Page 99 - Haematologica March 2020

Role of PIEZO1 during human erythropoiesis
YODA1 inhibits cell proliferation and blocks erythroid differentiation in a PIEZO1-dependent manner in UT7 cells
The UT7/GM cell line was used as a model of EPO- induced erythroid differentiation. Indeed, these cells expressed a low level of GPA under exposure to GM-CSF, and acquired GPA in the presence of EPO (Online Supplementary Figure S1A-C). We tested YODA1, a specif-
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ic PIEZO1 activator,25 at increasing concentrations and selected 5 mM for further experiments in these cells (Online Supplementary Figure S3A, B). At this concentration, YODA1 decreased cell proliferation (Figure 2A) and increased the percentage of cells in the G0/G1 phase (Figure 2B). No difference in cell mortality or apoptosis rate was noted (Online Supplementary Figure S3C, D). Under the differentiation condition using 5 U/mL EPO,
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Figure 2. Effect of PIEZO1 chemical activation on the proliferation and differentiation of UT7 cells. Glycophorin A (GPA) expression and cell proliferation were assessed after 72 h of culture in medium containing granulocyte-macrophage colony-stimulating factor (GMCSF) or erythropoietin (EPO). (A) In the UT7/GM cell line, stimulation with 5 mM YODA1 reduced cell expansion compared to that induced by dimethylsulfoxide (DMSO) (2.6 fold). (B) In the UT7/GM cell line exposure to 5 mM YODA1 in GMCSF-containing medium led to cell accumulation in the G0/G1 phase of the cycle (67±1% with DMSO vs. 75±3% with YODA1), and a significant decrease in cells in the G2/M phase (18±1% with DMSO vs. 12±2% with YODA1). (C) Stimulation with 5 U/mL EPO induced partial erythroid differentiation in UT7/GM cells, as shown by GPA acquisition (56±3% vs. 14±2% in GMCSF-containing medium), which was strongly inhibited after PIEZO1 chemical activation using 5 mM YODA1 (9±8% with YODA1 vs. 56±3% with EPO+DMSO). (D) Representative multiparametric flow cytometry histograms showing the repression of EPO-medi- ated GPA expression after exposure to YODA1 in UT7/GM cells. (E) GPA repression was also observed in UT7/EPO cells after exposure to 5 mM YODA1 for 3 days (82±6% vs. 46±7% with DMSO). (F) In UT7/EPO cells transduced with Sh-SCR, YODA1 repressed GPA expression (39±1.8% vs. 77±1.1% with DMSO) whereas infec- tion with a mixture of four different Sh-PIEZO1 abolished the YODA1-mediated inhibition of GPA (96±2% with YODA1 vs. 98±0.4%, P=NS). (n=3 for all experiments, ***P<0.001; **P<0.01).
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