Download 0605 - identification of a novel form of a soluble cytosolic cox

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ABSTRACT NO. 1045
PAPER NO. 0605
Corresponding Author:
Ashok
First Name
Amin
Last Name
Presenting Author:
Ashok
First Name
Amin
Last Name
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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