Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

Abstract

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

Red blood cell proteomics reveal remnant protein biosynthesis and folding pathways in PIEZO1-related hereditary xerocytosis

“…Of note, no retention of endoplasmic reticulum (ER) proteins has been observed in RBCs of PIEZO1-HX patients. It differs from observations reported in polycythemia vera (PV), where features shared with PIEZO1-HX as elevated levels of intra-cellular calcium and increased Gárdos activity, were associated with altered organelle sorting during enucleation and increased retention of ER proteins in reticulocytes and RBCs (Buks et al, 2021). Although sharing in part similar calciummediated pathways, the difference of behaviour in RBCs from PV and HX patients, as well as the absence of enrichment in proteins directly regulated by calcium, highlights the complexity of PIEZO1-HX, where calcium influxes fail to faithfully reveal the pathophysiology of the disease.…”

Deregulated calcium signaling in blood cancer: Underlying mechanisms and therapeutic potential

“…Changes in Ca 2+ homeostasis are seen in reactive ineffective erythropoiesis (e.g. in β-thalassemia) ( 109 ) or in neoplastic erythropoiesis driven by Janus kinase 2 (JAK2) V617F mutation in polycythemia vera (PV) ( 110 , 111 ). CALR is an ER-resident protein that regulates functions of other proteins by chaperoning them to their active sites in response to changing intracellular Ca 2+ levels ( 112 ).…”

“…Glucocorticoid receptor α is retained in the nucleus maintaining the expression of stress genes that increase proliferation of erythroblasts ( 110 ). Elevated levels of Ca 2+ may also impair actin reorganization required to extrude organelles during enucleation ( 111 ). Consequently, PV reticulocytes have a high content of organellar remnants e.g.…”

“…mitochondria, ER and ER-associated proteins including CALR. In mature RBCs from PV patients, high Ca 2+ levels increase the activity of the Gárdos channel leading to cell dehydration ( 111 ) ( Figure 2B ).…”

“…Increased levels of cytoplasmic Ca 2+ , cell dehydration and the presence of organelle remnants in RBCs have the potential to promote thrombosis in PV ( 111 ). Dehydrated RBCs are more rigid, thus less amenable to shape changes required to pass through narrow capillaries, and also more susceptible to hemolysis under high-shear rates that occur in arterial circulation ( 113 , 114 ).…”