Adenosine exerts paracrine and autocrine functions on most cell types. Pathophysiologic conditions of hypoxia/ischemia result in numerous adenine nucleotide metabolic changes, and adenosine has a demonstrated role in organ function under such conditions. In the present studies, we explored the mechanisms and impact of CD73 induction by hypoxia, a primary determinant of localized production of adenosine at tissue interfaces (39
). These studies revealed that ambient hypoxia transcriptionally regulates CD73 and that one mechanism of such induction involves HIF-1. Extensions of these studies revealed that CD73 induction may subserve intestinal permeability during hypoxia in vivo.
Adenosine is a critical mediator during ischemia and hypoxia (9
). While the source of interstitial adenosine in hypoxic tissue has been the basis of much debate, it is generally accepted that the dephosphorylation of AMP by CD73 represents the major pathway of extracellular adenosine formation during oxygen supply imbalances (9
). Extracellular adenosine production in the ischemic myocardium, for example, is attributable to activity of CD73 (40
), and both CD73 activity and adenosine metabolism have been demonstrated in cardiac preconditioning by brief periods of ischemia (41
). Increased CD73 activity in ischemic preconditioning has been attributed to a variety of acute activation pathways (10
), and a recent study provides direct evidence that CD73 is transcriptionally regulated by hypoxia in pheochromocytoma cells in vitro (13
). Once liberated in the extracellular space, adenosine either is taken up into the cell (through dipyridamole-sensitive carriers) or interacts with cell surface adenosine receptors (5
). Presently, four subtypes of G protein–coupled adenosine receptors exist, designated A1
, and A3
. These receptors are classified according to utilization of pertussis toxin–sensitive pathways (A1
) or adenylate cyclase activation pathways (A2A
). Epithelial cells of many origins constitutively express adenosine receptors (5
), primarily of the A2A
). As such, the present findings of functional CD39/CD73 induction during hypoxia help to clarify at least some of the issues related to increased enzyme activity during hypoxia. Much still remains unknown about these pathways. For example, we do not know whether intracellular nucleotidases are similarly regulated at the protein level. The antibody used here to determine protein levels (mAb 1E9) specifically recognizes the extracellular nucleotidase as opposed to other forms of the molecule (32
), and thus, no general conclusions can be drawn about generation of total cellular adenosine. Similarly, little is known about the regulation of CD39 at the transcriptional/posttranscriptional level, and whether similar HIF-1 pathways may also contribute to hypoxia-inducibility.
Given the temporal and robust hypoxia response observed in the induction of CD73, a candidate regulator was HIF-1, a member of the rapidly growing Per-ARNT-Sim family of basic helix-loop-helix transcription factors (47
). HIF-1 exists as an αβ heterodimer, the activation of which is dependent upon stabilization of an O2
-dependent degradation domain of the α subunit by the ubiquitin-proteasome pathway (49
). A search of the cloned CD73 gene promoter revealed a classic HIF-1–binding DNA consensus motif, 5′-CCGTG-3′, located at positions –367 to –371 relative to the major transcription start site (11
). However, the existence of an HIF-1α binding consensus is not evidence for an HIF-1α–mediated response; instead, the HRE is defined as a cis
-acting transcriptional regulatory sequence located within 5′-flanking, 3′-flanking, or intervening sequences of target genes (50
). Three approaches were used to define a role for HIF-1α in the induction of CD73. First, the use of previously published antisense oligonucleotides (23
), but not sense controls, resulted in a nearly complete blockade of CD73 induction. Second, the combination of antisense oligonucleotides and transient reporter construct transfections was used to add further evidence for HIF-1 and revealed a complete blockade of CD73 induction. A third approach using luciferase reporter constructs was used to identify the hypoxia-responsive region of the promoter. Results from these studies narrowed the region to –518 to +63, and mutations of this HIF-1α site resulted in a greater than 85% decrease in hypoxia-inducibility. A two-nucleotide mutation of this site resulted in a loss of hypoxia-inducibility and provided additional evidence for a functional HRE. Of note, studies with reporter constructs also indicated that repressor elements may also regulate CD73 expression in hypoxia. For example, the larger 5′ truncations of the promoter sequence to bp –518 to +63 (pGL20.57NT
) indicated increased hypoxia-inducibility compared with the full-length promoter, suggesting the presence of partial repressor activity (bp –993 to –518). Transcription factor binding analysis (e.g., TFSEARCH) (51
) of this region indicated consensus sites for CdxA, SRY, GATA-1, GATA-2, and HNF-3b. While we have not directly addressed this issue, at least two of these transcription factors (GATA-1 and GATA-2) have been recently implicated in repression of genes in hypoxia (52
). Thus, it is likely that both positive and negative regulatory pathways contribute to overall CD73 promoter activity.
Recent work from a number of laboratories has indicated that CREB may mediate hypoxia-elicited induction of a number of genes (14
). Based on these findings, and previous observations that the CRE site of the CD73 promoter is functional (30
), mutational analysis was employed to define the function of this site. Surprisingly, these studies revealed that CREB binding is critical for basal expression of CD73. These results may have broader implications. For example, it is not presently known how tightly CD73 expression is regulated and whether such regulation is transcriptionally coupled. In addition, it is not known what physiologically relevant mediators (e.g., hormones, chemokines, nucleotides, cytokines) might influence basal expression of CD73. It is possible that basal, low-level maintenance of CD73 expression occurs as a bystander process of adenosine A2A
receptor activation (i.e., activation of CREB). In this regard, and as we have hypothesized previously (30
), adenosine may serve as a feed-forward mechanism to regulate nucleotide metabolic enzymes, such as CD73.
Our previous studies suggested that surface CD73 likely represents a protective pathway for the maintenance of barrier function in epithelia (31
) and vascular endothelia (4
). We provide in vivo evidence here that CD73, and likely CD39, functions as an overall barrier-protective element in the intestine. In control animals exposed to luminal APCP, significantly increased permeability was observed, suggesting that CD73 provides a physiologic function in this regard, and this influence was enhanced in parallel to induction of CD73 mRNA in hypoxic animals. These findings are in line with previous studies indicating that the intestinal mucosa supports barrier-protective pathways during hypoxia in vivo. Work addressing the role of intestinal trefoil factor (ITF) suggested that hypoxia-induced ITF (via HIF-1 activation) contributes, in part, an endogenous protective mechanism for intestinal epithelia (15
). Those studies were noteworthy in that there were likely other important molecules with similar functions. It is possible that CD73 is an additional molecule with a similar function. Of note, it was recently reported that the MDR1
gene product P-glycoprotein could also contribute a similar function during hypoxia (54
), particularly since MDR has been associated with barrier abnormalities in intestinal disease (55
). Taken together, a number of molecules likely contribute to this interesting pathway, and the redundancy likely indicates the biologic importance of this protective mechanism.
While the present studies are the first, to our knowledge, to define a physiologic role for CD73 in barrier function in vivo, significant work has implicated CD73 in barrier regulation in vitro. During modeled inflammation, neutrophils release a number of biologically active mediators, including 5′-AMP (7
), and metabolism of 5′-AMP to adenosine requires CD73 (1
). Inhibitor-based studies have implicated CD73 in regulation of both epithelial and endothelial permeability during PMN transmigration and have suggested that the metabolism of 5′-AMP to adenosine by CD73 may be rate-limiting (i.e., increased CD73 results in parallel increases in 5′-AMP–mediated bioactivity) (4
). The competitive inhibitor APCP abolishes the influence of 5′-AMP, whereas the less potent, noncompetitive inhibitor mAb 1E9 substantially diminishes the influence of 5′-AMP (4
). Our findings that the CD73 antagonist APCP increases intestinal permeability are consistent with previous studies in rats showing that APCP inhibits ATP-dependent (i.e., cAMP-dependent) peripheral vasodilation. Importantly, in this regard, it will be necessary to define the exact role of CD39 in barrier function, particularly since a number of cell types, especially activated platelets, are able to release large quantities of CD39 substrates (i.e., ATP and ADP) at sites of inflammation or hypoxia (57
). Recent studies with stroke models in CD39–/–
mice suggest that CD39 provides a protective thromboregulatory function during ischemia and stroke (58
). Such observations suggest that the hypoxic microenvironment may liberate large amounts of both CD39 and CD73 substrates, and, as a result, large quantities of extracellular adenosine. While we do not know the exact mechanism(s) of barrier promotion by adenosine, recent studies suggest that phosphorylation of the tight junction–associated protein vasodilator-stimulated phosphoprotein (VASP) may be critical in both epithelial and endothelial permeability (20
In these studies, we assessed mucosal hypoxia using the nitroimidazole EF5. In the absence of adequate levels of oxygen, EF5 undergoes reduction to more reactive products, which then bind to cellular proteins and are thus retained and detected with antibodies directed against the adducts (37
). Noteworthy in these experiments was the nearly complete lack of staining in connective tissue under all experimental conditions, but evident staining in the epithelium of normoxic animals. This basal staining in normoxic animals was not evident in other mucosal tissues examined (e.g., liver and kidney; data not shown) and may be related to previous observations that the pO2
of the healthy intestine is relatively low (~30–35 mmHg, depending on the specific region examined) (60
). Given these features, it stands to reason that the relative hypoxia of the healthy intestine could be an endogenous mechanism to maintain expression of hypoxia-responsive genes, such as CD39 and CD73. This hypothesis has not been directly tested.
In summary, these results define hypoxia-regulated CD39 and CD73 expression in the intestinal mucosa and identify a previously unappreciated HIF-1 regulatory binding site in the CD73 promoter. This regulatory pathway extends to the transcriptional level in vitro and in vivo and identifies an important role for CD39/CD73 in the regulation of intestinal permeability during hypoxia.