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Synergystic induction of HIF-1α transcriptional activity by hypoxia and lipopolysaccharide in macrophages

Zenghui Mi, Annamaria Rapisarda, Lynn Taylor, Alan Brooks, Mark Creighton-Gutteridge, Giovanni Melillo and Luigi Varesio

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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 NFκB 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-1α protein levels or HIF-1 DNA binding activity relative to hypoxic induction. HIF-1α, but not HIF-2α, was critical for the synergistic induction of HRE-dependent transcriptional activity in macrophages, as indicated by experiments using siRNA targeting HIF-1α or HIF-2α. 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-1α 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-1α-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

Zenghui Mi

National Cancer Institute; Frederick, MD

Annamaria Rapisarda

Science Applications International Corporation𠅏rederick, Inc.; Frederick, MD

Lynn Taylor

National Cancer Institute; Frederick, MD

Alan Brooks

Science Applications International Corporation𠅏rederick, Inc.; Frederick, MD

Mark Creighton-Gutteridge

National Cancer Institute; Frederick, MD

Giovanni Melillo

Science Applications International Corporation Frederick, Inc.; Frederick, MD

Luigi Varesio

Istituto G. Gaslini; Genova, Italy

Purchase article for $19

Subscribe to this journal for $129/year