Page 107 - Haematologica March 2020

Role of PIEZO1 during human erythropoiesis
PIEZO1 inhibitors, which are much less specific than a Sh- RNA based strategy. The PIEZO1-Ca2+-ERK axis has been described in epithelial cells, in which it regulated the cell cycle and survival.15 Whether Ca2+ entry into erythroblasts through PIEZO1 was sufficient to activate the ERK path- way by itself or whether it triggered secondary pathways is under investigation. ERK1/2 activation has been shown to promote early human progenitor cell expansion and its constitutive activation in mice blocked differentiation of fetal liver erythroid progenitor cells.39-41 Alternatively, ERK activation through EPO has been shown to promote ery- throid differentiation in a MASL1-dependent manner.42 This discrepancy in the role of ERK1/2 in erythroid differ- entiation may depend on its activation level. Our results showed that PIEZO1 activation synergized both EPO- dependent ERK and STAT5 phosphorylation in primary erythroblasts, suggesting that PIEZO1 may act as a modu- lator of EPO signaling during human erythropoiesis. Previous data suggested that erythroid regulation involved balanced STAT5 and ERK1/2 phosphorylation, and we assume that PIEZO1 may modify this equilibrium to delay erythroid terminal differentiation.43 Of note, YODA1-sus- tained ERK1/2 activation in UT7/GM cells did not pro- mote cell proliferation, in contrast to other cell types, but instead led to accumulation in the G0/G1 phase of the cell cycle.41,44 This may be due to the fact that YODA1 phos- phorylated ERK1/2 but not STAT5 in UT7/GM cells in which proliferation was mainly dependent on STAT5. Alternatively, PIEZO1 activation could modulate cell cycle regulators as previously shown in canine epithelial cells.6 This highlights the heterogeneous role of PIEZO1 activa- tion, which differs depending on the type of cell. We also showed that the effects of YODA1 required NFAT activa- tion and nuclear translocation, which have been involved in mice erythropoiesis.45 Whether this pathway also depends on EPO signaling is under investigation.
Erythroid cells from HX patients carrying PIEZO1 gain- of-function mutations had a similar phenotype as the one observed after PIEZO1 chemical activation. This high- lights the pathophysiological relevance of this ion channel, proving that how PIEZO1 is activated (i.e., constitutive mutation vs. chemical activation) is not critical. Moreover, it suggests that the HX erythroid phenotype may involve a certain degree of impaired erythropoiesis, as evoked in two recent case reports, which may participate in the high
rate of iron overload described in these patients.8,19,22,32 However, the delay in erythroid differentiation does not translate into anemia since most patients have a totally compensated hemolysis. First, dyserythropoiesis may be mild in vivo. Second, it has been suggested that PIEZO1- HX red blood cells have an increased hemoglobin affinity for oxygen.46 Therefore, hemoglobin level could represent a balance between factors that tend to decrease it (hemo- lysis, dyserythropoiesis) and factors that tend to increase it (hemoglobin hyperaffinity). We were unable to find a clear correlation between in vivo erythrocyte parameters in HX patients and the in vitro erythroid phenotype. This may be due to different mechanisms involved, since mature red cell dehydration is known to be Gardos- dependent while the YODA1-induced erythroid delay was insensitive to Senicapoc. However, it would be of great interest to correlate, for each mutation, the intensity of the erythroid phenotype with functional tests such as high-throughput patch clamping on red cells.47 Alternatively, modeling the different HX mutations through viral transduction in normal erythroid progenitors would represent an interesting way to better understand the disease heterogeneity and to highlight the potential role of other genetic factors that may modulate the pheno- type.
In summary, we describe here a role for PIEZO1 during human erythroid differentiation, in erythroleukemic cell lines, in normal primary erythroblasts after exposure to YODA1, and in primary cells from HX patients carrying an activating PIEZO1 mutation. We observed a delay in terminal erythroid maturation which depended on Ca2+ entry, and NFAT, STAT5 and ERK1/2 pathway activation. We observed that PIEZO1 could synergize with EPO-sig- naling during human erythropoiesis and that its constitu- tive activation in HX led to impairment in proliferation and differentiation of erythroid progenitors, highlighting a new pathophysiological mechanism in this rare disorder.
Acknowledgments
The authors are grateful to the University Hospital of Amiens (CHU Amiens), the Club du Globule Rouge et du Fer (CGRF), and the Réseau Hématologie Picardie (RHEPI) for their support. CHU Amiens provided financial support through the HEMAS- TEM project. We thank Prof. Stephane Giraudier (Paris, France) for the MEK-DN plasmid.
References
1. Coste B, Mathur J, Schmidt M, et al. Piezo1 and Piezo2 are essential components of dis- tinct mechanically activated cation channels. Science. 2010;330(6000):55-60.
2. Saotome K, Murthy SE, Kefauver JM, Whitwam T, Patapoutian A, Ward AB. Structure of the mechanically activated ion channel Piezo1. Nature. 2018;554(7693):481- 486.
3. ZhaoQ,ZhouH,ChiS,etal.Structureand mechanogating mechanism of the Piezo1 channel. Nature. 2018;554(7693):487-492.
4. Cinar E, Zhou S, DeCourcey J, Wang Y, Waugh RE, Wan J. Piezo1 regulates
mechanotransductive release of ATP from human RBCs. Proc Natl Acad Sci U S A. 2015;112(38):11783-11788.
5. Cahalan SM, Lukacs V, Ranade SS, Chien S, Bandell M, Patapoutian A. Piezo1 links mechanical forces to red blood cell volume. Elife. 2015;4.
6. Wu J, Young M, Lewis AH, Martfeld AN, Kalmeta B, Grandl J. Inactivation of mechan- ically activated Piezo1 ion channels is deter- mined by the C-terminal extracellular domain and the inner pore helix. Cell Rep. 2017;21(9):2357-2366.
7. Gnanasambandam R, Bae C, Gottlieb PA, Sachs F. Ionic selectivity and permeation properties of human PIEZO1 channels. PLoS One. 2015;10(5):e0125503.
8. Andolfo I, Alper SL, De Franceschi L, et al.
Multiple clinical forms of dehydrated hered- itary stomatocytosis arise from mutations in PIEZO1. Blood. 2013;121(19):3925-3935.
9. ZarychanskiR,SchulzVP,HoustonBL,etal. Mutations in the mechanotransduction pro- tein PIEZO1 are associated with hereditary xerocytosis. Blood. 2012;120(9):1908-1915.
10. Albuisson J, Murthy SE, Bandell M, et al. Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion chan- nels. Nat Commun. 2013;4:1884.
11. Bae C, Gnanasambandam R, Nicolai C, Sachs F, Gottlieb PA. Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1. Proc Natl Acad Sci U S A. 2013;110(12):E1162- 1168.
haematologica | 2020; 105(3)
621