Adenosine and hypoxia-inducible factor signaling in intestinal injury and recovery

Abstract

The gastrointestinal mucosa has proven to be an interesting tissue in which to investigate disease-related metabolism. In this review, we outline some of the evidence that implicates hypoxia-mediated adenosine signaling as an important signature within both healthy and diseased mucosa. Studies derived from cultured cell systems, animal models, and human patients have revealed that hypoxia is a significant component of the inflammatory microenvironment. These studies have revealed a prominent role for hypoxia-induced factor (HIF) and hypoxia signaling at several steps along the adenine nucleotide metabolism and adenosine receptor signaling pathways. Likewise, studies to date in animal models of intestinal inflammation have demonstrated an almost uniformly beneficial influence of HIF stabilization on disease outcomes. Ongoing studies to define potential similarities with and differences between innate and adaptive immune responses will continue to teach us important lessons about the complexity of the gastrointestinal tract. Such information has provided new insights into disease pathogenesis and, importantly, will provide insights into new therapeutic targets.

Figure 1

Localization of hypoxia and mechanism of hypoxia-inducible factor (HIF) stabilization. (a) Tissue sections from healthy control or trinitrobenzene sulfonic acid (TNBS) colitis were examined for localization of the 2-nitroimidazole compound 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,-pentafluoropropyl) acetamide (EF5) (red). Nuclear counterstaining with 4′,6-diamidino-2-phenylindole (DAPI) is shown in blue. Hematoxylin and eosin staining of an adjacent section is shown for orientation. (b) General features of HIF. Overlapping functions of HIF-1α and HIF-2α include the regulation of tissue development, vascular angiogenesis, cell proliferation, and multiple inflammation targets and genes that promote or suppress tumorigenesis. Specific target genes for HIF-1α include those genes involved primarily in metabolism, including genes within the glycolytic pathways. HIF-2α-specific targets include erythropoietin and gene targets involved in duodenal iron transport. (c) The biochemical pathway of HIF hydroxylation by the combination of α-ketoglutarate (αKG), molecular oxygen (O₂), and the prolyl hydroxylase (PHD) enzymes in normoxia. When O₂ or prolyl hydroxylation becomes limiting (due to hypoxia or PHD inhibition), the HIF-1α subunit and/or HIF-2α subunit stabilize and bind to the HIF-1β subunit within the nucleus, where the dimer becomes transcriptionally active upon binding to the hypoxia-response element (HRE) DNA consensus sequence.

Figure 2

Platelet–polymorphonuclear leukocyte (PMN) cotransmigration in crypt abscesses from human inflammatory bowel disease. (a) A merged fluorescence image localizing PMNs in green [anti-myeloperoxidase (MPO)], platelets in red (anti-CD41), and nuclei 4′,6-diamidino-2-phenylindole (DAPI) stain in blue from a human ulcerative colitis crypt abscess. (b) A model of facilitated platelet translocation and activation of epithelial electrogenic Cl⁻ secretion during PMN transmigration. During active inflammation, platelets are caught in the flow of PMN transmigration. PMN- and platelet-derived ATP is selectively metabolized to adenosine by a two-step enzymatic reaction involving CD39 and CD73. Adenosine binding to apical adenosine A_2B receptors (A_2BAR) results in activation of electrogenic Cl⁻ secretion and the paracellular movement of water.

Figure 3

Biochemical analysis of intestinal epithelial CD73 activity. (a) A representative high-performance liquid chromatography (HPLC) tracing demonstrating peak resolution between etheno-AMP (structure shown in inset) and etheno-adenosine. (b) T84 intestinal epithelial monolayers were exposed to normoxia (pO₂ 147 torr) or hypoxia (pO₂ 20 torr) for 36 h and washed. Surface CD73 activity was determined by HPLC analysis of etheno-AMP conversion to etheno-adenosine at the indicated sampling times.

Figure 4

Analysis of colonic vascular leak in CD39-deficient (Cd39^−/−) mice. (a) Wild-type (Cd39^+/+) and Cd39^−/− mice were administered intravenous Evans blue (EB) solution (0.2 ml of 0.5% in phosphate-buffered solution) and were exposed to normobaric hypoxia (8% O₂, 92°N₂) or room air for 4 h. Animals were sacrificed, and their colons were harvested and imaged. (b) A plot of extracted EB, expressed as optical density (OD) at 610 nm mg⁻¹ tissue.

Figure 5

Model of cooperation between nucleotide and nucleoside receptors in inflammation. In areas of ongoing inflammation, diminished O₂ supply (inflammatory hypoxia) coordinates the metabolism of nucleotides to adenosine and subsequent signaling via P1 adenosine receptors. Much of the inflammatory nucleotide signaling occurs via P2 receptors, whereas P1 receptors contribute significantly to the resolution of ongoing inflammation.