Under conditions of limited
oxygen availability (
hypoxia), multiple cell types release
adenine nucleotides in the form of
ATP,
ADP, and
AMP. Extracellular
AMP is metabolized to
adenosine by surface-expressed
ecto-5'-nucleotidase (CD73) and subsequently activates surface
adenosine receptors regulating endothelial and epithelial barrier function. Therefore, we hypothesized that
hypoxia transcriptionally regulates CD73 expression. Microarray
RNA analysis revealed an increase in CD73 and
ecto-apyrase CD39 in hypoxic epithelial cells. Metabolic studies of CD39/CD73 function in intact epithelia revealed that
hypoxia enhances CD39/CD73 function as much
as 6 +/- 0.5-fold over normoxia. Examination of the CD73 gene promoter identified at least one binding site for
hypoxia-inducible factor-1 (HIF-1) and inhibition of HIF-1alpha expression by
antisense oligonucleotides resulted in significant inhibition of
hypoxia-inducible CD73 expression. Studies using
luciferase reporter constructs revealed a significant increase in activity in cells subjected to
hypoxia, which was lost in truncated constructs lacking the HIF-1 site. Mutagenesis of the HIF-1alpha binding site resulted in a nearly complete loss of
hypoxia-inducibility. In vivo studies in a murine
hypoxia model revealed that
hypoxia-induced CD73 may serve to protect the epithelial barrier, since the CD73 inhibitor
alpha,beta-methylene ADP promotes increased intestinal permeability. These results identify an HIF-1-dependent regulatory pathway for CD73 and indicate the likelihood that CD39/CD73 protects the epithelial barrier during
hypoxia.