A ROLE OF PIEZO1 IN ERYTHROID DIFFERENTIATION OF NORMAL AND HEREDITARY XEROCYTOSIS DERIVED HEMATOPOIETIC PROGENITORS.
Author(s): ,
Alexis Caulier
Affiliations:
EA HEMATIM,Université Picardie Jules Verne,Amiens,France
,
Nicolas Jankovsky
Affiliations:
EA HEMATIM,Université Picardie Jules Verne,Amiens,France
,
Hakim Ouled-Haddou
Affiliations:
EA HEMATIM,Université Picardie Jules Verne,Amiens,France
,
Yohann Demont
Affiliations:
EA HEMATIM,Université Picardie Jules Verne,Amiens,France
,
Dominique Bastit
Affiliations:
EFS Normandie,Bois-Guillaume,France
,
Christian Rose
Affiliations:
Hématologie,Hôpital Saint-Vincent,Lille,France
,
Hélène Guizouarn
Affiliations:
UMR 7277,Institut de Biologie Valrose ,Nice,France
,
Agnès Lahary
Affiliations:
Laboratoire d'hématologie,CHU Rouen,Rouen,France
,
Lavinia Merlusca
Affiliations:
Hematologie clinique,CHU Amiens,Amiens,France
,
Corinne Guitton
Affiliations:
Hématologie pédiatrique,Hôpital Kremlin-Bicêtre,Paris,France
,
Delphine Lebon
Affiliations:
EA HEMATIM,Université Picardie Jules Verne,Amiens,France
,
Marion Thuillier
Affiliations:
EA HEMATIM,Université Picardie Jules Verne,Amiens,France
,
Catherine Badens
Affiliations:
Département de Génétique Médicale,Hôpital de La Timone,Marseille,France
,
Jean-Pierre Marolleau
Affiliations:
EA HEMATIM,Université Picardie Jules Verne,Amiens,France
,
Véronique Picard
Affiliations:
Laboratoire d'hématologie,Hôpital Kremlin-Bicêtre,Paris,France
Loïc Garçon
Affiliations:
EA HEMATIM,Université Picardie Jules Verne,Amiens,France
EHA Library. Caulier A. 06/15/18; 214445; S144
Alexis Caulier
Alexis Caulier
Contributions
×
Abstract

Abstract: S144

Type: Oral Presentation

Presentation during EHA23: On Friday, June 15, 2018 from 11:30 - 11:45

Location: Room A10

Background

Activation of the mechanotransductor PIEZO1 plays a central role in the red cell dehydration observed in hereditary xerocytosis (HX). Data recently published in epithelial and endothelial cells suggested that PIEZO1 was involved in the balance between cell proliferation and differentiation. Preliminary expression study confirmed that PIEZO1 was expressed in immature erythroblasts. However, its role during erythropoiesis and the consequences of its gain-of-function mutations in HX have not been evaluated so far. 

Aims

We studied the role of PIEZO1 activation in erythroid differentiation of normal and hereditary xerocytosis derived hematopoietic progenitors.

Methods

In vitro human erythropoiesis was conducted during 21 days using normal peripheral CD34+ cells after PIEZO1 activation by Yoda1 (Y1, n=4) and CD34+ (n=6) or total mononuclear cells (MNC, n=9) from 10 HX patients displaying 9 different mutations. Cell proliferation, apoptosis, and differentiation were assessed using cell count, flow cytometry (FC) and cytology at day 10. The erythroleukemic cell line UT7/EPO was used in parallel for Piezo1 knock-down experiments using shRNA lentiviruses, for calcium signaling and for transduction pathways studies.

Results

PIEZO1 activation by Y1 1µM in normal CD34+ cells induced a slowdown in erythroid differentiation as shown by a decrease in mature CD71+/Glycophorin A (GPA)+ expressing cells at day 10 of culture (20.9%±10.8 vs 77.6%±8.5, p<10-3), with no effect on cell proliferation and a slight increase in the apoptosis rate (Annexin V: 14.7±2.3% vs 5.1±0.8, p=0.049). A drastic decrease in GPA expression was also observed in UT7/EPO cells after Y1 exposure (reduction by 88%±0.7; p<10-3). This decrease was transcriptional, as shown by RQ-PCR, and was abolished after shRNA-mediated PIEZO1 knock down or after PIEZO1-inhibition using Gadolinium. Functional studies using flow imaging revealed that Y1 induced a calcium influx inside UT7/EPO cells. EGTA reverted the Y1-induced GPA repression, showing that Ca2+ signaling mediated the phenotype. Of note, a secondary Ca2+ dependant activation of Gardos channel was not involved, since Senicapoc did not correct the phenotype.

Investigating the PIEZO1 downstream signaling pathways, we observed that phospho-ERK blockade by either chemical inhibitor UO126 or retroviral dominant-negative MEK transduction prevented Y1-induced GPA repression (76%±3 versus 9%±1.1; p<10-3), showing that a functional ERK was necessary to the PIEZO1-mediated effects in human erythropoiesis.

In vitro erythroid differentiation of progenitors from HX patients showed heterogeneous results. A similar phenotype than described after Y1 exposure was clearly observed for 5 mutations. Among them, 1 patient carrying the PIEZO1 mutation G1792A was tested in triplicate: the mature CD71+/GPA+ population at day 10 was significantly decreased in comparison with controls (13.8%±12.6 versus 62.2%±20.7; p<0.01). This delay was confirmed by cytology after MGG staining, showing a significant increase in the proportion of immature precursors (pro+basophilic erythroblasts) at day 10 (99%±1 versus 58.2%±19.3; p<0.01).

Conclusion

We described here for the first time a role of PIEZO1 during human erythroid differentiation. PIEZO1 activation repressed erythroid terminal maturation through Calcium entry and secondary phosphorylation of the ERK pathway. A similar phenotype was observed in the majority of PIEZO1-mutated HX patients but not all, underlying the high heterogeneity in the phenotype and the pathophysiology of this disease. 

Session topic: 29. Enzymopathies, membranopathies and other anemias

Keyword(s): Calcium, Erythroid differentiation, Erythropoieisis, MEK

By clicking “Accept Terms & all Cookies” or by continuing to browse, you agree to the storing of third-party cookies on your device to enhance your user experience and agree to the user terms and conditions of this learning management system (LMS).

Cookie Settings
Accept Terms & all Cookies