Anja Hillmann

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.

Cell surface-bound TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival pathways.

Background The matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitor of metalloproteinases (TIMPs 1–4) are responsible for the physiological remodeling of the extracellular matrix (ECM). Among all TIMPs, TIMP3 appears to play a unique role since TIMP3 is a secreted protein and, unlike the other TIMP family members, is tightly bound to the ECM. Moreover TIMP3 has been shown to be able to induce apoptotic cell death. As little is known about the underlying mechanisms, we set out to investigate the pro-apoptotic effect of TIMP3 in human mesenchymal cells. Methodology/Principal Findings Lentiviral overexpression of TIMP3 in mesenchymal cells led to a strong dose-dependent induction of ligand-independent apoptosis as reflected by a five-fold increase in caspase 3 and 7 activity compared to control (pLenti6/V5-GW/lacZ) or uninfected cells, whereas exogenous TIMP3 failed to induce apoptosis. Concordantly, increased cleavage of death substrate PARP and the caspases 3 and 7 was observed in TIMP3 overexpressing cultures. Notably, activation of caspase-8 but not caspase-9 was observed in TIMP3-overexpressing cells, indicating a death receptor-dependent mechanism. Moreover, overexpression of TIMP3 led to a further induction of apoptosis after stimulation with TNF-alpha, FasL and TRAIL. Most interestingly, TIMP3-overexpression was associated with a decrease in phosphorylation of cRaf, extracellular signal-regulated protein kinase (Erk1/2), ribosomal S6 kinase (RSK1) and Akt and serum deprivation of TIMP3-overexpressing cells resulted in a distinct enhancement of apoptosis, pointing to an impaired signaling of serum-derived survival factors. Finally, heparinase treatment of heparan sulfate proteoglycans led to the release of TIMP3 from the surface of overexpressing cells and to a significant decrease in apoptosis indicating that the binding of TIMP3 is necessary for apoptosis induction. Conclusion The results demonstrate that exclusively cell surface-bound endogenous TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival signaling pathways.

Uptake of SLE autoantibodies by podocytes

Backgroundand objectives Systemic lupus erythematosus (SLE) is an autoimmune disease, resulting in inflammation and tissue damage in several organs. This autoimmune reaction is caused by antibodies against dsDNA (adsDNAabs). Most patients with SLE develop joint pain and about 70% develop renal symptoms, partly with lupus nephritis leading to proteinuria. It is still unclear why the kidney and especially the podocytes (filtrating cells of the renal glomerulus) are involved in such a dramatic manner during SLE. Methods Anti dsDNA antibodies were isolated from SLE-patients, cloned and produced recombinantly in HEK293 cells. Podocytes were treated with equal amounts of different adsDNAabs and control antibodies in time and concentration dependent experiments and examined by immunofluorescence. To investigate the degradation mechanism, cells were treated with Bafilomycine A or MG132 at different time points of co-cultivation with the antibodies. Results Cultivated podocytes take up adsDNA-antibodies selectively and aggregate them in cytosolic speckles. The uptake of adsDNAabs occurs in a concentration and time dependent manner. The authors found podocytes with up to 80 aggregates per cell. The aggregates are eliminated over time in media without adsDNAabs (recovery experiment). The authors could show that the adsDNAabs are degraded by autophagy, which leads to a better survival of the cells. The number of aggregates after the inhibition of autophagy rises significantly up to twofold. The number of aggregates of podocytes treated with a control antibody is almost zero. Conclusion Podocytes are postmitotic and highly complex cells. Loss of these cells leads to kidney injury and proteinuria. During lupus-nephritis, podocytes are destroyed. The authors could show that these cells take up SLE-antibodies selectively and are harmed by these antibody-aggregates. The authors could show that induction of autophagy can help to eliminate the aggregates. This should be considered for future therapeutic approaches in lupus nephritis.

A2.13 DNA-antibody complexes are internalised by podocytes

Background and objectives Systemic lupus erythematosus (SLE) causes inflammation and damage of various organs, often of the kidney (Lupus nephritis, LN). The occurrence of antibodies against double stranded DNA (adsDNAabs) is a hallmark of the disease, and serum levels of adsDNAabs correlate directly with the risk for LN during SLE. Podocytes are the filtrating cells of the renal glomerulus, and podocyte damage is a key factor in the development of LN-associated proteinuria. However, the molecular pathogenesis of LN as well as the question of how dsDNAabs act on podocytes during LN are not completely understood. Methods Human adsDNAabs originally isolated from SLE patients were produced recombinantly, and podocytes (AB8 cell line) were incubated with these adsDNAabs as well as control antibodies. The interaction of the antibodies with the AB8 cells, their internalisation and subcellular localisation were analysed by immunofluorescence, Western blot, and electron microscopy. To decompose potential antibody-antigen complexes, adsDNAabs were pre-incubated with different amounts of DNaseI. To study potential internalisation mechanisms, endocytosis was inhibited by MDC, Nystatin, and Chlorpromazine. Primary human podocytes from urine of LN patients were harvested, fixed and stained for immunofluorescence. Results Podocytes internalised adsDNAabs in a time and dose dependent manner, whereas no uptake of non-selfreactive and anti-SSA/Ro antibodies was observed. These adsDNAabs were seen in intracellular vesicle-like structures, and internalisation depended on the formation of complexes of adsDNA antibodies with DNA. Inhibition of clathrin dependent endocytosis reduces the number of IgG positive vesicles within the cell by 75%, while inhibition of caveolin dependent endocytosis had no effects. Podocytes of LN patients were found in urine and exhibited human IgG positive aggregates, indicating a disease relevant role of antibody internalisation by podocytes. Conclusion Podocytes are postmitotic cells, therefore damage and loss of podocytes during LN is an irreversible process, leading to proteinuria. We could show that these cells internalise adsDNAabs selectively in complex with their target. A similar pattern of IgG positive structures could be found in podocytes isolated from the urine of LN patients. The internalisation takes place in a clathrin dependent manner and could be part of the pathogenesis of LN.

A8.12 Small Ubiquitin Related Modifier-1 (SUMO-1) Regulates Osteoclastogenesis

Background and Objectives Rheumatoid arthritis (RA) is a common autoimmune disease characterised by the hyperplastic transformation of synovium, its infiltration with different inflammatory cells and by stimulation of bone resorption through osteoclast activation leading to joint destruction. Posttranslational modification of proteins by SUMO has been shown for a number of target molecules including transcription factors and is involved in a variety of cellular processes, including protein localisation, transcriptional regulation, protein stability, cell survival and death. Previously, we have shown that the increased expression of SUMO-1 contributes to the inflammatory response in RA. Here, we investigated the role of SUMO-1 in osteoclastogenesis and studied the skeletal phenotype of SUMO-1 -/- mice under physiological conditions. Materials and Methods For all in vitro experiments, bone marrow macrophages were isolated from SUMO-1 -/- mice and wild type (WT) controls and were cultured in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor k-B ligand. Osteoclast differentiation was verified by tartrate-resistant acid phosphatase (TRAP) staining. Using real time PCR mRNA levels of DC-STAMP and Cathepsin K were analysed. Proliferation of preosteoclasts was determined using CyQuant proliferation assay. Osteoclast resorption capacity was analysed using a calcium phosphate bone resorption assay. The skeletal phenotype of 8-week old mice was investigated by µCT-analysis of trabecular bone in the lumbar spine and femora. The vertebral bodies L5 from each animal were dehydrated and embedded nondecalcified into methylmetacrylate for sectioning. Sections were stained using van Kossa and for TRAP activity. Results In PCR analyses, we found decreased expression of DC-STAMP and Cathepsin K in SUMO-1 -/- mice compared to wt mice during osteoclast differentiation. Proliferation of preosteoclasts was not affected by loss of SUMO-1. In osteoclast formation assays, the loss of SUMO-1 was associated with impaired osteoclast differentiation and with impaired bone resorption capacity. In addition, histological analyses revealed a reduced number of osteoclasts in SUMO-1 -/- mice. At 8-weeks old, SUMO-1 -/- mice had a 20% higher trabecular bone volume fraction compared with wt mice. Moreover, trabecular thickness was higher and trabecular separation was lower in SUMO-1 -/- mice. Conclusions In our study, we found that SUMO-1 -/- mice have high bone mass owing to a decrease in number, size and function of osteoclasts. Furthermore, osteoclast markers contributing to osteoclast fusion and to osteoclast resorption capacity were decreased. These data suggest that SUMO-1 is involved predominantly in the regulation of bone mass by osteoclast formation and activity, and therefore may be an interesting target for treating diseases associated with bone loss.

