MicroRNA199b regulates mouse hematopoietic stem cells maintenance

The preservation of hematopoieticstem cell pool in bone marrow (BM) is crucial for sustained hematopoiesisin adults. Studies assessing adult hematopoieticstem cells functionality had been shown that for example loss of quiescence impairs hematopoieticstem cells maintenance. Although, miR‐199b is frequently down‐regulated in acute myeloid leukemia, its role in hematopoieticstem cells quiescence, self‐renewal and differentiation is poorly understood. Our laboratory investigated the role of miR‐199b in hematopoieticstem and progenitor cells (HSPCs) fate using miR‐199b‐5p global deletion mouse model. Characterization of miR‐199b expression pattern among normal HSPC populations revealed that miR‐199b is enriched in LT‐HSCsand reduced upon myeloablativestress, suggesting its role in HSCsmaintenance. Indeed, our results reveal that loss of miR‐199b5p results in imbalance between long‐term hematopoieticstem cells (LT‐HSCs), short‐term hematopoieticstem cells (ST‐HSCs) and multipotentprogenitors (MMPs) pool. We found that during homeostasis, miR‐199b‐null HSCshave reduced capacity to maintain quiescent state and exhibit cell‐cycle deregulation. Cell cycle analyses showed that attenuation of miR‐199b controls HSCspool, causing defects in G1‐S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT‐HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS‐sorted LT‐HSCs(LineagenegSca1posc‐Kitpos CD48neg CD150pos) lacking miR‐199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS‐sorted LSK population of 52 weeks old miR‐199b KO mice revealed that loss of miR‐199b promotes accumulation of GMP‐like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age‐related pathway. We used noncompetitive repopulation studies to show that overall BM donor cellularitywas markedly elevated in the absence of miR‐199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR‐199b‐null LT‐HSC render enhanced self‐renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self‐renewal potential of miR‐199b‐null LT‐HSC by a secondary BM transplantation of unfractionatedBM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCspopulations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT‐HSC were similar between recipients reconstituted with wild‐type and miR‐199b‐KO chimeras, whereas miR‐199b‐null HSCscontributed relatively more into MPPs. Our data identify that attenuation of miR‐199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR‐199b promotes signals that govern differentiation of LT‐HSC to MPP leading to accumulation of highly proliferativeprogenitors during long‐term reconstitution. Hematopoieticregeneration via repopulation studies also revealed that miR‐199b‐deficient HSPCshave a lineage skewing potential toward myeloid lineage or clonalmyeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR‐199b in hematopoietic aging

Governing roles for Trib3 pseudokinase during stress erythropoiesis.

In response to anemia, the heightened production of erythropoietin (EPO) can sharply promote erythroid progenitor cell (EPC) formation. Specific mediators of such EPO- accelerated erythropoiesis, however, are not well understood. Presently, we first report that the expression of Trib3 in adult bone marrow EPCs in vivo is nominal at steady state, but strongly activated on EPO challenge. In a knockout mouse model, Trib3 disruption modestly increased steady-state erythrocyte numbers and decreased mean corpuscular volume. Following 5-fluorouracil myeloablation, however, rebound red blood cell production and hemoglobin levels were substantially (and selectively) compromised in Trib

Phospho-proteomic discovery of novel signal transducers including thioredoxin-interacting protein as mediators of erythropoietin-dependent human erythropoiesis.

