Regulated Extracellular Nucleotide Metabolism and Function at the Mucosa - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 127

or inflamed tissue [58, 68]. Classical HIF-regulated pathways have been identified
in developmental pathways where gene activation may be required as cell mass
and number increase in the developing organism [57]. This may be coupled with
an increased requirement for regulation of glycolytic genes to meet the energy
requirements of the cells, and an increase in oxygen delivery in tissues through
erythropoietin (EPO) induction and an increase in vascular networks through the
induction of vascular endothelial growth factor (VEGF). HIF activation may con-
tribute to inappropriate adaptation to hypoxic areas such as in the core of solid
tumors whereby HIF signaling permits evasion from apoptosis, increased cell pro-
liferation and increased angiogenesis [57]. Conversely, HIF-1 activation may serve
to resolve potentially injurious levels of either physiological tissue hypoxia, or the
high oxygen demand that is associated with pathologies such as inflammation [33,
67, 68]. Accordingly, it has been demonstrated that adenosine signaling is protec-
tive in inflammatory diseases such as glomerulonephritis [21] colitis [20, 41] and
during ischaemic preconditioning models of intestinal, hepatic and lung injury [13,
25, 26]. This protective phenotype afforded by increased levels of adenosine is
achieved through an upregulation of the ecto-nucleotideases and their subsequent
activity on extracellular ATP, ADP and AMP. Both CD39 and CD73 have been
shown to be upregulated in intestinal epithelial cells incubated under hypoxic con-
ditions (pO 2 20 torr) and the functional activity of both ecto-enzymes was shown
to increase under hypoxic conditions as demonstrated by increased conversion of
etheno-ATP (E-ATP) to etheno-AMP (E-AMP), and increased E-AMP conversion to
etheno-adenosine (E-Ado) for CD39 and CD73 activity, respectively. Furthermore,
mice subjected to ambient hypoxia (8% O 2 , 92% N 2 ) demonstrated an increase
in CD73 expression in intestinal mucosal scrapings [65]. Further analysis demon-
strated the ability for HIF-1 to associate with a HIF consensus site within the
CD73 gene promoter region, whose deletion resulted in a loss of luciferase reporter
activity [65].
Although CD39 appears to be functionally upregulated under hypoxic condi-
tions, there is no current evidence that it is directly regulated by interactions between
the HIF-1 transcription factor and the promoter region of CD39. There is evidence,
however, that other transcription factors may be involved in the regulation of genes
under hypoxic conditions, such as Sp1, early growth response-1 (Egr-1), activating
transcription factor-4 (ATF-4) and erythroblastosis virus E26 oncogene homolog 1
(Ets-1) [4, 48, 66, 73]. Truncation analysis of the promoter region of CD39 has
revealed the presence of a Sp1 binding site, which when deleted or mutated, results
in loss of promoter reporter activity using a firefly luciferase based assay [16].
Studies have identified HIF-1 dependent induction of Sp1, leading to indirect acti-
vation of hypoxic target genes. Mutation of either Egr1 or Sp1 binding sites, but
not the HIF-1 response element (HRE) in the promoter region of the gene encod-
ing the epinephrine-synthesizing enzyme, phenylethanolamine N-methyltransferase
(PNMT) results in an abrogation of HIF induction of PNMT [66]. More direct evi-
dence of HIF-1/Sp1 coactivation has been demonstrated with hypoxic regulation of
the stress response protein Redd1. In this model, both Sp1 and HIF-1 are required
for maximal activity of the Redd1 promoter, suggesting a coordinated response of