Extracellular Adenosine Signaling in Molecular Medicine

Adenosine – a purinergic signaling molecule

Biochemically, adenosine belongs to a group of molecules referred to as purines. Purines are heterocyclic aromatic molecules that are among the oldest and most influential biochemical compounds in evolutionary history [1]. They are critical building blocks of the genetic code, and therefore the substrate of life, as represented by DNA. These relatively simple molecules are composed from adenine and guanine, and without these molecules, life as we know it would not be possible [2]. In a wide sense, purines are central to the self-sustained and reproducible existence of nucleotide-protein systems, which form cells and tissues that ultimately resulted in an appearance of Homo sapiens [2]. As such, the purine adenosine is well recognized as molecular building block of the genetic code or as part of adenosine triphosphate (ATP) – the universal energy currency of biological reactions [3]. Beyond these function, Alan Drury and Albert Szent-Györgyi from the University of Cambridge introduced in 1929 the idea that purines could also function as extracellular signaling molecules. They injected extracts from cardiac tissues intravenously into a whole animal. They observed a transient slowing of the heart rate [4]. Following several purification steps, they came to the conclusion that the biologic activity in the extract was an “adenine compound”[4]. Today we have genetic evidence that the transient heart-block induced by intravascular adenosine injection is mediated by the activation of an adenosine receptor [5, 6]. Indeed, adenosine signaling can occur through four distinct adenosine receptors – the Adora1, Adora2a, Adora2b and Adora3 - all of them G-protein coupled receptors. Adenosine-induced heart block remains the most famous clinical application for adenosine signaling, as intravenous adenosine injection continues to be a mainstay therapy for the diagnosis and treatment of supraventricular tachycardia [7, 8].

The complex control of extracellular adenosine signaling

In order to better understand the control mechanism for extracellular adenosine signaling, many studies have addressed the question of where extracellular adenosine comes from. There are examples that extracellular adenosine can be released from intracellular stores [9–11]. However, many studies imply that during disease conditions such as hypoxia, ischemia or inflammation, extracellular adenosine predominantly stems from the breakdown of precursor nucleotides (for example ATP). Stressful conditions are associated with leakage or controlled release of ATP from the intracellular towards the extracellular compartment [12–14]. ATP is subsequently converted via the ectonucleoside triphosphate diphosphohydrolase 1 (CD39) to adenosine monophosphate (AMP) [15–20], and subsequently through the ecto-5′-nucleotidase CD73 to adenosine (Figure 1) [21–23]. Once released into the extracellular compartment, activation of adenosine receptors is responsible for adenosine’s biological activities as signaling molecule. For example, activation of the Adora2b receptor has been implicated in enhancing ischemia tolerance [24–26] and attenuating acute inflammatory responses [21, 25, 27–29].

Figure 1. Extracellular Adenosine Signaling and Its Termination.

In inflammatory conditions, extracellular adenosine is derived predominantly from the enzymatic conversion of the precursor nucleotides ATP and ADP to AMP through the enzymatic activity of the ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and the subsequent conversion of AMP to adenosine through ecto-5′-nucleotidase (CD73). Extracellular adenosine can signal through four distinct adenosine receptors: ADORA1 (A₁), ADORA2A (A_2A), ADORA2B (A_2B), and ADORA3 (A₃). An example of the functional role of extracellular adenosine signaling is A₁-receptor activation during intravenous administration of adenosine for the treatment of supraventricular tachycardia. In addition, experimental studies implicate activation of A_2A that is expressed on inflammatory cells such as neutrophils or lymphocytes in the attenuation of inflammation. Other experimental studies provide evidence of signaling events through A_2B in tissue adaptation to hypoxia and attenuation of ischemia and reperfusion. A clinical trial has shown that an oral agonist of the A₃ adenosine receptor may be useful in the treatment of the dry-eye syndrome. Adenosine signaling is terminated by adenosine uptake from the extracellular space toward the intracellular space, predominantly through equilibrative nucleoside transporter 1 (ENT1) and equilibrative nucleoside transporter 2 (ENT2), followed by metabolism of adenosine to AMP through the adenosine kinase or to inosine through the adenosine deaminase. Blockade of equilibrative nucleoside transporters by dipyridamole is associated with increased extracellular adenosine concentrations and signaling (e.g., during pharmacologic stress echocardiography or in protection of tissue from ischemia). From Eltzschig HK, Sitkovsky MV, Robson SC. Purinergic Signaling during Inflammation. New England Journal of Medicine 2012;367:2322–33, Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

There are many control steps regulating extracellular adenosine signaling events – such as ATP release, its conversion to adenosine via CD39 and CD73, or the expression of adenosine receptors (Figure 1). Moreover, there is evidence that alternative molecular pathways exist that can function to enhance extracellular adenosine signaling independent of adenosine, such as enhancement of purinergic signaling events through the neuronal guidance molecule netrin-1 [30–32]. Finally, the termination of extracellular adenosine signaling is a highly complex biological process with many steps that are independently regulated on a transcriptional level [33–35]. As such, adenosine is taken up from the extracellular into the intracellular compartment through adenosine transporters [24], and subsequently converted to inosine via the adenosine deaminase [36, 37], or via the adenosine kinase to AMP (Figure 1) [38]. These processes can function together to fine-tune extracellular adenosine levels and signaling functions [7].

Extracellular adenosine signaling during disease states

Due to the complexity of the system that regulates extracellular adenosine signaling, it has been challenging for many years to characterize biological functions of extracellular adenosine signaling during disease conditions. While first evidence was provided by pharmacological compounds that would specifically modulate signaling through individual adenosine receptors [39, 40], or studies utilizing compounds that would indirectly enhance extracellular adenosine concentrations [41], several breakthrough discoveries in the field were achieved during the past 15 years by generating and studying mice with genetic deletions of specific adenosine receptors or deletions of the enzymatic machinery that controls extracellular adenosine levels. In order to give an overview for the functional role of extracellular adenosine signaling during molecular medicine and its impact on human disease, five different research groups in this field are providing their perspective on recent advances in the field of extracellular adenosine signaling, and how it can be targeted for disease treatment. For example, a land-mark paper from the research group of Dr. Sitkovsky provided the first genetic evidence for an anti-inflammatory role for adenosine signaling as an endogenous feedback loop to limit collateral damage during uncontrolled inflammation via Adora2a receptors [42]. In extension of these findings this team of scientists found that excessive levels of extracellular adenosine may become detrimental during neoplastic disease states, thereby implicating adenosine receptor antagonists in the treatment of cancer [43]. As such, a review by Sitkovsky et al. in this issue is focused on how the hypoxia-adenosinergic signaling pathway can be targeted to improve the adoptive immunotherapy of cancer (49). Studies from the laboratory of Dr. Colgan implicated inflammatory hypoxia in the extracellular production and control of adenosine signaling, and identified hypoxia-induced increases in adenosine signaling as a control mechanism to dampen intestinal inflammation as occurs during inflammatory bowel disease (Figure 2) [29, 44, 45]. Therefore, a review by Colgan et al. is focused on adenosine and gastrointestinal inflammation (50). Studies from the laboratory of Dr. Robson utilized mice with genetic deletions of CD39 and CD73 to describe immunological roles of extracellular adenosine production by enhancing the anti-inflammtory functions of regulatory T-cells [46]. Indeed, a review from Longhi et al. describes biological functions of ecto-enzymes in regulating extracellular adenosine levels in neoplastic and inflammatory disease states [51]. Moreover, studies from the laboratory of Dr. Blackburn examined mice with genetic deletions of the adenosine deaminase [47]. These studies revealed dramatically increased levels of extracellular adenosine, and provide genetic evidence for a potentially detrimental role for prolonged adenosine elevations during chronic inflammatory disease states [36]. In the present issue, a review by Karmouty-Quintana et al. is focused on adenosine signaling during acute versus chronic disease states [52]. Finally, research work from our group has been interested over many years on the transcriptional regulation of adenosine responses, and particularly on the functional role of hypoxia-inducible factors to enhance acute adenosine protection during ischemia or inflammation. Indeed, a review by Poth et al. discusses the transcriptional control of adenosine signaling by hypoxiainducible transcription factors during ischemic or inflammatory disease states [53].

Figure 2. Hypoxia Control of Extracellular Adenosine Generation and Signaling in Intestinal Inflammation.

Histologic staining of intestinal sections for hypoxia shows that hypoxia is present within the apical surface of the intestinal mucosa (orange area in upper insert). This presence is most likely due to the fact that the intestinal lumen is anaerobic, which results in a steep oxygen gradient across the epithelial monolayer. In patients with intestinal inflammation such as that which occurs in the course of inflammatory bowel disease, a decrease in metabolic supply (e.g., due to thrombosed vessels) and profound increases in oxygen demand result in an imbalance in oxygen availability. This imbalance causes severe hypoxia of the inflamed mucosa, as indicated by histologic staining for tissue hypoxia (as shown in the lower insert, the orange staining that extends from the apical aspects of the mucosa into the crypts and submucosal tissues indicates severe tissue hypoxia). Release of ATP or ADP from inflammatory cells, platelets, or epithelial cells results in the activation of P2 receptors such as the P2X7 receptor, expressed on enteric neurons, thereby promoting tissue inflammation and injury. Hypoxia causes the activation of transcriptional programs that result in an Sp1-dependent induction of CD3917 and a hypoxia-inducible factor (HIF)–dependent induction of CD73 and the ADORA2B (A_2B) adenosine receptor. These transcriptional changes lead to an increased rate of turnover of the extracellular nucleotides ATP and ADP to AMP (through CD39) and subsequently to adenosine (through CD73). Experimental studies indicate that adenosine-receptor activation — particularly through Adora2a (A_2A)89 and A_2B90 — dampens intestinal inflammation and promotes epithelial integrity during intestinal inflammation. From Eltzschig HK, Sitkovsky MV, Robson SC. Purinergic Signaling during Inflammation. New England Journal of Medicine 2012;367:2322–33, Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.

As such, there are numerous examples for the importance of extracellular adenosine signaling in molecular medicine. In most instances, extracellular adenosine signaling has anti-inflammatory functions during acute disease states, such as acute lung injury, ischemia and reperfusion or intestinal inflammation [7, 48]. Under such circumstances, pharmacologic approaches to enhance adenosine signaling effects (e.g. via adenosine receptor agonists or adenosine uptake inhibitors) are investigated in preclinical studies [50,51,53]. In contrast, adenosine-elicited inhibition of immune responses during neoplastic disease states contributes to tumor growth and metastasis, thereby implicating adenosine receptor blockers in the treatment of cancer [49]. Similarly, inhibition of adenosine receptors is an evolving therapeutic concept for the treatment chronic disease states, such as pulmonary fibrosis or sickle cell disease. Here, it will be particularly important to identify biomarkers that will help physicians to judge when adenosine protection during an acute disease states turns into promoting its chronicity [52]. Much work will be required to determine the clinical contexts in which the activation or inhibition of specific adenosine receptors can be utilized therapeutically to improve outcomes of acute and chronic inflammatory diseases states, ischemia and reperfusion injury or cancer. The five articles in this special issue of The Journal of Molecular Medicine [49–53] provide a roadmap for further exploration of the field of purinergic signaling for the treatment of human disease states.