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