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)-1ß 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-1ß 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.

Small ubiquitin-like modifier 1 [corrected] 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.

Susceptibility of rheumatoid arthritis synovial fibroblasts to FasL- and TRAIL-induced apoptosis is cell cycle-dependent.

INTRODUCTION: The rheumatoid arthritis (RA) synovium is characterised by the presence of an aggressive population of activated synovial fibroblasts (RASFs) that are prominently involved in the destruction of articular cartilage and bone. Accumulating evidence suggests that RASFs are relatively resistant to Fas-ligand (FasL)-induced apoptosis, but the data concerning tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) have been conflicting. Here, we hypothesise that the susceptibility of RASFs to receptor-mediated apoptosis depends on the proliferation status of these cells and therefore analysed the cell cycle dependency of FasL- and TRAIL-induced programmed cell death of RASFs in vitro. METHODS: Synovial fibroblasts were isolated from patients with RA by enzymatic digestion and cultured under standard conditions. Cell cycle analysis was performed using flow cytometry and staining with propidium iodide. RASFs were synchronised or arrested in various phases of the cell cycle with 0.5 mM hydroxyurea or 2.5 microg/ml nocodazol and with foetal calf serum-free insulin-transferrin-sodium selenite supplemented medium. Apoptosis was induced by stimulation with 100 ng/ml FasL or 100 ng/ml TRAIL over 18 hours. The apoptotic response was measured using the Apo-ONE Homogenous Caspase-3/7 Assay (Promega GmbH, Mannheim, Germany) and the Cell Death Detection (ELISAPlus) (enzyme-linked immunosorbent assay) (Roche Diagnostics GmbH, Mannheim, Germany). Staurosporin-treated cells (1 microg/ml) served as a positive control. Expression of Fas and TRAIL receptors (TRAILR1-4) was determined by fluorescence-activated cell sorting analysis. RESULTS: Freshly isolated RASFs showed only low proliferation in vitro, and the rate decreased further over time, particularly when RASFs became confluent. RASFs expressed Fas, TRAIL receptor-1, and TRAIL receptor-2, and the expression levels were independent of the cell cycle. However, the proliferation rate significantly influenced the susceptibility to FasL- and TRAIL-induced apoptosis. Specifically, proliferating RASFs were less sensitive to FasL- and TRAIL-induced apoptosis than RASFs with a decreased proliferation rate. Furthermore, RASFs that were synchronised in S phase or G2/M phase were less sensitive to TRAIL-induced apoptosis than synchronised RASFs in G0/G1 phase. CONCLUSIONS: Our data indicate that the susceptibility of RASFs to FasL- and TRAIL-induced apoptosis depends on the cell cycle. These results may explain some conflicting data on the ability of RASFs to undergo FasL- and TRAIL-mediated cell death and suggest that strategies to sensitise RASFs to apoptosis may include the targeting of cell cycle-regulating genes.

The expression of collagenase 3 (MMP-13) mRNA in the synovial tissue is associated with histopathologic type II synovitis in rheumatoid arthritis.

The histopathologic analysis of the synovial tissue is important to distinguish rheumatoid arthritis (RA) from other forms of synovitis and to provide information about prognosis and therapeutic strategies at early stages of the disease. In this context, the present study was performed to investigate the correlation between immunohistopathological and morphological features of synovitis and the expression of collagenase 3 (MMP-13) known to contribute significantly to cartilage degradation in RA. In the histopathologic scoring system used in this study, type I synovitis is characterized by B lymphocyte infiltration and an intact lining, and is only mild destructive to cartilage and bone. Type II shows marked diffuse infiltrations of macrophages and T lymphocytes, an ulcerated lining, fibrin exudation, and invasive growth into cartilage and bone tissue. Investigating 36 patients with RA, 21 patients (58%) were positive for the expression of collagenase 3 mRNA in the synovial tissue. Among these patients, 19 showed a histopathologic type II synovitis and only 2 patients had undifferentiated synovitis. In contrast, synovial tissue samples from patients without collagenase 3 mRNA expression were characterized in 6 cases by type I, in 5 cases by type II and in 4 cases by undifferentiated synovitis. The analysis of the clinical data revealed that RA patients with a histopathologic type II synovitis and synovial tissue collagenase 3 mRNA expression had elevated levels of systemic markers of inflammation and received stronger therapies. The data suggest, that collagenase 3 expression and the histopathologic type II synovitis are associated with a severe and destructive course of RA.

[The influence of the drain suction in knee arthroplasty]. / Einfluss des Drainagesoges auf den postoperativen Verlauf nach Knieendoprothesen-Implantation.

PROBLEM: In a prospective randomised clinical study, we investigated the impact of drain-suction on the post-operative blood loss and on both clinical and laboratory parameters after knee replacement operations. PATIENTS AND METHOD: In this study, 116 patients with unilateral implantation of knee replacements were evaluated. The patients' average age was 71.2 years. The operation was carried out mostly without tourniquet application. All patients received two wound drains, 57 with and 59 without suction. The postoperative blood loss as well as clinical and laboratory parameters were assessed. RESULTS: The average peri-operative blood loss amounted to 338 ml. The post-operative blood loss in the group without drain suction was 436 ml and 528 ml in the group with suction. No significant differences could be found concerning the hemoglobin values pre-operatively and on the first and seventh post-operative day, the drainage quantity 12, 24, 36, and 48 hours post-operatively, the wound healing and the CRP. Six patients in the group without and five patients in the group with drain-suction had to receive blood transfusions. DISCUSSION: The application of suction on the drainage system had no significant impact on the post-operative blood loss and the postoperative course. In nine out of ten cases no homologous blood was needed.