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.

Keywords

N-methyl-D-aspartate receptor - endoplasmic reticulum stress - intracellular calcium - megakaryocyte - erythropoiesis

Authors' Contributions

M.L.K.Z designed the study. J.I.H., T.N.G., M.C., Y.N.S.N., L.L. and M.L.K.Z generated and analysed data. N.C., C.B., R.C.P., D.C.S., S.K.B. and M.L.K.Z provided methodology advice, supervision and mentorship. J.I.H., T.N.G. and M.L.K.Z wrote the manuscript. All authors edited the manuscript and approved the final version for submission.

Note

J.I.H. received PhD scholarship from Anne and Victoria Norman, supplemented by payments from the Marijana Kumerich Trust.

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