USF-1 and USF-2 trans-repress IL-1beta-induced iNOS transcription in mesangial cells.
Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682-1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region -904/-883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the -893/-888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the -893/-888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1beta (IL-1beta) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the -893/-888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1beta-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the -904/-883 region augmented IL-1beta stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at -893/-888 serves to trans-repress basal expression and IL-1beta induction of the iNOS promoter.
Gupta
Ashish K
AK
Department of Internal Medicine, The University of Texas Medical School at Houston, 6431 Fannin, Houston, TX 77030, USA.
Kone
Bruce C
BC
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D017930:Genes, Reporter; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000502:physiology; D012045:Regulatory Sequences, Nucleic Acid / Q000187:drug effects / Q000502:physiology; D012097:Repressor Proteins / Q000235:genetics / Q000494:pharmacology / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000502:physiology; D051720:Upstream Stimulatory Factors
D004268:DNA-Binding Proteins; D007375:Interleukin-1; D012097:Repressor Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; C497183:Usf1 protein, mouse; C497186:Usf2 protein, mouse; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse