Background/Purpose:

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). Here we investigated the role of p62 in the activation of apoptosis and nuclear factor-κB (NF-κB) activity in RASF.

Methods:

The expression of p62 in synovial tissues from RA and osteoarthritis (OA) patients was verified by immunohistochemistry using antibodies against p62. RASF were incubated with and without TNF-α (10 ng/ml), IL-1β (1 ng/ml) and IL-6 (50 ng/ml) in presence or absence of soluble IL-6 receptor (30 ng/ml) for 24 hours and p62 expression was evaluated by quantitative Real-time PCR and immunoblotting. RASF were transfected with siRNA targeting p62 and knockdown was verified by immunoblotting. To induce cell death, RASF were treated with TRAIL (100 ng/ml) in presence or absence of a pan-caspase inhibitor (20 μM Z-VAD) for 24 hours. Apoptosis was evaluated by flow cytometry using annexin V/ propidium iodide staining and a caspase-3 activity assay (NucView 488, Biotium). RASF were transfected with reporter-gene vectors containing NF-κB response elements and control vectors (GAPDH). NF-κB activity was measured by Dual-Luciferase reporter assays.

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 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) or patients treated with tocilizumab (n=4, p=0.008) or abatacept (n=3, p=0.02). Consistently, p62 mRNA (n=4, p=0.02) and protein (n=5, p=0.002) were induced in RASF by TNF-α, but not by IL-1β or by IL-6 stimulation even in the presence of soluble IL-6 receptor. p62 knockdown in RASF promoted TRAIL-induced cell death (n=6, p=0.02) that was accompanied by caspase-3 activation and was inhibited by a pan-caspase inhibitor. Furthermore, p62 knockdown strongly suppressed basal NF-κB activity (68.0 ± 10.6% reduction, n = 3, p = 0.008) in RASF.

Conclusion:

Our data indicate that the scaffold protein p62 is involved in the regulation of multiple pathways in RASF, including NF-κB signaling, as well as the induction of caspase-3 dependent and -independent cell death and autophagy. p62 is positively regulated by TNF-α and plays a protective role against apoptosis in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-scaffold-protein-p62-is-involved-in-nf-%ce%bab-signaling-caspase-3-dependent-and-independent-cell-death-and-autophagy-in-rheumatoid-arthritis-synovial-fibroblasts/