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gene transcription in ANA-1 murine macrophages
Department of Surgery, Georgetown University Medical Center, Washington, District of Columbia 20007
On the basis of
previous work demonstrating nitric oxide (NO)-mediated inhibition of
nuclear factor-
B (NF-B) DNA binding, we hypothesized that NO
downregulates NF-B-dependent interleukin-1
(IL-1) production
in an ANA-1 macrophage model of lipopolysaccharide (LPS)
stimulation. In the presence of LPS (100 ng/ml), levels of
IL-1 protein and mRNA were significantly upregulated with NO
synthase inhibition. Using nuclear run-on analysis and transient transfection studies, IL-1 gene transcription and IL-1 promoter activity were also found to be increased with inhibition of NO production. Parallel transfection studies using an NF-B long terminal repeat-reporter plasmid exhibited similar
findings, suggesting an NO-mediated effect on NF-B activity. Gel
shift studies showed that LPS-associated NF-B DNA binding was
increased, both in the setting of NO synthase inhibition and in a
reducing environment. Repletion of NO by addition of an
S-nitrosothiol restored IL-1 protein synthesis, mRNA levels, gene transcription, promoter activity, and NF-B DNA binding to levels noted in the presence of LPS alone. Our studies indicate that NO may regulate LPS-associated inflammation by downregulating IL-1 gene transcription through
S-nitrosation of NF-B.
S-nitrosation; inducible nitric
oxide synthase; cytokine; nuclear factor-B; lipopolysaccharide; interleukin-1
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