Cooperative interaction between hypoxia and lipopolysaccharide (LPS) in the activation of the inducible nitric oxide synthase (INOS) promoter

We have recently reported that a hypoxia-responsive element (iNOS-HRE) is required for the synergistic activation of the iNOS promoter by hypoxia (1% oxygen) plus IFN-y. To investigate the role of hypoxic conditions in the LPS-dependent induction of iNOS expression, ANA-1 M were stimulated with sub-optimal concentrations of LPS (10 ng/ml) cultured in hypoxic or normoxic (20% oxygen) conditions. We found that hypoxia, although ineffective by itself, caused a significant increase in iNOS mRNA expression induced by LPS alone. ANA-1 M transiently transfected with a plasmid containing the fall-length iNOS promoter linked to a CAT reporter gene expressed significantly higher levels of CAT activity when stimulated with LPS plus hypoxia, versus LPS alone. Deletion of either the iNOS-HRE or the NF-KB consensus sequence from the full-length iNOS promoter caused a dramatic decrease (> 80%) of CAT expression induced by LPS plus hypoxia In contrast, iNOS promoter constructs deleted of the IFN-y-responsive region remained inducible by LPS plus hypoxia. These data demonstrate that hypoxia is a costimulus with LPS for the induction of iNOS expression and they suggest the existence of a cooperative interaction between iNOS-HRE and NF-KB in the regulation of iNOS transcription.