Background It has been shown previousely that posttranslational protein modification through SUMOylation is enhanced in rheumatoid athritis synovial fibroblasts (RASF) compared to osteoathritis synovial fibroblasts (OASF) and that higher levels of SUMOylation result in a reduced capability of RASF to undergo FAS induced apoptosis. Objectives Based on these findings, we here focussed on the expression of the SUMO specific proteases SENP5 and SENP7, both of which are essential for the maturation and deconjugation of SUMO2/3 from its target proteins. Methods Synovial tissue samples were obtained from RA and OA patients and used for cell culture experiments and histological analysis. The expression of SENP5 and 7 was analysed by PCR and Western Blot. With specific antibodies, the expression of SENP5 and 7 was analyzed by immunohistochemistry in synovial tissue specimens. The subcellular localization of SENP5 and 7 was studied by immunocytochemical costaining with cellcompartment specific markers and confocal laser scanning microscopy. Results We where able to show an imbalance in both SUMO specific protease 5 and 7. Both were strongly upregulated in RASF in comparison to OASF. SENP5 showed a clear nuclear localization whereas SENP7 was found also in the cytoplasm. In line with our findings in tissue samples we could show a expression of SENP5 and 7 in RASF compared to OASF. Furthermore we where able to confirm these findings in the hTNFtg mouse model of human RA. Here, we found a strong upregulation of SENP5 and 7 on protein level. However the mRNA level showed only a difference for SENP7. In immuncytochemistry experiments the nuclear and cytoplasmic staining for Senp7 could be proven. Conclusions Our findings underline the role of posttranslational protein modifications in RASF. Strong unbalances in the SUMOylations in hand with a striking overexpression of SENP’s adds to the characteristic features of RASF that promote the diseases processes in RA fibroblasts. Disclosure of Interest None Declared

Background Posttranslational modification of SUMO to target proteins regulates diverse cellular functions, including transcription, nuclear translocation, stress response and protein stability. Previously, we showed that the increased expression of SUMO-1 in RA fibroblasts regulates the susceptibility to apoptosis through a SUMO-1/SENP1 dependent mechanism. Objectives As SUMO-2/3 is a structurally similar but functionally distinct member of the SUMO family, we investigated the expression of SUMO-2/3 in human RA and in hTNFtg mice and studied its role in regulating both apoptosis and the expression of disease specific MMPs. Methods Synovial tissue was obtained from RA and osteoarthritis (OA) patients at joint replacement surgery and used for histological analysis as well as for the isolation of synovial fibroblasts (SFs). Using specific antibodies in immunohistochemical and Western blot analyses, we studied the expression of SUMO-2/3 in fibroblasts from human RA as well as from hTNFtg and wt mice. Knockdown of SUMO-2/3 was performed using specific siRNA against both SUMO-2 and -3. Total RNAs were isolated from RASF/OASF and from hTNFtg/wt SFs. The apoptotic response of the fibroblasts was measured using a Caspase-3/7 assay after induction of cell death with 100ng/ml Fas ligand over 13h. MMP-1, MMP-3 and MMP-13 production in SFs from RA and OA patients was measured by ELISA following the stimulation with TNF-alpha and IL-1beta. Nuclear protein extracts from HeLa cells were used for NFkB p65 transcription factor assay. Results Immunohistochemistry and Western blot analyses revealed a clear upregulation of SUMO-2/3 expression in all RA synovial tissue samples and in RASF compared to OA control samples. In line with these data, tissue sections of hTNFtg mice, as well as SFs from these mice confirmed increased expression of SUMO-2/3. Interestingly, real-time PCR showed elevated expression of SUMO-2 mRNA in RASF and hTNFtg SFs, but not of SUMO-3. Further, expression of SUMO-2/3 proteins were increased after stimulation with TNF-alpha in RASF. At the mRNA level SUMO-2 expression was increased after TNF-alpha stimulation, but not SUMO-3. Knockdown of SUMO-2/3 by siRNA sensitized RASF to Fas-mediated apoptosis. Surprisingly, TNF-alpha and IL-1beta induced upregulation of MMP-3 and MMP-13 expression was significantly stronger after knockdown of SUMO-2/3 in RA- and OASF. Whereas, the expression of MMP-1 was not affected. In addition, downregulation of SUMO-2/3 resulted in enhanced activity of NF-kB (p65) after TNF-alpha stimulation. Conclusions We conclude that posttranslational modification of target proteins by SUMO-2/3 and specifically increased levels of SUMO-2/3 in RASF contribute to the resistance of these cells against Fas-mediated apoptosis. Moreover, they indicate that SUMO-2/3 is regulated by TNF-alpha. Furthermore, SUMO-2/3 positively regulates TNF-alpha induced NF-kB transcriptional activity and hence increases expression of MMP-3 and MMP-13. Therefore, we hypothesize that SUMO-2/3 are novel players contributing to the specific activation of RASF and, thus, to the disease process of RA. Disclosure of Interest None Declared

SUMO-2/3 belongs to the small ubiquitin-related modifier family of proteins that have been implicated in many biological functions such as intracellular protein transport, control of gene expression and stability of proteins. The authors have previously shown that elevated expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (RASF) confers the resistance against Fas-induced apoptosis through SUMO-1/SENP1-dependent mechanisms. Based on these findings, the authors set out to analyse the expression of SUMO-2/3 in human RA and in a tumour necrosis …

Small ubiquitin-related modifiers (SUMOs) are key players in post-translational protein modifications and regulate various cellular processes such as cell division or apoptosis. SUMOylation is regulated by a family of SUMO specific proteases (SENPs) known to activate SUMO precursors and regulate deSUMOylation processes of various target proteins. Our previous studies showed that enhanced SUMO expression in rheumatoid arthritis (RA) altered their apoptotic behaviour. Based on these findings and the fact that SENP7 is localised on a susceptibility locus …

Integrins are the main receptors for cell-matrix interactions and integrin signalling is critical for a variety of cellular functions such as adhesion, cell spreading and inflammatory responses. α2β1 integrin functions as a major receptor for type I collagen on a number of different cells, including fibroblasts and inflammatory cells. Although α2-deficient mice appear normal apart from mild platelet dysfunction, it was shown that α2β1 integrin contributes to the induction of matrix metalloproteinases (MMPs) in tissue remodelling. Based on the hypothesis …