Marvin A. Peters

Modification of nuclear PML protein by SUMO-1 regulates Fas-induced apoptosis in rheumatoid arthritis synovial fibroblasts

The small ubiquitin-like modifier (SUMO)-1 is an important posttranslational regulator of different signaling pathways and involved in the formation of promyelocytic leukemia (PML) protein nuclear bodies (NBs). Overexpression of SUMO-1 has been associated with alterations in apoptosis, but the underlying mechanisms and their relevance for human diseases are not clear. Here, we show that the increased expression of SUMO-1 in rheumatoid arthritis (RA) synovial fibroblasts (SFs) contributes to the resistance of these cells against Fas-induced apoptosis through increased SUMOylation of nuclear PML protein and increased recruitment of the transcriptional repressor DAXX to PML NBs. We also show that the nuclear SUMO-protease SENP1, which is found at lower levels in RA SFs, can revert the apoptosis-inhibiting effects of SUMO-1 by releasing DAXX from PML NBs. Our findings indicate that in RA SFs overexpression of SENP1 can alter the SUMO-1-mediated recruitment of DAXX to PML NBs, thus influencing the proapoptotic effects of DAXX. Accumulation of DAXX in PML NBs by SUMO-1 may, therefore, contribute to the pathogenesis of inflammatory disorders.

Epigenetic modifications in rheumatoid arthritis

Over the last decades, genetic factors for rheumatoid diseases like the HLA haplotypes have been studied extensively. However, during the past years of research, it has become more and more evident that the influence of epigenetic processes on the development of rheumatic diseases is probably as strong as the genetic background of a patient. Epigenetic processes are heritable changes in gene expression without alteration of the nucleotide sequence. Such modifications include chromatin methylation and post-translational modification of histones or other chromatin-associated proteins. The latter comprise the addition of methyl, acetyl, and phosphoryl groups or even larger moieties such as binding of ubiquitin or small ubiquitin-like modifier. The combinatory nature of these processes forms a complex network of epigenetic modifications that regulate gene expression through activation or silencing of genes. This review provides insight into the role of epigenetic alterations in the pathogenesis of rheumatoid arthritis and points out how a better understanding of such mechanisms may lead to novel therapeutic strategies.

The loss of α2β1 integrin suppresses joint inflammation and cartilage destruction in mouse models of rheumatoid arthritis.

OBJECTIVE Integrin α2β1 functions as a major receptor for type I collagen on different cell types, including fibroblasts and inflammatory cells. Although in vitro data suggest a role for α2β1 integrin in regulating both cell attachment and expression of matrix-degrading enzymes such as matrix metalloproteinases (MMPs), mice that lack the α2 integrin subunit (Itga2(-/-) mice) develop normally and are fertile. We undertook this study to investigate the effect of Itga2 deficiency in 2 different mouse models of destructive arthritis: the antigen-induced arthritis (AIA) mouse model and the human tumor necrosis factor α (TNFα)-transgenic mouse model. METHODS AIA was induced in the knee joints of Itga2(-/-) mice and wild-type controls. Human TNF-transgenic mice were crossed with Itga2(-/-) mice and were assessed clinically and histopathologically for signs of arthritis, inflammation, bone erosion, and cartilage damage. MMP expression, proliferation, fibroblast attachment, and ERK activation were determined. RESULTS Under arthritic conditions, Itga2 deficiency led to decreased severity of joint pathology. Specifically, Itga2(-/-) mice showed less severe clinical symptoms and dramatically reduced pannus formation and cartilage erosion. Mice lacking α2β1 integrin exhibited reduced MMP-3 expression, both in their sera and in fibroblast-like synoviocytes (FLS), due to impaired ERK activation. Further, both the proliferation and attachment of FLS to cartilage were partially dependent on α2β1 integrin in vitro and in vivo. CONCLUSION Our findings suggest that α2β1 integrin contributes significantly to inflammatory cartilage destruction by promoting fibroblast proliferation and attachment and MMP expression.

Regulation of matrixmetalloproteinase-3 and matrixmetalloproteinase-13 by SUMO-2/3 through the transcription factor NF-κB

Objective Based on previous data that have linked the small ubiquitin-like modifier-1 (SUMO-1) to the pathogenesis of rheumatoid arthritis (RA), we have investigated the expression of the highly homologous SUMO family members SUMO-2/3 in human RA and in the human tumour necrosis factor α transgenic (hTNFtg) mouse model of RA and studied their role in regulating disease specific matrixmetalloproteinases (MMPs). Methods Synovial tissue was obtained from RA and osteoarthritis (OA) patients and used for histological analyses as well as for the isolation of synovial fibroblasts (SFs). The expression of SUMO-2/3 in RA and OA patients as well as in hTNFtg and wild type mice was studied by PCR, western blot and immunostaining. SUMO-2/3 was knocked down using small interfering RNA in SFs, and TNF-α induced MMP production was determined by ELISA. Activation of nuclear factor-κB (NF-κB) was determined by a luciferase activity assay and a transcription factor assay in the presence of the NF-κB inhibitor BAY 11-7082. Results Expression of SUMO-2 and to a lesser extent of SUMO-3 was higher in RA tissues and RASFs compared with OA controls. Similarly, there was increased expression of SUMO-2 in the synovium and in SFs of hTNFtg mice compared with wild type animals. In vitro, the expression of SUMO-2 but not of SUMO-3 was induced by TNF-α. The knockdown of SUMO-2/3 significantly increased the TNF-α and interleukin (IL)-1β induced expression of MMP-3 and MMP-13, accompanied by increased NF-κB activity. Induction of MMP-3 and MMP-13 was inhibited by blockade of the NF-κB pathway. TNF-α and IL-1β mediated MMP-1 expression was not regulated by SUMO-2/3. Conclusions Collectively, we show that despite their high homology, SUMO-2/3 are differentially regulated by TNF-α and selectively control TNF-α mediated MMP expression via the NF-κB pathway. Therefore, we hypothesise that SUMO-2 contributes to the specific activation of RASF.

The small ubiquitin‐like modifier mediates the resistance of prosthesis‐loosening fibroblast‐like synoviocytes against fas‐induced apoptosis

OBJECTIVE To study the expression of small ubiquitin-like modifier 1 (SUMO-1) in aseptic loosening of prosthesis implants and to investigate its role in regulating the susceptibility of prosthesis-loosening fibroblast-like synoviocytes (FLS) to Fas-induced apoptosis. METHODS Specimens of aseptically loosened tissue were obtained at revision surgery, and the expression of SUMO-1 was analyzed by in situ hybridization. SUMO-1 levels in FLS were determined by quantitative polymerase chain reaction and Western blot analysis. Immunohistochemistry and confocal microscopy were used to study the subcellular localization of SUMO-1. The functional role of SUMO-1 in Fas-induced apoptosis of prosthesis-loosening FLS was investigated by small interfering RNA-mediated knockdown of SUMO-1 and by gene transfer of the nuclear SUMO-specific protease SENP1. RESULTS SUMO-1 was expressed strongly in aseptically loosened tissue and was found prominently at sites adjacent to bone. Prosthesis-loosening FLS expressed levels of SUMO-1 similar to the levels expressed by rheumatoid arthritis (RA) FLS, with SUMO-1 being found mainly in promyelocytic leukemia protein nuclear bodies. Knockdown of SUMO-1 had no effect on spontaneous apoptosis but significantly increased the susceptibility of prosthesis-loosening FLS to Fas-induced apoptosis. Gene transfer of the nuclear SUMO-specific protease SENP1 reverted the apoptosis-inhibiting effects of SUMO-1. CONCLUSION These data suggest that SUMO-1 is involved in the activation of both RA FLS and prosthesis-loosening FLS by preventing these cells from undergoing apoptosis. Modification of nuclear proteins by SUMO-1 contributes to the antiapoptotic effects of SUMO-1 in prosthesis-loosening FLS, providing evidence for the specific activation of sumoylation during their differentiation. Therefore, SUMO-1 may be an interesting target for novel strategies to prevent aseptic prosthesis loosening.

[Gene therapy in rheumatoid arthritis: new aspects].

Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease of unknown aetiology that is characterized by the progressive destruction of articular structures. The accumulation of inflammatory cells in the joint and the transformation of the healthy synovial membrane into a hyperplasic and aggressive pannus tissue constitute important steps in the pathology of RA. The synthesis and secretion of inflammatory factors such as cytokines and chemokines as well as the release of matrix degrading enzymes play a decisive role and have, therefore, become of interest for novel therapeutic strategies, among the gene therapy. This article summarizes the principles and current developments of gene therapy in RA and gives an overview about available vector systems and target genes.

SAT0077 Sumo-2/3 regulates apoptosis and MMP expression in rheumatoid arthritis synovial fibroblasts

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

THU0018 Sumo specific proteases 5 and 7 contribute to the inbalanced sumoylation level of rheumatoid athritis synovial fibroblasts

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

SUMO-specific protease 7 (SENP7) regulates matrix metalloproteinase-9 expression in synovial fibroblasts

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 …

Upregulated SUMO-2/3 expression is involved in the regulation of apoptosis and matrix metalloproteinase expression in rheumatoid arthritis synovial fibroblasts

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 …