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

James I. Hearn; Taryn N. Green; Martin Chopra; Yohanes N. S. Nursalim; Leandro Ladvanszky; Nicholas Knowlton; Cherie Blenkiron; Raewyn C. Poulsen; Dean C. Singleton; Stefan K. Bohlander; Maggie L. Kalev-Zylinska

doi:10.1055/s-0040-1708483

Thrombosis and Haemostasis, Table of Contents

CC BY-NC-ND 4.0 · Thromb Haemost 2020; 120(04): 671-686
DOI: 10.1055/s-0040-1708483

Cellular Signaling and Proteolysis

Georg Thieme Verlag KG Stuttgart · New York

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

James I. Hearn

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Taryn N. Green

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Martin Chopra

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Yohanes N. S. Nursalim

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Leandro Ladvanszky

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Nicholas Knowlton

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Cherie Blenkiron

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Raewyn C. Poulsen

²Department of Medicine, School of Medicine, University of Auckland, Auckland, New Zealand

³Department of Pharmacology and Clinical Pharmacology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Dean C. Singleton

⁴Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand

,

Stefan K. Bohlander

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

,

Maggie L. Kalev-Zylinska

¹Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand

⁵LabPlus Haematology, Auckland City Hospital, Auckland, New Zealand

› Author Affiliations

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

Full Text

References

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