Marianne Heitzmann

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.

Transport mechanisms and their pathology-induced regulation govern tyrosine kinase inhibitor delivery in rheumatoid arthritis.

Background Tyrosine kinase inhibitors (TKIs) are effective in treating malignant disorders and were lately suggested to have an impact on non-malignant diseases. However, in some inflammatory conditions like rheumatoid arthritis (RA) the in vivo effect seemed to be moderate. As most TKIs are taken up actively into cells by cell membrane transporters, this study aimed to evaluate the role of such transporters for the accumulation of the TKI Imatinib mesylates in RA synovial fibroblasts as well as their regulation under inflammatory conditions. Methodology/Principal Findings The transport and accumulation of Imatinib was investigated in transporter-transfected HEK293 cells and human RA synovial fibroblasts (hRASF). Transporter expression was quantified by qRT-PCR. In transfection experiments, hMATE1 showed the highest apparent affinity for Imatinib among all known Imatinib transporters. Experiments quantifying the Imatinib uptake in the presence of specific transporter inhibitors and after siRNA knockdown of hMATE1 indeed identified hMATE1 to mediate Imatinib transport in hRASF. The anti-proliferative effect of Imatinib on PDGF stimulated hRASF was quantified by cell counting and directly correlated with the uptake activity of hMATE1. Expression of hMATE1 was investigated by Western blot and immuno-fluorescence. Imatinib transport under disease-relevant conditions, such as an altered pH and following stimulation with different cytokines, was also investigated by HPLC. The uptake was significantly reduced by an acidic extracellular pH as well as by the cytokines TNFα, IL-1β and IL-6, which all decreased the expression of hMATE1-mRNA and protein. Conclusion/Significance The regulation of Imatinib uptake via hMATE1 in hRASF and resulting effects on their proliferation may explain moderate in vivo effects on RA. Moreover, our results suggest that investigating transporter mediated drug processing under normal and pathological conditions is important for developing intracellular acting drugs used in inflammatory diseases.

Structural cartilage damage attracts circulating rheumatoid arthritis synovial fibroblasts into affected joints

BackgroundRheumatoid arthritis synovial fibroblasts (RASFs) are known to travel via the bloodstream from sites of cartilage destruction to new locations where they reinitiate the destructive processes at distant articular cartilage surfaces. In this study, we examined the role of interleukin (IL)-1-induced cartilage changes and their chemotactic effect on RASF transmigratory capacity.MethodsTo investigate synovial fibroblast (SF) transmigration through endothelial layers, we used a modified Boyden chamber with an endothelioma cell layer (bEnd.5) as a barrier and IL-1-treated murine cartilage explants as a chemotactic stimulus for SFs from human tumor necrosis factor–transgenic (hTNFtg) mice. We injected recombinant IL-1 or collagenase into knee joints of wild-type mice, followed by tail vein injection of fluorescence-labeled hTNFtg SFs. The distribution and intensity of transmigrating hTNFtg SFs were measured by fluorescence reflectance imaging with X-ray coregistration. Toluidine blue staining was performed to evaluate the amount of cartilage destruction.ResultsHistomorphometric analyses and in vivo imaging revealed a high degree of cartilage proteoglycan loss after intra-articular IL-1 and collagenase injection, accompanied by an enhanced in vivo extravasation of hTNFtg SFs into the respective knee joints, suggesting that structural cartilage damage contributes significantly to the attraction of hTNFtg SFs into these joints. In vitro results showed that degraded cartilage was directly responsible for the enhanced transmigratory capacity because stimulation with IL-1-treated cartilage, but not with IL-1 or cartilage alone, was required to increase hTNFtg SF migration.ConclusionsThe present data indicate that structural cartilage damage facilitates the migration of arthritic SF into affected joints. The prevention of early inflammatory cartilage damage may therefore help prevent the progression of rheumatoid arthritis and its spread to previously unaffected joints.

A10.15 LASP-1 modifies ECM-synovial fibroblast interactions in a mouse model of ra

Background and objectives The LIM-and-SH3-domain-protein-1 (Lasp-1) is an actin-associated protein and is localised at focal adhesion sites where it is involved in organisation of actin polymerization and focal adhesion turnover prozesses. We investigated its role in regulating synovial fibroblast (SF) interaction with components of the extracellular matrix (ECM) and in establishing cell-cell contacts during RA. Materials and methods Lasp-1 expression was analysed in tissue from RA patients and in the hind paws of arthritic hTNFtg mice by Western blot, immunofluorescence and immunohistochemistry. Furthermore, Lasp-1-/- mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Migration characteristics of SF derived from wild type (wt), Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg mice were analysed in a modified scratch assay and by live cell imaging. Cell-matrix interactions and cell-cell contacts of isolated SF from all different genotypes were investigated using fibronectin coating in an electrical cell/substrate impedance sensing assay (ECIS). Additionally, we used an in vitro three-dimensional organ culture system for functional analyes. Results Lasp-1 expression levels were increased in human RA tissue and hTNFtg mice in comparison to healthy controls. Evaluation of Lasp-1-/-/hTNFtg mice revealed milder arthritis score compared to hTNFtg mice,which was confirmed by immunohistochemistry. Results of the scratch assays demonstrated a significantly reduced migration rate of Lasp-1-/- SF (-43,7% vs wt SF) and Lasp-1-/-/hTNFtg SF (-69,11% vs hTNFtg SF). Furthermore, live cell imaging studies demonstrated striking differences in the migration velocity and in migration edge formation of Lasp-1-/-/hTNFtg SF compared to hTNFtg SF. ECIS analysis demonstrated an increased cell-cell contact formation in Lasp1-/- compared to wt SF (+22% versus wt SF) and prolonged cell-cell interaction remodelling of Lasp-1-/-/hTNFtg SF in comparison to hTNFtg SF. Histological analysis of 3D matrices showed that Lasp-1 deletion in the hTNFtg background resulted in an organised cellular lining layer at the interface between the matrix and fluid phases comparable with wild type SF. In contrast, hTNFtg SF formed unorganised cellular condensations with no synovial architecture evident. Conclusion Lasp-1 regulates the migratory behaviour of synovial fibroblasts in rheumatoid arthritis by controlling the dynamics of cell-matrix and cell-cell contacts.

A6.7 The junctional adhesion molecule JAM-3 regulates the motility of fibroblast-like synoviocytes

Background and objectives Rheumatoid arthritis (RA) is a disease of a continuous activation and expansion of the synovium involving migration and dissemination of fibroblast-like synoviocytes (FLS). Several lines of evidence indicate that adhesion molecules play a crucial role in this process. We have focused on the junctional adhesion molecule 3 (JAM-3), known to be involved in adhesion, polarity and migration of other cells. So far it has been demonstrated that blocking of JAM-3 in a serum transfer model of RA ameliorates the disease. However, the question of whether JAM-3 is involved in fibroblast motility or primarily regulates the influx of inflammatory cells remains unknown. Therefore, our objective was to clarify the involvement of JAM-3 in the establishment of cell-cell and cell-matrix contacts as well as in the migration of FLS. Materials and methods As an arthritis model we use human TNFalpha transgenic (hTNFtg) mice that due to high levels of human TNFalpha develop a RA-like disease. FLS form these animals as well as form JAM-3 floxed mice CreERT2 (tamoxifen inducible cre line) and control mice were isolated by collagenase digestion of the hind paws. Cells were subjected to electrical cell-substrate impedance sensing (ECIS) surface staining (FACS), immunofluorescence, in vitro cell migration assays and standard molecular biology methods. Results JAM-3 was found on both wild type and hTNFtg synoviocytes. Blocking or silencing of JAM-3 significantly increased cell spreading in an modified scratch assay but decreased migration in an in vitro transmigration assay, indicating that JAM-3 is involved in the establishment of cell-cell contacts between FLS. In line with this notion ECIS experiments revealed altered adhesion properties of these cells. Explaining these findings, we found that JAM-3 co-localises with the tight junction molecule zo-1 at cell contact sites, which are connected to actin cell filaments. The ultrastructure of these structures and an influence of Jam-3 on the cytoskeleton dynamics is under investigation. Conclusions JAM-3 modulates migration of FLS in vitro, which may contribute to the beneficial effects of JAM-3 inhibition as seen in the serum transfer model of RA in vivo. We suggest that Jam-3 is an adhesion molecule, responsible for controlling motility by organising the cytoskeleton in FLS.

