Tissue
hypoxemia is common in several pathological diseases, including vaso-occlusion in
sickle cell disease and
myocardial infarction. One finds increased presence of leukocytes during
lung injury and at sites of
inflammation in vascular endothelium. In this study, we used human pulmonary microvascular endothelial cells and human dermal microvascular endothelial immortalized cell line to delineate the cellular signaling mechanism of
hypoxia- and CoCl2 (a mimetic of
hypoxia)-induced
IL-8 expression, and the latter's role in chemotaxis of polmorphonuclear neutrophils. We show that
hypoxia- and CoCl2-induced
IL-8 mRNA and
protein expression involved activation of PI3K/Akt and
p38 MAPK, but not
MEK kinase. Analysis of some
transcription factors associated with
IL-8 promoter revealed that
hypoxia and CoCl2 increased
DNA-binding activity of
hypoxia-inducible factor-1alpha (HIF-1alpha),
NF-kappaB, and
AP-1. In addition, we show that
hypoxia- and CoCl2-induced
IL-8 expression requires activation of HIF as demonstrated by the following: 1) EMSA; 2) transfection studies with IL-8 promoter reporter constructs with mutation in HIF-1alpha binding site; 3) attenuation of IL-8 expression by both HIF-1alpha
small interfering RNA and
R59949; 4) augmentation of IL-8 expression by either transfection with HIF-
prolyl hydroxylase-2
small interfering RNA or overexpression of HIF-1alpha; and 5)
chromatin immunoprecipitation analysis. Moreover,
conditioned medium from
hypoxia-treated endothelial cells augmented chemotaxis of neutrophils, due to release of IL-8. These data indicate that
hypoxia-induced signaling in vascular endothelium for transcriptional activation of IL-8 involves PI3K/Akt,
p38 MAPK, and HIF-1alpha. Pharmacological agents, which inhibit HIF-1alpha, may possibly ameliorate
inflammation associated with
hypoxia in pathological diseases.