Erythroid cell formation critically depends on signals transduced via erythropoietin (EPO)/EPO receptor (EPOR)/JAK2 complexes. This includes not only core response modules (e.g., JAK2/STAT5, RAS/MEK/ERK), but also specialized effectors (e.g., erythroferrone, ASCT2 glutamine transport, Spi2A). By using phospho-proteomics and a human erythroblastic cell model, we identify 121 new EPO target proteins, together with their EPO-modulated domains and phosphosites. Gene ontology (GO) enrichment for Molecular Function identified adaptor proteins as one top EPO target category. This includes a novel EPOR/JAK2-coupled network of actin assemblage modifiers, with adaptors DLG-1, DLG-3, WAS, WASL, and CD2AP as prime components. Cellular Component GO analysis further identified 19 new EPO-modulated cytoskeletal targets including the erythroid cytoskeletal targets spectrin A, spectrin B, adducin 2, and glycophorin C. In each, EPO-induced phosphorylation occurred at pY sites and subdomains, which suggests coordinated regulation by EPO of the erythroid cytoskeleton. GO analysis of Biological Processes further revealed metabolic regulators as a likewise unexpected EPO target set. Targets included aldolase A, pyruvate dehydrogenase α1, and thioredoxin-interacting protein (TXNIP), with EPO-modulated p-Y sites in each occurring within functional subdomains. In TXNIP, EPO-induced phosphorylation occurred at novel p-T349 and p-S358 sites, and was paralleled by rapid increases in TXNIP levels. In UT7epo-E and primary human stem cell (HSC)-derived erythroid progenitor cells, lentivirus-mediated short hairpin RNA knockdown studies revealed novel pro-erythropoietic roles for TXNIP. Specifically, TXNIP\u27s knockdown sharply inhibited c-KIT expression; compromised EPO dose-dependent erythroblast proliferation and survival; and delayed late-stage erythroblast formation. Overall, new insight is provided into EPO\u27s diverse action mechanisms and TXNIP\u27s contributions to EPO-dependent human erythropoiesis

Brief report: serpin Spi2A as a novel modulator of hematopoietic progenitor cell formation.

Prime regulation over hematopoietic progenitor cell (HPC) production is exerted by hematopoietins (HPs) and their Janus kinase-coupled receptors (HP-Rs). For HP/HP-R studies, one central challenge in determining specific effects involves the delineation of nonredundant signal transduction factors and their lineage restricted actions. Via loss-of-function studies, we define roles for an HP-regulated Serpina3g/Spi2A intracellular serpin during granulomyelocytic, B-cell, and hematopoietic stem cell (HSC) formation. In granulomyelocytic progenitors, granulocyte macrophage colony stimulating factor (GMCSF) strongly induced Serpina3g expression with Stat5 dependency. Spi2A-knockout (KO) led to 20-fold decreased CFU-GM formation, limited GMCSF-dependent granulocyte formation, and compromised neutrophil survival upon tumor necrosis factor alpha (TNF-α) exposure. In B-cell progenitors, Serpina3g was an interleukin-7 (IL7) target. Spi2A-KO elevated CFU-preB greater than sixfold and altered B-cell formation in competitive bone marrow transplant (BMT), and CpG challenge experiments. In HSCs, Serpina3g/Spi2A expression was also elevated. Spi2A-KO compromised LT-HSC proliferation (as well as lineage(neg) Sca1(pos) Kit(pos) (LSK) cell lysosomal integrity), and skewed LSK recovery post 5-FU. Spi2A therefore functions to modulate HP-regulated immune cell and HSC formation post-5-FU challenge

Novel roles for podocalyxin in regulating stress myelopoiesis, Rap1a, and neutrophil migration.

Podocalyxin (Podxl) is a CD34 orthologue and cell surface sialomucin reported to have roles in renal podocyte diaphragm slit development; vascular cell integrity; and the progression of blood, breast, and prostate cancers. Roles for Podxl during nonmalignant hematopoiesis, however, are largely undefined. We have developed a Vav-Cre Podxl knockout (KO) mouse model, and report on novel roles for Podxl in governing stress myelopoiesis. At steady state, Podxl expression among hematopoietic progenitor cells was low level but was induced by granulocyte colony-stimulating factor (G-CSF) in myeloid progenitors and by thrombopoietin in human stem cells. In keeping with low-level Podxl expression at steady state, Vav-Cre deletion of Podxl did not markedly alter peripheral blood cell levels. A G-CSF challenge in Podxl-KO mice, in contrast, hyperelevated peripheral blood neutrophil and monocyte levels. Podxl-KO also substantially heightened neutrophil levels after 5-fluorouracil myeloablation. These loss-of-function phenotypes were selective, and Podxl-KO did not alter lymphocyte, basophil, or eosinophil levels. Within bone marrow (and after G-CSF challenge), Podxl deletion moderately decreased colony forming units-granulocytes, eyrthrocytes, monocyte/macrophages, megakaryocytes and CD16/3

Brief report: Serpin Spi2A as a novel modulator of hematopoietic progenitor cell formation

Prime regulation over hematopoietic progenitor cell (HPC) production is exerted by hematopoietins (HP’s) and their Janus kinase-coupled receptors (HP-R’s). For HP/HP-R studies, one central challenge in determining specific effects involves the delineation of non-redundant signal transduction factors and their lineage restricted actions. Via loss-of-function (LOF) studies, we define roles for an HP-regulated Serpina3g/Spi2A intracellular serpin during granulomyelocytic, B-cell and HSC formation. In granulomyelocytic progenitors, GMCSF strongly induced Serpina3g expression with Stat5-dependency. Spi2A-KO led to 20-fold decreased CFU-GM formation, limited GMCSF-dependent granulocyte formation, and compromised neutrophil survival upon TNF-α exposure. In B-cell progenitors, Serpina3g was an IL7 target. Spi2A-KO elevated CFU-preB >6-fold, and altered B-cell formation in competitive BMT, and CpG challenge experiments. In HSCs, Serpina3g/Spi2A expression was also elevated. Spi2A-KO compromised LT-HSC proliferation (as well as LSK cell lysosomal integrity), and skewed LSK recovery post 5-FU. Spi2A therefore functions to modulate HP-regulated immune cell, and HSC formation post 5-FU challenge

ErbB2 promotes endothelial phenotype of human left ventricular epicardial highly proliferative cells (eHiPC).

The adult human heart contains a subpopulation of highly proliferative cells. The role of ErbB receptors in these cells has not been studied. From human left ventricular (LV) epicardial biopsies, we isolated highly proliferative cells (eHiPC) to characterize the cell surface expression and function of ErbB receptors in the regulation of cell proliferation and phenotype. We found that human LV eHiPC express all four ErbB receptor subtypes. However, the expression of ErbB receptors varied widely among eHiPC isolated from different subjects. eHiPC with higher cell surface expression of ErbB2 reproduced the phenotype of endothelial cells and were characterized by endothelial cell-like functional properties. We also found that EGF/ErbB1 induces VEGFR2 expression, while ligands for both ErbB1 and ErbB3/4 induce expression of Tie2. The number of CD3

Targeting EPO and EPO receptor pathways in anemia and dysregulated erythropoiesis.

INTRODUCTION: Recombinant human erythropoietin (rhEPO) is a first-line therapeutic for the anemia of chronic kidney disease, cancer chemotherapy, AIDS (Zidovudine therapy), and lower-risk myelodysplastic syndrome. However, rhEPO frequently elevates hypertension, is costly, and may affect cancer progression. Potentially high merit therefore exists for defining new targets for anti-anemia agents within erythropoietin (EPO) and EPO receptor (EPOR) regulatory circuits. AREAS COVERED: EPO production by renal interstitial fibroblasts is subject to modulation by several regulators of hypoxia-inducible factor 2a (HIF2a) including Iron Response Protein-1, prolyl hydroxylases, and HIF2a acetylases, each of which holds potential as anti-anemia drug targets. The cell surface receptor for EPO (EPOR) preassembles as a homodimer, together with Janus Kinase 2 (JAK2), and therefore it remains attractive to develop novel agents that trigger EPOR complex activation (activating antibodies, mimetics, small-molecule agonists). Additionally, certain downstream transducers of EPOR/JAK2 signaling may be druggable, including Erythroferrone (a hepcidin regulator), a cytoprotective Spi2a serpin, and select EPOR-associated protein tyrosine phosphatases. EXPERT OPINION: While rhEPO (and biosimilars) are presently important mainstay erythropoiesis-stimulating agents (ESAs), impetus exists for studies of novel ESAs that fortify HIF2a\u27s effects, act as EPOR agonists, and/or bolster select downstream EPOR pathways to erythroid cell formation. Such agents could lessen rhEPO dosing, side effects, and/or costs