Isocitrate ameliorates anemia by suppressing the erythroid iron restriction response

• Text
• PDF

Abstract

The unique sensitivity of early red cell progenitors to iron deprivation, known as the erythroid iron restriction response, serves as a basis for human anemias globally. This response impairs erythropoietin-driven erythropoiesis and underlies erythropoietic repression in iron deficiency anemia. Mechanistically, the erythroid iron restriction response results from inactivation of aconitase enzymes and can be suppressed by providing the aconitase product isocitrate. Recent studies have implicated the erythroid iron restriction response in anemia of chronic disease and inflammation (ACDI), offering new therapeutic avenues for a major clinical problem; however, inflammatory signals may also directly repress erythropoiesis in ACDI. Here, we show that suppression of the erythroid iron restriction response by isocitrate administration corrected anemia and erythropoietic defects in rats with ACDI. In vitro studies demonstrated that erythroid repression by inflammatory signaling is potently modulated by the erythroid iron restriction response in a kinase-dependent pathway involving induction of the erythroid-inhibitory transcription factor PU.1. These results reveal the integration of iron and inflammatory inputs in a therapeutically tractable erythropoietic regulatory circuit.

Authors

Chanté L. Richardson, Lorrie L. Delehanty, Grant C. Bullock, Claudia M. Rival, Kenneth S. Tung, Donald L. Kimpel, Sara Gardenghi, Stefano Rivella, Adam N. Goldfarb

The nucleotide sugar UDP-glucose mobilizes long-term repopulating primitive hematopoietic cells

• Text
• PDF

Abstract

Hematopoietic stem progenitor cells (HSPCs) are present in very small numbers in the circulating blood in steady-state conditions. In response to stress or injury, HSPCs are primed to migrate out of their niche to peripheral blood. Mobilized HSPCs are now commonly used as stem cell sources due to faster engraftment and reduced risk of posttransplant infection. In this study, we demonstrated that a nucleotide sugar, UDP-glucose, which is released into extracellular fluids in response to stress, mediates HSPC mobilization. UDP-glucose–mobilized cells possessed the capacity to achieve long-term repopulation in lethally irradiated animals and the ability to differentiate into multi-lineage blood cells. Compared with G-CSF–mobilized cells, UDP-glucose–mobilized cells preferentially supported long-term repopulation and exhibited lymphoid-biased differentiation, suggesting that UDP-glucose triggers the mobilization of functionally distinct subsets of HSPCs. Furthermore, co-administration of UDP-glucose and G-CSF led to greater HSPC mobilization than G-CSF alone. Administration of the antioxidant agent NAC significantly reduced UDP-glucose–induced mobilization, coinciding with a reduction in RANKL and osteoclastogenesis. These findings provide direct evidence demonstrating a potential role for UDP-glucose in HSPC mobilization and may provide an attractive strategy to improve the yield of stem cells in poor-mobilizing allogeneic or autologous donors.

Authors

Sungho Kook, Joonseok Cho, Sean Bong Lee, Byeong-Chel Lee

Gray platelet syndrome and defective thrombo-inflammation in Nbeal2-deficient mice

• Text
• PDF

Abstract

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.

Authors

Carsten Deppermann, Deya Cherpokova, Paquita Nurden, Jan-Niklas Schulz, Ina Thielmann, Peter Kraft, Timo Vögtle, Christoph Kleinschnitz, Sebastian Dütting, Georg Krohne, Sabine A. Eming, Alan T. Nurden, Beate Eckes, Guido Stoll, David Stegner, Bernhard Nieswandt

Developmental differences in IFN signaling affect GATA1s-induced megakaryocyte hyperproliferation

• Text
• PDF

Abstract

About 10% of Down syndrome (DS) infants are born with a transient myeloproliferative disorder (DS-TMD) that spontaneously resolves within the first few months of life. About 20%–30% of these infants subsequently develop acute megakaryoblastic leukemia (DS-AMKL). Somatic mutations leading to the exclusive production of a short GATA1 isoform (GATA1s) occur in all cases of DS-TMD and DS-AMKL. Mice engineered to exclusively produce GATA1s have marked megakaryocytic progenitor (MkP) hyperproliferation during early fetal liver (FL) hematopoiesis, but not during postnatal BM hematopoiesis, mirroring the spontaneous resolution of DS-TMD. The mechanisms that underlie these developmental stage–specific effects are incompletely understood. Here, we report a striking upregulation of type I IFN–responsive gene expression in prospectively isolated mouse BM- versus FL-derived MkPs. Exogenous IFN-α markedly reduced the hyperproliferation FL-derived MkPs of GATA1s mice in vitro. Conversely, deletion of the α/β IFN receptor 1 (Ifnar1) gene or injection of neutralizing IFN-α/β antibodies increased the proliferation of BM-derived MkPs of GATA1s mice beyond the initial postnatal period. We also found that these differences existed in human FL versus BM megakaryocytes and that primary DS-TMD cells expressed type I IFN–responsive genes. We propose that increased type I IFN signaling contributes to the developmental stage–specific effects of GATA1s and possibly the spontaneous resolution of DS-TMD.

Authors

Andrew J. Woo, Karen Wieland, Hui Huang, Thomas E. Akie, Taylor Piers, Jonghwan Kim, Alan B. Cantor

β-globin gene transfer to human bone marrow for sickle cell disease

• Text
• PDF

Abstract

Autologous hematopoietic stem cell gene therapy is an approach to treating sickle cell disease (SCD) patients that may result in lower morbidity than allogeneic transplantation. We examined the potential of a lentiviral vector (LV) (CCL-βAS3-FB) encoding a human hemoglobin (HBB) gene engineered to impede sickle hemoglobin polymerization (HBBAS3) to transduce human BM CD34⁺ cells from SCD donors and prevent sickling of red blood cells produced by in vitro differentiation. The CCL-βAS3-FB LV transduced BM CD34⁺ cells from either healthy or SCD donors at similar levels, based on quantitative PCR and colony-forming unit progenitor analysis. Consistent expression of HBBAS3 mRNA and HbAS3 protein compromised a fourth of the total β-globin–like transcripts and hemoglobin (Hb) tetramers. Upon deoxygenation, a lower percentage of HBBAS3-transduced red blood cells exhibited sickling compared with mock-transduced cells from sickle donors. Transduced BM CD34⁺ cells were transplanted into immunodeficient mice, and the human cells recovered after 2–3 months were cultured for erythroid differentiation, which showed levels of HBBAS3 mRNA similar to those seen in the CD34⁺ cells that were directly differentiated in vitro. These results demonstrate that the CCL-βAS3-FB LV is capable of efficient transfer and consistent expression of an effective anti-sickling β-globin gene in human SCD BM CD34⁺ progenitor cells, improving physiologic parameters of the resulting red blood cells.

Authors

Zulema Romero, Fabrizia Urbinati, Sabine Geiger, Aaron R. Cooper, Jennifer Wherley, Michael L. Kaufman, Roger P. Hollis, Rafael Ruiz de Assin, Shantha Senadheera, Arineh Sahagian, Xiangyang Jin, Alyse Gellis, Xiaoyan Wang, David Gjertson, Satiro DeOliveira, Pamela Kempert, Sally Shupien, Hisham Abdel-Azim, Mark C. Walters, Herbert J. Meiselman, Rosalinda B. Wenby, Theresa Gruber, Victor Marder, Thomas D. Coates, Donald B. Kohn

HDAC3 is essential for DNA replication in hematopoietic progenitor cells

• Text
• PDF

Abstract

Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3–5 times more multipotent progenitor cells. Hdac3^–/– stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3^–/– stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3^–/– stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.

Authors

Alyssa R. Summers, Melissa A. Fischer, Kristy R. Stengel, Yue Zhao, Jonathan F. Kaiser, Christina E. Wells, Aubrey Hunt, Srividya Bhaskara, Jessica W. Luzwick, Shilpa Sampathi, Xi Chen, Mary Ann Thompson, David Cortez, Scott W. Hiebert

Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs

• Text
• PDF

Abstract

Critically short telomeres activate p53-mediated apoptosis, resulting in organ failure and leading to malignant transformation. Mutations in genes responsible for telomere maintenance are linked to a number of human diseases. We derived induced pluripotent stem cells (iPSCs) from 4 patients with aplastic anemia or hypocellular bone marrow carrying heterozygous mutations in the telomerase reverse transcriptase (TERT) or the telomerase RNA component (TERC) telomerase genes. Both mutant and control iPSCs upregulated TERT and TERC expression compared with parental fibroblasts, but mutant iPSCs elongated telomeres at a lower rate compared with healthy iPSCs, and the deficit correlated with the mutations’ impact on telomerase activity. There was no evidence for alternative lengthening of telomere (ALT) pathway activation. Elongation varied among iPSC clones derived from the same patient and among clones from siblings harboring identical mutations. Clonal heterogeneity was linked to genetic and environmental factors, but was not influenced by residual expression of reprogramming transgenes. Hypoxia increased telomere extension in both mutant and normal iPSCs. Additionally, telomerase-mutant iPSCs showed defective hematopoietic differentiation in vitro, mirroring the clinical phenotype observed in patients and demonstrating that human telomere diseases can be modeled utilizing iPSCs. Our data support the necessity of studying multiple clones when using iPSCs to model disease.

Authors

Thomas Winkler, So Gun Hong, Jake E. Decker, Mary J. Morgan, Chuanfeng Wu, William M. Hughes V, Yanqin Yang, Danny Wangsa, Hesed M. Padilla-Nash, Thomas Ried, Neal S. Young, Cynthia E. Dunbar, Rodrigo T. Calado

GSK3β regulates physiological migration of stem/progenitor cells via cytoskeletal rearrangement

• Text
• PDF

Abstract

Regulation of hematopoietic stem and progenitor cell (HSPC) steady-state egress from the bone marrow (BM) to the circulation is poorly understood. While glycogen synthase kinase-3β (GSK3β) is known to participate in HSPC proliferation, we revealed an unexpected role in the preferential regulation of CXCL12-induced migration and steady-state egress of murine HSPCs, including long-term repopulating HSCs, over mature leukocytes. HSPC egress, regulated by circadian rhythms of CXCL12 and CXCR4 levels, correlated with dynamic expression of GSK3β in the BM. Nevertheless, GSK3β signaling was CXCL12/CXCR4 independent, suggesting that synchronization of both pathways is required for HSPC motility. Chemotaxis of HSPCs expressing higher levels of GSK3β compared with mature cells was selectively enhanced by stem cell factor–induced activation of GSK3β. Moreover, HSPC motility was regulated by norepinephrine and insulin-like growth factor-1 (IGF-1), which increased or reduced, respectively, GSK3β expression in BM HSPCs and their subsequent egress. Mechanistically, GSK3β signaling promoted preferential HSPC migration by regulating actin rearrangement and microtubuli turnover, including CXCL12-induced actin polarization and polymerization. Our study identifies a previously unknown role for GSK3β in physiological HSPC motility, dictating an active, rather than a passive, nature for homeostatic egress from the BM reservoir to the blood circulation.

Authors

Kfir Lapid, Tomer Itkin, Gabriele D’Uva, Yossi Ovadya, Aya Ludin, Giulia Caglio, Alexander Kalinkovich, Karin Golan, Ziv Porat, Massimo Zollo, Tsvee Lapidot

Smap1 deficiency perturbs receptor trafficking and predisposes mice to myelodysplasia

• Text
• PDF

Abstract

The formation of clathrin-coated vesicles is essential for intracellular membrane trafficking between subcellular compartments and is triggered by the ARF family of small GTPases. We previously identified SMAP1 as an ARF6 GTPase-activating protein that functions in clathrin-dependent endocytosis. Because abnormalities in clathrin-dependent trafficking are often associated with oncogenesis, we targeted Smap1 in mice to examine its physiological and pathological significance. Smap1-deficent mice exhibited healthy growth, but their erythroblasts showed enhanced transferrin endocytosis. In mast cells cultured in SCF, Smap1 deficiency did not affect the internalization of c-KIT but impaired the sorting of internalized c-KIT from multivesicular bodies to lysosomes, resulting in intracellular accumulation of undegraded c-KIT that was accompanied by enhanced activation of ERK and increased cell growth. Interestingly, approximately 50% of aged Smap1-deficient mice developed anemia associated with morphologically dysplastic cells of erythroid-myeloid lineage, which are hematological abnormalities similar to myelodysplastic syndrome (MDS) in humans. Furthermore, some Smap1-deficient mice developed acute myeloid leukemia (AML) of various subtypes. Collectively, to our knowledge these results provide the first evidence in a mouse model that the deregulation of clathrin-dependent membrane trafficking may be involved in the development of MDS and subsequent AML.

Authors

Shunsuke Kon, Naoko Minegishi, Kenji Tanabe, Toshio Watanabe, Tomo Funaki, Won Fen Wong, Daisuke Sakamoto, Yudai Higuchi, Hiroshi Kiyonari, Katsutoshi Asano, Yoichiro Iwakura, Manabu Fukumoto, Motomi Osato, Masashi Sanada, Seishi Ogawa, Takuro Nakamura, Masanobu Satake

Platelet ITAM signaling is critical for vascular integrity in inflammation

• Text
• PDF

Abstract

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

Authors

Yacine Boulaftali, Paul R. Hess, Todd M. Getz, Agnieszka Cholka, Moritz Stolla, Nigel Mackman, A. Phillip Owens III, Jerry Ware, Mark L. Kahn, Wolfgang Bergmeier