Metabolic sensor O-GlcNAcylation regulates megakaryopoiesis and thrombopoiesis through c-Myc stabilization and integrin perturbation

Abstract

Metabolic state of hematopoietic stem cells (HSCs) is an important regulator of self-renewal and lineage-specific differentiation. Posttranslational modification of proteins via O-GlcNAcylation is an ideal metabolic sensor, but how it contributes to megakaryopoiesis and thrombopoiesis remains unknown. Here, we reveal for the first time that cellular O-GlcNAcylation levels decline along the course of megakaryocyte (MK) differentiation from human-derived hematopoietic stem and progenitor cells (HSPCs). Inhibition of O-GlcNAc transferase (OGT) that catalyzes O-GlcNAcylation prolongedly decreases O-GlcNAcylation and induces the acquisition of CD34+CD41a+ MK-like progenitors and its progeny CD34−CD41a+/CD42b+ megakaryoblasts (MBs)/MKs from HSPCs, consequently resulting in increased CD41a+ and CD42b+ platelets. Using correlation and co-immunoprecipitation analyses, we further identify c-Myc as a direct downstream target of O-GlcNAcylation in MBs/MKs and provide compelling evidence on the regulation of platelets by novel O-GlcNAc/c-Myc axis. Our data indicate that O-GlcNAcylation posttranslationally regulates c-Myc stability by interfering with its ubiquitin-mediated proteasomal degradation. Depletion of c-Myc upon inhibition of OGT promotes platelet formation in part through the perturbation of cell adhesion molecules, that is, integrin-α4 and integrin-β7, as advised by gene ontology and enrichment analysis for RNA sequencing and validated herein. Together, our findings provide a novel basic knowledge on the regulatory role of O-GlcNAcylation in megakaryopoiesis and thrombopoiesis that could be important in understanding hematologic disorders whose etiology are related to impaired platelet production and may have clinical applications toward an ex vivo platelet production for transfusion.

Metabolic sensor O-GlcNAcylation regulates erythroid differentiation and globin production via BCL11A

“…During human hematopoiesis, both erythrocytes and megakaryocytes are derived from a committed progenitor, called megakaryocyte–erythroid progenitors (MEPs) [ 38 , 39 ]. We have demonstrated in our recent study that metabolic sensor O -GlcNAcylation plays an essential role in megakaryopoiesis from HSPCs and platelet production from megakaryoblasts/megakaryocytes via the regulation of c-Myc-mediated integrin-α4 and integrin-β7 [ 40 ]. In this study, we extend the knowledge on the roles of O -GlcNAcylation in erythropoiesis, starting from the lineage specification of HSPCs to terminal differentiation and erythroid maturation.…”

OGT and OGA gene-edited human induced pluripotent stem cells for dissecting the functional roles of O-GlcNAcylation in hematopoiesis

“…O -GlcNAcylation, mediated by the cycling enzymes OGT and OGA, contributes to the regulation of hematopoiesis, which produces and replenishes various cell types comprising the blood system, by means of HSC fate decision and lineage specification ( Zhang et al, 2019 ; Luanpitpong et al, 2021 ; Luanpitpong et al, 2022a ; Luanpitpong et al, 2022b ). Recent studies have demonstrated that O -GlcNAcylation also affects the functions of red blood cells and various immune cells.…”

“…The extent of O -GlcNAcylation generally reflects the global metabolic dynamics in the cells, and hence, it serves as an ideal metabolic sensor that participates in the regulation of various cellular processes through changes in protein function, gene expression, and cellular signaling. We previously reported that O -GlcNAcylation is a key determinant of human HSC fate decision and certain lineage-specific differentiation, including megakaryopoiesis ( Luanpitpong et al, 2021 ), erythropoiesis ( Luanpitpong et al, 2022a ), and dendritic cell development ( Luanpitpong et al, 2022b ). Inhibition of OGT that causes a prolonged decrease in O -GlcNAcylation promotes megakaryopoiesis and subsequent thrombopoiesis through the perturbation of cell adhesion molecules.…”

Generation and Functional Characterization of Anti-CD19 Chimeric Antigen Receptor-Natural Killer Cells from Human Induced Pluripotent Stem Cells

“…Given that BMP4, SCF, and VEGF have already been supplemented in the used hematopoietic differentiation medium, we may additionally include growth factors or small molecule inhibitors that manipulate WNT/β-catenin and Activin/Nodal signaling, e.g., Activin A, CHIR99021 (GSK-3 inhibitor), and SB-431542 (TGFβ inhibitor) [ 14 ]. Moreover, we are currently investigating the roles of O -GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, in HPC differentiation from iPSCs, as we previously found that O -GlcNAcylation is a key determinant of hematopoietic stem cell (HSC) fate decision and certain lineage-specific differentiation processes, including megakaryopoiesis [ 38 ], erythropoiesis [ 39 ], and dendritic cell differentiation [ 40 ]. For improving NK commitment, we may further increase the concentrations of major cytokines, such as IL-7 and IL-15, or introduce an additional stage of lymphoid progenitor expansion before NK cell commitment.…”