Original ArticlesMolecular Cloning and Analysis of the Rat Inducible Nitric Oxide Synthase Gene Promoter in Aortic Smooth Muscle Cells 1
Section snippets
Reagents
Human recombinant IL-1β was obtained from Boehringer. TNF-α was purchased from R & D Systems. LPS, l-NMMA and l-NAME were purchased from Sigma. Tissue culture media and Lipofectamine were obtained from Life Technologies.
Cell Culture
RASMC were harvested from Wistar rats (Harlan) by enzymatic dissociation, using standard methods [20]. The cells were identified as smooth muscle by indirect immunofluorescent staining for α-actin, using mouse anti-α-actin antibody and anti-mouse IgG FITC conjugate. The positive
Cloning, Sequence, and Analysis of the 5′-Flanking Region of the Rat iNOS
Four DNA fragments with 0.404, 0.572, 3.28, and 5.2 kb of the 5′-flanking region of the rat iNOS were obtained by polymerase chain reaction amplification from PvuII, SspI, DraI, and ScaI DNA pools, respectively. The EcoRV DNA pool was not amplified. All DNA fragments were cloned into a luciferase containing vector, PGL3-Basic. The DNA sequences of the −3.2-kb (−3196 to +88) construct, as well as of the 404 (−316 to +88) and 572 (−484 to +88) bp, were determined by dye terminator chemistry on
Discussion
Type II NOS can be induced in many species and cell types, and the molecular regulation of this gene appears to differ among cell types. For example, in a mouse macrophage cell line, RAW 264.7, the promoter region that responds to LPS contains only a single downstream NF-κB binding site [11]. However, in the VSMC cell line A7r5, it is the −890-to −1002-bp region (also containing another NF-κB site) that best responds to stimulation by cytokines [18]. Still, in RASMC it is the −234 to +31 region
Acknowledgements
This work was supported by Grant HL52958. We thank Dr. Richard C. Venema for helpful discussion and suggestions and Ms. Aretha Bogan for preparing the manuscript.
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The nucleotide sequence data reported in this paper have been submitted to the GenBank with the accession number AF042085.