Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Science 23 December 2005:
Vol. 310. no. 5756, pp. 1966 - 1970
DOI: 10.1126/science.1119407
Prev | Table of Contents | Next

Reports

Inducible Nitric Oxide Synthase Binds, S-Nitrosylates, and Activates Cyclooxygenase-2

Sangwon F. Kim1, Daniel A. Huri1 and Solomon H. Snyder1,2,3*

1 Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
2 Department of pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
3 Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.


Fig. 1. COX-2 and iNOS bind selectively in vitro and in intact cells. (A) RAW264.7 cells were treated with LPS (2 µg/ml) and IFN-{gamma} (100 U/ml). COX-2 was immunoprecipitated by COX-2–specific antibody and analyzed by Western blot with antibodies against COX-2 and iNOS. Control indicates untreated cells. (B and C) RAW264.7 cells were treated with LPS–IFN-{gamma} with or without an iNOS inhibitor 1400W (100 µM) or COX-2 inhibitor SC58125 (100 µM). Cell lysates were subjected to immunoprecipitation (IP) and Western blot analysis with antibodies against COX-2 and iNOS. (D) The fragments of iNOS denoted in red bind to full-length COX-2, whereas fragments labeled purple do not, as determined by coimmunoprecipitation of full-length COX-2 by iNOS fragment fused to glutathione S-transferase (GST). The numbers represent the number of the amino acid sequence. (E) Transfected HEK293T cells expressing COX-2 and iNOS fragments expressed as fusion proteins with GST were precipitated with glutathione-conjugated beads. Proteins were detected by Western blot with antibodies against GST or COX-2. (F) Transfected HEK293T cells expressing COX-2 and epitope-tagged (Myc) iNOS fragments were immunoprecipitated with Myc-specific antibody and then analyzed by Western blot. (mock: The cells were treated with transfection reagent without the plasmid.) (G) Generated fragments of COX-2 that bind to full-length iNOS are labeled in red; those that do not bind are labeled in yellow. (MBD, membrane-binding domain). (H) Transfected HEK293T cells expressing iNOS and Myc-tagged COX-2 fragments were immunoprecipitated with Myc-specific antibody and analyzed by Western blot. (mock: The cells were treated with transfection reagent without the plasmid.) [View Larger Version of this Image (23K GIF file)]
 

Fig. 2. S-Nitrosylation of COX-2 enhances enzyme activity. (A) COX-2 expressed in transfected HEK293T cells is S-nitrosylated in the presence of GSNO (100 µM) or glutathione (reduced form) (100 µM) as determined by biotin-switch assay. All the S-nitrosylated proteins were precipitated and COX-2 was detected by Western blot with COX-2–specific antibody. COX-2 was selectively S-nitrosylated by GSNO. (B) LPS–IFN-{gamma} treatment of RAW264.7 cells elicits S-nitrosylation of COX-2, which is prevented by the iNOS inhibitor 1400W (100 µM). COX-2 was selectively S-nitrosylated by endogenously generated NO. (C) COX-2enzyme activity was measured from the cell lysate of transfected HEK293T cells expressing COX-2–Myc in the presence or absence of SNP and ascorbate. Bars represent the mean ± SEM of three independent cell cultures performed in triplicate (*statistically significant by Student's t test). (D) COX-2–Myc expressed in transfected HEK293T cells is S-nitrosylated by various concentrations of GSNO. The dose-dependence of GSNO-mediated activation of PGE2 was measured. Data were pooled from at least three independent determinations, each in triplicate. (E) COX-2–Myc expressed in transfected HEK293T cells is S-nitrosylated in the presence of SNP and reversed by the addition of ASC. All the S-nitrosylated proteins were precipitated, and COX-2 was detected by Western blot with COX-2–specific antibody. (F) Recombinant human COX-2 was treated with SNP, and COX-2 activity was measured (n = 3, control: Vmax = 81.3 ± 4.8 nmol/min per mg, Km = 16.2 ± 2.2 µM; SNP: Vmax = 132 ± 6.5 nmol/min per mg, Km = 17.0 ± 2.0 µM). (G) Recombinant human COX-2 was treated with SNP, and its turnover rate (kcat) was measured in the presence of various concentrations of sucrose. Data were expressed as kcat-control over kcat in each viscosity versus viscosity ratio. [View Larger Version of this Image (29K GIF file)]
 

Fig. 3. Endogenously generated NO enhances COX-2 activity. (A) RAW264.7 cells were activated by LPS–IFN-{gamma} and treated with various concentrations of iNOS inhibitor 1400W for 18 hours. The dose dependence of 1400W-mediated suppression of PGE2 and nitrite was then measured. Data were pooled from at least three independent determinations, each in triplicate (*statistically significant by Student's t test). (B) Combinations of L-NAME (500 µM), L-NAME + L-Arg (1 mM) or D-Arg (1 mM), and D-NAME (500 µM) were added to RAW264.7 cells treated with LPS–IFN-{gamma}. PGE2 was measured. The data were pooled from three independent experiments performed, each in triplicate. (C) PGE2 and nitrite were measured from primary peritoneal macrophages isolated from wild-type (WT) or iNOS knockout (KO) mice. Macrophages were treated with LPS–IFN-{gamma} or untreated (*statistically significant by Student's t test). (D) S-Nitrosylation of COX-2 of WT primary peritoneal macrophages treated with LPS–IFN-{gamma} is abolished in iNOS KO macrophages. All the S-nitrosylated proteins were precipitated, and COX-2 was detected by Western blot with COX-2–specific antibody. [View Larger Version of this Image (19K GIF file)]
 

Fig. 4. COX-2–Myc fragment attenuates iNOS binding to COX-2– and NO-mediated activation of PGE2 production. Transfected RAW264.7 cells expressing COX-2–Myc fragments 1 to 483 or 484 to 604 were treated with LPS–IFN-{gamma}. (A) Cell lysates were immunoprecipitated with rabbit iNOS-specific antibody and analyzed by Western blot with antibodies against mouse iNOS, goat COX-2, and mouse Myc. (B) COX-2–Myc fragment (484 to 604) decreases S-nitrosylation of COX-2 in RAW264.7 cells. All the S-nitrosylated proteins were precipitated and COX-2 was detected by Western blot with COX-2–specific antibody. (C) Transfected RAW264.7 cells expressing the indicated COX-2 fragments were treated with LPS–IFN-{gamma}. PGE2 levels were measured and the data were pooled from three independent experiments performed, each in triplicate (*statistically significant by Student's t test). (D) PGE2 and the indicated COX-2 fragments were visualized with confocal microscopy using antibodies against mouse Myc and rabbit PGE2. Images of COX-2 (red) and PGE2 (green) were superimposed to show colocalization. Nuclei were visualized with Hoechst staining (blue). In D1, arrows point to two RAW264.7 cells, only one of which is expressing the COX-2 fragment 484 to 604 (red). In D2, the same two cells are analyzed for presence of endogenous PGE2 after activation of RAW264.7 cells by LPS–IFN-{gamma} treatment. Immunofluorescent staining shows a reduction in the PGE2 expression in cells expressing COX-2(484–604) compared with the nontransfected cell (D2). This observation contrasts with D4, where the arrows point to a nontransfected cell and a transfected cell expressing COX-2(1–483). D5 does not show a reduction of PGE2 in the transfected cell as compared with the nontransfected cell. [View Larger Version of this Image (33K GIF file)]
 

To Advertise     Find Products

Science. ISSN 0036-8075 (print), 1095-9203 (online)