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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 
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Taryn N. Green
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Martin Chopra
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Yohanes N. S. Nursalim
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Leandro Ladvanszky
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Nicholas Knowlton
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Cherie Blenkiron
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Raewyn C. Poulsen
2   Department of Medicine, School of Medicine, University of Auckland, Auckland, New Zealand
3   Department of Pharmacology and Clinical Pharmacology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Dean C. Singleton
4   Auckland Cancer Society Research Centre, University of Auckland, Auckland, New Zealand
,
Stefan K. Bohlander
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
,
Maggie L. Kalev-Zylinska 
1   Department of Molecular Medicine and Pathology, School of Medical Sciences, University of Auckland, Auckland, New Zealand
5   LabPlus Haematology, Auckland City Hospital, Auckland, New Zealand
› Author Affiliations Funding This work was funded by Auckland Medical Research Foundation, Leukemia & Blood Cancer New Zealand, and Cancer Research Trust.
Further Information

Publication History

21 September 2019

04 February 2020

Publication Date:
14 April 2020 (online)

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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.


Supplementary Material

 

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