A2.11 LASP-1 deficiency is changing synovial fibroblast interaction with cartilage matrix in TNFα mediated arthritis

Background and objectives Lasp-1 (LIM-and-SH3-domain-protein-1) is an actin-binding protein that modifies cytoskeleton organisation and is localised at cell-matrix interaction sites where it is involved in cell adhesion, migration and metastatic invasion. Its role in regulating synovial fibroblast (SF) interaction with components of extracellular matrix (ECM) and cell-cell contacts during RA is unknown. Furthermore, involved signalling pathways have to be elucidated. Materials and methods Lasp-1 expression in RA tissue and hind paws of arthritic hTNFtg mice was analysed via Western blot and immunohistochemistry. Furthermore, Lasp-1 ko mice were interbred with hTNFtg mice and offsprings were analysed for the progression of joint destruction by clinical evaluation and histopathology. Cell migration properties of SF derived from wild type (wt), Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg mice were measured using a modified scratch assay and by live cell imaging. Extracellular matrix structure and organisation from wt, Lasp-1-/-, hTNFtg and Lasp-1-/-/hTNFtg SF were analysed by electron microscopy (EM). Cell-matrix interactions as well as cell-cell contacts of isolated SF from mice of all genotypes were investigated using fibronectin and self-purified collagen matrix in an electrical cell/substrate impedance sensing assay (ECIS). Via pull down assays and EM components of integrin mediated cell-matrix interaction complexes were examined. In addition, src-phosphorylation levels were evaluated by Western blot. Results Lasp-1 expression levels were increased in human RA and in hTNFtg mice compared to healthy controls. Lasp-1-/-/hTNFtg mice displayed milder clinical symptoms confirmed by immunohistochemistry. Migration assays presented a significantly reduced migration rate of Lasp-1-/- and Lasp-1-/-/hTNFtg SF compared to SF from wt and hTNFtg mice. Analyses of SF ECM showed thickened collagen fibrils with less crosslinking of Lasp-1-/- SF in comparison to wt controls. In ECIS, Lasp-1-/- SF adhered to both fibronectin and self purified ECM faster than other genotypes (-50% vs. hTNFtg and wt SF). Additionally, Lasp-1-/- SF formed a higher number of cell-cell contacts than wt and hTNFtg SF (-17% vs. hTNFtg and -22% wt vs. SF). Pull down assays identified cortactin, uPar and vinculin as components of cell-matrix interaction complexes with increased levels of vinculin found in complexes from Lasp-1-/-compared to wtSF. Finally, both Lasp-1-/- and Lasp-1-/-/hTNFtg SF showed reduced src-phosphorylation compared to wt and hTNFtg SF. Conclusion The loss of Lasp-1 leads to altered src-phosphorylation and changes in cell-matrix interaction complexes as well as altered extracellular matrix organisation. Furthermore, Lasp-1 deficiency leads to significantly reduced migration rates of RASF and hTNFtg SF in vitro and to decreased cartilage degradation in hTNFtg mice in vivo.

A1.23 Lasp-1 deficiency modifies synovial fibroblast migration and cartilage destruction in a model of HTNF alpha mediated arthritis

Background and Objectives Lasp-1 is an important actin-crosslinking protein. It’s localises at focal adhesions, along stress fibres and leading edges and is involved in cellular migration and metastatic dissemination of different cancers. Rheumatoid arthritis synovial fibroblasts (RASF) are also able to enter the bloodstream from sites of cartilage destruction to new locations where they re-initiate the destructive processes. The underlying mechanisms of this process are of special interest, but currently unclear. Therefore we have investigated the role of Lasp-1 in synovial fibroblast migration during RA. Materials and Methods Lasp-1 expression and its subcellular location was analysed using Western blotting and immunofluorescence microscopy of cells seeded on various coatings, including fibronectin, laminin and a self-purified collagen matrix. Furthermore, Lasp-1 deficient mice were generated, which were interbred with hTNFtg mice, to form a model of inflammatory arthritis. Mice of all genotypes were analysed using a clinical score, measuring weight, grip strength and paw swelling over a time course of 14 weeks. Hind paws from each genotype were isolated, paraffin-embedded and toluidine blue stained in order to assess synovial inflammation, cartilage degradation and SF attachment. Cell migration properties of SFs derived from WT, Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg were measured using a modified scratch assay and by live cell imaging. Results Lasp-1 expression was up regulated in RASF and in SF from hTNFtg mice compared to healthy controls. In immunofluorescence, Lasp-1 was co-localised with cortactin, a marker for structures of cell adhesion and invasion, particularly when cultured on a purified collagen matrix. Lasp1-/-/hTNFtg mice showed milder clinical symptoms in comparison to hTNFtg mice. Notably, histopathological analyses revealed less cartilage destruction (0.4 mm vs. 1.6 mm, p<0.05) and less attachment of synovial tissue to the cartilage surface (5% vs. 35%, p<0.05) in Lasp1-/-/hTNFtg mice at 14 weeks of age. Lasp-1-/- SFs and Lasp1-/-/hTNFtg SFs exhibited a significantly reduced migration rate in comparison to WT and hTNFtg SFs, with significant differences observed in cell migration speed, cell spreading and in the formation of stress fibres and filopodia. Conclusion Lasp-1 deficiency leads to significantly reduced migration rates of RASF and hTNFtg SF in vitro and results in a decreased cartilage degradation and destruction and less SF attachment to articular cartilage in hTNFtg mice in vivo. Therefore, Lasp-1 is an important target for therapeutic strategies aimed to reduce the invasive and migratory behaviour of synovial fibroblast in rheumatoid arthritis.

A8.15 The Focal Contact Protein Lasp-1 Modulates the Migration Capacity of Synovial Fibroblasts

