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KMID : 0857020050200010120
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Kosin Medical Journal
2005 Volume.20 No. 1 p.120 ~ p.131
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Regulatory Mechanisms for the Expression of Inducible Nitric Oxide Synthase ( iNOS)Gene in Rat Vascular Smooth Muscle cells
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An Ho-Jun
Rhew Hyun-Yul
Kim Taek-Sang
Chung Hun-Taeg
Kang Ju-Seok
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Abstract
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Background: The expression of inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells(VSMCs) is dependent on the multiple regulatory mechanisms of which are largely unknown. In this study, we focused on the role of IFN-γ in IL-1β-mediated expression of the VSMCs iNOS gene.
Materials and MEthods: VSMCs were isolated from rat thoracic aorta by enzymatic dissociation. Phosphorothioate-modified ODNs were designed to block initiation of target mRNA translation. Nitrite concentrations were measured by mixing 100μL of clulture medium with 100μL of Griess reagent. The biological activity of IFN-γ was demonstrated by testing the effect of supernatants collected from the IL-1 β-treated VSMCs on the production of NO in RAW264.7 cells stimulated with LPS. Supernatant levels of IFN-γ were measured by Endogen Rat Interferon gamma enzyme-linked immunosorbent assay kit. Total RNA was prepared from cultured VSMCs using RNAzol (Tel-Test) 12 hours after IL-1 β treatment. RNA (1㎍) from each sample was reverse-transcribed with Oligo-dT as the first-strand cDNA primer and Maloney murine leukemia virus reverse transcriptase. Western blotting and electrophoretic mobility shift assay were performed.
Results: In a system of rat VSMCs in primary culture, we found that IL-1 βinduced the productions of both NO and IFN-γ. IFN-γ protein and mRNA were expressed in the IL-1 β-stimulated VSMCs. Anti-IFN-γ antibody or antisense IFN-γ oligodeoxynucleotide (ODN) markedly inhibited NO production and iNOS protein expression as well as IFN-γ synthesis in the IL-1 β-stimulated cells. Furthermore, antisense IFN-γ ODN abolished the IL-1 β-induced activation of interferon regulatory factor-1(IRF-1) without affecting NF-κB activity as determined by electronic mobility shift assay. The repletion of IFN-γ to the system restored NO production and IRF-1 activity.
Conclusion>: These results suggest that the expression of iNOS gene in the IL-1 β-stimulated VSMCs is dependent on the endogenous IFN-γ synthesis and regulated through the activations of both IRF-1 and NF-κB.
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KEYWORD
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VSMCs, iNOS, IL-1, IFN-r, IRF-1
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