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PC 8 ABSTRACT NO. 1045 PAPER NO. 0605 Corresponding Author: Ashok First Name Amin Last Name Presenting Author: Ashok First Name Amin Last Name I prefer a Poster Presentation Please consider for New Investigator Recognition Awards Please consider for the American Geriatrics Society Award REQUIRED - Supply first keyword from one of these lists: Infection/Inflammation Supply 4 remaining keywords from the list in the Instructions: None Prostaglandin E2 Nitric Oxide Macrophage Inflammatory Mediators IDENTIFICATION OF A NOVEL FORM OF A SOLUBLE CYTOSOLIC COX-2 LIKE PROTEIN REGULATED BY NITRIC OXIDE: IS THIS ANOTHER COX-2, ISOFORM OR COX-3 *+Amin, A.R., Patel, R., Attur, M.G., Dave, M., Abramson, S.B. *NYU Medical Center, Department of Pathology, Medicine, Rheumatology, New York, NY. +Dept. of Rheumatology, Rm. 1600, Hospital for Joint Diseases, 301 E. 17th Street, New York, NY 10003. Tel: 212-598-6537. Fax: 212-598-7604 Relevance to Musculoskeletal Conditions: PGE2 (produced by membrane bound COX-2) and nitric oxide are overexpressed in arthritis [1, 2]. These proinflammatory mediators are involved in joint inflammation. The present study identifies a second form of COX: cytosolic COX-2 which may be involved in the pathophysiology of joints in arthritis Introduction: Prostaglandin E2 and nitric oxide are two pleiotropic mediators produced at inflammatory sites by the inducible enzymes, cyclooxygenase-2 (COX-2) and nitric oxide synthase (iNOS) respectively. A complex relationship is merging with regard to “cross-talk” between the nitric oxide and cyclooxygenase (COX) pathways. We have recently reported that explants of human OA cartilage obtained at the time of joint replacement surgery express COX-2 and OA-NOS. These explants spontaneously produced PGE2 and NO, both can be augmented by treatment with IL-1β + TNFα and LPS [2[] We have also observed that inhibition, spontaneous or induced NO production in OA-explants resulted in marked increases in PGE2 release. This derepression of PGE2 in OA-explants was observed upon inhibition of NO by a conventional NOS inhibitor, such as L-NMMA. In view of the above observations, we chose to perform experiments in murine macrophages (RAW 264.7) in order to further examine the mechanisms by which NO attenuates PGE2 production and the type of COX involved in this process. Previous immunofluorescent studies have indicated that COX-2 is localized in the endoplasmic reticulum (ER), Golgi complex and nuclear envelops (NE). The C-terminal-S/PTEL sequences of COX 1 and 2 target these enzymes to the membrane. In addition, the Nterminal of both enzymes have sequences characteristic of membrane targeting signal peptides. Therefore, based on immunofluroscent studies and characteristic membrane targeting amino acid sequences, our detection of a soluble form of COX-2 like protein as it cross reacts to antibodies that bind to COX-2 is a novel finding. The finding of a soluble COX protein, the amount of which is regulated by endogenous nitric oxide production, suggests the following: 1) an alternatively spliced COX-2 isoform lacking key membrane targeting peptides, or 2) intracellular trafficking between ER, golgi and cytoplasm or stabilization of COX-2 protein due to post-translational modification, 3) a new form of soluble COX. References: [1] Amin AR et al. J. Exp. Med. 1995; 182:2097-2102 [2] Amin AR et al. J. Clin. Inv. 1997; 99:1231-1237 [3] Amin AR et al. J. Inflammation 1997; 47:190-205 Methods: Nitric Oxide and PGE2 Analysis:. Nitric oxide was estimated by its stable end product: nitrite as previously described and PGE2 was estimated by RIA as described previous [2]. Northern blot analysis of NOS and COX was as previously described [2] Cell-free extracts were prepared [3] by lysing the cells in a Polytron PA 1200 homogenizer after 3 cycles of rapid freeze-thawing. Alterntively the extracts were also prepared by a dounce homogenizer. The lysate was centrifuged at 18,000 rpm for 60 min at 4°C in an eppendoff centrifuge to generate a soluble cytosolic fraction. The resulting pellet was comprised of the nuclear fraction. Results: Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of COX-2 in the nuclear envelope, and approximately 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 g indicates that the COX-2 like is distributed both in the soluble fraction and membrane fraction. The soluble COX-2 like protein was identified using cross-reacting antiCOX-2 antibodies and various markers in the sub-cellular fractions of the cell. Stimulation of RAW 264.7 cells with LPS in the preseence of iNOS inhibitor L-NMMA, at concentrations that inhibits nitrite accumulation by ≤80% is inadequate to augment PGE2 production. However, inhibition of nitrite accumulation by ≥ 85% with higher concentration of L-NMMA (> 100 µM) shows (a) upregulation of PGE2 production, (b) accumulation of COX-2 protein in the 100,000 g soluble and membrane fractions of the cytosolic fraction as examined by western blot analysis (c) with no signifricant effects on the accumluation of COX-2 mRNA. These experiments suggest that low concentration of nitric oxide (10-15% of the total) produced in repsponse to LPS attenuates induced PGE2 production in RAW 264.7 cells. This inhibition is, in part due to decreased expression of cytosolic COX-2 like protein and not the membrane COX-2 Discussion: A novel observation in this study is that PGE2 production (due to decrease in nitric oxide) is associated with significant increase in the amount of cytosolic COX-2 like protein and not the mRNA accumulations or the membrane bound COX-2. This soluble cytosolic COX-2 like may be susceptible to pharmacological intervention and may pose as a novel target regulated by other important mediators such as nitric oxide. One or more of the authors have received something of value from a commercial or other party related directly or indirectly to the subject of my presentation. The authors have not received anything of value from a commercial or other party related directly or indirectly to the subject of my presentation. 45th Annual Meeting, Orthopaedic Research Society, February 1-4, 1999, Anaheim, California 605