Background and Objectives RA synovial fibroblasts (SF) have been suggested to contribute to the spreading of disease through their ability to leave cartilage destruction sites, migrate via the bloodstream and re-initiate the destructive process at distant articular cartilage surfaces. In this context, the actin-crosslinking protein Lasp-1 is of interest, because it is localised at leading edges of migrating cells and regulates metastatic dissemination of different tumours. Therefore, it is particularly important to investigate the role of Lasp-1 in SF migration and its effects on RA. Materials and Methods To identify different Lasp-1 expression levels in the hind paws of wt and hTNFtg mice, an established model for human RA, Western- blot analyses were performed. In parallel, Lasp-1 expression and its sub-cellular distribution was investigated in SF from wt and hTNFtg mice by Western-blot analyses and immunofluorescence. The migratory capacity of SFs derived from wild-type, Lasp-1-/-, hTNFtg and Lasp1-/-/hTNFtg mice was studied in a modified scratch assay as well as in live cell imaging studies. Furthermore, a transmigration assay using SF from all four genotypes and murine endothelioma cells (bEnd.5) as an endothelial barrier was carried out. For more detailed information, SF transmigration was evaluated when endothelial cells were also pre-treated with TNF-alpha, mimicking inflammatory conditions. Results Lasp-1 expression is upregulated in SF from hTNFtg mice and localises to structures of cell adhesion and invasion. In the scratch assay, a significantly reduced migration rate was detected in Lasp-1-/- SFs after 24 hrs (–43.7% versus wt, p < 0.05) and in Lasp1-/-/hTNFtg, respectively (–69.11% versus hTNFtg, p < 0.05). Live cell imaging studies showed a slower migration and striking differences in migration morphology of Lasp1-/-/hTNFtg compared to hTNFtg SF. Furthermore, analyses showed a significant reduction of transmigration of Lasp1-/-/hTNFtg compared to hTNFtg SF that was even enhanced by TNF-alpha stimulation of the endothelial cells. Interestingly, interbred Lasp1-/-/hTNFtg mice presented milder clinical symptoms and analyses of histopathology revealed less cartilage degradation and less attachment of synovial tissue to the cartilage than hTNFtg mice at an age of 14 weeks. Conclusions Our data provide that the migratory capacity of SF is regulated by Lasp-1 and influences the severity of arthritis in hTNFtg mice. SF – when activated – migrate through the formation of invasive and adhesive membrane structures such as invadopodia, where Lasp-1 is prominently localised. Thus, targeting Lasp-1 may be a promising strategy to reduce the invasive and migratory behaviour of synovial fibroblasts in RA.

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

A8.5 Fibroblast Activation Protein Alpha in Inflammatory Bone Destruction

Background The Fibroblast Activation Protein alpha (FAPα) is an integral membrane serine protease that plays a major role in migration, wound healing, and metastasis. Based on recent studies that have implicated membrane-bound serine proteases in osteoclast migration, we studied the expression of FAPα in rheumatoid arthritis (RA) and analysed its role in osteoclast development under inflammatory conditions. Methods FAPα expression in vivo was determined by immunohistochemistry in human synovial tissues of patients with RA and osteoarthritis (OA) as well as in hind paws of tumour necrosis factor-alpha transgenic (hTNFtg) mice, which develop a RA-like destructive arthritis. In vitro expression and regulation of FAPα was analysed in differentiated osteoclasts cocultured with osteoblasts, RA or OA synovial fibroblasts (SF) by PCR. To scrutinise the role of FAPα in osteoclastogenesis, we analysed the in vitro differentiation of osteoclasts from wildtype (WT) and FAP-/- mice (Oncology Research, Boehringer Ingelheim RCV, Vienna) by TRAP staining. In order to assess the role of FAP in arthritis severity, we crossed FAP-/- and hTNFtg mice and performed TRAP staining. Results RA synovial tissues demonstrated a high expression of FAPα throughout the tissue whereas in OA samples FAPα was expressed only in the lining layer. In vitro, no expression of FAPα was found in differentiated preosteoclasts and osteoclasts, but coculture experiments showed that RASF, but not OASF or osteoblasts, induce the expression of FAPα in preosteoclasts and osteoclasts. Consistent with the selective induction of FAPα in osteoclasts by RASF, FAPα expression was detected in osteoclasts at the invasion front of the hyperplastic synovial tissues in joints of hTNFtg mice. FAP-/- mice show a severely diminished osteoclast formation compared to WT. We also found a lesser amount of osteoclasts in the hind paws of FAP-/-hTNFtg compared to hTNFtg. Conclusions The disease-dependent expression of FAPα by osteoclasts in human RA and hTNFtg mice suggests an important role of FAPα in joint destruction in RA. The selective induction of FAPα in preosteoclasts and osteoclasts by RASF indicate that FAPα may be regulated through the interaction with the pannus tissue. The fact, that under inflammatory conditions the loss of FAP led to a reduced number of osteoclasts in the hind paws and FAP deficient bone marrow derived macrophages showed a reduced osteoclast formation, suggest a role of this serine protease in macrophage/osteoclast precursor migration and differentiation.