Transcriptional control of adenosine signaling by hypoxia-inducible transcription factors during ischemic or inflammatory disease
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- Poth, J.M., Brodsky, K., Ehrentraut, H. et al. J Mol Med (2013) 91: 183. doi:10.1007/s00109-012-0988-7
Inflammatory lesions, ischemic tissues, or solid tumors are characterized by the occurrence of severe tissue hypoxia within the diseased tissue. Subsequent stabilization of hypoxia-inducible transcription factors—particularly of hypoxia-inducible factor 1α (HIF1A)—results in significant alterations of gene expression of resident cells or inflammatory cells that have been recruited into such lesions. Interestingly, studies of hypoxia-induced changes of gene expression identified a transcriptional program that promotes extracellular adenosine signaling. Adenosine is a signaling molecule that functions through the activation of four distinct adenosine receptors—the ADORA1, ADORA2A, ADORA2B, and ADORA3 receptors. Extracellular adenosine is predominantly derived from the phosphohydrolysis of precursor nucleotides, such as adenosine triphosphate or adenosine monophosphate. HIF1A-elicited alterations in gene expression enhance the enzymatic capacity within inflamed tissues to produce extracellular adenosine. Moreover, hypoxia-elicited induction of adenosine receptors—particularly of ADORA2B—results in increased signal transduction. Functional studies in genetic models for HIF1A or adenosine receptors implicate this pathway in an endogenous feedback loop that dampens excessive inflammation and promotes injury resolution, while at the same time enhancing ischemia tolerance. Therefore, pharmacological strategies to enhance HIF-elicited adenosine production or to promote adenosine signaling through adenosine receptors are being investigated for the treatment of acute inflammatory or ischemic diseases characterized by tissue hypoxia.
KeywordsAdenosineA1A2AA2BA3IschemiaCancerHypoxia-inducible factorHIF1HIF2Equilibrative nucleoside transportersENT1ENT2Adenosine kinaseAdenosine deaminaseCD73EctonucleotidaseCD39ApyraseAMPATPAcute lung injuryColitisInflammatory bowel diseaseIschemiaSepsis
The field of hypoxia changed its direction dramatically when Gregg Semenza performed studies of the erythropoietin promoter that led to the subsequent identification of hypoxia-inducible factor 1α (HIF1A)—the key transcription factor of hypoxia adaptation [1–5]. It turned out that this transcription factor controls numerous hypoxia-inducible genes and has been implicated in a number of physiological and pathological changes that are closely associated with hypoxic conditions such as those that occur during ischemic or inflammatory diseases . Subsequently, it has been appreciated that tissue hypoxia is a distinct feature of a wide variety of diseases, including ischemia, inflammatory diseases, and cancer [7, 8], and that the induction of hypoxia-inducible transcription factors under these disease conditions is not simply a bystander effect, but significantly impacts the inflammatory or ischemic microenvironment [9–12]. One of the important functions of HIF1A-dependent alterations of gene expression during hypoxia is its transcriptional effect on extracellular adenosine signaling. In addition, there are also transcription factors other than HIF involved in the adaptive response to hypoxia (for example, the Sp1 transcription factor) [13, 14]. In the present review, we discuss the mechanisms of how HIF-dependent changes in gene expression are associated with enhanced adenosine responses. Moreover, we give examples of how hypoxia-elicited increases in extracellular adenosine provide an endogenous feedback signal that dampens excessive inflammation and promotes tissue repair and healing. This pathway has been implicated in tissue protection during acute lung injury (ALI), inflammatory bowel disease, or ischemic disorders (discussed and referenced in the succeeding sections).
Tissue hypoxia occurs in a wide range of clinical disorders
Hypoxia-induced transcription factors
Since PHDs and FIH require oxygen as cofactor for their individual hydroxylation reactions, hypoxia is associated with a functional inhibition of FIH and PHDs, respectively. Therefore, hypoxic conditions are associated with the posttranslational stabilization of HIF1A and HIF2A. It should be noted that the levels of other cosubstrates and products can also modulate FIH and PHD activities (e.g., 2-oxoglutarate, succinate) . While HIF1A is expressed ubiquitously, HIF2A expression is limited to certain tissues (e.g., vascular endothelia). Understanding the differential effects of HIF1A versus HIF2A is currently an area of intense investigation. Indeed, there is emerging evidence that a specific hypoxia-elicited response is predominantly mediated by either HIF1A or HIF2A . For example, a study in patients with familial erythrocytosis discovered a gain-of-function mutation in the HIF2A gene as cause for the disease .
Following stabilization of HIF1A/HIF2A and coactivator binding, the alpha-subunit forms a heterodimer with HIF1B, translocates into the nucleus, and binds to promoter regions within HIF target genes. This promoter region is conserved and referred to as hypoxia response element (HRE). The consensus core sequence for an HRE is RCGTG (where R is A or G) . In most instances, binding of the HIF heterodimer to an HRE causes transcriptional induction of the gene [7, 8, 49]. However, there are also examples for HIF-mediated gene repression either by HIF-dependent induction of a gene repressor—such as microRNAs  or other inhibitor pathways—or by the same mechanism that also induces expression: direct binding of HIF to an HRE within the promoter region of the gene [17, 51–54]. It is presently not understood why direct binding of HIF to an HRE can, in some instances, function as gene repressor. HIF-dependent alteration in gene expression have been implicated in many hypoxia-adaptive responses, such as the induction of erythropoietin during anemia or the induction of vascular endothelial growth factor in hypoxic tissues. In addition to hypoxia-dependent stabilization of HIF, there are also examples of hypoxia-independent HIF stabilization during inflammatory or infectious diseases [28, 29]. In the present review, we discuss how HIF functions increase extracellular adenosine signaling events and concomitant tissue protection from hypoxia, ischemia, and inflammation.
Influence of hypoxia on extracellular adenosine signaling events
Extracellular adenosine production
During injurious conditions, many cell types release adenosine precursor molecules into the extracellular compartment [40, 76]. This occurs predominantly in the form of the adenosine precursor nucleotides ATP and adenosine diphosphate (ADP). Since intracellular ATP concentrations are generally high, many cells release ATP upon stimulation. For example, inflammatory cells or vascular endothelia release ATP during inflammation or hypoxia [68, 76–78]. Other sources for extracellular nucleotides include platelets that release ADP via granular release . ATP can function as a signaling molecule itself, via activation of ATP receptors . In many instances, ATP signaling drives proinflammatory responses , and hypoxia-dependent enhancement of ATP conversion to adenosine functions as an endogenous feedback loop to dampen excessive inflammatory injury [6, 24]. Extracellular ATP and ADP are rapidly converted to adenosine through a two-step enzymatic process, including CD39-dependent conversion of ATP/ADP to adenosine monophosphate (AMP) and CD73-dependent conversion of AMP to adenosine [72, 81–83]. Studies on the transcriptional effects of hypoxia revealed that CD39 is transcriptionally induced by hypoxia through an SP1-dependent transcriptional pathway [13, 14]. Similarly, CD73 is transcriptionally induced by HIF1A binding to the CD73 promoter and subsequent induction of CD73 transcript and protein levels [67, 68, 84, 85] (Fig. 5a). As such, hypoxia and inflammation are associated with increased production of adenosine from its precursor molecules, thereby shifting the balance from proinflammatory ATP signaling towards anti-inflammatory adenosine signaling.
Adenosine receptor expression during hypoxia
Comparative studies of adenosine receptor expression revealed that ADORA2B is selectively induced in different tissues during inflammation or hypoxia, including vascular endothelia , intestinal epithelia [66, 72–74], the kidneys [17, 70], the heart [15, 16, 69], and the lungs . Similarly, studies from human tissues—e.g., biopsies from ischemic myocardial tissues—revealed a selective induction of ADORA2B transcript levels . HIF1A binding to an HRE within the ADORA2B promoter results in increased ADORA2B transcription, protein expression, and function during conditions of hypoxia [66, 68]. Other studies identified a transcriptional pathway for ADORA2A, indicating that this receptor is transcriptionally induced in pulmonary endothelial cells during hypoxic conditions via a transcriptional pathway under the control of HIF2A . Taken together, these studies demonstrate that hypoxia drives extracellular adenosine signaling events on the receptor level by HIF-dependent induction of ADORA2A and ADORA2B [38–40].
Influence of hypoxia on the termination of adenosine signaling
Extracellular adenosine signaling is terminated by transport of adenosine from the extracellular towards the intracellular compartment through equilibrative nucleoside transporters (ENT1 and ENT2) [17, 51, 53, 54], followed by intracellular metabolism to inosine (via enzymatic activity of intracellular adenosine deaminase)  or to AMP (via enzymatic activity of adenosine kinase (ADK)) . Conditions of hypoxia or inflammation result in a functional attenuation of adenosine breakdown by repressing adenosine transporter activity and metabolism. Indeed, studies on the transcriptional control of ENTs demonstrate an HIF1A-dependent pathway of ENT1 [17, 54] and ENT2 repression [51, 53, 89], resulting in attenuated adenosine uptake and prolonged signaling effects during conditions of hypoxia. In addition, intracellular adenosine metabolism via ADK is attenuated due to HIF1A-dependent repression of ADK transcript, protein, and function  (Fig. 5b). Taken together, hypoxia-elicited repression of adenosine transporters (particularly ENT1 and ENT2) and ADK results in enhanced extracellular adenosine levels and signaling events.
Hypoxia enhances adenosine signaling through netrin-1
Several reports indicate that, during conditions of hypoxia, additional molecular signals can influence adenosine signaling. For example, hypoxia-elicited induction of netrin-1 (NTN1) has been implicated in enhancing extracellular adenosine signaling during inflammatory hypoxia [71, 90, 91]. NTN1 was originally described as a neuronal guidance molecule. Recent studies, however, also discovered an anti-inflammatory role of NTN1 in different diseases (e.g., inflammatory bowel disease, ALI) [55, 71, 91–94]. Just like hypoxia increases levels of extracellular adenosine, so it increases NTN1 levels, albeit by direct transcriptional induction of gene expression . Surprisingly, several studies indicate that NTN1 exerts its immunological effects by enhancing signaling events through the ADORA2B receptor . As such, migration of leukocytes is inhibited, making NTN1 a strong chemorepulsive molecule, which can dampen the influx of inflammatory cells during conditions of limited oxygen availability. The mechanism by which NTN1 signaling enhances extracellular adenosine signaling remains unclear (e.g., direct activation of the receptor, indirect enhancement of adenosine levels, or allosteric enhancement of Adora2b signaling).
Functional role of hypoxia-elicited adenosine elevations during acute inflammatory diseases
Inflammatory bowel disease
Many studies have implicated hypoxia-elicited elevations of extracellular adenosine levels in an endogenous feedback loop to dampen intestinal inflammation as it occurs during inflammatory bowel disease . The intestinal mucosa is already under physiologic conditions among the more “hypoxic” organs of the body. This phenomenon is referred to as “physiological hypoxia” and relates to the fact that the intestinal lumen is anaerobic, thereby causing an extremely steep oxygen gradient across the intestinal epithelial monolayer covering the mucosal surface. Due to alterations in oxygen supply and demand caused by intestinal inflammation, the degree of hypoxia becomes far more severe during intestinal inflammation . Interestingly, genetic studies on the role of HIF uncovered that stabilization of HIF provides a protective and anti-inflammatory pathway during intestinal inflammation [10, 11, 25, 95–97]. Moreover, studies with pharmacologic compounds that achieve HIF stabilization (PHD inhibitors) demonstrate robust protection from intestinal inflammation. As discussed previously, HIF stabilization results in enhanced extracellular adenosine production and signaling. As such, several studies show that mice with failure to produce extracellular adenosine (CD39−/−or CD73−/−mice) develop a more severe course of disease when exposed to experimentally induced inflammatory bowel disease [98, 99]. Moreover, extracellular adenosine signaling through ADORA2B or ADORA2B has been shown to protect from intestinal inflammation [71, 74, 100–102].
Similar to intestinal inflammation, intestinal ischemia is characterized by profound intestinal tissue hypoxia in the context of intermittent loss of perfusion to the gut, resulting in dramatic increases in morbidity and mortality . In this context, different studies have provided evidence for an anti-inflammatory role for HIF1A-elicited enhancement of extracellular adenosine production via CD73  and signaling through ADORA2B . In addition, a recent study targeted HIF1A during intestinal ischemia using pharmacological or genetic approaches. Initial studies with the pharmacological HIF activator (PHD inhibitor) dimethyloxallyl glycine (DMOG) indicated attenuation of intestinal injury with DMOG treatment in intestinal ischemia and reperfusion injury (IR) . In this murine model, DMOG treatment was associated with induction of CD73 and ADORA2B transcript and protein levels, while DMOG protection was abolished in CD73−/− or Adora2b−/− mice. Finally, studies of mice with tissue-specific deletion of Hif1a in intestinal epithelia or pharmacological inhibition of Hif1a with 17-dimethylaminoethylamino-17-demethoxygeldanamycin revealed enhanced tissue injury during IR, thereby providing strong evidence for the Hif–adenosine pathway in gut protection from ischemia .
