Abstract
Sickle Cell Disease (SCD) is a devastating genetic disorder attacking red blood cells (RBCs) and affecting millions of humans worldwide. The Glu/Val mutation in the sixth amino acid of β-globin leads to the polymerization of deoxygenated sickle hemoglobin and subsequent sickling, which initiates the disease. Although SCD has been studied for more than a century, factors that contribute to sickling remain unclear. Our lab recently conducted non-biased metabolomic screening and identified that the levels of a small signaling lipid, sphingosine 1-phosphate (S1P), were dramatically increased in SCD transgenic (Tg) mice and patients. Although S1P is enriched and stored in erythrocytes, the role of S1P in normal and sickle erythrocytes remains unknown. Here we revealed that elevated S1P is a previously unrecognized allosteric modulator collaboratively working with 2, 3-bisiphosphoglycerate (2, 3-BPG) to facilitate oxygen release and thereby triggers sickling. Subsequently, we found that the enzymatic activity of sphingosine kinase 1 (Sphk1), which is the only enzyme producing S1P inside red blood cell (RBCs), is significantly elevated in SCD Tg mice and patients. Intriguingly, we found that hypoxia condition significantly increases Sphk1 activity and decreases hemoglobin oxygen (O2) binding affinity in WT mice but not in Sphk1–deficient mice. In a view of 1) our finding that SphK1 activity is induced by hypoxia; 2) our recent finding that excessive adenosine signaling through the A2B adenosine receptor (ADORA2B) promoting sickling by induction 2,3-BPG (Zhang, et al, Nature Medicine, 2011) and 3) the fact that adenosine is a signaling nucleoside that elicits many physiological effects via its receptors under hypoxic conditions, we hypothesized that adenosine functions via its receptors regulating Sphk1 activity and S1P production in erythrocytes. To test this hypothesis, we conducted both pharmacological and genetic studies. First, we found that adenosine directly induces SphK1 activities in cultured primary mouse and human normal erythrocytes. Next, we found that genetic deletion or inhibition of ADORA2B significantly reduces adenosine-induced SphK1 activities in cultured RBCs. Extending in vitro studies to in vivo experiments, we showed that excess circulating adenosine in adenosine deaminase (ADA, adenosine degrading enzyme)-deficient mice leads to significantly increased erythrocyte SphK1 activities. Similar to in vitro studies, we further found that specific deletion of ADORA2B in ado-/-(i.e ado-/-/adora2b-/- double deficient mice) abolishes excess adenosine-induced SphK1 activities in RBCs. Mechanistically, we further revealed that extralcellular signal regulated kinase (ERK) and protein kinase A (PKA) function downstream of ADORA2B underlying adenosine-induced erythrocyte Sphk1 activity. Overall, our studies demonstrate that 1) S1P is an allosteric modulator to induce O2 release and trigger sickling; 2) elevated adenosine functions via ADORA2B coupled with PKA and ERK signaling network responsible for elevated erythrocyte SphK1 activities and S1P production. Therefore, our findings reveal a previously unrecognized role of SphK1-S1P in erythrocyte physiology and novel mechanisms regulating its activities, add a new insight to the pathophysiology of SCD and open up new therapies for the disease.
Disclosures:
No relevant conflicts of interest to declare.
P-selectin–deficient mice to study pathophysiology of sickle cell disease
