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Abstract Number: 1406

The Scaffold Protein p62 Is Involved In NF-κB Signaling, Caspase-3 Dependent and -Independent Cell Death and Autophagy In Rheumatoid Arthritis Synovial Fibroblasts

Masaru Kato1, Caroline Ospelt1, Christoph Kolling2, Beat A. Michel3, Renate E. Gay4, Steffen Gay5 and Kerstin Klein1, 1Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland, 2Schulthess Clinic, Zurich, Switzerland, 3Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland, 4Center of Experimental Rheumatology, Zurich University Hospital, Zurich, Switzerland, 5Center of Experimental Rheumatology, University Hospital Zurich and Zurich Center of Integrative Human Physiology (ZIHP), Zurich, Switzerland

Meeting: 2013 ACR/ARHP Annual Meeting

Keywords: Apoptosis

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Session Information

Session Title: Rheumatoid Arthritis - Human Etiology and Pathogenesis

Session Type: Abstract Submissions (ACR)

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.


Disclosure:

M. Kato,

EURO-TEAM, IMI-BT Cure, IAR,

2;

C. Ospelt,

EURO-TEAM, IMI-BT Cure, IAR,

2;

C. Kolling,

EURO-TEAM, IMI-BT Cure, IAR,

2;

B. A. Michel,
None;

R. E. Gay,

EURO-TEAM, IMI BTCure, IAR Epalinges,

2;

S. Gay,
None;

K. Klein,

EURO-TEAM, IMI-BT Cure, IAR,

2.

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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/

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