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Regulation of Cytosolic COX-2 and Prostaglandin E₂ Production by Nitric Oxide in Activated Murine Macrophages

Rajesh Patel^*, Mukundan G. Attur^*, Mandar Dave^*, Steven B. Abramson^*, and Ashok R. Amin^1,*,,

^* Department of Rheumatology, Hospital for Joint Diseases, New York, NY 10003; and Departments of Medicine and Pathology, Kaplan Cancer Center, New York University Medical Center, New York, NY 10016.

	Abstract

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Murine macrophages (RAW 264.7) when stimulated with LPS show 90% distribution of cyclooxygenase-2 (COX-2) in the nuclear fraction and 10% in the cytosolic fraction. Further analysis of this cytosolic fraction at 100,000 x g indicates that the COX-2 is distributed both in the 100,000 x g soluble fraction and membrane fraction. Stimulation of RAW 264.7 cells with LPS in the presence of inducible nitric oxide synthase inhibitor L-NMMA at concentrations that inhibit nitrite accumulation by 80% is inadequate to augment PGE₂ production. However, inhibition of nitrite accumulation by 85% with higher concentrations of L-NMMA shows 1) up-regulation of PGE₂ production, 2) accumulation of COX-2 protein in the 100,000 x g soluble and membrane fractions of the cytosolic fraction, and 3) with no significant effects on the accumulation of COX-2 mRNA. These experiments suggest that low concentrations of nitric oxide (10–15% of the total) attenuate PGE₂ production in response to LPS in RAW 264.7 cells. This inhibition is, in part, due to decreased expression of cytosolic COX-2 protein.

	Introduction

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Prostaglandin E₂ and nitric oxide are two pleiotropic mediators produced at inflammatory sites by the inducible enzymes, cyclooxygenase-2 (COX-2)² and nitric oxide synthase (iNOS), respectively. A variety of cells (e.g., endothelium, macrophages, chondrocytes) up-regulate both inducible enzyme isoforms and produce NO and PG simultaneously in response to cytokines and other activators. The paracrine effects of these molecules are often similar and include the capacity to relax smooth muscle, inhibit platelet and neutrophil adhesion, and inhibit neutrophil oxidant production (1). Increased production of both mediators has been reported in rheumatoid arthritis and osteoarthritis (2, 3).

A complex relationship is emerging with regard to "cross-talk" between the NO and COX pathways. NO, for example, has been reported to stimulate COX activity in selected cell types by a heme-independent mechanism (4). Conversely, Vane and coworkers have reported in cultured murine macrophages that NO attenuates PGE₂ production (5). One of the mechanism of NO-mediated PGE₂ attenuation was elucidated by Habib et al. (6), who reported that NO suppressed the activity and expression of COX-2 mRNA in LPS-stimulated rat peritoneal macrophages.

We have recently observed that explants of human osteoarthritis (OA) cartilage obtained at the time of joint replacement surgery express COX-2 and OA-NOS. These explants spontaneously produced PGE₂ and NO, and both can be augmented by treatment with IL-1ß plus TNF and LPS (7, 8). Consistent with the findings of Habib et al. (6) and Swierkosz et al. (5), we could demonstrate in these OA explants that the inhibition of both spontaneous and induced NO production resulted in marked increases in PGE₂ production. This derepression of PGE₂ in OA-explants was observed upon inhibition of NO by a conventional NOS inhibitor, such as L-NMMA (8).

In view of the above observations, we chose to perform experiments in murine macrophages (RAW 264.7) to further examine the mechanisms by which NO attenuates PGE₂ production. Our data indicate that low concentrations of inducible endogenous NO suppresses the production of PGE₂ in LPS-stimulated RAW 264.7 cells. A novel observation in this study is that increased PGE₂ production is associated with significant increase in the amount of cytosolic COX-2 protein and not mRNA accumulation: an observation distinct from that described by Habib et al. (6). Furthermore, the effect of NO on COX-2 and PGE₂ production seems to be biphasic.

	Materials and Methods

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Cell lines and reagents

Murine macrophages (RAW 264.7) were obtained from the American Type Culture Collection (Manassas, VA). Anti-murine iNOS and COX-2 Abs were obtained from Transduction Laboratories (Lexington, KY). Hydrocortisone, cycloheximide, and LPS were obtained from Sigma (St. Louis, MO). Abs to RXR and histone deacetylase (HDACI; C-19) were purchased from Santa Cruz Biotech (Santa Cruz, CA).

NO and PGE₂ analysis

NO was estimated by its stable end product: nitrite as previously described (9). PGE₂ was estimated by RIA as described previously (8).

Fractionation of cell lysate by Dounce method

The cells (2 x 10⁷) were lysed by using a Dounce homogenizer (Pyrex; Fisher Scientific, Springfield, NJ) to prepare nuclear and cytoplasmic (S-100) fraction as previously reported (10). The lysate was spun down at 3,000 x g to remove unlysed cells in an Eppendorf centrifuge (Fisher Scientific). The resulting supernatant was subjected to 18,000 x g in an Eppendorf centrifuge. This step isolated the intact nuclei from which nuclear extracts were prepared. The supernatant of the nuclear fraction (i.e., cytosolic fraction) was further centrifuged at 100,000 x g in an ultracentrifuge, which resulted in a membrane pellet that included the endoplasmic reticulum and Golgi bodies. The resulting supernatant was the soluble fraction. All fractions were analyzed by SDS-PAGE and western blotted for the COX-2 protein.

Preparation of cell-free extracts using a Polytron homogenizer

The cells were pelleted at 4°C and resuspended in Tris buffer (10 mM, pH 7.4) containing 10 µg/ml each, chymostatin, antipain, leupeptin, and pepstatin, 1 mM DTT, and 1 mM PMSF (lysis buffer mixture). Cells were lysed in a Polytron PA 1200 homogenizer (Kinematica AG, Switzerland) after three cycles of rapid freeze-thawing. This lysate was designated as total lysate. The total lysate was centrifuged at 18,000 x g for 60 min at 4°C in an Eppendorf centrifuge. The resulting supernatant was designated as the cytosolic fraction. The pellet (which constitutes mainly the nuclear fraction and membranes) was resuspended in 50 mM Tris-HCl, pH 6.8, and 2% SDS. The protein was measured by bicinchoninic acid assay reagent using bicinchoninic acid as standard (11).

Cell-free enzyme assay for COX-2

The cells were lysed by a Polytron homogenizer as shown above in Tris buffer with a mixture of protease inhibitors. The enzyme assay was performed as previously described by Vane et al. (12) using total unfractionated cell extracts; the PGE₂ was estimated by RIA. The specific enzyme activity was defined as nanograms of PGE₂ released/micrograms protein/37°C for 20 min.

Western blot analysis

Equal amounts of protein (50 µg) estimated by bicinchoninic acid reagent were loaded onto SDS-PAGE gels and examined by Western blot analysis with a specific anti-iNOS or anti-COX-2 murine mAb as specified by Transduction Laboratories. Membranes were reused for Western blot analysis. Briefly, membranes with bound Abs (e.g., anti-iNOS) were stripped by submersion of the blots in stripping buffer (100 mM 2-ME/12% SDS/62.5 mM Tris-HCl, pH 6.7) and incubation at 50°C for 30 min with occasional agitation. Membranes were then washed twice for 10 min at room temperature using large volumes of wash buffer before using them again. This filter was then reprobed with an anti-RXR, anti-HDACI (nuclear markers), anti-glucose-6-phosphate dehydrogenase (G6PDH), anti-actin Ab (provided by James L. Lessard, Children’s Hospital Medical Center, Cincinnati, OH), or anti-glucosidase Ab (provided by Dr. Inder Vijay, University of Maryland, College Park, MD). Blots were developed using the enhanced chemiluminescence Western blot system (Amersham, Arlington Heights, IL). Quantitation of the bands was performed using a personal densitometer (Molecular Dynamics, Sunnyvale, CA).

Northern blot analysis

Total RNA was isolated using TRI reagent (Molecular Research Center, Cincinnati, OH). Poly(A)⁺ RNA was prepared using an Oligotex mRNA kit (Qiagen, Chatsworth, CA). Northern blot analysis was conducted as described (13). Briefly, 20 µg of RNA was subjected to electrophoresis and transferred by capillary action onto a nylon membrane (Zeta-Probe, Bio-Rad, Hercules, CA). The membrane was hybridized with [³²P]dCTP-labeled COX-2 or COX-1 cDNA, and the blot was exposed to Kodak x-ray film (Rochester, NY) for 24–48 h with intensifying screens at -70°C. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was purchased from Clontech (Palo Alto, CA) and probed as described above. Quantitation was performed using a personal densitometer (PDSI-pc) (Molecular Dynamics).

Statistical analysis

Data are expressed as mean ± SD, and statistical analysis was performed using GraphPad Software (V1.14) (San Diego, CA). The Student’s t test or nonparametric (Mann-Whitney or Wilcoxon) test was performed to analyze the data.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effect of NOS inhibitor on PGE₂ production in murine macrophages stimulated with LPS

Murine macrophages (RAW 264.7) were stimulated with LPS to induce iNOS and COX-2 by 10- to 20-fold above control. The cells were incubated in the presence or absence of L-NMMA. In these studies, 25–100 µM of L-NMMA inhibited nitrite accumulation by 20–70%, but this level of NOS inhibition did not result in a significant effect on the accumulation of PGE₂ (Fig. 1). However, higher concentrations of L-NMMA (200–500 µM), which were sufficient to inhibit the accumulation of nitrite by 85%, significantly augmented the production of PGE₂. Hydrocortisone at 10 µM, as expected, blocked both NO and PGE₂ production. These experiments indicate that relatively low levels of endogenous NO production is sufficient to suppress PGE₂ production in murine macrophages and that derepression of PGE₂ production is observed only when 85% NOS inhibition is achieved.

View larger version (24K): [in this window] [in a new window]   FIGURE 1. Regulation of PGE2 by NOS inhibitor in murine macrophages. Murine macrophages (RAW 264.7) were stimulated with 1 µg/ml LPS for 16 h in the presence and absence of L-NMMA (100–500 µM) or 10 µM of hydrocortisone. The levels of nitrite and PGE2 accumulated were determined as described in Materials and Methods. The data are representative of one of the three experiments performed in triplicate. The p values for nitrite and PGE2 accumulation are compared between LPS-stimulated cells and experiments as follows: a 0.5; b 0.09; c 0.01; d 0.001; e 0.0001.

The cells were homogenized using two different methods: Dounce and Polytron (Fig. 2A). A relatively mild method of cell lysis using a Dounce homogenizer was used for the first set of experiments. This lysate was centrifuged at 3,000 x g to remove the unlysed cells. The homogenate (fraction I) was further fractionated (at 18,000 x g) into cytosolic/soluble fraction II and nuclear/membrane fraction III. The cytosolic/soluble fraction II was further separated (at 100,000 x g) into a soluble fraction IV and a membrane fraction V. Equal amounts of protein from all the fractions were analyzed by Western blot analysis using an anti-COX-2 Ab (Fig. 2B). The data shows that at 18,000 x g separation, 90% of the COX-2 was localized in the nuclear/membrane fraction III and 10% in the soluble/cytosolic fraction II. We designate this COX-2 as "Cy-COX-2". Analysis of the soluble/cytosolic (at 100,000 x g) indicate an 7:3 distribution of Cy-COX-2 in the membrane and soluble fractions, respectively. We conducted experiments to rule out the possibility of contamination of the nuclear/membrane COX-2 fraction III into the soluble/cytosolic fraction II and soluble fraction IV. Fig. 2C shows Western blot analysis of unfractionated fraction I, soluble/cytosolic fraction II, and nuclear membrane fraction III using a nuclear marker RXR known to be localized in the nucleus (14). The COX-2 and ß-actin (15) was observed in all three fractions. The RXR was seen in the total unfractionated fraction I and the nuclear/membrane fraction III and not the 18,000 x g soluble/cytosolic fraction II. To further confirm the localization of soluble COX-2, we lysed the cells with the Dounce method and analyzed unfractionated fraction I, 18,000 x g nuclear/membrane fraction III, and 100,000 x g soluble fraction IV as shown in Fig. 2, A and D. Equal amounts of protein was Western blotted using Abs to COX-2, ß-actin, and another nuclear marker: histone deacetylase (HDACI) known to be associated with the nucleus (16). ß-actin expression was observed in all the fractions (15). The nuclear fraction showed the presence of COX-2 and HDACI, whereas the 100,000 x g soluble fraction showed the presence of COX-2 but not HDACI. These experiments show that the COX-2 observed in the 18,000 x g soluble/cytosolic fraction II and 100,000 x g soluble fraction IV is not due to the contamination of the nuclear/membrane fraction III.

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Localization of COX-2 in subcellular fractions. Murine macrophages were stimulated with 1 µg/ml of LPS for 16–18 h. The cells were harvested and fractionated using two different methods. In the first method, the cells were homogenized using a Dounce homogenizer. The unlysed cells were removed by centrifuging the lysate at 3,000 x g (fraction I), and the resulting supernatant was centrifuged further at 18,000 x g. This resulted into a 18,000 x g soluble/cytosolic fraction II and a nuclear/membrane fraction III. The soluble/cytosolic fraction was further separated at 100,000 x g into a soluble fraction IV and membrane fraction V. In an alternate method, the cells were subjected to freeze-thaw cycles followed by usage of a Polytron homogenizer as previously described (9). The resulting unfractionated fraction I was centrifuged at 18,000 x g into a soluble/cytosolic fraction II and nuclear/membrane fraction III. Alternatively, the total unfractionated cell lysate was also centrifuged at 100,000 x g to generate a 100,000 x g soluble fraction IV and membrane fraction V. A total of 50 µg of protein was loaded in all the lanes in the experiments described below. B shows Western blot analysis of COX-2 of cell fraction (I to V) prepared by Dounce method. C shows Western blot analysis of fractions I, II, and III (prepared by Dounce method) using anti-COX-2, anti-actin, and anti-RXR Abs. D shows Western blot analysis of fractions I, III, and IV (prepared by Dounce method) using anti-COX-2, anti-iNOS, and anti-HDACI Abs. E shows the Western blot analysis of fractions I, II, and III using the polytron method. The blot were probed with anti-COX-2, anti-G6PDH, and anti-actin and anti-iNOS Abs. F shows Western blot analysis of fractions IV and V prepared by the Polytron method using anti-COX-2 and anti-glucosidase Abs.

Regulation of COX-2 protein by NO

The expression of iNOS and COX-2 at the level of transcription, translation, and the enzyme level has been extensively studied in murine macrophages (21, 22). Therefore, we continued our studies in murine macrophages to further analyze the regulation of cytosolic iNOS and COX-2 in the presence of NOS inhibitors.

Western blot analysis. RAW 264.7 cells were stimulated with LPS in the presence of NOS inhibitor: L-NMMA. We performed Western blot analysis of iNOS, COX-2, and ß-actin from the soluble/cytosolic fraction. As expected, there was a decrease in nitrite accumulation in the medium (Fig. 1). As recently reported by Peng et al. (23) and Kim et al. (24), L-NMMA increased the accumulation of iNOS protein. Similarly, in the present study L-NMMA at 25–500 mM increased the accumulation of iNOS protein by 30–50% above LPS-stimulated cells. There was no significant effect observed on the levels of ß-actin in the presence of 25–500 µM of L-NMMA (Fig. 3, A and B). L-NMMA caused a dose-dependent increase in the levels of soluble/cytosolic COX-2 as shown in Fig. 3C. Interestingly, although there was a measurable increase in the accumulation of soluble/cytosolic COX-2 in the presence of 25–100 µM of L-NMMA, there was no significant increase in the accumulation of PGE₂ at these concentrations. A significant increase in PGE₂ accumulation was seen in cells treated with 200–500 µM of L-NMMA as previously described in Fig. 1, accompanied with a two- to threefold increase in the COX-2 protein.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Western blot analysis of cytosolic iNOS and COX-2 in murine macrophages. Murine macrophages (RAW 264.7) were stimulated with 1 µg/ml of LPS for 16–18 h in the presence and absence of various concentrations of iNOS inhibitor, L-NMMA. The cells were harvested and washed twice with PBS before they were homogenized by repeated freeze-thaw cycles followed by Polytron homogenization as described in Materials and Methods. A total of 50 µg of protein from the 18,000 x g cytosol fraction was analyzed by Western blot analysis using specific anti-iNOS (A), anti-COX-2 (B), and ß-actin (C) Abs on the same blot as described in Materials and Methods. D, A total of 20 µg of protein from the 18,000 x g nuclear/membrane fraction III and 100 µg of protein from 100,000 x g membrane fraction V and soluble fraction IV were analyzed for COX-2 in the presence of LPS ± 500 µM L-NMMA. The percent increase in the COX-2 signal was compared with the LPS-stimulated cells using a densitometer. Nitrite (Griess method) and PGE2 (RIA) were estimated from the medium as previously described in Materials and Methods.

In vitro effects of L-NMMA. We analyzed the effect of L-NMMA on COX-2 activity in vitro enzyme assay in cell-free extracts. Previous studies have shown that L-NMMA and L-arginine use the same transporters. The IC₅₀ for L-NMMA uptake in the presence of L-arginine is 1–37 µM (25). Therefore, 1–25 µM of L-NMMA was selected to examine the effect of L-NMMA on COX-2 activity in cell-free extracts. L-NMMA at 1–25 µM had no significant effect (2%) on PGE₂ production, whereas 25 µg of indomethacin or COX-2-specific inhibitor NS-398 (26) inhibited COX-2 activity by 94 and 78%, respectively (Table I). These experiments rule out the direct effect of L-NMMA on COX-2 activity.

We examined the effect of cycloheximide on L-NMMA-induced PGE₂ nitrite production (Table II). L-NMMA, as expected, inhibited NO and augmented PGE₂ production in LPS-stimulated cells. A total of 1 µg/ml of cycloheximide significantly inhibited both LPS-induced and LPS plus L-NMMA (500 µM)-induced PGE₂ production by 60–70%. These experiments indicate that de novo protein synthesis is required for up-regulation of L-NMMA-induced PGE₂ production in activated murine macrophages, similar to that reported by Habib et al. (6), who used relatively high concentrations (1–3 mM) of L-NMMA to augment PGE₂ production.

NO has been implicated in the regulation of various genes, these include iNOS itself (27), heat shock protein-70 (28), superoxide dismutase (29), and Fe²⁺ metabolism (30). We examined the expression of COX-2 mRNA in murine macrophages stimulated with LPS in the presence or absence of various concentration of L-NMMA for 4 h (data not shown) or 16 h (Fig. 4). Northern blot analysis showed no significant effect on COX-2 mRNA accumulation (at 4 and 16 h) upon inhibition of NO production at concentrations of L-NMMA, which augmented PGE₂ production and Cy-COX-2 accumulation. In a separate experiment (not shown), hydrocortisone (10 µM), as previously reported (9), inhibited COX-2 mRNA accumulation by 60%. These experiments suggest that the effect of intracellular NO in murine RAW 264.7 cells does not appear to be on increased transcription or accumulation of COX-2 mRNA.

View larger version (52K): [in this window] [in a new window]   FIGURE 4. Northern blot analysis of COX-1 and COX-2. Murine macrophages were stimulated for 16 h with 1 µg/ml LPS ± L-NMMA. Total RNA was extracted as described. The filters were probed with COX-2 and GAPDH cDNA, and the signal was quantitated using both a phosphoimager and a densitometer. The percent modulation of COX-2 mRNA was normalized with GAPDH signal and compared with the value of the LPS-stimulated cells. Data reflects one of the two representative experiments (A). Total RNA from murine macrophages RAW 264.7 and NIH3T3 was analyzed by Northern blot analysis for COX-1 as shown (B).