THU0026 P62/SQSTM1 links reactive oxygen species formation and obesity to increased tnfalpha-mediated joint destruction via its signalling domains

Background The multidomain protein p62/SQSTM1 modulates important signalling pathways responsible for osteoclastogenesis, cell proliferation and polarity. Furthermore, it also regulates protein degradation by autophagy, the ubiquitin proteasome system or sequestration. Although there is a link between p62 mutations and Paget’s disease of bone, it is unclear how p62 acts as a signaling scaffold between the different pathways and how the single domains coordinate the protein functions and link different processes during chronic inflammation. Methods Therefore, we generated mice carrying a truncated form of p62, which lacks the binding sites for TRAF6 and aPKC, but still contains a functional ubiquitin binding site (p62Delta69-251). We characterized these mice and crossed them with arthritic hTNFtg animals to study the effects of the p62 signaling domain on the onset, severity and histologicals changes of a TNFalpha dependent arthritis. Isolated cells of these mice were analysed for alterations in their differentiation capacity, ROS production and MAPK signaling. Results The truncated p62Delta69-251 protein was ubiquitously expressed and was still able to oligomerize and to bind LC3, ubiquitinated proteins, and ERK1 whereas TRAF6 binding was missing. Cells of homozygote p62Delta/69-251mice showed an increased production of ROS and elevated levels of autophagy-related acidic membrane vesicles. Stimulation of p62Delta69-251 cells with the autophagy-inhibitor Bafilomycin and the proteasome-inhibitor MG132 still resulted in an accumulation of p62 and aggregate formation. Furthermore, the lack of the signaling module was sufficient to produce an obese phenotype accompanied by the enlargement of organs, e.g. liver and kidney. PET/CT studies revealed an increased bone metabolism and BMDMs of p62Delta69-251 mice showed a significantly increased osteoclastogenesis in vitro, particularly when stimulated with TNFalpha. WB analysis revealed an increased TNFalpha- induced p38 phosphorylation in cells from p62Delta69-251 compared to wt mice. Crossing of p62Delta69-251mice with hTNFtg animals resulted in a dramatic increase in the severity of joint damage conceivable by an increased number and size of osteoclasts. Interestingly, p62Delta69-251/+hTNFtg mice showed a significant increased weight at week 12 compared to hTNFtg mice. Conclusions The loss of p62 signaling domains has important consequences both for metabolic activation and for osteoclastogenesis under inflammatory conditions. Disclosure of Interest None Declared

SAT0159 Podocyte damage by autoantibodies in lupus nephritis

Background Systemic lupus erythematosus (SLE) is an autoimmune disease, resulting in inflammation and tissue damage in several organs. This autoimmune reaction is caused by antibodies against dsDNA (adsDNAabs). Most patients with SLE develop joint pain and about 70% develop renal symptoms, partly with lupus nephritis leading to proteinuria. It is still unclear why the kidney and especially the podocytes (filtrating cells of the renal glomerulus) are involved in such a dramatic manner during SLE. Methods Anti dsDNA antibodies were isolated from SLE-patients, cloned and produced recombinantly in HEK293 cells. Podocytes were treated with equal amounts of different adsDNAabs and control antibodies in time and concentration dependent experiments and examined by immunofluorescence. To investigate the degradation mechanism, cells were treated with Bafilomycine A or MG132 at different time points of co-cultivation with the antibodies. Results adsDNA-antibodies are taken up by cultivated podocytes selectively. They turn up in cytosolic aggregates. The uptake of adsDNAabs occurs in a concentration and time dependent manner. We found podocytes with up to 80 aggregates per cell. In a recovery experiment, we could show that these aggregates are eliminated over time in media without adsDNAabs. The degradation of these adsDNAabs is reduced in experiments under autophagy inhibiting conditions (treatment with Bafilomycin A) and number of aggregates after the inhibition of autophagy rises significantly up to twofold. Stimulation of Autophagy by MG132 leads to a better survival of the cells. The number of aggregates of podocytes treated with a control antibody is almost zero. Conclusions Podocytes are postmitotic and highly complex cells. Loss of these cells leads to kidney injury and proteinuria. During lupus-nephritis, podocytes are destroyed. We could show that these cells take up SLE-antibodies selectively and are harmed by these antibody-aggregates. Furthermore, we have first evidence that the induction of autophagy can help to eliminate the aggregates. This should be considered for future therapeutic approaches in lupus nephritis. 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

The signalling domain of the multiadaptor protein p62/SQSTM1 links reactive oxygen species formation and obesity to increased TNFα-mediated joint damage

Background The multidomain protein p62 plays a key role in signal transduction through interaction with TRAF6, ERK1 and aPKC. It also contributes to protein degradation by autophagy, the ubiquitin proteasome system or sequestration. Despite an established role as in Pagets disease, it is unclear how p62 acts as a signalling hub between different pathways and how the single domains coordinate the protein functions and link different pathological processes. Methods Therefore, the authors generated a mouse line carrying a shortened variant of p62, which lacks the binding sites for TRAF6 and aPKC, but still contains all protein degradation modules (p62Delta69-251). The authors analysed the phenotype of these mice and crossed them with hTNFtg arthritic animals to study the effects of the p62 signalling domain on the onset, severity and histologicals changes of a TNFα dependent arthritis. Isolated cells of these mice were analysed for alterations in their differentiation capacity, ROS production and MAPK signalling. Results The truncated p62Delta69-251 protein was ubiquitously expressed and was still able to oligomerise and to bind LC3, ubiquitinated proteins and ERK1. As expected, p62Detla69-251 failed to interact with TRAF6. Of note, cells of homozygote p62Delta/69-251 mice showed an increased production of ROS, and this effect correlated with elevated levels of autophagy-related acidic membrane vesicles. Stimulation of p62Delta69-251 cells with the autophagy-inhibitor Bafilomycin and the proteasome-inhibitor MG132 still resulted in an accumulation of p62 and aggregate formation. Interestingly, the lack of the signaling module was sufficient to produce an obese phenotype accompanied by the enlargement of organs, for example, liver and kidney. PET/CT studies revealed an increased bone metabolism and BMDMs of p62Delta69-251 mice showed a significantly increased osteoclastogenesis in vitro, particularly when stimulated with tumour necrosis factor α (TNFα). WB analysis revealed an increased TNFα- induced p38 phosphorylation in cells from p62Delta69-251 compared to wt mice. Crossing of p62Delta69-251 mice with hTNFtg animals resulted in a dramatic increase in the severity of joint damage conceivable by an increased number and size of osteoclasts. Interestingly, p62Delta69-251/+ hTNFtg mice showed a significant increased weight at week 12 compared to hTNFtg mice. Conclusion The authors show for the first time, that the signalling module of p62 can act as an intracellular antiobesity-factor and that this function does not require the modules for autophagy and ubiquitin-binding. Moreover, p62 is an important regulator of TNFα mediated joint damage indicating that the loss of p62 signalling domains has important consequences both for metabolic activation and for osteoclastogenesis under inflammatory conditions.

The TRAF6 binding molecule p62/SQSTM1 is a critical regulator of inflammatory bone destruction

The activation of nuclear factor κB (NFκB) via RANK is essential in regulating osteoclastogenesis. Under inflammatory conditions this process is enhanced by cytokines such as tumour necrosis factor α (TNFα). P62/SQSTM1 directly modulates these pathways through complex formation with TRAF6, aPKCs and ubiquitin. However, the role of p62/SQSTM1 in regulating bone turnover under inflammatory conditions, and specifically the role of the signal transduction domains of p62/SQSTM1, is unclear. Mice were generated that carry a shortened but functional mutant of p62 with …