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By Differential Display, Expression of the Active Arthritis-Affected Cartilage TNF-α Convertase Enzyme from Human Arthritis-Affected Cartilage: Isolation of cDNA by Differential Display, Expression of the Active Enzyme, and Regulation of TNF- α This information is current as of September 27, 2021. Indravadan R. Patel, Mukundan G. Attur, Rajesh N. Patel, Steven A. Stuchin, Ruben A. Abagyan, Steven B. Abramson and Ashok R. Amin J Immunol 1998; 160:4570-4579; ; http://www.jimmunol.org/content/160/9/4570 Downloaded from References This article cites 48 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/160/9/4570.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. TNF-a Convertase Enzyme from Human Arthritis-Affected Cartilage: Isolation of cDNA by Differential Display, Expression of the Active Enzyme, and Regulation of TNF-a1 Indravadan R. Patel,2* Mukundan G. Attur,2* Rajesh N. Patel,* Steven A. Stuchin,† Ruben A. Abagyan,‡ Steven B. Abramson,*§ and Ashok R. Amin3*§¶i A snake venom-like protease isolated by a differential display screen between normal and osteoarthritis (OA)-affected cartilage (designated as cSVP) has a cDNA sequence identical to TNF-a convertase enzyme (TACE). TACE shows the presence of an unknown prodomain, a cysteine switch, a catalytic domain, a zinc binding region, a disintegrin region, an EGF-like domain, a transmembrane domain, and a unique cytoplasmic region. A TACE construct harboring the signal 1 prodomain 1 catalytic region (TACE-SPCDDETCy), expressed in baculovirus could cleave preferentially (;12-fold) the TNF-specific peptide over the Downloaded from matrix metalloproteases peptide in vitro. This recombinant protein also cleaved the natural substrate GST-ProTNF-a to TNF-a (17 kDa) in vitro. The mRNA for TACE, which is broadly distributed and differentially expressed in a variety of human tissues, is up-regulated in arthritis-affected cartilage, but not normal cartilage. OA-affected cartilage also expressed TNF-a mRNA that was not detected in normal cartilage. The OA-affected cartilage (in explant assays) spontaneously released TNF-a and IL-8 in ex vivo conditions. Addition of TNF-aR fused to IgG Fc fragment (TNF-aR:Fc) in the presence or absence of soluble IL-1R (with http://www.jimmunol.org/ which it acted additively) significantly attenuated the spontaneous/autocrine release of articular IL-8 in this assay. These exper- iments demonstrate a functional paracrine/autocrine role of TNF-a in OA-affected cartilage that may depend, in part, on up- regulated levels of chondrocyte-derived TACE. The Journal of Immunology, 1998, 160: 4570–4579. umor necrosis factor a, a pleiotropic cytokine, produces a anti-TNF-a Abs (8) or soluble TNF-aR (9) has proven to be highly broad spectrum of injurious effects, which makes it an effective in reducing clinical score, paw swelling, and histologic T important target for therapeutic intervention. TNF-a is severity of the disease. Extension of these studies in patients with involved in the pathophysiology of arthritis, AIDS, cancer, auto- rheumatoid arthritis in phase II and III clinical trials has yielded immune diseases (immune complex diseases), lung fibrosis, mul- very encouraging results. These early clinical results indicate that by guest on September 27, 2021 tiple sclerosis, skin delayed-type hypersensitivity reactions, and neutralizing the effects of TNF-a may have profound effects on bacterial and parasitic infections (1–6). clinical symptoms and signs (10, 11). The gene for human TNF-a encodes a prohormone that is in- The matrix metalloproteases (MMPs)4 have long been impli- serted into the cell membrane as a polypeptide with a molecular cated in cartilage degradation in both rheumatoid arthritis (RA) mass of 26 kDa (5). This membrane-bound form of TNF-a is and osteoarthritis (OA), although their precise role in disease pro- bioactive as assayed by cell cytotoxicity and has been implicated gression remains to be determined (12). The two main families of in the paracrine activities of TNF-a in various tissues (6). In re- MMPs believed to be responsible for cartilage degradation are col- sponse to LPS and other stimuli, the 26-kDa form of proTNF-a is lagenases and proteoglycanases (12). proteolytically cleaved (by a metalloprotease referred to as TNF-a The synthesis of TNF is known to be regulated at transcrip- convertase) into a soluble 17-kDa polypeptide (6). TNF-a binds its tional, translational, and posttranslational levels (6). Three inde- cognate receptors (p55 and p75) as a bioactive trimer and triggers pendent groups have demonstrated that broad-spectrum inhibitors complex intracellular signaling pathways (6, 7). of MMPs can specifically inhibit the release of membrane pro- Studies in animal models of arthritis indicate that TNF-a may be TNF-a (but not IL-1b or IL-6) from various cell surfaces, includ- a pivotal cytokine involved in these disease processes. Injection of ing RA synovial cell cultures (13–17). This inhibitor of proTNF-a processing could protect mice against a lethal dose of endotoxin administered to them (14). The “TNF-a convertase” activity was *Department of Rheumatology and Medicine, and †Department of Orthopedic Sur- isolated using these inhibitors as ligands by affinity purification, gery, Hospital for Joint Diseases, New York, NY; ‡Skirball Institute of Biomolecular Medicine, Departments of §Medicine and ¶Pathology, and iKaplan Cancer Research which resulted in identification of an 80-kDa protein with the ca- Center, New York University Medical Center, New York, NY 10016. pacity to cleave the Gln-Ala-2-Val-Arg sequence of proTNF- Received for publication October 1, 1997. Accepted for publication January 6, 1998. a (15, 16). The costs of publication of this article were defrayed in part by the payment of page In the present study we report the following: a) the full-length charges. This article must therefore be hereby marked advertisement in accordance cDNA sequence of a snake venom-like protease from human ar- with 18 U.S.C. Section 1734 solely to indicate this fact. thritis-affected cartilage (cSVP) is identical to TACE cDNA (15, 1 The authors appreciate the generosity of the Abeles Fund and the Falk Fund in supporting these studies. The DNA sequence of cSVP (TACE) has been submitted to GenBank under accession number U92649. 4 Abbreviations used in this paper: MMP, matrix metalloprotease; cSVP, cartilage- 2 The first two authors have contributed equally to the paper. derived snake venom-like protease; OA, osteoarthritis; RA, rheumatoid arthritis; sIL- 3 Address correspondence and reprint requests to Dr. Ashok R. Amin, Department of 1R, soluble IL-1R; GST-proTNF-a, glutathione-S-transferase-proTNF-a; TNF-aR: Rheumatology, Room 1600, Hospital for Joint Diseases, 301 E. 17th St., New York, Fc, TNF-a receptor fused to IgG Fc fragment; TACE, TNF-a convertase enzyme; NY 10003. EGF, epidermal growth facor; RACE, rapid amplification of cDNA ends. Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 4571 16); b) the functional expression of a truncated recombinant TACE branes were hybridized with 32P-labeled probes overnight at 65°C. After showing TNF-a convertase activity; c) the up-regulation of both hybridization, the membranes were washed at high stringency twice for 10 a min at 65°C. The blots were exposed to Kodak x-ray films (Kodak, Roch- TACE and proTNF- mRNA in OA- and RA-affected, but not in 2 a ester, NY) at 70°C. All methods were conducted according to Sambrook, normal cartilage; and d) the intraarticular TNF- expressed in OA- et al. (23). affected cartilage is sufficient to modulate the autocrine cartilage- a derived IL-8 production. We conclude from these data that TNF- , Colony hybridization which is induced in arthritis-affected cartilage, may be influenced by the up-regulated TNF-a convertase to promote cartilage cata- White colonies, including one positive control each for collagenase and ; bolic activities. stromelysin, were taken on grids on 150-mm plates ( 400 colonies per plate) and incubated overnight at 37°C. Colonies were transferred onto nitrocellulose membrane. The filters were then fixed with a UV Crosslinker Materials and Methods (Stratagene, La Jolla, CA). Treatment of membranes, prehybridization, hy- Procurement of human cartilage bridization, and washing conditions used were according to published pro- tocols (22). Membranes were exposed to an autoradiographic film at Cartilage slices were taken from the knees of patients with the diagnosis of 270°C. The collagenase1/stromelysin1 clones were excluded from the advanced OA or RA who were undergoing knee replacement surgery, and grids before sequencing the inserts of some of the other clones. from nonarthritic knees. The OA/RA patients were free of nonsteroidal anti-inflammatory drugs for at least 2 wk before surgery. Nonarthritic knee 9 9 cartilage was obtained within 12 h from accident victims who were un- Cloning of full-length cSVP (TACE) by 3 and 5 rapid dergoing knee amputation. Some of the samples were provided by the amplification of cDNA ends (RACE) National Disease Research Interchange (Philadelphia, PA). Downloaded from The 39 and 59 extensions of cSVP were cloned using the 39RACE and OA cartilage organ culture 59RACE methods according to manufacturer’s instructions (Life Technol- ogies).
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