IL-6 induction of protein-DNA complexes via a novel regulatory region of the inducible nitric oxide synthase gene promoter: role of octamer binding proteins.
Macrophage inducible nitric oxide synthase (iNOS) catalyzes the synthesis of NO. IL-6-stimulated macrophage differentiation of murine myeloid M1 cells is accompanied by iNOS gene induction and steady-state mRNA expression. Two regions within the iNOS promoter mediate transcriptional responsiveness to LPS and IFN-gamma. Region I contains several essential transcription factor binding motifs and promotes responsiveness to LPS, whereas region II potentiates the LPS response by IFN-gamma. Because region I possesses basal promoter activity and directly mediates iNOS gene activation, we attempted to identify the trans-acting factors involved in IL-6-stimulated induction of the murine iNOS gene through this region. Using an electrophoretic mobility shift assay and methylation interference, we show that IL-6 induced reciprocal changes in the binding activity of POU family members to the candidate nonconsensus octamer sequence of region I that correlated, temporally, with iNOS steady-state mRNA expression. Although DNA-protein binding activity of IL-6-stimulated whole-cell extracts also interacted with a radiolabeled canonical octamer motif, such DNA-protein complexes were not eliminated in competition assays using consensus nuclear factor kappaB or IL-6 oligonucleotides. Specifically, our studies show that octamer binding protein-1-related protein binding activity decreased, while binding of octamer binding protein-2-related proteins increased during differentiation. Mutation of the octamer motif disrupted both binding of the IL-6-induced protein-DNA interactions and transcriptional activation through region I, revealing that this motif is absolutely essential for IL-6 induction of iNOS. Thus, differential activation of octamer binding transcriptional modulators from the POU family may be a novel mechanism of IL-6-mediated iNOS gene regulation.
Sawada
T
T
Center for Biologics Evaluation Research, U.S. Food and Drug Administration, Bethesda, MD 20892, USA.
Falk
L A
LA
Rao
P
P
Murphy
W J
WJ
Pluznik
D H
DH
D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015850:Interleukin-6 / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D012333:RNA, Messenger / Q000032:analysis
D004268:DNA-Binding Proteins; D015850:Interleukin-6; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D004247:DNA; D019001:Nitric Oxide Synthase