Acute kidney injury
Similar to intestinal IR, several studies have shown a protective role of hypoxia-elicited increases in extracellular adenosine production and signaling during IR in other organs, including the heart [7, 14–16, 69, 104] and the liver [13, 82, 89]. Particularly during ischemic tissue injury of the kidneys—a life-threatening condition that frequently complicates the care of hospitalized patients—hypoxia-elicited increases in adenosine production have been shown to protect from AKI. Studies in mice deficient in the pathway for extracellular adenosine production or deficient for signaling through Adora2b experience more severe kidney injury [70, 105–107]. Additional studies on the termination of extracellular adenosine signaling revealed a functional role for hypoxia-elicited elevations of extracellular adenosine in preventing a no-reflow phenomenon . Indeed, a complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed during ischemic AKI . Due to its role in adapting tissues to hypoxia, one of our recent studies pursued the hypothesis that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion . Consistent with the notion that ENTs terminate adenosine signaling, this study found that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1−/− mice. Comprehensive examination of adenosine receptor knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves Adora2b. Subsequent studies in mice with tissue-specific deletion of the Adora2b and Adora2b reporter mice revealed a crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia to effectively prevent a postischemic no-reflow phenomenon. Together, these studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI .
Acute lung injury
ALI is characterized by alveolar injury and uncontrolled inflammation [108, 109]. Several studies have provided evidence for a protective role for the hypoxia–adenosine pathway also during ALI [86, 110–112]. Very compelling evidence comes from the laboratory of Michail Sitkovsky and his team [32, 33, 113]. The group examined the consequences of different levels of oxygenation and concomitant alterations of the adenosine pathway on outcome parameters during murine ALI . ALI frequently requires symptomatic supportive therapy by intubation and mechanical ventilation with the supplemental use of high oxygen concentrations. Although oxygen therapy represents a life-saving measure, the discovery of hypoxia-elicited adenosine production and concomitant lung protection would predict that administration of oxygen to ALI patients with uncontrolled pulmonary inflammation could have dangerous side effects . As such, oxygenation could weaken tissue hypoxia-driven adenosine production and signaling, and thereby dampen the hypoxia-mediated anti-inflammatory pathway and further exacerbate lung injury. To examine this hypothesis, the authors used a mouse model of ALI induced by bacterial infection. Thiel et al. exposed one group of mice to 100 % oxygen, mimicking therapeutic oxygenation, and left another group at normal ambient levels (21 % oxygen). Five times more mice died after receiving 100 % oxygen than died breathing normal oxygen levels . Mice given 60 % oxygen—considered clinically safe—developed severe pneumonia, but did not die. Indeed, the authors went on to demonstrate that hypoxia-dependent lung protection during ALI involves the enhancement of extracellular adenosine signaling through Adora2a.
Other studies from the laboratory of Sean Colgan have implicated extracellular adenosine production and signaling through ADORA2B in lung protection during hypoxia—as occurs in the setting of hypoxic preconditioning. They found that ADORA2B-dependent adenosine signaling helps to protect the lungs via inhibition of the proinflammatory transcription factor nuclear factor NF-κB . Indeed, adenosine-dependent inhibition of NF-κB involved the proteasomal pathway and was mediated by adenosine-mediated cullin-1 deneddylation . As such, these findings implicate hypoxia-elicited elevations of extracellular adenosine and ADORA2B-dependent adenosine signaling in lung protection from excessive inflammation [114, 115].
Research over the past decade indicates that several disease conditions, such as IR, inflammatory bowel disease, or ALI are associated with the stabilization of hypoxia-inducible transcription factors. One of the key outcomes of HIF activation during these disease conditions includes the activation of extracellular adenosine signaling—particularly through ADORA2B. Many functional studies demonstrate that this pathway resembles an endogenous feedback loop to dampen hypoxia-induced inflammation and to provide organ protection during conditions of limited oxygen availability. It will be critical for the next years to translate these basic research findings from the bench towards patient treatment. For example, this could be achieved by pharmacological means of HIF activation (such as PHD inhibitors) or adenosine receptor agonists. We anticipate that targeting hypoxia-induced increases in extracellular adenosine signaling will provide novel therapies for a wide range of acute inflammatory diseases.
The present research work was supported by Grant D/10/52531 from the German Academic Exchange Service (DAAD) to J.M.P., a German Research Foundation (DFG) Grant (EH401/1-1) to H.E., an American Heart Association Grant to A.G., and National Heart Institute Grants R01-HL0921, R01-DK083385, and R01-HL098294 and a grant by the Crohn’s and Colitis Foundation of America (CCFA) to H.K.E.
Conflict of interest
The authors have declared no conflict of interest.