Expression of COX-1 in RAW 264.7 cells and NIH3T3 cells

We examined the expression of COX-1 mRNA in RAW 264.7 and NIH3T3 cells. Total RNA (20 µg) was analyzed by Northern blot analysis. NIH3T3 as previously reported showed detectable amounts of COX-1 mRNA (31). RAW 264.7 RNA showed a significant signal of the 2.8-kb COX-1 mRNA. We have observed low levels of PGE₂ (0.3 ng/ml) in unstimulated RAW 264.7 cells, which may be released by low levels of COX-1 expression. It should be noted that the Northern blots were exposed at -70°C for 10 days to detect these signals of COX-1 as observed in Fig. 4B, as compared with an overnight exposure for COX-2 and GAPDH seen in Fig. 4A.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have previously demonstrated that NO inhibits production of PGE₂ in human OA cartilage cultures in ex vivo conditions (8). In the current studies, we demonstrate similar suppression of PGE₂ production by NO in LPS-stimulated cultures of murine macrophages. The findings are consistent with those of Stadler et al. (32) and Swierkosz et al. (5), who demonstrated that endogenous NO inhibits the synthesis of COX products in rodent Kupffer cells and macrophages, respectively. Habib et al. (6) and Vane and coworkers (5) have made similar observations in rat peritoneal macrophages and murine macrophage cell line: RAW 264.7. However, others have observed that NO enhances COX activity and PGE₂ production in various cell types (4, 33, 34, 35). This controversy in the literature may be due to tissue-specific expression and regulation of NOS and COX-2 in various cell types, as well as the influence of other mediators in the microenvironment that may influence the production of NO and PGE₂. An example of cell-cell variation has been illustrated by studies of Janabi et al. (36) in which endogenous NO activated PGF₂ production in human microglial cells but not in astrocytes.

With respect to mechanisms by which NO suppressed PGE₂ production in our studies, several observations are worth noting. First, the augmentation of PGE₂ synthesis by the NOS inhibitor, L-NMMA, was dose dependent, requiring >85% inhibition of NO produced in response to LPS. These effects were not limited to inhibition by L-NMMA and could be reproduced with other NOS inhibitors, such as L-N⁶-(1-imino ethyl)-lysine · HCl (L-NIL · HCl), L-N⁵-(1-imino ethyl-ornithine · HCL (L-NIO · HCL), and thiocitrulline.³

Furthermore, L-NMMA had no significant direct effect on COX-2 enzyme activity in vitro. A second important observation was that the increased PGE₂ production in RAW 264.7 cells was associated with a greater than twofold increase of Cy-COX-2 protein detected (in the presence of 200 µM of L-NMMA) in the cellular cytosolic fraction by immunoblot analysis. Inhibition of NO by L-NMMA shows dose-dependent accumulation of Cy-COX-2 protein with no significant increase in PGE₂ accumulation, although 100 µM of L-NMMA inhibits 70% of nitrite accumulation. This observation may be explained as follows: 1) "low concentrations" of NO and peroxynitrite as previously described (37, 38) may augment COX-2 enzyme activity when the inhibition of NO by L-NMMA exceeds >85% with 200 µM L-NMMA; 2) "high levels" of intracellular NO may inhibit directly COX-2 enzyme activity (or an essential cofactor) more than the accumulation of Cy-COX-2. Therefore, the intracellular concentration of NO has complex (direct or indirect) effects on COX-2 mediated PGE₂ accumulation. We observed increases in both, membrane (V) and soluble fraction (IV) of COX-2, which could be due to increased stability or else trafficking of the COX-2 protein. It is tempting to speculate that the increase in Cy-COX-2 accumulation may be due to post-translational modifications, which may render it loosely bound to the membrane; a mechanism not previously recognized for the regulation of COX-2 protein, although previous studies have suggested that NO may not have an effect on the stability of total COX-2 protein (6). Our studies indicated that the source of Cy-COX-2 may not be nuclear membrane-associated COX-2 but de novo synthesized Cy-COX-2. Thirdly, increased levels in immunodetectable COX-2 protein was not accompanied by increases in mRNA: an observation that is distinct in murine macrophage cell line (RAW 264.7) and peritoneal murine macrophages under similar conditions (6). It should be noted that the increase in the soluble/cytosolic COX-2 constitutes 10% of total COX-2, and such subtle changes in total mRNA may not be evident, although no obvious spliced form of COX-2 was observed. These experiments together indicate that a minor fraction of the COX-2 expressed in macrophages is sensitive to intracellular signaling molecules such as NO and probably peroxynitrite, but NO independent of the above effect may also modulate COX-2 enzyme activity.

Our detection of a soluble form of COX-2 is a novel finding because previous immunofluorescent studies by Smith et al. (39) has indicated that COX-2 is localized in the endoplasmic reticulum, Golgi complex, and nuclear envelope (39). The C-terminal-S/PTEL sequences of COX 1 and 2 target these enzymes to the membrane. In addition, the N-terminal of both enzymes have sequences characteristic of membrane-targeting signal peptides (20).

An intriguing aspect of this study is that a pleiotropic molecule such as NO regulates another multifunctional mediator, PGE₂ (40, 41). While, it is generally held that NO exerts predominately catabolic effects in cartilage, the role of PGE₂ is less clear. A conflicting literature to date suggests that PGE₂ may be a "double-edged" sword with respect to its effects on cartilage metabolism. Anabolic or reparative effects of PGE₂ include its capacity to inhibit IL-1-induced proteoglycan degradation and to enhance type II collagen and proteoglycan synthesis (42, 43, 44, 45). As reported by DiBattista (46), increased matrix synthesis may be due to a PGE₂-dependent increase of insulin growth factor I and its binding protein-3 synthesis. Conversely, PGE₂ may exert catabolic effects on cartilage via its capacity to activate metalloproteinases (41), including gelatinase and stromelysin. In addition, studies by Blanco and Lotz (47) have reported that IL-1ß-induced NO inhibits chondrocyte proliferation via PGE₂.

In summary, we have shown in both OA cartilage (8) and murine macrophages that endogenous NO suppresses COX-2-dependent PGE₂ production in a biphasic manner as suggested by Vane and coworkers (37). This study also indicates that this effect of NO is due in part to an increase of a previously unrecognized soluble/cytosolic form of COX-2.

	Acknowledgments

 
We thank Ms. Una Yearwood for preparation of the manuscript. We also thank Dr. Paul Worley (John Hopkins University, Baltimore, MD) and Dr. T. Hla (University of Connecticut, School of Medicine, Hartford, CT) for generously providing the COX-2 and COX-1 cDNA, respectively; Dr. James L. Lessard (Children’s Hospital Medical Center, Cincinnati, OH) for providing us with the anti-actin Abs; and Dr. Inder Vijay (University of Maryland, College Park, MD) for providing us with the anti-glucosidase Abs.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Ashok R. Amin, Department of Rheumatology, Room 1600, Hospital for Joint Diseases, 301 E. 17^th Street, New York, NY 10003. E-mail address:

² Abbreviations used in this paper: COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase; NO, nitric oxide; OA, osteoarthritis; OA-NOS, osteoarthritis affected cartilage-induced NOS; Cy-COX-2, cytosolic COX-2; Sol-COX-2, soluble COX-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; G6PDH, glucose-6-phosphate dehydrogenase; HDACI, histone deacetylase.

³ Attur, M. G., R. Patel, S. B. Abramson, A. R. Amin. 1998. The perplexing role of nitric oxide on prostaglandin production: inhibitors and cell type variation. Submitted for publication.

Received for publication April 23, 1998. Accepted for publication December 18, 1998.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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