Role of Ets-2 in the regulation of heme oxygenase-1 by endotoxin.
Ets proteins play a vital role in the regulation of mammalian immunity, and family members Ets-1 and Ets-2 regulate a variety of genes that participate in the propagation of an inflammatory response. Heme oxygenase (HO)-1, although acutely induced by inflammatory stimuli, has cytoprotective properties and prevents an exaggerated inflammatory response. Ets-1 and Ets-2 both induce HO-1 promoter activity; however, Ets-2 was a more potent transactivator of HO-1 in macrophages. A potent inflammatory mediator, bacterial lipopolysaccharide (LPS), induced Ets-2 at the mRNA and protein level, and this induction preceded the up-regulation of HO-1. To further delineate the role of Ets-2 in regulating HO-1 transcription, we performed HO-1 promoter analysis studies in macrophages. Deletion mutants down to -137/+74 maintained an activity analogous to that of the largest construct, -4045/+74. Further deletion constructs (starting with -117/+74) showed a significant reduction in promoter activity when co-transfected with Ets-2 or exposed to LPS. Promoter sequence analysis revealed two putative Ets binding sites (EBSs) in this region, and mutation of these sites showed that EBS -93, more than EBS -125, was critical for full HO-1 promoter activity. Additional studies showed that EBS -93 binds Ets-2 and that mutation of the DNA binding domain of Ets-2 entirely prevented transactivation of HO-1. Finally, overexpression of a dominant negative form of Ets-2 blunted HO-1 promoter induction by LPS, and kinase inhibitors (PI3K more than JNK) that reduced Ets-2 expression markedly decreased endogenous HO-1 expression. Our data provide evidence that Ets-2 contributes to the up-regulation of HO-1 by the potent inflammatory stimulus LPS in macrophages.
Chung
Su Wol
SW
Pulmonary and Critical Care Division, Brigham and Women's Hospital, Boston, Massachusetts, 02115, USA.
Chen
Yen-Hsu
YH
Perrella
Mark A
MA
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism*; D017353:Gene Deletion; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D005822:Genetic Vectors; D006419:Heme Oxygenase (Decyclizing) / Q000378:metabolism*; D051547:Heme Oxygenase-1; D007249:Inflammation; D008070:Lipopolysaccharides / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008565:Membrane Proteins; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D050783:Proto-Oncogene Proteins c-ets; D012313:RNA / Q000378:metabolism; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation
D004731:Endotoxins; C494042:Ets1 protein, mouse; C494070:Ets2 protein, mouse; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D050784:Proto-Oncogene Protein c-ets-1; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D015534:Trans-Activators; D014157:Transcription Factors; D012313:RNA; D008156:Luciferases; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse