Mechanical Stress Induces Ca2+-Dependent Signal Transduction in Erythroblasts and Modulates Erythropoiesis.

Bioreactors are increasingly implemented for large scale cultures of various mammalian cells, which requires optimization of culture conditions. Such upscaling is also required to produce red blood cells (RBC) for transfusion and therapy purposes. However, the physiological suitability of RBC cultures to be transferred to stirred bioreactors is not well understood. PIEZO1 is the most abundantly expressed known mechanosensor on erythroid cells. It is a cation channel that translates mechanical forces directly into a physiological response. We investigated signaling cascades downstream of PIEZO1 activated upon transitioning stationary cultures to orbital shaking associated with mechanical stress, and compared the results to direct activation of PIEZO1 by the chemical agonist Yoda1. Erythroblasts subjected to orbital shaking displayed decreased proliferation, comparable to incubation in the presence of a low dose of Yoda1. Epo (Erythropoietin)-dependent STAT5 phosphorylation, and Calcineurin-dependent NFAT dephosphorylation was enhanced. Phosphorylation of ERK was also induced by both orbital shaking and Yoda1 treatment. Activation of these pathways was inhibited by intracellular Ca2+ chelation (BAPTA-AM) in the orbital shaker. Our results suggest that PIEZO1 is functional and could be activated by the mechanical forces in a bioreactor setup, and results in the induction of Ca2+-dependent signaling cascades regulating various aspects of erythropoiesis. With this study, we showed that Yoda1 treatment and mechanical stress induced via orbital shaking results in comparable activation of some Ca2+-dependent pathways, exhibiting that there are direct physiological outcomes of mechanical stress on erythroblasts.

SMIM1 missense mutations exert their effect on wild type Vel expression late in erythroid differentiation.

BACKGROUND: Vel expression on erythrocytes is variable due to polymorphisms, complicating Vel typing. Weak Vel expression can be caused by mutations within SMIM1 in a heterozygous setting, suggesting a dominant negative effect of SMIM1 mutants on wild type (wt)SMIM1 expression. Here we report how SMIM1 expression is regulated during erythropoiesis, to understand its variable expression on erythrocytes. STUDY DESIGN AND METHODS: Peripheral blood reticulocytes at different stages, cultured erythroid precursors and HEK293T cells were used to investigate expression and putative competition between wtSMIM1 and mutated SMIM1 VEL*01W.01, (c.152T>A (p.Met51Lys)), VEL*01W.02 (c.152T>G (p.Met51Arg)), and VEL*01W.03 (c.161T>C (p.Leu54Pro)). RESULTS: Depending on the mutations in SMIM1 an effect on total and membrane expression of SMIM1 was observed in transfected HEK293T cells, but co-expression of wtSMIM1 and mutatedSMIM1 did not have an effect on wtSMIM1 membrane expression. During differentiation of donors expressing VEL*01W.01, VEL*01W.03, Vel-positive, Vel-negative (homozygote SMIM1*64_80del), and Vel-heterozygote SMIM1*64_80del primary human erythroblasts no overt defect was found in Vel expression dynamics or total SMIM1 expression levels when compared with wtSMIM1 erythroblasts. However, during enucleation, total Vel expression was significantly lower on reticulocytes of Vel-weak donors expressing heterozygote mutated SMIM1 compared to Vel-positive or Vel-heterozygote SMIM1*64_80del donors, while Vel expression on extruded nuclei was maintained. In addition, reticulocyte maturation in vivo showed further loss of Vel expression in these individuals and nearly absent on erythrocytes. CONCLUSION: These results suggest that SMIM1 mutations exert a dominant negative effect on wtSMIM1 probably by affecting SMIM1 multimerization and thereby Vel epitope presentation at the latest stages of erythroid differentiation.

Inside Out Integrin Activation Mediated by PIEZO1 Signaling in Erythroblasts.

The non-selective mechanosensitive ion channel PIEZO1 controls erythrocyte volume homeostasis. Different missense gain-of-function mutations in PIEZO1 gene have been identified that cause Hereditary Xerocytosis (HX), a rare autosomal dominant haemolytic anemia. PIEZO1 expression is not limited to erythrocytes and expression levels are significantly higher in erythroid precursors, hinting to a role in erythropoiesis. During erythropoiesis, interactions between erythroblasts, central macrophages, and extracellular matrix within erythroblastic islands are important. Integrin α4ß1 and α5ß1 present on erythroblasts facilitate such interactions in erythroblastic islands. Here we found that chemical activation of PIEZO1 using Yoda1 leads to increased adhesion to VCAM1 and fibronectin in flowing conditions. Integrin α4, α5, and ß1 blocking antibodies prevented this PIEZO1-induced adhesion suggesting inside-out activation of integrin on erythroblasts. Blocking the Ca2+ dependent Calpain and PKC pathways by using specific inhibitors also blocked increased erythroid adhesion to VCAM1 and fibronectins. Cleavage of Talin was observed as a result of Calpain and PKC activity. In conclusion, PIEZO1 activation results in inside-out integrin activation, facilitated by calcium-dependent activation of PKC and Calpain. The data introduces novel concepts in Ca2+ signaling during erythropoiesis with ramification on erythroblastic island homeostasis in health and disease like Hereditary Xerocytosis.

Circular RNA expression in human hematopoietic cells is widespread and cell-type specific.

Hematopoietic stem cells differentiate into a broad range of specialized blood cells. This process is tightly regulated and depends on transcription factors, micro-RNAs, and long non-coding RNAs. Recently, also circular RNA (circRNA) were found to regulate cellular processes. Their expression pattern and their identity is however less well defined. Here, we provide the first comprehensive analysis of circRNA expression in human hematopoietic progenitors, and in differentiated lymphoid and myeloid cells. We here show that the expression of circRNA is cell-type specific, and increases upon maturation. CircRNA splicing variants can also be cell-type specific. Furthermore, nucleated hematopoietic cells contain circRNA that have higher expression levels than the corresponding linear RNA. Enucleated blood cells, i.e. platelets and erythrocytes, were suggested to use RNA to maintain their function, respond to environmental factors or to transmit signals to other cells via microvesicles. Here we show that platelets and erythrocytes contain the highest number of circRNA of all hematopoietic cells, and that the type and numbers of circRNA changes during maturation. This cell-type specific expression pattern of circRNA in hematopoietic cells suggests a hithero unappreciated role in differentiation and cellular function.

The Shape Shifting Story of Reticulocyte Maturation.

The final steps of erythropoiesis involve unique cellular processes including enucleation and reorganization of membrane proteins and the cytoskeleton to produce biconcave erythrocytes. Surprisingly this process is still poorly understood. In vitro erythropoiesis protocols currently produce reticulocytes rather than biconcave erythrocytes. In addition, immortalized lines and iPSC-derived erythroid cell suffer from low enucleation and suboptimal final maturation potential. In light of the increasing prospect to use in vitro produced erythrocytes as (personalized) transfusion products or as therapeutic delivery agents, the mechanisms driving this last step of erythropoiesis are in dire need of resolving. Here we review the elusive last steps of reticulocyte maturation with an emphasis on protein sorting during the defining steps of reticulocyte formation during enucleation and maturation.

DARC extracellular domain remodeling in maturating reticulocytes explains Plasmodium vivax tropism.

Plasmodium vivax is the most prevalent parasite species that causes malaria in humans and exclusively infects reticulocytes. Reticulocyte infection is facilitated by P vivax Duffy binding protein (DBP), which utilizes DARC (Duffy antigen receptor for chemokines) as an entry point. However, the selective tropism of P vivax for transferrin receptor (CD71)-positive reticulocytes remained unexplained, given the constitutive expression of DARC during reticulocyte maturation. CD71/RNA double staining of reticulocytes enriched from adult peripheral blood reveals 4 distinct reticulocyte populations: CD71high/RNAhigh (∼0.016%), CD71low/RNAhigh (∼0.059%), CD71neg/RNAhigh (∼0.37%), CD71neg/RNAlow (∼0.55%), and erythrocytes CD71neg/RNAneg (∼99%). We hypothesized that selective association of DBP with a small population of immature reticulocytes could explain the preference of P vivax for reticulocytes. Binding of specific monoclonal anti-DARC antibodies and recombinant DBP to CD71high/RNAhigh reticulocytes was significantly higher compared with other reticulocyte populations and erythrocytes. Interestingly, the total DARC protein throughout reticulocyte maturation was constant. The data suggest that selective exposure of the DBP binding site within DARC is key to the preferential binding of DBP to immature reticulocytes, which is the potential mechanism underlying the preferential infection of a reticulocyte subset by P vivax.

LFA-1 integrin antibodies inhibit leukocyte α4ß1-mediated adhesion by intracellular signaling.

Binding of intercellular adhesion molecule-1 to the ß2-integrin leukocyte function associated antigen-1 (LFA-1) is known to induce cross-talk to the α4ß1 integrin. Using different LFA-1 monoclonal antibodies, we have been able to study the requirement and mechanism of action for the cross-talk in considerable detail. LFA-1-activating antibodies and those inhibitory antibodies that signal to α4ß1 induce phosphorylation of Thr-758 on the ß2-chain, which is followed by binding of 14-3-3 proteins and signaling through the G protein exchange factor Tiam1. This results in dephosphorylation of Thr-788/789 on the ß1-chain of α4ß1 and loss of binding to its ligand vascular cell adhesion molecule-1. The results show that with LFA-1 antibodies, we can activate LFA-1 and inhibit α4ß1, inhibit both LFA-1 and α4ß1, inhibit LFA-1 but not α4ß1, or not affect LFA-1 or α4ß1 These findings are important for the understanding of integrin regulation and for the interpretation of the effect of integrin antibodies and their use in clinical applications.