sphingosine-kinase and Anemia--Sickle-Cell

There is substantial evidence that the enzymes, sphingosine kinase 1 and 2, which catalyse the formation of the bioactive lipid sphingosine 1-phosphate, are involved in pathophysiological processes. In this chapter, we appraise the evidence that both enzymes are druggable and describe how isoform-specific inhibitors can be developed based on the plasticity of the sphingosine-binding site. This is contextualised with the effect of sphingosine kinase inhibitors in cancer, pulmonary hypertension, neurodegeneration, inflammation and sickling.

Topics: Anemia, Sickle Cell; Binding Sites; Enzyme Inhibitors; Humans; Hypertension, Pulmonary; Inflammation; Lysophospholipids; Neoplasms; Neurodegenerative Diseases; Phosphotransferases (Alcohol Group Acceptor); Sphingosine

Sickle cell disease is a destructive genetic disorder characterized by the formation of fibrils of deoxygenated hemoglobin, leading to the red blood cell (RBC) morphology changes that underlie the clinical manifestations of this disease. Using cryogenic soft X-ray tomography (SXT), we characterized the morphology of sickled RBCs in terms of volume and the number of protrusions per cell. We were able to identify statistically a relationship between the number of protrusions and the volume of the cell, which is known to correlate to the severity of sickling. This structural polymorphism allows for the classification of the stages of the sickling process. Recent studies have shown that elevated sphingosine kinase 1 (Sphk1)-mediated sphingosine 1-phosphate production contributes to sickling. Here, we further demonstrate that compound 5C, an inhibitor of Sphk1, has anti-sickling properties. Additionally, the variation in cellular morphology upon treatment suggests that this drug acts to delay the sickling process. SXT is an effective tool that can be used to identify the morphology of the sickling process and assess the effectiveness of potential therapeutics.

Topics: Anemia, Sickle Cell; Animals; Cell Surface Extensions; Erythrocytes; Humans; Mice; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase Inhibitors; Tomography, X-Ray

Erythrocyte possesses high sphingosine kinase 1 (SphK1) activity and is the major cell type supplying plasma sphingosine-1-phosphate, a signaling lipid regulating multiple physiological and pathological functions. Recent studies revealed that erythrocyte SphK1 activity is upregulated in sickle cell disease (SCD) and contributes to sickling and disease progression. However, how erythrocyte SphK1 activity is regulated remains unknown. Here we report that adenosine induces SphK1 activity in human and mouse sickle and normal erythrocytes in vitro. Next, using 4 adenosine receptor-deficient mice and pharmacological approaches, we determined that the A2B adenosine receptor (ADORA2B) is essential for adenosine-induced SphK1 activity in human and mouse normal and sickle erythrocytes in vitro. Subsequently, we provide 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. Finally, we revealed that protein kinase A-mediated extracellular signal-regulated kinase 1/2 activation functioning downstream of ADORA2B underlies adenosine-induced erythrocyte SphK1 activity. Overall, our findings reveal a novel signaling network regulating erythrocyte SphK1 and highlight innovative mechanisms regulating SphK1 activity in normal and SCD.

Topics: Adenosine; Adenosine Deaminase; Adenosine-5'-(N-ethylcarboxamide); Agammaglobulinemia; Anemia, Sickle Cell; Animals; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Erythrocytes; Erythrocytes, Abnormal; Hemoglobin, Sickle; Humans; MAP Kinase Signaling System; Mice; Mice, Knockout; Mice, Transgenic; Models, Biological; Phosphotransferases (Alcohol Group Acceptor); Receptor, Adenosine A2B; Severe Combined Immunodeficiency; Signal Transduction

Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates multicellular functions through interactions with its receptors on cell surfaces. S1P is enriched and stored in erythrocytes; however, it is not clear whether alterations in S1P are involved in the prevalent and debilitating hemolytic disorder sickle cell disease (SCD). Here, using metabolomic screening, we found that S1P is highly elevated in the blood of mice and humans with SCD. In murine models of SCD, we demonstrated that elevated erythrocyte sphingosine kinase 1 (SPHK1) underlies sickling and disease progression by increasing S1P levels in the blood. Additionally, we observed elevated SPHK1 activity in erythrocytes and increased S1P in blood collected from patients with SCD and demonstrated a direct impact of elevated SPHK1-mediated production of S1P on sickling that was independent of S1P receptor activation in isolated erythrocytes. Together, our findings provide insights into erythrocyte pathophysiology, revealing that a SPHK1-mediated elevation of S1P contributes to sickling and promotes disease progression, and highlight potential therapeutic opportunities for SCD.

Topics: Anemia, Sickle Cell; Animals; Antisickling Agents; Disease Models, Animal; Disease Progression; Enzyme Inhibitors; Erythrocytes, Abnormal; Gene Knockdown Techniques; Hemolysis; Humans; Lysophospholipids; Metabolomics; Methanol; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Mice, Transgenic; Phosphotransferases (Alcohol Group Acceptor); Pyrrolidines; Signal Transduction; Sphingosine; Sulfones