PF425 PIEZO1 MEDIATED CALCIUM‐INDUCED SIGNAL TRANSDUCTION IN ERYTHROBLASTS MODULATES ERYTHROPOIESIS

Background: In erythrocytes, the mechanosensitive ion transporter PIEZO1 is crucial to maintain cellular volume homeostasis during circulation. Gain of function mutations in PIEZO1 gene lead to prolonged Ca 2+ influx resulting in hereditary xerocytosis (HX). This rare disorder of erythrocyte hydration is characterized by dehydration caused by increased Gardos channel activation. HX patients display a mild to moderate degree of hemolysis, which is generally compensated by an increased reticulocyte count. In addition to its function in erythrocytes we found that PIEZO1 RNA is also expressed at the proerythroblast stage suggesting a role during erythropoiesis. Aims: We hypothesize that disturbed Ca 2+ homeostasis due to inappropriate PIEZO1 channel function leads to defects in erythroid differentiation. Methods: HX patient samples with different mutations in the PIEZO1 gene, c.6262C>G p.(Arg2088Gly), c.7367G>A p.(Arg2456His) and c.1792G>A p.(Val598Met) were used in this study. Peripheral blood mononuclear cells were isolated from patient and controls and differentiated to erythroblasts. Cell viability and proliferation of erythroblasts was assessed and flow cytometry was used to monitor cellular death, expression of erythroid markers and intracellular calcium levels. Western blot was used to assess expression of PIEZO1, as well as proteins involved in signaling pathways (e.g. NFAT, JNK, ERK). Results: RNA sequencing analysis during erythroblast differentiation to enucleated reticulocytes showed that PIEZO1 RNA is highest expressed at the proerythroblast stage and decreases during differentiation. PIEZO1 protein expression was assessed by western blot in erythroblasts and PIEZO1 expression was unchanged between HX patients and healthy controls. During erythropoietin, stem cell factor and glucocorticoid mediated erythroblast proliferation, HX primary erythroblasts displayed a twofold decreased proliferation compared to healthy controls. In agreement with this, incubating erythroblasts from a healthy control with the PIEZO1 agonist Yoda1 led to a dose dependent decrease in erythroblast proliferation, thereby recapitulating the patient's phenotype. In addition, Yoda1 treatment was accompanied with increased cell death and reduced enucleation. In HX patient's erythroblasts basal Ca 2+ levels were higher compared to control cells. In agreement with this, Yoda1‐induced PIEZO1 activation led to a dose dependent Ca 2+ influx. Yoda1 caused increased ERK and JNK phosphorylation, which was also increased in the patient erythroblasts. In addition, NFATC2 dephosphorylation was observed, suggesting that PIEZO1 triggers calcium‐dependent signal transduction, possibly via calcineurin activation. Indeed, Yoda1‐induced NFATC2 dephosphorylation was blocked and JNK phosphorylation was inhibited upon pre‐incubation of erythroblasts with the calcineurin inhibitor FK506. Summary/Conclusion: In conclusion, we found that PIEZO1 has a role in regulating Ca2+ homeostasis during erythropoiesis possibly through activation of calcineurins. Inappropriate PIEZO1 channel activation in erythroid cells results in cell death and proliferation/differentiation defects. This novel feature adds to the complexity of the phenotypic expression of HX.