Elevated Adenosine Signaling Via Adenosine A2B Receptor Induces Normal and Sickle Erythrocyte Sphingosine Kinase 1 Activity

Abstract

Sickle Cell Disease (SCD) is one of the most devastating hemolytic genetic disorders affecting millions worldwide. Erythrocytes possess high sphingosine kinase 1 (Sphk1) activity and are considered to be the major cell type for supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is up-regulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte Sphk1 activity is regulated in SCD remains unknown. In an effort to identify specific factors and signaling pathways involved in regulation of erythrocyte SphK1 activities in SCD, we first chose to screen the effects of molecules known to induce SphK1 activities in other cell types and/or reported to be elevated in the circulation of SCD including tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF), S1P and adenosine, on the activities of SphK1 in cultured primary mouse normal erythrocytes. Among all of those molecules tested, we found that adenosine is a previously unidentified hypoxia inducible molecule directly inducing SphK1 activity in vitro in a time and dosage-dependent manner. Next, using four adenosine receptor deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in cultured primary mouse normal and sickle erythrocytes. Subsequently, we provided in vivo genetic evidence that adenosine deaminase (ADA)-deficiency leads to excess plasma adenosine and elevated erythrocyte SphK1 activity. Lowering adenosine by ADA enzyme therapy or genetic deletion of ADORA2B significantly reduced excess adenosine-induced erythrocyte SphK1 activity in ADA-deficient mice. Mechanistically, we revealed that PKA functions downstream of ADORA2B mediating ERK activation and subsequently underlying adenosine-induced SphK1 activities in cultured mouse erythrocytes. Finally, we conducted human translational studies and reported that adenosine signaling via ADORA2B directly increases SphK1 activity in cultured primary human normal and sickle erythrocytes in a PKA/ERK-dependent manner. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms to control SphK1 activity in normal and sickle setting. DisclosuresNo relevant conflicts of interest to declare.