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ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity [RNA-seq Human]

ABSTRACT: The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

ORGANISM(S): Homo sapiens

PROVIDER: GSE234560 | GEO | 2024/01/05

REPOSITORIES: GEO

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Project description:The hematopoietic system gives rise to a heterogeneous population of terminally differentiated cells that reside in the bone marrow (BM) to fulfill their roles in immunity, blood clotting and tissue oxygenation. Hematopoietic stem and progenitor cells are at the apex of a hierarchically organized maturation cascade constantly replenishing the pool of differentiated cells to maintain blood cell homeostasis. The bone marrow microenvironment is functionally compartmentalized by heterogeneous niche cells that provide physical and soluble signals to spatio-temporally organize hematopoiesis. Megakaryocytes (MKs) are niche cells of hematopoietic origin that support hematopoietic stem cell (HSCs) homeostasis and generate circulating platelets. Platelet production involves the release of MK fragments while their cell body is entirely consumed in a process termed thrombopoiesis. Consequently, replenishment of fragmented MKs from MK progenitors (termed megakaryopoiesis) is required to ensure sufficient platelet production, but also to maintain MK homeostasis within the HSC niche. Here, we used intravital imaging of the megakaryocytic lineage to identify the spatio-temporal patterns of megakaryopoiesis during steady state and thrombocytopenia. We show that MK consumption during thrombopoiesis is compensated by immediate and local proliferation of MK progenitors. MK homeostasis is controlled by plasmacytoid dendritic cells (pDCs) that scan the BM and secrete Interferon alpha (INFa) upon detection of exhausted megakaryocytes to trigger megakaryopoiesis. We identified local pDC-derived INFa release as a physiological inflammatory stimulus of the bone marrow niche that synchronizes megakaryopoiesis and thrombopoiesis to maintain homeostasis of MKs and platelets in steady state and under stress. Viral infection with SARS-CoV-2 can manipulate pDC-driven MK proliferation leading to inappropriate megakaryopoiesis, which has been associated with thrombotic complications during COVID-19.

Project description:d9 and d12 Mks were either cultured statically or subjected to shear flow for 30 min; at d9, half the Mks were placed back in culture for 30 min (60 min time point) Megakaryocytes (Mks) are exposed to shear flow as they migrate from the bone marrow hematopoietic compartment into circulation thus releasing platelets and pro/preplatelets directly into the blood stream. Shear forces have been now established as promoting Mk maturation and platelet biogenesis. In order to understand the underlying mechanisms that modulate the response of Mks to shear forces, we carried out transcriptional analysis on immature and mature stem cell-derived Mks that were exposed to physiologically-relevant shear (2.5 dyn/cm2). In immature (d9) Mks, shear exposure upregulated genes related to growth and Mk maturation, while in mature (d12) Mks, it upregulated genes involved in apoptosis and intracellular transport. Following shear-flow exposure, 6 AP-1 transcripts (ATF4, JUNB, JUN, FOSB, FOS, and JUND) were upregulated at d9 and two AP-1 proteins (JunD and c-Fos) were upregulated both at d9 and d12. Our data show that MAPK signaling is linked to both the shear-stress response and AP-1 upregulation. JNK phosphorylation increased significantly following shear stimulation, while JNK inhibition reduced shear-induced JunD protein expression. Although p38 phosphorylation did not increase following shear flow, its inhibition reduced shear-induced JunD and c-Fos protein expression. JNK inhibition reduced fibrinogen binding of d9 and d12 platelet-like particle s (PLPs) and P-selectin expression at d12 PLPs, while p38 inhibition reduced fibrinogen binding of d12 PLPs. Here we show that mechanotransduction of shear forces in Mks results in JNK activation, AP-1 upregulation, and downstream transcriptional changes that promote maturation of immature Mks and platelet biogenesis in mature Mks. Two- and Three-condition experiment (flow vs. static culture condition, d9 vs. d12, and 30 min vs. 60 min at d9); Biological replicates: 3; Technical replicates: 1 (dye-swap)

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ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity [RNA-seq Human]

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