Hypoxia-Mediated Expression of 5-Lipoxygenase-Activating Protein Involves HIF-1α and NF-κB and MicroRNAs 135a and 199a-5p

Hypoxia occurs in a number of pathological states, such as pulmonary, hematological, and cardiovascular disorders. In this study, we examined the molecular mechanism by which hypoxia contributes to increased leukotriene formation. Our studies showed hypoxia augmented the expression of 5-lipoxygenase activating protein (FLAP), a key enzyme in leukotriene formation, in both human pulmonary microvascular endothelial cells and a transformed human brain endothelial cell line. Hypoxia-induced FLAP mRNA expression involved activation of NADPH-oxidase, PI-3 kinase, mitogen-activated protein kinase, NF-kappa B, and hypoxia-inducible factor (HIF)-1 alpha. Hypoxia-induced FLAP promoter activity was attenuated on mutation of hypoxia-response elements (HREs) and NF-kappa B binding motif in the FLAP promoter. Hypoxia also augmented binding of HIF-1 alpha to HREs in FLAP promoter as demonstrated by EMSA with nuclear extracts. Furthermore, chromain immunoprecipitation analysis showed HIF-1 alpha bound to HREs in native chromatin obtained from hypoxia-treated cells. Next, we examined the role of HIF-1 alpha regulated microRNAs on FLAP expression. Our studies showed decreased expression of miR-135a and miR-199a-5p in response to hypoxia. However, overexpression of anti-miR-135a and anti-miR-199a-5p oligonucleotides led to a several fold increased FLAP mRNA and protein expression. These studies demonstrate for the first time that hypoxia-mediated FLAP expression is regulated by HREs and NF-kappa B site in its promoter, and negatively regulated by miR-135a and miR-199a-5p, which target the 3'-UTR of FLAP mRNA. An understanding of these regulatory pathways provides new avenues to ameliorate leukotriene formation and hence reactive airway disease, and inflammation in individuals who have sickle cell disease. The Journal of Immunology, 2010, 184: 3878-3888.