Page 97 - Haematologica March 2020

Role of PIEZO1 during human erythropoiesis
expressed FAM38A gene2,3 (GTEx Project) (https://gtexpor- tal.org/home/gene/PIEZO1). Activation of the mechanosensitive ion channel PIEZO1 has a significant effect on red blood cell physiology. Under physiological conditions, PIEZO1 regulates ATP release from human mature erythrocytes,4 and has been shown to control red blood cell volume and hydration homeostasis through ion balance.5 PIEZO1 induces a cationic current that develops quickly after activation of the protein by a mechanical stress; this activation is followed by a rapid inactivation determined by the C-terminal extracellular domain and the inner pore helix.6 PIEZO1 is a non-selec- tive channel although it presents a preferential conduc- tance of monovalent cations, and a significant permeabil- ity to calcium (Ca2+).1,7 PIEZO1 gain-of-function muta- tions have been associated with most cases of hereditary xerocytosis (HX), leading to either a slower inactivation or altered channel kinetics.8–11 These mutations induce excessive Ca2+ influx and secondary activation of the Gardos channel in red cells, thereby causing potassium (K+) leakage, water loss, and erythrocyte dehydration.12,13
So far, the role of PIEZO1 during erythropoiesis has only been described in mature erythrocytes. However, it is also expressed earlier in human erythroid progeni- tors.8,14 In many cell types such as epithelial, urothelial and endothelial cells, PIEZO1 has been involved in regu- lation of the cell cycle, proliferation and differentiation.15- 18 Prompted by a recent report that a PIEZO1 mutation could mimic myelodysplastic syndrome with mega- loblastic features,19 we performed an extensive and com- prehensive investigation of PIEZO1 expression and func- tion using primary human erythroid progenitor cells. We investigated consequences of its activation either by the selective activator YODA1 in normal human erythroid progenitors or by activating mutations in HX–derived hematopoietic progenitors from 14 patients carrying ten different mutations. We observed that PIEZO1 activation in our models modified the kinetics of erythropoiesis, inducing a delay in terminal erythroid differentiation. Our results suggest that PIEZO1 plays a key role during human erythroid differentiation.
Methods
The primary cell culture protocol, multiparametric flow cytometry (MFC), live imaging flow cytometry (IFC), western blot, immunofluorescence, quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) analysis and reagents are detailed in the Online Supplementary Methods.
Cell lines culture
Patients’ samples and primary cell cultures
HX hematopoietic cells were obtained from blood samples collected into EDTA or from phlebotomy bags after patients’ informed consent in accordance with the Helsinki declaration. This study was conducted in compliance with French legislation on non-interventional studies. Patients’ data were collected directly from their medical records or through the HX French Cohort registry, approved by the National Commission on Informatics and Liberty. The diagnosis of HX was based on clin- ical and biological features and a typical osmotic gradient ekta- cytometry curve before genetic testing. Thirteen of the patients in this study were recently extensively described.22 Control sam- ples were obtained from blood or mobilized peripheral blood mononuclear cells (MNC) of healthy subjects. The MNC were isolated using density gradient-centrifugation (Ficoll-Paque PLUS, GE Healthcare) and CD34+ cells were sorted by magnetic microbead separation on MACS columns (AutoMACS Separator). The complete protocol for erythroid differentiation is described in Online Supplementary Figures S1 and S2 and detailed in the Online Supplementary Methods.
Lenti/retroviral production and cell infection
Four short hairpin (Sh) RNA against PIEZO1 (Sh-PIEZO1) and one control scrambled ShRNA (Sh-SCR) cloned in pLKO.1- CMV-tGFP vector were designed using the Mission® shRNA tool and purchased from Sigma-Aldrich (detailed sequences are pro- vided in Online Supplementary Table S1). Specific anti-PIEZO1 targeting was verified using an online alignment research tool (nucleotide BLAST®, NCBI). Viral production was ensured in the HEK293T cell line, after transfection using Lipofectamine® LTX with PlusTM reagent (Thermo Fisher Scientific) in antibiotic-free, high-glucose Dulbecco modified Eagle medium (Dominique Dutscher). Lentiviral supernatant was harvested from day 2 to 4, and filtered through a 0.45 mM polyvinylidene fluoride mem- brane (Millex-HV 0.45 mM 33 mm, Merck-Millipore) before ultracentrifugation on day 4 (100,000 g for 90 min at 4°C, Optima L-80XP, Beckman-Coulter). We used a mix of the four ShRNA to knockdown PIEZO1 in the UT7/EPO cell line. Infection was performed overnight with 8 mg/mL polybrene (Sigma-Aldrich). In UT7/EPO cells, 10 mL of each supernatant were used to infect 5x105 cells, and were sufficient to induce >90% GFP, both with the Sh-SCR and Sh-PIEZO1 mix. Forty- eight hours after transduction, cells were washed in 50 mL 1x phosphate-buffered saline and cultured for an additional 3 days in the presence of dimethylsulfoxide (DMSO) or YODA1 before MFC staining. The retroviral MigR vector containing dominant- negative MEK was a generous gift from Prof. S. Giraudier (Hôpital Saint-Louis, Paris, France).
Statistical analysis
Statistical analyses were performed using two-tailed P values and parametric tests. The α value for statistical significance was set at 0.05. For quantitative variables we used a Student t-test for paired or unpaired samples depending on the experiment under consideration. All numerical values are expressed as mean values ± standard deviation.
Results
PIEZO1 is expressed at an early stage during in vitro erythropoiesis of human CD34+ cells
We first assessed PIEZO1 expression during synchro- nized human in vitro erythroid differentiation as described in Online Supplementary Figure S2B. PIEZO1 mRNA was
The UT7 cell line was maintained in α-minimum essential medium (Dominique Dutscher) supplemented with 10% fetal calf serum (Eurobio) and cytokines. Two subclones were used: the UT7/GM clone and the UT7/EPO clone. The UT7/GM clone was used as a model of erythropoietin (EPO)-driven dif- ferentiation, proliferating under 5 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF; Miltenyi) and differentiating under 5 U/mL EPO after GM-CSF had been removed by two washes in 1x phosphate-buffered saline.20 The UT7/EPO clone (a gift from Dr. Y.Zermati, Institut Cochin, Paris, France) was used as a model of EPO-driven pro- liferation, and cultured with 2 U/mL EPO (Online Supplementary Figure S1).21
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