title: Synergystic induction of HIF-1alpha transcriptional activity by hypoxia and lipopolysaccharide in macrophages.
abstract: Hypoxia Inducible Factor-1 (HIF-1) is activated by a variety of stimuli, including inflammatory mediators. In this report we investigated the role that bacterial lipopolysaccharide (LPS) and hypoxia play in the regulation of HIF-1-dependent gene expression in macrophages. We report that murine macrophages stimulated with low concentrations of LPS (1-10 ng/ml) expressed significantly higher levels of inducible nitric oxide synthase (iNOS) mRNA when cultured under hypoxic compared to normoxic conditions. Functional studies of the iNOS promoter demonstrated that the synergistic interaction between LPS and hypoxia was mediated, at least in part, by the NFkappaB and the HIF-1 binding sites. In addition, transient transfection experiments using a Hypoxia Response Element (HRE)-containing plasmid showed that LPS and hypoxia synergistically induced HIF-1-dependent transcriptional activity. Interestingly, LPS did not significantly affect HIF-1alpha protein levels or HIF-1 DNA binding activity relative to hypoxic induction. HIF-1alpha, but not HIF-2alpha, was critical for the synergistic induction of HRE-dependent transcriptional activity in macrophages, as indicated by experiments using siRNA targeting HIF-1alpha or HIF-2alpha. Addition of ROS-scavengers completely abrogated the synergistic induction of HIF-1 transcriptional activity by LPS and hypoxia, but neither inhibited HIF-1 transcriptional activity induced by hypoxia alone nor affected HIF-1alpha protein levels or HIF-1 DNA binding induced by hypoxia alone or hypoxia plus LPS. Taken together, our results demonstrate that LPS and hypoxia act synergistically to induce HIF-1alpha-transcriptional activity and they emphasize the existence of a cross talk between hypoxic and non-hypoxic signaling pathways in the regulation of macrophages gene expression.
authors.affiliation: Developmental Therapeutics Program, Istituto G. Gaslini, Genoa, Italy.
authors.forename: Zenghui
authors.initials: Z
authors.lastname: Mi
authors.forename: Annamaria
authors.initials: A
authors.lastname: Rapisarda
authors.forename: Lynn
authors.initials: L
authors.lastname: Taylor
authors.forename: Alan
authors.initials: A
authors.lastname: Brooks
authors.forename: Mark
authors.initials: M
authors.lastname: Creighton-Gutteridge
authors.forename: Giovanni
authors.initials: G
authors.lastname: Melillo
authors.forename: Luigi
authors.initials: L
authors.lastname: Varesio
mesh_terms: D000818:Animals; D015687:Cell Hypoxia; D002460:Cell Line; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D020218:Response Elements; D015533:Transcriptional Activation
chemical_list: C497443:Hif1a protein, mouse; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008070:Lipopolysaccharides; D016328:NF-kappa B; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse