ABSTRACT
Although excessive plasma adenosine is detrimental in sickle cell disease (SCD), the molecular mechanism underlying elevated circulating adenosine remains unclear. Here we report that the activity of soluble CD73, an ectonucleotidase producing extracellular adenosine, was significantly elevated in a murine model of SCD and correlated with increased plasma adenosine. Mouse genetic studies demonstrated that CD73 activity contributes to excessive induction of plasma adenosine and thereby promotes sickling, hemolysis, multiorgan damage, and disease progression. Mechanistically, we showed that erythrocyte adenosine 5'-monophosphate-activated protein kinase (AMPK) was activated both in SCD patients and in the murine model of SCD. AMPK functions downstream of adenosine receptor ADORA2B signaling and contributes to sickling by regulating the production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity. Preclinically, we reported that treatment of α,ß-methylene adenosine 5'-diphosphate, a potent CD73 specific inhibitor, significantly decreased sickling, hemolysis, multiorgan damage, and disease progression in the murine model of SCD. Taken together, both human and mouse studies reveal a novel molecular mechanism contributing to the pathophysiology of SCD and identify potential therapeutic strategies to treat SCD.
Subject(s)
5'-Nucleotidase , Adenosine Triphosphate/analogs & derivatives , Anemia, Sickle Cell , Erythrocytes/enzymology , 2,3-Diphosphoglycerate/metabolism , 5'-Nucleotidase/antagonists & inhibitors , 5'-Nucleotidase/genetics , 5'-Nucleotidase/metabolism , AMP-Activated Protein Kinases/genetics , AMP-Activated Protein Kinases/metabolism , Adenosine/metabolism , Adenosine Triphosphate/pharmacology , Anemia, Sickle Cell/drug therapy , Anemia, Sickle Cell/enzymology , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/pathology , Animals , Erythrocytes/pathology , Female , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Humans , Male , Mice , Mice, Knockout , Receptor, Adenosine A2B/genetics , Receptor, Adenosine A2B/metabolismABSTRACT
Elevated sphingosine 1-phosphate (S1P) is detrimental in Sickle Cell Disease (SCD), but the mechanistic basis remains obscure. Here, we report that increased erythrocyte S1P binds to deoxygenated sickle Hb (deoxyHbS), facilitates deoxyHbS anchoring to the membrane, induces release of membrane-bound glycolytic enzymes and in turn switches glucose flux towards glycolysis relative to the pentose phosphate pathway (PPP). Suppressed PPP causes compromised glutathione homeostasis and increased oxidative stress, while enhanced glycolysis induces production of 2,3-bisphosphoglycerate (2,3-BPG) and thus increases deoxyHbS polymerization, sickling, hemolysis and disease progression. Functional studies revealed that S1P and 2,3-BPG work synergistically to decrease both HbA and HbS oxygen binding affinity. The crystal structure at 1.9 Å resolution deciphered that S1P binds to the surface of 2,3-BPG-deoxyHbA and causes additional conformation changes to the T-state Hb. Phosphate moiety of the surface bound S1P engages in a highly positive region close to α1-heme while its aliphatic chain snakes along a shallow cavity making hydrophobic interactions in the "switch region", as well as with α2-heme like a molecular "sticky tape" with the last 3-4 carbon atoms sticking out into bulk solvent. Altogether, our findings provide functional and structural bases underlying S1P-mediated pathogenic metabolic reprogramming in SCD and novel therapeutic avenues.
Subject(s)
Anemia, Sickle Cell/metabolism , Erythrocytes, Abnormal/metabolism , Hemoglobin A/metabolism , Hemoglobin, Sickle/metabolism , Lysophospholipids/metabolism , Sphingosine/analogs & derivatives , 2,3-Diphosphoglycerate/chemistry , 2,3-Diphosphoglycerate/metabolism , Anemia, Sickle Cell/pathology , Animals , Erythrocytes, Abnormal/pathology , Female , Hemoglobin A/chemistry , Hemoglobin, Sickle/chemistry , Hemolysis , Humans , Lysophospholipids/chemistry , Male , Mice , Mice, Transgenic , Oxidative Stress , Pentose Phosphate Pathway , Sphingosine/chemistry , Sphingosine/metabolismABSTRACT
The molecular mechanisms of chronic pain are poorly understood and effective mechanism-based treatments are lacking. Here, we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected chronic mechanical and thermal hypersensitivity due to sustained elevated circulating adenosine. Extending from Ada(-/-) mice, we further discovered that prolonged elevated adenosine contributed to chronic pain behaviors in two additional independent animal models: sickle cell disease mice, a model of severe pain with limited treatment, and complete Freund's adjuvant paw-injected mice, a well-accepted inflammatory model of chronic pain. Mechanistically, we revealed that activation of adenosine A2B receptors on myeloid cells caused nociceptor hyperexcitability and promoted chronic pain via soluble IL-6 receptor trans-signaling, and our findings determined that prolonged accumulated circulating adenosine contributes to chronic pain by promoting immune-neuronal interaction and revealed multiple therapeutic targets.