Transcriptional control of adenosine signaling by hypoxia-inducible transcription factors during ischemic or inflammatory disease

Abstract

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.

Figure 1. 2-Nitroimidazole compounds (NO₂-R) stain hypoxic tissues in vivo

(A) Schematic of NO₂-R metabolism in the cell: After passive uptake (top left) NO₂-R undergoes a single-electron reduction to a nitro radical anion intermediate (lower left). Oxygen regenerates the native compound by electron-uptake (marked in red) and subsequent reaction to H₂O₂ (not shown). In the absence of oxygen, the activated compound intermediate is processed to a hydroxylamine intermediate (bottom middle) which then stably binds SH-containing molecules such as proteins (bottom right). These adducts accumulate in the cell and can be visualized using labeled antibodies (right). (B) C57bl/6 mice were subjected to sham procedure (upper panel) or kidney ischemia (occlusion of the renal artery for 30 minutes; lower panel). After 5 minutes of reperfusion, mice were injected with pimonidazole. Antibody staining was performed after additional 15 minutes.

Figure 2. Clinical examples for diseases characterized by hypoxia

In the schematic, disorders and diseases in which either increased consumption or decreased supply of oxygen dominate exemplify the interdependence of hypoxia and inflammation.

Figure 3. Hypoxia-dependent stabilization of the transcription factor hypoxia-inducible factor HIF

In normoxic conditions (left side of schematic), hydroxylases inactivate HIFA-subunits. FIH hydroxylates an asparaginyl residue in the carboxy-terminal activation domain (CAD), preventing co-activator (p300) recruitment. PHDs hydroxylate a proline residues in the N-terminal activation domain (in the oxygen dependent degradation domain (ODD)), facilitating pVHL-dependent ubiquitination and proteasomal degradation. In hypoxia, PHDs and FIH are inhibited and the co-activator p300 is recruited to the HIFα-subunit, which forms a heterodimer with HIFβ. This complex is transcriptionally active.

Figure 4. Hypoxia induces signaling through the ADORA2A and ADORA2B adenosine receptors

Four adenosine receptors (AR) have been described, mediating the effects of extracellular adenosine: ADORA1, ADORA2A, ADORA2B and ADORA3. All of these receptors modulate intracellular cAMP levels. ADORA1 and ADORA3 signaling lower cAMP concentrations, signaling through ADORA2A and ADORA2B – which are both transcriptionally induced in hypoxia – increases cAMP level. The schematic gives examples for the biological effects of AR signaling.

Figure 5. Hypoxia attenuates adenosine transport and metabolism, thereby enhancing extracellular adenosine signaling

(A) Extracellular ATP and ADP are released from different cell types upon stimulation (e. g., thrombocytes, neutrophils) or when they undergo necrosis or pyroptosis (danger associated molecular pattern molecules, DAMPs). The ecto-apyrase CD39 – which is expressed on epithelia, endothelia and immune cells – converts ATP and ADP to AMP. Ecto-5’-nucleotidase (CD73) rapidly converts extracellular AMP to ADO. Both enzymes are transcriptionally upregulated in hypoxic conditions, thereby promoting extracellular adenosine production during hypoxia. (B) Breakdown of extracellular adenosine is initiated by its uptake into the cell by equilibrative nucleoside transporters (ENT1 and ENT2). Intracellular ADO is either phosphorylated by adenosine kinase (ADK) or processed to inosine by adenosine deaminase (ADA). ENTs and AK are transcriptionally repressed during hypoxia, thereby prolonging adenosine signaling effects during conditions of hypoxia.