Role of the mechanotransductor PIEZO1 in megakaryocyte differentiation

Abstract

From haematopoietic stem cells to megakaryocytes (Mks), cells undergo various mechanical forces that affect Mk differentiation, maturation and proplatelet formation. The mechanotransductor PIEZO1 appears to be a natural candidate for sensing these mechanical forces and regulating megakaryopoiesis and thrombopoiesis. Gain-of-function mutations of PIEZO1 cause hereditary xerocytosis, a haemolytic anaemia associated with thrombotic events. If some functions of PIEZO1 have been reported in platelets, few data exist on PIEZO1 role in megakaryopoiesis. To address this subject, we used an in vitro model of Mk differentiation from CD34⁺ cells and studied step-by-step the effects of PIEZO1 activation by the chemical activator YODA1 during Mk differentiation and maturation. We report that PIEZO1 activation by 4 μM YODA1 at early stages of culture induced cytosolic calcium ion influx and reduced cell maturation. Indeed, CD41⁺CD42⁺ numbers were reduced by around 1.5-fold, with no effects on proliferation. At later stages of Mk differentiation, PIEZO1 activation promoted endomitosis and proplatelet formation that was reversed by PIEZO1 gene invalidation with a shRNA-PIEZO1. Same observations on endomitosis were reproduced in HEL cells induced into Mks by PMA and treated with YODA1. We provide for the first time results suggesting a dual role of PIEZO1 mechanotransductor during megakaryopoiesis.

Keywords: PIEZO1; megakaryocytes; proplatelet formation.

FIGURE 1

PIEZO1 expression in human primary CD34+ and Mks and Calcium flux activity. (A) Relative expression of PIEZO1 mRNA in CD41⁺ mature Mks at Day 12 of culture normalized on HPRT in Mks relative to CD34⁺ cells (n = 3). (B) Expression of PIEZO1 protein in CD34⁺ cells and in CD42⁺ mature Mks normalized on GAPDH in Mks (n = 4). (C) Representative western‐blot showing PIEZO1 and GAPDH protein expression in CD34⁺ cells and CD42⁺ mature. (D, E) Time course of cytosolic Ca²⁺ in response to four (dotted line) or 10 μM (solid line) YODA1 at Day 3 (D) or Day 12 (E) of megakaryocytic culture differentiation. Cells were labelled using Ca²⁺ probe Fluo3‐AM and mean fluorescence intensity (MFI) was recorded and expressed as fold change (normalized on DMSO) (n = 3 from 4 cord blood samples). *p < 0.05.

FIGURE 2

Effect of PIEZO1 activation on cell proliferation and megakaryocyte differentiation in human cord blood CD34⁺ cells. (A–E) Cell cultures were exposed from Day 0 to Day 12 to DMSO (control) or to 2 μM, 4 μM or 10 μM of YODA1 and were evaluated for: (A) cell proliferation at Days 4, 6, 7, 10, 11 and 12. Results are expressed as cell amplification fold compared to Day 0 (n = 6 from 12 cord blood samples); (B) mortality rate assessed by trypan blue staining at Day 12 (n = 6 from n = 12 cord blood samples); and percentage of (C) CD41⁺CD42⁺ and (D) CD41⁺CD42⁻ cells assessed by flow cytometry at Days 7, 10, 11 and 12 (n = 5 from n = 10 cord blood samples). (E) Representative dot plots of CD41⁺ and CD42⁺ Mks staining at Day 12 of culture. *p < 0.05, **p < 0.01. (F, G) After sorting CD41⁺CD42⁻ MKs at Day 7, cells were exposed to DMSO or to 4 μM YODA1 from Day 7 to Day 12 and were evaluated for: (F) percentage CD41⁺CD42⁺ Mks at D12 (n = 4 from n = 6 cord blood samples), and (G) cell proliferation (n = 5 from n = 10 cord blood samples). *p < 0.05, **p < 0.01.

FIGURE 3

Effect of PIEZO1 activation (A–G) and silencing (H–K) on CD41⁺CD42⁺ megakaryocytes obtained from CD34⁺ originating from human cord blood (A–C) or from leukapheresis (D–K). CD41⁺CD42⁺ Mks were sorted at Day 7 and then exposed from Day 7 to 12 to DMSO (black bars) or to 4 μM YODA1 (grey bars). At Day 12 were evaluated respectively for human cord blood or leukapheresis CD34⁺ cells: (A) and (D) Cell proliferation; (B) and (E) Ploidy and (C) and (F) Mean ploidy (n = 3 from n = 5 cord blood or leukapheresis samples). (G) Representative histogram of the CD41⁺CD42⁺ Mks ploidy from leukapheresis culture at D12 with DMSO (in red) and upon 4 μM YODA1 activation (in blue). At Day 7, CD41⁺CD42⁺ Mks were sorted and infected with Sh‐PIEZO1 or Sh‐scramble. (H) Relative expression of PIEZO1 mRNA normalized on HPRT in primary cells transfected with Sh1‐PIEZO1 or Sh2‐PIEZO1 (n = 3 from n = 3 leukapheresis CD34⁺ samples). (I) Mean ploidy and (J) Ploidy, at Day 12 of CD41⁺CD42⁺ cells (n = 5 from n = 6 leukapheresis CD34+ samples). Similar experiments were reproduced in Mk transfected cells, and (K) Mean ploidy was evaluated (n = 3 from n = 4 leukapheresis CD34+ samples) in the presence of 4 μM YODA1 or vehicle (DMSO). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Effect of PIEZO1 activation on PMA‐induced HEL. HEL cell line was cultured with 10 nM of PMA and YODA1 concentrations ranging from 0 μM (DMSO) to 10 μM. After 48 h were evaluated: (A) Mortality rate by DAPI staining (n = 5); (B) Cell proliferation as assessed by cell counting in 1 mL; (C) Mean ploidy and (D) Ploïdy as cell rate. (E) Representative histogram of the PMA‐induced HEL ploidy at H48 in the presence of DMSO (control) or 10 μM YODA1. HEL cell line transducted either with Sh1‐PIEZO1, Sh2‐PIEZO1 or Sh‐control were cultivated for 48 h in the presence of 10 nM PMA and 5 μM YODA1. Then, (F and G) PIEZO1 knockdown efficiency at protein level assessed by Western blot in HEL cells (n = 3) as well as (H) Mean ploidy, n = 3 and (I) Ploidy (n = 3). *p value <0.05, **p value <0.01, ***p value <0.001, ****p value <0.0001.

FIGURE 5

PIEZO1 activation induces proplatelet (PPT)‐bearing MKs in CD41⁺CD42⁺ Mks. At Day 7 culture of CD34⁺ cell from human cord blood (A) or leukapheresis (B and C) CD41⁺CD42⁺ Mks were sorted and plated in 96 well‐plates in the presence of vehicle (DMSO, black bars) or 4 μM YODA1 (grey bars) and, from Day 10 to 12, PPT‐bearing Mks obtained from (A) cord blood samples (n = 3 from n = 5 cord blood samples) and (B) leukapheresis samples (n = 4 from n = 4 leukapheresis samples) were quantified (Left panels). Right panels: Representative white light microscopy images of Mks with proplatelets at D12 in the presence of DMSO (control) or 4 μM YODA1. Similar experiments were performed on CD41⁺CD42⁺ Mks transducted with Sh1‐PIEZO1, Sh2‐PIEZO1 or Sh‐scramble: (C) Left panel: Percentage of PPT‐bearing MKs at D12. Right panel: Representative white light microscopy images of megakaryocytes transducted with Sh1‐PIEZO1, Sh2‐PIEZO1 or Sh‐scramble. (n = 3 from n = 3 leukapheresis samples). *p < 0.05, **p < 0.01, ***p < 0.001.