N-Methyl-D-Aspartate Receptor Hypofunction in Meg-01 Cells Reveals a Role for Intracellular Calcium Homeostasis in Balancing Megakaryocytic-Erythroid Differentiation

Abstract

The release of calcium ions (Ca2+) from the endoplasmic reticulum (ER) and related store-operated calcium entry (SOCE) regulate maturation of normal megakaryocytes. The N-methyl-D-aspartate (NMDA) receptor (NMDAR) provides an additional mechanism for Ca2+ influx in megakaryocytic cells, but its role remains unclear. We created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene, which encodes an obligate, GluN1 subunit of the NMDAR. We found that compared with unmodified Meg-01 cells, Meg-01-GRIN1 −/− cells underwent atypical differentiation biased toward erythropoiesis, associated with increased basal ER stress and cell death. Resting cytoplasmic Ca2+ levels were higher in Meg-01-GRIN1 −/− cells, but ER Ca2+ release and SOCE were lower after activation. Lysosome-related organelles accumulated including immature dense granules that may have contributed an alternative source of intracellular Ca2+. Microarray analysis revealed that Meg-01-GRIN1 −/− cells had deregulated expression of transcripts involved in Ca2+ metabolism, together with a shift in the pattern of hematopoietic transcription factors toward erythropoiesis. In keeping with the observed pro-cell death phenotype induced by GRIN1 deletion, memantine (NMDAR inhibitor) increased cytotoxic effects of cytarabine in unmodified Meg-01 cells. In conclusion, NMDARs comprise an integral component of the Ca2+ regulatory network in Meg-01 cells that help balance ER stress and megakaryocytic-erythroid differentiation. We also provide the first evidence that megakaryocytic NMDARs regulate biogenesis of lysosome-related organelles, including dense granules. Our results argue that intracellular Ca2+ homeostasis may be more important for normal megakaryocytic and erythroid differentiation than currently recognized; thus, modulation may offer therapeutic opportunities.

N-Methyl-D-Aspartate Receptors in Hematopoietic Cells: What Have We Learned?

“…In fact, altered neuregulin 1-ErbB4 signaling is a wellestablished mechanism of NMDAR hypofunction in schizophrenia (Hahn et al, 2006). Encouragingly, we also found that in Meg-01-GRIN1 −/− cells, transcripts for neuregulin 1 and ErbB receptor feedback inhibitor 1 were up-regulated (1.98-and 2.05-fold, respectively), implying the ErbB4-NMDAR link is maintained during megakaryocytic-erythroid differentiation (Hearn et al, 2020).…”

“…In keeping with the pro-erythroid pattern of transcription factors, Meg-01-GRIN1 −/− cells displayed features of erythroid differentiation (Figure 1Biii). Our data provide the first evidence that NMDARs comprise an integral component of the Ca 2+ toolkit in megakaryocytic cells, and argue that intracellular Ca 2+ homeostasis may be more important than currently recognized for balancing megakaryocytic with erythroid differentiation at the level of a common progenitor (Hearn et al, 2020).…”

“…To get more insights into the mechanism of this divergence, we recently created a model of NMDAR hypofunction in Meg-01 cells using CRISPR-Cas9 mediated knockout of the GRIN1 gene that encodes the obligate, GluN1 subunit of the NMDAR (Hearn et al, 2020). We found that GRIN1 deletion caused marked changes in the intracellular Ca 2+ homeostasis, including higher cytosolic Ca 2+ levels at baseline but lower ER Ca 2+ release after activation.…”

“…Nevertheless, some progress has been achieved in the characterization of the subunit composition and currents mediated by non-neuronal NMDAR, in particular in red blood cells (RBC) (Makhro et al, 2010), platelets (Kalev-Zylinska et al, 2014), lymphocytes (Fenninger and Jefferies, 2019), and hematopoietic precursors, erythroblasts (Makhro et al, 2013;Hanggi et al, 2014Hanggi et al, , 2015 and megakaryocytes (Genever et al, 1999;Kamal et al, 2015). Information on the potential physiological role of these receptors is accumulating as well, including in erythroid cells (Makhro et al, 2013(Makhro et al, , 2016, and megakaryocytes (Hitchcock et al, 2003;Green et al, 2017;Kamal et al, 2018;Hearn et al, 2020). The subsequent sections will focus on the subunit composition, properties and the roles of NMDAR in megakaryocytic and erythroid precursors, and their mature progeny, platelets and RBCs.…”

“…Other glutamate receptors and mature blood cells are not discussed in detail but the appropriate background is provided to place this emerging field of research in a meaningful context. We describe NMDAR effects on hematopoietic differentiation, including some of our recent observations that suggest a novel role for the receptor in balancing megakaryocytic and erythroid cell fates (Hearn et al, 2020).…”