Abstract
Background The discovery of PIEZO1 gain-of-function mutations in hereditary xerocytosis (HX) has highlighted the role of mechanotransduction in the regulation of erythrocyte hydration. However, the pathophysiology of erythroid defects in HX is not restricted to mature red blood cells. Indeed, we have previously described that activation of PIEZO1 strongly delays the kinetics of erythroid differentiation, mainly during progenitor maturation, in a calcium-dependent manner. However, little is known about the downstream mechanisms that inhibit erythropoiesis. As we had identified the ERK/MAPK pathway as an essential regulator of PIEZO1-dependent signaling, we performed a comparative phosphoproteome analysis on erythroid cells with and without chemical activation of PIEZO1 and identified phospho-calpastatin (CAST) as a potential downstream effector of PIEZO1. CAST is an endogenous inhibitor of calpains (CPN), a family of calcium-activated proteases, and therefore a seducing candidate to link calcium influx through PIEZO1 with the defective erythropoiesis we described after PIEZO1 activation. Here, we extensively investigated the role of the PIEZO1/CAST axis in the regulation of human erythropoiesis.
Methods Experiments were conducted using the erythroleukemic cell line UT-7/Epo and human CD34-positive primary cells undergoing in vitro erythroid differentiation. PIEZO1 was activated by exposing cells to the chemical activator Yoda1. Phosphoproteomic analysis was performed on UT-7/Epo cells starved overnight in serum and cytokines then restimulated with or without EPO and/or Yoda1. Erythroid differentiation was followed using flow cytometry, protein expression was assessed using Western Blots. KD experiments were performed using either a pLKO.1 shRNA based lentiviral strategy (CAST KD in UT-7/Epo and CD34+ cells), either a siRNA strategy, delivered by electroporation (HSP70 KD in UT-7/Epo).
Results Phosphoproteome performed on UT-7/Epo cells identified CAST, isoform 4, as phosphorylated on residues Ser 323 and 325 after Yoda1-induced PIEZO1 activation. CAST phophorylation in response to PIEZO1 activation was confirmed by immunoprecipitation and we found that CAST protein level was also drastically decreased in UT-7/Epo and human erythroid cells, suggesting that phosphorylation induces protein degradation. However, proteasome inhibition using Carfilzomib blocked CAST degradation but did not reverse the defective erythropoiesis upon Yoda1 exposure, suggesting that CAST phosphorylation is sufficient to mediate the phenotypic effects of PIEZO1 activation. A shRNA-based CAST knockdown (KD) during in vitro human erythroid differentiation faithfully recapitulated the effects of PIEZO1 activation by inducing a strong inhibition of cell proliferation and a significant delay in the acquisition of glycophorin A (GPA) expression. We confirmed that CAST-KD led to CPN activation. Moreover, UT-7/Epo exposure to Dibucaine, a CPN activator, decreased GPA expression in the same way than CAST KD or Yoda1 exposure, suggesting a direct role of CPN activation in PIEZO1-mediated erythroid phenotype. Previous reports have identified HSP70, a key regulator of erythropoiesis, as a potential subtract for CPN cleavage. We found that HSP70 protein level was decreased in both UT-7/Epo and CD34+ cells after Yoda1 exposure and after CAST-KD. Subcellular analysis in UT-7/Epo cells showed a decrease in both the nucleus and cytoplasm, suggesting a global downregulation rather than a cytoplasmic sequestration as observed in beta-thalassemia. Inhibition of HSP70 using siRNA in UT-7/Epo cells reproduced the erythroid defects seen upon exposure to Yoda1, suggesting that the PIEZO1 repressive effects on erythroid differentiation are related to HSP70 downregulation. Given that HSP70 acts as a chaperone protecting the transcription factor GATA1 from caspase-mediated cleavage during erythropoiesis, we quantified GATA1 protein expression and found that it was decreased after PIEZO1 activation, implying a loss of the protection provided by HSP70.
Conclusion Our work demonstrates that activation of PIEZO1 during erythropoiesis induces phosphorylation of CAST, followed by its degradation and activation of CPN. This leads to downregulation of HSP70 and GATA1, ultimately altering human erythroid differentiation. These results highlight a novel PIEZO1/calcium/CAPN/HSP70/GATA1 axis, providing new insights into how mechanotransduction can regulate human erythropoiesis.
