Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 52, No. 7, July 2005, pp 2010–2014 DOI 10.1002/art.21156 © 2005, American College of Rheumatology Retroviral Gene Transfer of an Antisense Construct Against Membrane Type 1 Matrix Metalloproteinase Reduces the Invasiveness of Rheumatoid Arthritis Synovial Fibroblasts Edita Rutkauskaite,1 Dagmar Volkmer,1 Yukio Shigeyama,2 Jörg Schedel,3 Geza Pap,1 Ulf Müller-Ladner,4 Ingmar Meinecke,5 Dorothea Alexander,6 Renate E. Gay,7 Susanne Drynda,1 Wolfram Neumann,1 Beat A. Michel,7 Wilhelm K. Aicher,6 Steffen Gay,7 and Thomas Pap5 MMP␣S on the invasiveness of RASFs were analyzed in the SCID mouse coimplantation model of RA. Results. MT1-MMP␣S–transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by nontransduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1MMP␣S–transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). Conclusion. The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs. Objective. Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblastmediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. Methods. Retroviral gene transfer of a pLXIN vector–based antisense RNA expression construct (MT1-MMP␣S) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMP␣S–transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1- Supported by the Swiss National Science Foundation (grant 32-64142.00) and the DFG (grants Pa 698/1-1, Pa 698/3-1, Mu 1383/ 3-3, and Ai 16/10-1). 1 Edita Rutkauskaite, MD, Dagmar Volkmer, MD, Geza Pap, MD, Susanne Drynda, MD, Wolfram Neumann, MD: Otto-vonGuericke-University, Magdeburg, Germany; 2Yukio Shigeyama, MD: Okayame Red Cross General Hospital, Okayame, Japan, and University Hospital, Zurich, Switzerland; 3Jörg Schedel, MD: University Hospital, Zurich, Switzerland, and University Hospital, Regensburg, Germany; 4Ulf Müller-Ladner, MD: University Hospital, Regensburg, Germany; 5Ingmar Meinecke, MD, Thomas Pap, MD: University of Munster, Munster, Germany; 6Dorothea Alexander, PhD, Wilhelm K. Aicher, PhD: University Hospital, Tubingen, Germany; 7Renate E. Gay, MD, Beat A. Michel, MD, Steffen Gay, MD: University Hospital, Zurich, Switzerland. Address correspondence and reprint requests to Thomas Pap, MD, Division of Molecular Medicine of Musculoskeletal Tissue, Department of Orthopaedic Surgery, University of Munster, D-48149 Munster, Germany. E-mail: email@example.com. Submitted for publication July 16, 2004; accepted in revised form April 14, 2005. Membrane-type matrix metalloproteinases (MTMMPs) are cell membrane–anchored MMPs that have been associated with both normal tissue remodeling and various diseases. Six different MT-MMPs have thus far been described, of which membrane type 1 MMP (MT1MMP) has been studied most intensively. MT1-MMP (also called MMP-14) has a specific structural organization that is characterized by a hydrophobic transmembrane domain and a short cytoplasmic tail. It also contains a recognition site for furin-like proprotein convertases, which, by furin-dependent cleavage, activate MT1-MMP intracellularly (1). MT1-MMP digests interstitial collagens as well as other extracellular matrix components, including fibronectin, laminin, aggrecan, 2010 MT1-MMP INHIBITION AND REDUCED INVASIVENESS OF RASFs and gelatin (2). In addition, MT1-MMP has been identified as an important activator of other MMPs, such as MMP-2 and MMP-13 (3,4). MT1-MMP has been shown to be indispensable for normal growth and development, in that MT1MMP–deficient mice are known to exhibit a short life span and a variety of pathologic abnormalities in the connective tissue that ultimately lead to the development of arthritis, dwarfism, and premature death (5). Although these findings point to a role for MT1-MMP in normal development, its contribution to pathologic development in adults is also evident. High levels of MT1-MMP have been detected in a number of malignant cells, and the importance of MT1-MMP in tumor cell migration and invasion is well established. Interestingly, high expression of MT1-MMP has also been found in nonmalignant diseases that are associated with progressive matrix degradation, such as aseptic prosthesis loosening (6) and rheumatoid arthritis (RA) (7). However, the degree to which MT1-MMP contributes to the progressive destruction of articular cartilage in RA is poorly understood. Herein we studied the effects of retroviral gene transfer of an antisense RNA expression construct against MT1-MMP (MT1MMP␣S) on the long-term production of MT1-MMP in RA synovial fibroblasts (RASFs), and investigated whether inhibition of MT1-MMP affects the invasiveness of RASFs into human articular cartilage in vivo. PATIENTS AND METHODS Isolation and culture of RASFs. Synovial tissues were obtained at joint replacement surgery (at the Schulthess Clinic, Zurich, Switzerland and Department of Orthopedic Surgery, University Hospital, Magdeburg, Germany) from 5 patients with active RA according to the revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) (8). RASFs were obtained by enzymatic digestion as previously described (9) and grown in Dulbecco’s modified Eagle’s medium (Biochrom, Berlin, Germany) with 10% fetal calf serum (Biochrom) in a humidified atmosphere containing 5% CO2 for 4–6 passages, before being used in the experiments. Generation of the MT1-MMP antisense construct and retroviral gene transfer. A 502-bp MT1-MMP–specific complementary DNA (cDNA) fragment with known binding capability to MT1-MMP messenger RNA (mRNA) (7) was amplified from RASFs by polymerase chain reaction (PCR) and cloned into the Hpa I site of the retroviral pLXIN vector. Following transformation of competent Escherichia coli, individual clones were selected, and the retrieved plasmids were analyzed for identity and orientation of the inserts by automated sequencing. PT67 amphotropic packaging cells (Clontech, Heidelberg, Germany) were transfected with the retroviral vector carrying the MT1-MMP fragment in antisense 2011 orientation (MT1-MMP␣S) by lipofectin (SuperFect; Roche Diagnostics, Mannheim, Germany) and selected with G418. RASFs were transduced by incubation with the retroviral supernatants for 12 hours in the presence of 8 g/ml polybrene. The procedure was repeated after an interval of 12 hours to increase retroviral infection. Successfully transduced RASFs were selected with G418 (800 g/ml; Clontech) for at least 10 days. As controls, mock transduction with the empty pLXIN vector as well as the pLEIN retroviral vector expressing enhanced green fluorescent protein were used, along with nontransduced RASFs. Analysis of MT1-MMP production. Levels of MT1MMP RNA were analyzed by quantitative real-time PCR using a fluorogenic 5⬘-nuclease assay (TaqMan; Applied Biosystems, Weiterstadt, Germany) on an ABI Prism 7900 HT Sequence Detection system. For each experiment, total RNA was extracted from 105 cells using the RNeasy system (Qiagen, Hilden, Germany) and reverse transcribed using random hexamer primers. For quantitative PCR, the primers and FAMTAMRA–labeled probes were as follows: MT1-MMP forward primer, 5⬘-TGG-AGG-AGA-CAC-CCA-CTT-TGA-3⬘, MT1MMP reverse primer, 5⬘-GCC-ACC-AGG-AAG-ATG-TCATTT-C-3⬘, MT1-MMP TaqMan probe, 5⬘-5⬘FAM-CCT-GACAGT-CCA-AGG-CTC-GGC-AGA-3⬘TAMRA. Primers and probes were designed to detect both endogenous MT1-MMP mRNA and RNA derived from the MT1-MMP␣S construct. MT1-MMP expression was determined relative to the 18S ribosomal RNA that was coamplified as an internal standard, with expression levels calculated with the ⌬⌬Ct method as previously described (10). The expression of MT1-MMP protein was analyzed by Western blotting. Cells (5 ⫻ 105) were lysed with lysis buffer and protein concentrations were determined with a commercially available protein quantification kit (Uptima Interchim, Montluçon, France). Identical amounts of protein (50 g) were loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Amersham Biosciences, Freiburg, Germany). The membrane was probed with rabbit anti-human MT1-MMP polyclonal antibodies (1:1,000; Dianova, Hamburg, Germany) for 1 hour at room temperature. Detection was carried out using horseradish peroxidase–conjugated donkey anti-rabbit (IgG) secondary antibodies and an enhanced chemiluminescence kit (Amersham Biosciences). Blots were stripped and reprobed with antibodies to ␤-actin (Abcam, Cambridge, UK) as loading control. Long-term expression of MT1-MMP was monitored by indirect immunofluorescence (11). Briefly, RASFs were fixed in acetone/methanol (1:1) and incubated with a monoclonal antibody to MT1-MMP (clone 114-6G6; Oncogene Research Products, Cambridge, MA) at a dilution of 1:200 for 1 hour at room temperature. Cy3-conjugated sheep anti-mouse sera (Dianova) were used as secondary antibodies. SCID mouse coimplantation experiments and histologic evaluation. Four-week-old SCID mice were provided by Charles River (Sulzfeld, Germany). Normal human articular cartilage was obtained from patients undergoing joint replacement surgery at the Department of Orthopedic Surgery, University Hospital, Tübingen, Germany. Implantation of RASFs together with normal human cartilage was performed as described previously (10). After 60 days, the implants were 2012 RUTKAUSKAITE ET AL removed at the time of death and the tissues were embedded into paraffin. Five animals were used per group (MT1MMP␣S, mock, and nontransduced), resulting in a total of 15 mice. Hematoxylin and eosin staining of the sections was carried out and histologic evaluation was performed using semiquantitative scores for invasion as previously described (10). Statistical analysis. Results are expressed as the mean and SEM. Differences between groups were tested for statistical significance using the Mann-Whitney U test. Differences were considered statistically significant at a P value less than 0.05. RESULTS Reduction of MT1-MMP by an MT1-MMP antisense RNA expression construct in RASFs. The expression of MT1-MMP RNA was analyzed by quantitative real-time PCR using a combination of primers and probes that detected both endogenous MT1-MMP mRNA and RNA derived from the MT1-MMP␣S construct. Stable transduction of RASFs with the MT1MMP␣S construct resulted in a 15-fold increase in MT1-MMP RNA as compared with that in both nontransduced and mock-transduced cells (Figure 1A), indicating a significant excess of MT1-MMP antisense RNA over endogenous MT1-MMP mRNA. In the next step, Western blotting was performed to analyze the expression of MT1-MMP at the protein level. As shown in Figure 1B, both the 63-kd proMT1MMP and the 60-kd active MT1-MMP forms (12) were found prominently in the nontransduced and mocktransduced RASFs. In contrast, RASFs that were stably transduced with MT1-MMP␣S exhibited a markedly reduced expression of MT1-MMP. Immunocytochemistry demonstrated that the decreased expression of MT1MMP was maintained over at least 60 days (Figure 1C). When compared with both nontransduced and mocktransduced RASFs, a markedly reduced staining for MT1-MMP was observed in MT1-MMP␣S–transduced cells at 60 days after the establishment of stable cell cultures. The expression of other MMPs (MMP-2, MMP-3, and MMP-13) was not changed by MT1MMP␣S, as determined by quantitative PCR and enzyme-linked immunosorbent assay (results not shown). Decreased invasiveness of RASFs by gene transfer of MT1-MMP␣S in the SCID mouse in vivo model of RA. To investigate the effects of MT1-MMP inhibition on the invasiveness of RASFs, we used the SCID mouse coimplantation in vivo model that has been described in detail before. For this purpose, RASFs were coimplanted together with normal human articular cartilage Figure 1. Inhibition of membrane type 1 matrix metalloproteinase (MT1-MMP) by an antisense RNA expression construct (MT1MMP␣S) in rheumatoid arthritis synovial fibroblasts (RASFs). A, Quantitative real-time polymerase chain reaction, with a combination of primers and probes detecting both endogenous MT1-MMP mRNA and RNA derived from the MT1-MMP␣S construct, showed a significant excess of MT1-MMP RNA in MT1-MMP␣S–transduced cells as compared with nontransduced (⫺) and pLEIN-transduced (mock) cells (n ⫽ 3 in different RASF cultures) (ⴱ ⫽ P ⬍ 0.05). rRNA ⫽ ribosomal RNA. Bars show the mean and SEM. B, Using Western blot, a reduced expression of MT1-MMP was found in MT1-MMP␣S– transduced cells compared with nontransduced and mock-transduced cells. No differences were seen in the levels of ␤-actin (used as loading control). C, Sixty days after the establishment of stable cell cultures, immunocytochemistry showed that expression of MT1-MMP was still markedly reduced in MT1-MMP␣S–transduced RASFs compared with the respective controls. into SCID mice for 60 days. It has been shown in a number of studies that RASFs deeply invade the coimplanted cartilage in the absence of human inflammatory MT1-MMP INHIBITION AND REDUCED INVASIVENESS OF RASFs Figure 2. Reduction of invasiveness of RASFs by MT1-MMP␣S in the SCID mouse model of rheumatoid arthritis. A, At histologic evaluation of hematoxylin and eosin–stained sections, deep invasion of RASFs into the cartilage was seen with the control nontransduced (⫺) and mock cells, whereas transduction with MT1-MMP␣S resulted in a markedly reduced invasion (representative picture of n ⫽ 5 in each group). B, A semiquantitative scoring system was used to measure the depth of invasion. In the MT1-MMP␣S group, the RASF invasiveness was significantly reduced as compared with that in the control groups (ⴱ ⫽ P ⬍ 0.05). Bars show the mean and SEM. See Figure 1 for definitions. cells in this model, whereas normal, non-RA synovial fibroblasts do not exhibit such behavior. Similar to the results in previous studies, nontransduced and mock-transduced RASFs showed deep cartilage invasion, with mean ⫾ SEM invasion scores of 3.0 ⫾ 0.03 and 3.1 ⫾ 0.08, respectively (Figure 2). Stable retroviral expression of MT1-MMP␣S significantly reduced the invasiveness of the RASFs (mean invasion score 1.8 ⫾ 0.62; P ⬍ 0.05). Although MT1-MMP␣S failed to protect the cartilage completely, the highest invasion score in the MT1-MMP␣S group was only 2.5, which was clearly below that seen in the mocktransduced and nontransduced groups (maximum invasion scores of 4.0 and 3.5, respectively). DISCUSSION MMPs are critically involved in both normal tissue remodeling and the degradation of extracellular matrix under disease conditions. Among the MMPs, the membrane-type MMPs are of special importance. They act on the surface of cells, which results in a close 2013 association of MT-MMP effects with cellular function. In addition, MT-MMPs contribute not only to the degradation of extracellular matrix, but also to the activation of other MMPs. MT1-MMP is the bestcharacterized member of the MT-MMP family and has been associated with the invasiveness of cancer cells (13,14). To date, most studies using antisense strategies to inhibit MT1-MMP have been performed in conditions involving malignancies and have focused on MMP activation as well as cell trafficking. Monea and coworkers found that HT-1080 cells transfected with antisense cDNA failed to activate proMMP-2, which was accompanied by a considerable suppression of the 60-kd and 58-kd active forms of MT1-MMP in the antisense transfectants (15). On the basis of the hypothesis that furin is essential for proMT1-MMP activation, Sato and coworkers used antisense oligonucleotides against furin in human fibrosarcoma HT-1080 cells, human uterine cervical fibroblasts (HUCFs), and rabbit dermal fibroblasts (RDFs); they found that furin antisense constructs inhibited the concanavalin A–induced activation of proMMP-2 in HUCFs but not in RDFs, suggesting that there are different mechanisms of proMT1-MMP activation (16). Focusing on cell migration, Udayakumar et al demonstrated that MT1-MMP antisense oligonucleotides inhibited the migration of Du-145 prostate carcinoma cells, which was dependent on the cleavage of laminin-5 by MT1-MMP (17). These studies have clearly demonstrated a functional involvement of MT1-MMP in tumor progression and metastasis. It has also been demonstrated that MT1-MMP is expressed at elevated levels in the RA synovial membrane and particularly at sites of joint destruction (7,18,19). In addition, data obtained by Honda and coworkers (20) have shown convincingly that proinflammatory cytokines such as interleukin-1␤ up-regulate the expression of MT1-MMP in RASFs, thus potentially contributing to MT1-MMP–mediated matrix degradation in RA. However, the specific contribution of MT1MMP to direct, fibroblast-mediated destruction of articular extracellular matrix is incompletely understood (21). In the present study we used gene transfer of an MT1-MMP antisense expression construct to study its effect on the invasiveness of RASFs into cartilaginous matrix. Retroviral gene transfer of MT1-MMP␣S resulted in a sustained overexpression of MT1-MMP antisense RNA that down-regulated MT1-MMP production for at least 60 days. This was seen most clearly in Western blot analysis and immunocytochemistry, in 2014 RUTKAUSKAITE ET AL which MT1-MMP was found in nontransduced and mock-transduced RASFs but could be detected in only negligible amounts in MT1-MMP␣S–transduced cells. Of interest, inhibition of MT1-MMP production in RASFs was accompanied by a significant reduction of their invasiveness in the SCID mouse model. Whereas prominent invasion into cartilage was observed with nontransduced and mock-transduced RASFs, cartilage destruction was reduced significantly in MT1-MMP␣S– transduced cells. It needs to be emphasized that our data do not allow us to distinguish between direct and indirect effects of MT1-MMP inhibition, i.e., between the inhibition of MT1-MMP–mediated matrix degradation per se and the reduced activation of other disease-relevant MMPs such as MMP-2 and MMP-13. However, our results do show clearly that MT1-MMP is involved functionally in the direct destruction of articular cartilage in RA. Although there is evidence that MT1-MMP is indispensable for normal embryonic development (5), it is difficult to predict whether inhibition of MT1-MMP will lead to unwanted effects in adults that may hamper the use of approaches that involve MT1-MMP inhibition. In this context, our data also demonstrate that viral gene transfer of antisense expression constructs may inhibit MMPs over extended periods of time, and thus constitutes a feasible tool for studying the individual contributions of MMPs (including MT-MMPs) to cartilage destruction in RA. 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