Session Information
Session Type: Abstract Submissions (ACR)
Background/Purpose:
Protein Tyrosine Phosphatase Non-receptor Type 2 (PTPN2) is a protein phosphatase that has been associated with the development of autoimmune diseases in GWA studies. Here we analyse its contribution to inflammation in RA before and after anti-TNFα therapy.
Methods:
Immunohistochemistry was used to detect PTPN2 in synovial tissues from patients with osteoarthritis (OA) and RA with and without anti-TNF treatment. The mRNA expression of PTPN2 was measured in synovial tissues using real-time PCR. RASF and OASF were stimulated with TNFα for short (10 ng/ml, 24 h) or long term (10 ng/ml, in 7 days), TNFα and IL1-β (1 ng/ml, 24 h), LPS (100 µg/ml, 24 h), and hypoxia (1%, 24 h). The levels of PTPN2 transcripts and protein were measured with real-time PCR or Western blotting. PTPN2 was silenced with three silencing RNAs. Commercially available ELISA was used to measure IL-6 and IL-8 production. TRAIL (20 ng/ml, 24h) was used to induce apoptosis. Apoptotic cells were detected by flow cytometry, using AnnexinV staining. Thapsigargin-induced (5µM, 24 h) autophagy was measured by Western blot using antibodies against LC3B-I and LC3B-II.
Results:
Levels of PTPN2 expression were higher in the lining and sublining layers of RA compared to OA synovial tissues (OA n=11, RA n=30, 1.75-fold, p<0.001). This was also confirmed with real-time PCR on mRNA level (2.0 fold, RA tissue n=4, OA tissue n=5). Most interestingly, in synovial tissues from patients who were treated with anti-TNFα, 30% less PTPN2 staining could be detected by immunohistochemistry than in patients without anti-TNFα treatment (n=8, p<0.05). In synovial fibroblasts, the constitutive expression of PTPN2 was higher in RASF compared to OASF on mRNA (1.6 fold, p<0.01, n=10-16) and on the protein level (2.0 fold, p<0.05, n=3-7). The transcript levels of PTPN2 could be upregulated after stimulation with TNFα (3.1 fold, p<0.05, n=4), TNFα and IL-1β (2.3 fold, n=5), LPS (1.9 fold, n=5) and hypoxia (1.3 fold, n=3). The upregulation after stimulation with TNFα could also be confirmed on the protein level (1.7 fold, p<0.05, n=7). Moreover, PTPN2 protein expression was further increased after long term stimulation with TNFα (2.4 fold, n=3). Next, the function of PTPN2 was studied in synovial fibroblasts. Using siRNAs, PTPN2 was silenced by 80%. PTPN2 silenced cells produced more IL-6 (2.1 fold, n=4) than scrambled control cells, whereas levels of IL-8 did not change. TRAIL-induced apoptosis was increased by 37 % (n=5) and TG induced autophagy was decreased by 20% (n=5) after PTPN2 silencing.
Conclusion:
We show here for the first time that PTPN2 is expressed in a TNF-dependent manner in RA synovial tissues. PTPN2 counter regulates the expression of the inflammatory cytokine IL-6 and regulates apoptosis and autophagy in RASF.
Disclosure:
B. Aradi,
IMI BTCure, IAR, EU-TEAM, EU- Osteoimmune, Masterswitch-FP7 and ZIHP,
2;
M. Kato,
IMI BTCure, IAR, EU-TEAM, EU- Osteoimmune, Masterswitch-FP7 and ZIHP,
2;
M. Filková,
paid by the institution,
9;
S. Kasper,
ZIHP,
2;
K. Klein,
None;
M. Bader,
None;
M. Scharl,
ZIHP,
2;
B. A. Michel,
None;
R. E. Gay,
IMI BTCure, IAR, EU-TEAM, EU- Osteoimmune, Masterswitch-FP7 and ZIHP,
2;
E. I. Buzas,
None;
S. Gay,
None;
A. Jüngel,
IMI BTCure, IAR, EU-TEAM, EU- Osteoimmune, Masterswitch-FP7 and ZIHP,
2.
« Back to 2013 ACR/ARHP Annual Meeting
ACR Meeting Abstracts - https://acrabstracts.org/abstract/protein-tyrosine-phosphatase-non-receptor-type-2-ptpn2-is-expressed-in-a-tnf-dependent-manner-in-ra-synovial-tissues/