Docosahexaenoic acid decreases IRF-1 mRNA and thus inhibits activation of both the IRF-E and NFkappa d response elements of the iNOS promoter.
BACKGROUND
Nitric oxide produced by inducible nitric oxide synthase (iNOS) may be cytotoxic during cardiac, hepatic, and renal acute allograft rejection. Because the incidence of rejection is decreased by fish oils that contain docosahexaenoic acid (DHA), we investigated the effects of DHA on iNOS. Using nuclear run-on assays and iNOS-promoter constructs, we previously showed that docosahexaenoic acid (DHA) inhibits activation of the iNOS gene by murine macrophages that had been stimulated in vitro by IFNgamma plus lipopolysaccharide.


METHODS
In our current investigation, our purpose has been to determine how DHA inhibits iNOS gene activation in murine macrophages, by using gel retardation and Northern blotting techniques. We studied the effects of DHA on the formation nuclear protein complexes that interact with the critical iNOS promoter's response elements for IRF-1 (IRF-E -923 to -913 bp) and NF-kappaB (NFkappa d -85 to -75 bp).


RESULTS
We now show that DHA inhibited increases of IRF-1 mRNA abundance in response to IFNgamma plus lipopolysaccharide. As expected, we found that this prevented formation of the nuclear protein complex that binds to the IRF-E DNA response element. We also found that inhibition of IRF-1 inhibited formation of the nuclear protein complex that binds to the NFkappa d DNA response element.


CONCLUSIONS
DHA decreases the abundance of IRF-1 mRNA in stimulated cells. That, in turn, results in the decreased nuclear protein binding to the major iNOS promoter response elements (IRF-E and NF-kappaB). We found that this occurred because IRF-1 is a component of both the nuclear protein complex that binds to IRF-E and the nuclear protein complex that binds to NFkappa d.
Kielar
M L
ML
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8856, USA.
Jeyarajah
D R
DR
Penfield
J G
JG
Lu
C Y
CY
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D004281:Docosahexaenoic Acids / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D050856:Interferon Regulatory Factor-1; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052247:Nitric Oxide Synthase Type II; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics; D012709:Serum Albumin; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; C494258:Irf1 protein, mouse; D008070:Lipopolysaccharides; D016328:NF-kappa B; D010750:Phosphoproteins; D012333:RNA, Messenger; D012709:Serum Albumin; D014157:Transcription Factors; D004281:Docosahexaenoic Acids; D007371:Interferon-gamma; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse