Abstract
Interactions and possible cross talk between inducible nitricoxide synthase (iNOS), cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9), were studied in rat aortic vascular smooth muscle cells stimulated with bacterial lipopolysaccharide (LPS), interferon-γ (IFN-γ), and phorbol 12-myristate13-acetate (PMA). The expression and activity of iNOS, COX-2, and MMP-9 were characterized at the transcriptional, protein, and enzyme activity levels. The NOS inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) was used to investigate the effects of NO on COX-2 and MMP-9 at the transcriptional level. The measurements of mRNAs for these enzymes using real-time polymerase chain reaction (PCR) showed that COX-2 mRNA was up-regulated 2.3-fold, whereas MMP-9 mRNA up-regulation was 11.7-fold in the presence of LPS, IFN-γ, and PMA. Real-time PCR results indicated that l-NAME exerted an inhibitory effect on COX-2 and MMP-9 mRNA synthesis. Both superoxide dismutase (SOD) and the SOD mimetic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) did not modify significantly the up-regulation of these enzymes, indicating that neither superoxide nor peroxynitrite are involved in this mechanism. Furthermore, NO-mediated up-regulation of MMP-9 was cGMP-dependent since 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, blocked, in a concentration-dependent manner, the increased expression of MMP-9, an effect reversed by 8-bromo-cGMP, a soluble analog of cGMP. Our findings suggest that NO and cGMP are necessary to up-regulate the expression of MMP-9.
Footnotes
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This work was supported by funds from the Canadian Institutes for Health Research (M.W.R. is a CIHR scientist). A.D.B. is the holder of the AstraZeneca Canada, Inc. Chair in Asthma Research.
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DOI: 10.1124/jpet.103.050385.
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ABBREVIATIONS: LPS, lipopolysaccharide; NO, nitric oxide; iNOS, inducible NO synthase; COX-2, cyclooxygenase-2; MMP, matrix metalloproteinase; PGE2, prostaglandin E2; IFN-γ, interferon γ; PMA, phorbol 12-myristate 13-acetate; VSM, vascular smooth muscle; Dex, dexamethasone; RT-PCR, reverse transcription-polymerase chain reaction; l-NAME, Nω-nitro-l-arginine methyl ester; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; SOD, superoxide dismutase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; bp, base pair(s); MnTMPyP, Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; DAF-FM, diaminofluorescein-fluorometry; PKG, cGMP-dependent protein kinase.
- Received February 10, 2003.
- Accepted August 5, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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