TNF-a, but not by IL-1b or by IL-6 stimulation even in the presence of soluble IL-6 receptor. Silencing of p62 in RASF resulted in the accumulation of poly-ubiquitinated proteins, however, the formation of poly-ubiquitinated protein aggregates was decreased. Treatment of RASF with bafilomycin A1 (n=5, p=0.002) or MG132 (n=5, p=0.006) increased the accumulation of p62 protein without inducing its mRNA expression indicating that the turnover of p62 is regulated not only by autophagy but also by the proteasome. p62 knockdown in RASF increased cell death induced by TRAIL (n=8, p=0.004) that was accompanied by caspase-3 activation and was inhibited by a pan-caspase inhibitor but not by a necroptosis inhibitor or by an autophagy inhibitor.
Conclusions Our data indicate that the scaffold protein p62 promotes the degradation of misfolded proteins through both, the autophagy-lysosome pathway and the ubiquitin-proteasome system in RASF. p62 is positively regulated by TNF-a and contributes to the apoptosis-resistant phenotype of RASF.
Acknowledgements EURO-TEAM, IMI-BT Cure, IAR.
Disclosure of Interest M. Kato Grant/research support: EURO-TEAM, IAR, T. Atsumi Grant/research support: Kyowa Hakko Kirin, Chugai, Novartis, Mitsubishi Tanabe,Teijin, MSD, Astellas, Sanofi, Takeda, Novo Nordisk, Otsuka, Boehringer Ingelheim, Avvi,Kissey, Pfizer, Astellas, Shionogi, Dainippon,TaisyoToyama, Bristol-Myers, GlaxoSmithKline, Acterion, Paid instructor for: Astellas, Acterion, Avvi, Ezai, MSD, Asahikasei, Otsuka, Daiichi-Sankyo, Mitsubishi Tanabe,Takeda, Chugai, Pfizer, Bristol-Myers, C. Kolling: None declared, R. Gay Grant/research support: EURO-TEAM, IMI-BT Cure, IAR, S. Gay Grant/research support: EURO-TEAM, IMI-BT Cure, IAR, K. Klein Grant/research support: EURO-TEAM, IMI-BT Cure, IAR