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PIEZO1 Activation Delays Erythroid Differentiation of Normal and Hereditary Xerocytosis-Derived Human Progenitor Cells

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PIEZO1 Activation Delays Erythroid Differentiation of Normal and Hereditary Xerocytosis-Derived Human Progenitor Cells ARTICLE Red Cell Biology & its Disorders PIEZO1 activation delays erythroid Ferrata Storti Foundation differentiation of normal and hereditary xerocytosis-derived human progenitor cells Alexis Caulier,1,2* Nicolas Jankovsky,1* Yohann Demont,3 Hakim Ouled-Haddou,1 Julien Demagny,4 Corinne Guitton,5 Lavinia Merlusca,2 Delphine Lebon,1,2 Pascal Vong,1 Aurélien Aubry,1 Agnès Lahary,6 Christian Rose,7 Sandrine Gréaume,8 Emilie Cardon,1 Jessica Platon,1 Halima Ouadid-Ahidouch,9 Jacques Haematologica 2020 Rochette,1,10 Jean-Pierre Marolleau,1,2 Véronique Picard11 and Loïc Garçon1,4,10 Volume 105(3):610-622 1EA4666 HEMATIM, Université Picardie Jules Verne, Amiens; 2Service des Maladies du Sang, CHU Amiens, Amiens; 3Unité de Thérapie Cellulaire, CHU Amiens, Amiens; 4Service d’Hématologie Biologique, CHU Amiens; 5Service de Pédiatrie Générale, CHU Bicêtre, AP- HP, Le Kremlin-Bicêtre; 6Laboratoire d’Hématologie, CHU Rouen, Rouen; 7Service d’Oncologie et d’Hématologie, Hôpital Saint Vincent de Paul, Lille; 8Etablissement Français du Sang (EFS) de Normandie, Bois-Guillaume; 9EA4667 Laboratoire de Physiologie Cellulaire et Moléculaire, Université Picardie Jules Verne, Amiens; 10Laboratoire de Génétique Moléculaire, CHU Amiens, Amiens and 11Laboratoire d’Hématologie, AP-HP, Le Kremlin-Bicêtre, France *AC and NJ contributed equally to this work. ABSTRACT ereditary xerocytosis is a dominantly inherited red cell membrane disorder caused in most cases by gain-of-function mutations in HPIEZO1, encoding a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 was expressed early in erythroid progenitor cells, and investigated whether it could be involved in erythropoiesis, besides having a role in the homeostasis Correspondence: of mature red cell hydration. In UT7 cells, chemical PIEZO1 activation using LOÏC GARÇON YODA1 repressed glycophorin A expression by 75%. This effect was [email protected] PIEZO1-dependent since it was reverted using specific short hairpin-RNA knockdown. The effect of PIEZO1 activation was confirmed in human pri- mary progenitor cells, maintaining cells at an immature stage for longer and Received: February 11, 2019. modifying the transcriptional balance in favor of genes associated with early Accepted: August 9, 2019. erythropoiesis, as shown by a high GATA2/GATA1 ratio and decreased α/β- Pre-published: August 14, 2019. globin expression. The cell proliferation rate was also reduced, with accumu- lation of cells in G0/G1 of the cell cycle. The PIEZO1-mediated effect on UT7 cells required calcium-dependent activation of the NFAT and ERK1/2 doi:10.3324/haematol.2019.218503 pathways. In primary erythroid cells, PIEZO1 activation synergized with Check the online version for the most updated erythropoietin to activate STAT5 and ERK, indicating that it may modulate information on this article, online supplements, signaling pathways downstream of erythropoietin receptor activation. and information on authorship & disclosures: Finally, we studied the in-vitro erythroid differentiation of primary cells www.haematologica.org/content/105/3/610 obtained from 14 PIEZO1-mutated patients, from 11 families, carrying ten different mutations. We observed a delay in erythroid differentiation in all cases, ranging from mild (n=3) to marked (n=8). Overall, these data demon- ©2020 Ferrata Storti Foundation strate a role for PIEZO1 during erythropoiesis, since activation of PIEZO1 - Material published in Haematologica is covered by copyright. both chemically and through activating mutations - delays erythroid matu- All rights are reserved to the Ferrata Storti Foundation. Use of published material is allowed under the following terms and ration, providing new insights into the pathophysiology of hereditary xero- conditions: cytosis. https://creativecommons.org/licenses/by-nc/4.0/legalcode. Copies of published material are allowed for personal or inter- nal use. Sharing published material for non-commercial pur- poses is subject to the following conditions: Introduction https://creativecommons.org/licenses/by-nc/4.0/legalcode, sect. 3. Reproducing and sharing published material for com- mercial purposes is not allowed without permission in writing PIEZO proteins were identified as a family of mechanically activated transduc- from the publisher. tors, first described in neuronal cell lines, which convert mechanical forces into biological signals.1 PIEZO1 is a large, three-blade propeller-shaped protein dis- playing 38 transmembrane-helix domains, and is encoded by the broadly 610 haematologica | 2020; 105(3) Role of PIEZO1 during human erythropoiesis expressed FAM38A gene2,3 (GTEx Project) (https://gtexpor- Patients’ samples and primary cell cultures tal.org/home/gene/PIEZO1). Activation of the HX hematopoietic cells were obtained from blood samples mechanosensitive ion channel PIEZO1 has a significant collected into EDTA or from phlebotomy bags after patients’ effect on red blood cell physiology. Under physiological informed consent in accordance with the Helsinki declaration. conditions, PIEZO1 regulates ATP release from human This study was conducted in compliance with French legislation mature erythrocytes,4 and has been shown to control red on non-interventional studies. Patients’ data were collected blood cell volume and hydration homeostasis through directly from their medical records or through the HX French ion balance.5 PIEZO1 induces a cationic current that Cohort registry, approved by the National Commission on develops quickly after activation of the protein by a Informatics and Liberty. The diagnosis of HX was based on clin- mechanical stress; this activation is followed by a rapid ical and biological features and a typical osmotic gradient ekta- inactivation determined by the C-terminal extracellular cytometry curve before genetic testing. Thirteen of the patients domain and the inner pore helix.6 PIEZO1 is a non-selec- in this study were recently extensively described.22 Control sam- tive channel although it presents a preferential conduc- ples were obtained from blood or mobilized peripheral blood tance of monovalent cations, and a significant permeabil- mononuclear cells (MNC) of healthy subjects. The MNC were ity to calcium (Ca2+).1,7 PIEZO1 gain-of-function muta- isolated using density gradient-centrifugation (Ficoll-Paque tions have been associated with most cases of hereditary PLUS, GE Healthcare) and CD34+ cells were sorted by magnetic xerocytosis (HX), leading to either a slower inactivation microbead separation on MACS columns (AutoMACS or altered channel kinetics.8–11 These mutations induce Separator). The complete protocol for erythroid differentiation excessive Ca2+ influx and secondary activation of the is described in Online Supplementary Figures S1 and S2 and Gardos channel in red cells, thereby causing potassium detailed in the Online Supplementary Methods. (K+) leakage, water loss, and erythrocyte dehydration.12,13 So far, the role of PIEZO1 during erythropoiesis has Lenti/retroviral production and cell infection only been described in mature erythrocytes. However, it Four short hairpin (Sh) RNA against PIEZO1 (Sh-PIEZO1) and is also expressed earlier in human erythroid progeni- one control scrambled ShRNA (Sh-SCR) cloned in pLKO.1- tors.8,14 In many cell types such as epithelial, urothelial CMV-tGFP vector were designed using the Mission® shRNA tool and endothelial cells, PIEZO1 has been involved in regu- and purchased from Sigma-Aldrich (detailed sequences are pro- lation of the cell cycle, proliferation and differentiation.15- vided in Online Supplementary Table S1). Specific anti-PIEZO1 18 Prompted by a recent report that a PIEZO1 mutation targeting was verified using an online alignment research tool could mimic myelodysplastic syndrome with mega- (nucleotide BLAST®, NCBI). Viral production was ensured in the loblastic features,19 we performed an extensive and com- HEK293T cell line, after transfection using Lipofectamine® LTX prehensive investigation of PIEZO1 expression and func- with Plus™ reagent (Thermo Fisher Scientific) in antibiotic-free, tion using primary human erythroid progenitor cells. We high-glucose Dulbecco modified Eagle medium (Dominique investigated consequences of its activation either by the Dutscher). Lentiviral supernatant was harvested from day 2 to 4, selective activator YODA1 in normal human erythroid and filtered through a 0.45 mM polyvinylidene fluoride mem- progenitors or by activating mutations in HX–derived brane (Millex-HV 0.45 mM 33 mm, Merck-Millipore) before hematopoietic progenitors from 14 patients carrying ten ultracentrifugation on day 4 (100,000 g for 90 min at 4°C, different mutations. We observed that PIEZO1 activation Optima L-80XP, Beckman-Coulter). We used a mix of the four in our models modified the kinetics of erythropoiesis, ShRNA to knockdown PIEZO1 in the UT7/EPO cell line. inducing a delay in terminal erythroid differentiation. Infection was performed overnight with 8 mg/mL polybrene Our results suggest that PIEZO1 plays a key role during (Sigma-Aldrich). In UT7/EPO cells, 10 mL of each supernatant human erythroid differentiation. 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 Methods phosphate-buffered saline and cultured for an additional 3 days in the presence of dimethylsulfoxide (DMSO) or YODA1 before The primary cell culture protocol, multiparametric flow
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