Dataset Information

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.

SUBMITTER: Hearn JI

PROVIDER: S-EPMC7286128 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundProper control of the lineage bias of megakaryocytic and erythroid progenitor cells (MEPs) is of significant importance, the disorder of which will lead to abnormalities in the number and function of platelets and erythrocytes. Unfortunately, the signaling pathways regulating MEP differentiation largely remain to be elucidated. This study aimed to analyze the role and the underlying molecular mechanism of miR-1915-3p in megakaryocytic and erythroid differentiation.MethodsWe utilized miRNA mimics and miRNA sponge to alter the expression of miR-1915-3p in megakaryocytic and/or erythroid potential cells; siRNA and overexpression plasmid to change the expression of SOCS4, a potential target of miR-1915-3p. The expression of relevant surface markers was detected by flow cytometry. We scanned for miR-1915-3p target genes by mRNA expression profiling and bioinformatic analysis, and confirmed the targeting by dual-luciferase reporter assay, western blot and gain- and lost-of-function studies. One-way ANOVA and t-test were used to analyze the statistical significance.ResultsIn this study, overexpression or knockdown of miR-1915-3p inhibited or promoted erythroid differentiation, respectively. Accordingly, we scanned for miR-1915-3p target genes and confirmed that SOCS4 is one of the direct targets of miR-1915-3p. An attentive examination of the endogenous expression of SOCS4 during megakaryocytic and erythroid differentiation suggested the involvement of SOCS4 in erythroid/megakaryocytic lineage determination. SOCS4 knockdown lessened erythroid surface markers expression, as well as improved megakaryocytic differentiation, similar to the effects of miR-1915-3p overexpression. While SOCS4 overexpression resulted in reversed effects. SOCS4 overexpression in miR-1915-3p upregulated cells rescued the effect of miR-1915-3p.ConclusionsmiR-1915-3p acts as a negative regulator of erythropoiesis, and positively in thrombopoiesis. SOCS4 is one of the key mediators of miR-1915-3p during the differentiation of MEPs.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets