Inhibition of Retinoic Acid Signaling by the cdx-hox Pathway Is Essential for Blood Cell Formation during Embryogenesis.

Abstract The zebrafish mutant kugelig (kgg) is caused by a defect in the caudal-related homeobox gene cdx4 and has a deficit in both ‘primitive’ and ‘definitive’ hematopoietic stem cells. The embryonic anemia in the mutants can be rescued by overexpressing hox genes such as hoxb7a and hoxa9a, but not hoxb8a. This suggests that specific hox genes are required to make mesoderm competent to form blood. To further explore this, we undertook a microarray analysis to identify differentially expressed genes in kgg mutants and wild-type embryos. We found that raldh2, an enzyme required for retinoic acid (RA) production, is overexpressed in kgg mutants during the early stages of blood formation. This data led us to hypothesize that RA may act to suppress blood formation and that the cdx-hox pathway functions to limit RA production, thereby permitting blood formation to occur. To test this, we treated wild-type zebrafish embryos with RA and found that they became severely anemic. Treating kgg embryos with DEAB, a chemical that blocks raldh2 activity, restored hematopoiesis in kgg mutants. Expression of hoxa9a was not rescued in these treated embryos, indicating that RA acts downstream of the hox genes. DEAB also induced an expansion of erythroid cells in wild-type embryos, thus supporting the notion that the levels of RA during development are a critical determinant for blood formation. By performing a time-course rescue experiment, we determined that DEAB is effective when scl + hematopoietic progenitors are first formed from mesoderm, suggesting that RA acts upstream of the blood-inducer scl. In support of this, SCL overexpression rescues GATA-1 expression in embryos treated with RA. We next looked at the effects of DEAB and RA on the formation of mouse hematopoietic progenitors arising from ES cell-derived embryoid bodies (EBs). Addition of DEAB to EBs between days 2 to 3 of development resulted in a 5–8 fold increase in ‘primitive’ erythroid colonies (CFU-Ep), analogous to our results in zebrafish embryos. In contrast, RA treatment caused a general inhibition in the growth of all colony types. Taken together, these results suggest a new model in which suppression of RA by the cdx-hox pathway is necessary for yolk sac hematopoiesis to occur. This model provides an explanation for how hox genes control the spatiotemporal formation of hematopoietic tissue during organogenesis and may shed new light on the pathogenesis of leukemias involving translocations of the cdx , hox, and retinoic acid receptors.

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Retinoic Acid Blockade Increases Primitive Blood Cell Formation in cdx4 Mutant Zebrafish Embryos, Murine Yolk Sac Explants and Differentiated Embryonic Stem Cells.

Blood ◽

10.1182/blood.v110.11.201.201 ◽

2007 ◽

Vol 110 (11) ◽

pp. 201-201

Author(s):

Jill L. de Jong ◽

Alan J. Davidson ◽

Yuan Wang ◽

James Palis ◽

George Q. Daley ◽

...

Keyword(s):

Stem Cells ◽

Retinoic Acid ◽

Embryonic Stem Cells ◽

Embryoid Body ◽

Embryonic Stem ◽

Yolk Sac ◽

Zebrafish Embryos ◽

Retinoic Acid Signaling ◽

Wild Type ◽

Hematopoietic Stem

Abstract Retinoic acid signaling is critical for proper development, as well as the regulation of hematopoiesis in murine and human cells. It has been reported that retinoic acid expands definitive hematopoietic stem cells and confers an advantage in transplantation of murine HSCs. We demonstrate here that all trans retinoic acid (ATRA) blocks early hematopoiesis in zebrafish embryos as measured by decreased expression of scl and gata1. This loss of gata1 expression after exposure to ATRA is reminiscent of the lack of gata1 expression in cdx4 mutant zebrafish embryos, prompting the hypothesis that retinoic acid and cdx4 share a common signaling pathway regulating the emergence of the hematopoietic system, and that inhibition of ATRA might rescue blood development in cdx4 mutants. We tested 4-diethylamino benzaldehyde (DEAB), an inhibitor of retinoic acid biosynthesis, and AGN193109, a pan-RAR antagonist, for their ability to rescue gata1 expression in cdx4 mutant embryos. We found that treatment with either DEAB or AGN193109 results in increased gata1 expression in the posterior mesoderm of both cdx4 mutants and wild type zebrafish embryos. This rescue of gata1 expression in the cdx4 mutants is observed only when the DEAB treatment occurs during late gastrulation. Mutant embryos exposed to DEAB only during early gastrulation or somitogenesis did not have increased gata1 expression. While this observation may reflect the complex activity of ATRA in both hematopoiesis and in mesodermal patterning, we conclude that it is also consistent with a switch to the stimulatory effect of retinoic acid on hematopoiesis observed by others in adult mice. Similarly, DEAB treatment increased primitive erythroid cells in murine hematopoietic cultures. We show that E7.5 murine yolk sac explant cultures exposed to DEAB have increased primitive erythroid colony forming cells (EryP-CFC) and definitive erythroid burst forming units (BFU-E). In contrast, ATRA markedly inhibits all erythroid colony formation in murine yolk sac explants. Likewise, murine embryonic stem cells treated with DEAB during embryoid body development have an 8-fold expansion of EryP-CFC, and a 2-fold expansion of multipotent GEMM colonies. Previously we published that overexpression of hoxA9 mRNA rescues gata1 expression in cdx4 mutant embryos. However, the hematopoietic defect in cdx4 mutant embryos is not rescued by overexpression of scl. To test the hypothesis that hoxA9 and scl are both signaling downstream of retinoic acid in the stimulation of primitive erythroid development, we overexpressed hoxA9 or scl by microinjecting mRNA into single-cell wild type zebrafish embryos followed by treatment with ATRA. We find that overexpression of either hoxA9 or scl partially rescues the block of hematopoiesis induced by ATRA. Taken together, these data indicate that the commitment of mesodermal cells to hematopoietic fates is inhibited by retinoic acid, and that the retinoic acid signal is acting downstream of cdx4 in the zebrafish embryo, while scl and hoxA9 are acting downstream of retinoic acid signaling.

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Scl is required for dorsal aorta as well as blood formation in zebrafish embryos

Blood ◽

10.1182/blood-2004-09-3547 ◽

2005 ◽

Vol 105 (9) ◽

pp. 3502-3511 ◽

Cited By ~ 113

Author(s):

Lucy J. Patterson ◽

Martin Gering ◽

Roger Patient

Keyword(s):

Close Association ◽

Embryoid Bodies ◽

Dorsal Aorta ◽

Zebrafish Embryos ◽

Hemogenic Endothelium ◽

Cardinal Vein ◽

Hematopoietic Stem ◽

Blood Formation ◽

Endothelial Development

AbstractBlood and endothelial cells arise in close association in developing embryos, possibly from a shared precursor, the hemangioblast, or as hemogenic endothelium. The transcription factor, Scl/Tal1 (stem cell leukemia protein), is essential for hematopoiesis but thought to be required only for remodeling of endothelium in mouse embryos. By contrast, it has been implicated in hemangioblast formation in embryoid bodies. To resolve the role of scl in endothelial development, we knocked down its synthesis in zebrafish embryos where early precursors and later phenotypes can be more easily monitored. With respect to blood, the zebrafish morphants phenocopied the mouse knockout and positioned scl in the genetic hierarchy. Importantly, endothelial development was also clearly disrupted. Dorsal aorta formation was substantially compromised and gene expression in the posterior cardinal vein was abnormal. We conclude that scl is especially critical for the development of arteries where adult hematopoietic stem cells emerge, implicating scl in the formation of hemogenic endothelium.

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Role of the HOXA cluster in HSC emergence and blood cancer

Biochemical Society Transactions ◽

10.1042/bst20210234 ◽

2021 ◽

Author(s):

Mays Abuhantash ◽

Emma M. Collins ◽

Alexander Thompson

Keyword(s):

Embryonic Development ◽

Hox Genes ◽

Hoxa Cluster ◽

Hematopoietic Stem ◽

Blood Cancer ◽

Non Coding Rna ◽

Blood Formation ◽

Peripheral Cells ◽

Normal Embryonic Development

Hematopoiesis, the process of blood formation, is controlled by a complex developmental program that involves intrinsic and extrinsic regulators. Blood formation is critical to normal embryonic development and during embryogenesis distinct waves of hematopoiesis have been defined that represent the emergence of hematopoietic stem or progenitor cells. The Class I family of homeobox (HOX) genes are also critical for normal embryonic development, whereby mutations are associated with malformations and deformity. Recently, members of the HOXA cluster (comprising 11 genes and non-coding RNA elements) have been associated with the emergence and maintenance of long-term repopulating HSCs. Previous studies identified a gradient of HOXA expression from high in HSCs to low in circulating peripheral cells, indicating their importance in maintaining blood cell numbers and differentiation state. Indeed, dysregulation of HOXA genes either directly or by genetic lesions of upstream regulators correlates with a malignant phenotype. This review discusses the role of the HOXA cluster in both HSC emergence and blood cancer formation highlighting the need for further research to identify specific roles of these master regulators in normal and malignant hematopoiesis.

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Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo

Blood ◽

10.1182/blood-2002-06-1699 ◽

2003 ◽

Vol 101 (2) ◽

pp. 508-516 ◽

Cited By ~ 240

Author(s):

Hanna K. A. Mikkola ◽

Yuko Fujiwara ◽

Thorsten M. Schlaeger ◽

David Traver ◽

Stuart H. Orkin

Keyword(s):

Endothelial Cells ◽

Mouse Embryo ◽

Fetal Liver ◽

Stem Cell Differentiation ◽

Yolk Sac ◽

Embryoid Bodies ◽

Embryonic Stem Cell Differentiation ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Definitive Hematopoiesis

Murine hematopoietic stem cells (HSCs) originate from mesoderm in a process that requires the transcription factor SCL/Tal1. To define steps in the commitment to blood cell fate, we compared wild-type and SCL−/− embryonic stem cell differentiation in vitro and identified CD41 (GpIIb) as the earliest surface marker missing from SCL−/− embryoid bodies (EBs). Culture of fluorescence-activated cell sorter (FACS) purified cells from EBs showed that definitive hematopoietic progenitors were highly enriched in the CD41+ fraction, whereas endothelial cells developed from CD41− cells. In the mouse embryo, expression of CD41 was detected in yolk sac blood islands and in fetal liver. In yolk sac and EBs, the panhematopoietic marker CD45 appeared in a subpopulation of CD41+ cells. However, multilineage hematopoietic colonies developed not only from CD45+CD41+ cells but also from CD45−CD41+ cells, suggesting that CD41 rather than CD45 marks the definitive culture colony-forming unit (CFU-C) at the embryonic stage. In contrast, fetal liver CFU-C was CD45+, and only a subfraction expressed CD41, demonstrating down-regulation of CD41 by the fetal liver stage. In yolk sac and EBs, CD41 was coexpressed with embryonic HSC markers c-kit and CD34. Sorting for CD41 and c-kit expression resulted in enrichment of definitive hematopoietic progenitors. Furthermore, the CD41+c-kit+ population was missing from runx1/AML1−/− EBs that lack definitive hematopoiesis. These results suggest that the expression of CD41, a candidate target gene of SCL/Tal1, and c-kit define the divergence of definitive hematopoiesis from endothelial cells during development. Although CD41 is commonly referred to as megakaryocyte–platelet integrin in adult hematopoiesis, these results implicate a wider role for CD41 during murine ontogeny.

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BMP Signaling Via the Cdx-Hox Pathway Allocates Mesoderm to Hematopoietic vs Cardiac Fates.

Blood ◽

10.1182/blood.v108.11.4183.4183 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4183-4183

Author(s):

Claudia Lengerke ◽

Shannon McKinney-Freeman ◽

Yuan Wang ◽

Il-ho Jang ◽

Jeremy Green ◽

...

Keyword(s):

Hox Genes ◽

Es Cells ◽

Primitive Streak ◽

Bmp Signaling ◽

Model Organisms ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Hox Gene ◽

Bmp Receptors ◽

Bmp Pathway

Abstract Studies in model organisms have implicated the BMP pathway in allocating mesodermal tissues to blood fates. BMP receptors are widely expressed in the AGM region, implying BMP signaling in formation of definitive hematopoietic stem cells (HSCs). To dissect the role of the BMP pathway in murine hematopoietic development, we have exploited embryonic stem (ES) cells as a model system allowing experimental modulation of BMP4 protein and the BMP inhibitor Noggin during in vitro differentiation. Distinct mesodermal progenitors formed in response to a BMP gradient: progressively higher concentrations promoted cells expressing transcription factors of the posterior (caudal) primitive streak (e.g. Evx1, Cdx1, Mesp1), at the expense of anterior (rostral) markers (e.g. Cerberus, Goosecoid, FoxA2). Noggin addition during d0–2.5 was sufficient to inhibit hematopoiesis, data consistent with the observation that hemangioblasts form almost exclusively from the posterior primitive streak in vivo (Huber et al, 2004). Noggin addition after d2.5 abrogated hematopoietic fate, demonstrating a subsequent requirement for BMPs. At a molecular level, BMP4 added at d2.5 induced posterior Hox genes of the A (A7, A9, A10), and B cluster (B7, B8), while Noggin promoted an anterior Hox profile (A2, A4, B1). BMP4 patterns populations within the posterior primitive streak to blood by activating specific Hox genes. Cdx4 and Cdx1 are homeodomain-containing transcriptional regulators implicated in blood development in the zebrafish (Davidson et al, 2004Davidson et al, 2006), and hypothesized to convey positional information from morphogens to Hox genes. We have observed that Cdx genes show enhanced expression after BMP4, and suppression after Noggin treatment. Moreover, Cdx4 expression could bypass the inhibitory effect of Noggin on hematopoieisis after d2.5 of EB development. In serum-free conditions, either BMP4 addition or Cdx4 expression was sufficient to specify hematopoietic progenitors at equal or greater levels than serum cultures. These data demonstrate that Cdx4 complements the BMP requirement for specification of primitive streak cells to blood, presumably by activating the Hox gene signature for blood. We are currently exploring whether direct biochemical interactions can be established between the BMP/Smad pathway and Cdx genes. We investigated what alternative fates the posterior primitive streak cells might adopt, if not specified to blood by BMPs. Angioblasts were not affected, but cardiac differentiation displayed a striking inverse relationship to hematopoietic fate. Using quantitative assays of hematopoietic and cardiac potential, we determined that conditions promoting hematopoiesis (BMP4 addition or Cdx4 activation), simultaneously strongly suppressed cardiac development. We reproduced these observations with an ES cell line carrying a GFP reporter gene driven by the cardiac-specific NKX2.5 promoter (Wu et al., submitted). Hox gene analysis in purified cardiac (NKX2.5-GFP+) and hematopoietic progenitors (CD41+ckit+), showed posterior Hox genes to be suppressed in cardiac, and elevated in hematopoietic cells, supporting our hypothesis of positional patterning by differential Hox gene activation. We conclude that BMPs act on common progenitors via the Cdx-Hox pathway to pattern hematopoietic and cardiac fates within the primitive streak.

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A Conserved Cis-Regulatory Retinoic Acid Responsive Element Is Essential for Maintenance of Primitive Hematopoietic Stem Cells through Regulation of Hoxb Cluster

Blood ◽

10.1182/blood.v126.23.2377.2377 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2377-2377

Author(s):

Pengxu Qian ◽

Youngwook Ahn ◽

Bony De Kumar ◽

Christof Nolte ◽

Xi C. He ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Retinoic Acid ◽

Hox Genes ◽

Cell Types ◽

Hematopoietic Stem ◽

Fold Reduction ◽

Responsive Element ◽

Hox Clusters

Abstract Hematopoietic stem cells (HSCs) sustain lifelong production of multiple blood cell types through a finely-tuned balance between stem cell maintenance and activation to prevent bone marrow exhaustion or overgrowth. The highly conserved Hox family of homeodomain containing transcription factors have been identified as key regulators and contributors in both normal hematopoiesis and leukemogenesis. Most previous work has focused on individual Hox genes; however, it remains largely unknown whether and how multiple Hox genes in a cluster are regulated and function in hematopoiesis. We initiated a study to perform systematic, high-throughput transcriptome analysis in the following 17 cell types from the bone marrow (BM) of C57BL/6J mice: 4 hematopoietic stem and progenitor cells (CD49blo long-term (LT)-HSC, CD49bhi intermediate-term (IT)-HSC, short-term (ST)-HSC, and MPP); and 4 committed progenitors (CLP, CMP, GMP and MEP); and 9 mature lineage cells (B cell, T cell, NK cell, dendritic cell, monocyte, macrophage, granulocyte, megakaryocyte and nucleated erythrocyte). Intriguingly, as part of a unique fingerprint observed in the most primitive CD49blo LT-HSCs, we detected expression from the Hoxb cluster. Further analysis on all the four Hox clusters revealed that most of the genes from the Hoxb cluster, and not from the other Hox clusters, were predominantly expressed in the CD49blo LT-HSCs. This suggests that they might function as a cluster to maintain CD49blo LT-HSCs. A previous study has shown that one cis -regulatory retinoic acid responsive element (RARE), is conserved among vertebrate species and regulates multiple Hoxb gene expression in central nervous system development. Thus, we asked whether RARE is essential for maintenance of primitive CD49blo LT-HSCs by regulation of Hoxb cluster. To test this hypothesis, we utilized a RAREΔ knockout mouse model and assayed for HSC numbers in BM. We observed that homozygous deletion of RARE led to 2-fold reduction in both the frequency and absolute number of CD49blo LT-HSCs. Functionally, we first conducted limiting dilution, competitive repopulating unit (CRU) assays by transplanting 2.5×104, 7.5×104 or 2×105 of BM cells from RAREΔ mutants and their control littermates, together with 2×105 recipient BM cells derived from the Ptprc mutant strain, into lethally irradiated recipient mice. Our data showed a 2.5-fold decrease in functional HSCs in RAREΔ HSCs (1/20,326) compared to control (1/50,839). To further evaluate the long-term effect of RARE on HSCs, we performed serial BM transplantation and observed a 12.9-fold reduction of reconstitution ability after secondary transplantation. These data indicate that deletion of RARE compromised HSC long-term reconstitution capacity. Collectively, our work provides evidence showing that RARE is essential for maintenance of the primitive HSCs by regulation of Hoxb cluster genes. Disclosures No relevant conflicts of interest to declare.

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Lentiviral Gene Therapy for the Correction of Congenital Dyserythropoietic Anemia Type II

Blood ◽

10.1182/blood-2021-152332 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1994-1994

Author(s):

Mercedes Dessy-Rodriguez ◽

Sara Fañanas-Baquero ◽

Veronica Venturi ◽

Salvador Payan ◽

Cristian Tornador ◽

...

Keyword(s):

Gene Therapy ◽

Erythroid Differentiation ◽

Type Ii ◽

Wild Type ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Knock Out ◽

Cd34 Cells ◽

Cda Ii

Abstract Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythroid lineage. CDA type II is the most common one: it accounts for around 60% of all cases, and more than 600 cases have been reported so far. CDA II is caused by biallelic mutations in the SEC23B gene and is characterized by ineffective erythropoiesis with morphologic abnormalities of erythroblasts, hemolysis, and secondary iron overload, which is the most frequent complication. Patients usually suffer from variable degrees of jaundice, splenomegaly, and absolute reticulocyte count inadequate depending on the degree of anemia. Hydrops fetalis, aplastic crisis and gallstones are other associated clinical signs. CDA II bone marrow is characterized by the presence of more than 10% mature binucleated erythroblasts. Another distinctive feature of CDA II erythrocytes is hypoglycosylation of membrane proteins. The management of CDA II is generally limited to blood transfusion and iron chelation. Splenectomy has proved to reduce the number of transfusions in CDA II patients. However, allogenic hematopoietic stem cell transplant (HSCT) represents the only curative option for this disease. Autologous HSCT of genetically corrected cells will mean a definitive treatment for CDA II, overcoming the limitations of allogeneic HSCT, such as limited availability of HLA-matched donors, infections linked to immunosuppression or development of graft versus host disease. This strategy has been used to treat many inherited hematological diseases, including red blood cell diseases such as β-thalassemia, sickle cell disease or pyruvate kinase deficiency. Therefore, we have addressed a similar strategy to be applied to CDAII patients. Two different lentiviral vectors carrying either wild type or codon optimized versions of SEC23B cDNA (wtSEC23B LV or coSEC23B LV, respectively) under the control of human phosphoglycerate kinase promoter (PGK) have been developed. Taking advantage of a CDA II model, in which SEC23B knock-out was done in human hematopoietic progenitors through gene editing, we have determined the most effective SEC23B LV version and the most suitable multiplicity of infection (MOI) to compensate protein deficiency. SEC23B knock out human hematopoietic progenitors (CD34 + cells; 80% frame shift mutations; SEC23BKO) showed a sharp reduction in SEC23B protein level. Those SEC23BKO hematopoietic progenitors were transduced with both lentiviral vectors at MOIs ranged from 3 to 25. We observed that SEC23B protein reached physiological or even supraphysiological levels. In addition, the reduction in the number of erythroid colony forming units (CFUs) identified in SEC23BKO CD34 + cells, was partially restored in the LV transduced SEC23BKO progenitors. Significantly, we observed a clear correlation between the used MOI and the vector copy number (VCN) in the CFUs derived from transduced SEC23BKO CD34 + cells. Furthermore, SEC23BKO hematopoietic progenitors were subjected to an in vitro erythroid differentiation protocol. A sharp decrease in the cell growth throughout erythroid differentiation was observed in SEC23BKO condition. However, the transduction with any of SEC23B LVs at MOIs above 10 was able to recover cell expansion to values equal to wild type cells. Interestingly, total level of protein glycosylation during erythroid differentiation was enhanced after SEC23B LV transduction. Glycosylation level in wtSEC23B LV transduced SEC23BKO cells was most similar to the level in wild type cells. Then, we transduced peripheral blood-derived hematopoietic progenitors (PB-CD34 + cells) from a CDA II patient with wtSEC23B LV at MOI 25 and differentiated in vitro to erythroid cells. A complete restauration of SEC23B protein expression and a cell growth increase of wtSEC23B transduced CDAII was observed with vector copy numbers of 0.3 after 14 days under erythroid conditions. More importantly, we could find a decrease in the percentage of bi-/multinucleated erythroid cells generated in vitro after wtSEC23B LV transduction. In summary, SEC23B LV compensate the SEC23B deficiency in SEC23BKO and in CDAII hematopoietic progenitor cells, paving the way for gene therapy of autologous hematopoietic stem and progenitor cell as an alternative and feasible treatment for CDA II. Disclosures Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanchez: Bloodgenetics: Other: Co-Founder and promoter; UIC: Current Employment. Ramirez: VIVEBiotech: Current Employment. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company.

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Oxidative Stress-Mediated Activation of AKT/mTOR Signaling Pathway Leads to Myeloproliferative Syndrome in FoxO3 Null Mice: A Role for Lnk Adaptor Protein

Blood ◽

10.1182/blood.v112.11.509.509 ◽

2008 ◽

Vol 112 (11) ◽

pp. 509-509 ◽

Cited By ~ 2

Author(s):

Safak Yalcin ◽

Sathish Kumar Mungamuri ◽

Dragan Marinkovic ◽

Xin Zhang ◽

Wei Tong ◽

...

Keyword(s):

Progenitor Cells ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Hematopoietic Progenitor Cells ◽

Cytokine Signaling ◽

Wild Type ◽

Hematopoietic Progenitors ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem

Abstract Reactive oxygen species (ROS) are toxic byproducts of oxidative metabolism implicated in many debilitating human disorders including hematological malignancies and aging. ROS are also generated by growth factors and cytokine stimulation and play critical functions in normal cellular signaling. However, not much is known of how ROS impact physiological processes in normal and diseased states. We and others have recently shown critical functions for box (O) family of forkhead transcription factors (Fox)O in the regulation of physiological ROS in primitive hematopoietic cells. In particular, FoxO3 has emerged as the principal FoxO whose regulation of ROS is essential for the maintenance of hematopoietic stem cell pool. Although FoxO3’s activity is constitutively repressed by several oncoproteins that play critical roles in myeloproliferative disorders the role of FoxO3 in the regulation of primitive hematopoietic progenitors remains elusive. FoxO’s function is restrained by AKT serine threonine protein kinase. AKT supports growth, survival and proliferation by promoting inhibition of FoxO and activation of the mammalian target of rapamycin (mTOR) and its downstream target p70 S6 Kinase (S6K) through phosphorylation. We demonstrate that loss of FoxO3 leads to a myeloproliferative-like syndrome characterized by leukocytosis, splenomegaly, enhanced generation of primitive progenitors including colony-forming-unit-spleen (CFU-S) in hematopoietic organs and hypersensitivity of hematopoietic progenitor cells to cytokines in FoxO3 null mice. These findings were intriguing since we had not found FoxO3 null hematopoietic stem cells to exhibit enhanced cycling in vivo or to generate excessive hematopoietic progenitors ex vivo (Yalcin et al., JBC, 2008). To investigate the mechanism of enhanced myeloproliferation, we interrogated cytokine-mediated activation of signaling pathways in freshly isolated FoxO3 null versus wild type bone marrow cells enriched for hematopoietic progenitors. To our surprise we found that stimulation with cytokines including IL-3 led to hyperphosphorylation of AKT, mTOR and S6K but not STAT5 proteins in FoxO3 null as compared to wild type cytokine-starved hematopoietic progenitors. In agreement with these results, in vivo administration of the mTOR inhibitor rapamycin resulted in significant reduction of FoxO3 null- but not wild type-derived CFU-Sd12 in lethally irradiated hosts. These unexpected results suggested that AKT/mTOR signaling pathway is specifically overactivated as part of a feedback loop mechanism and mediates enhanced generation of FoxO3 null primitive multipotential hematopoietic progenitors in vivo. We further showed that phosphorylation of AKT/mTOR/S6K is highly sensitive to ROS scavenger N-Acetyl-Cysteine (NAC) in vivo and ex vivo in both wild type and FoxO3 null primitive hematopoietic progenitors indicating that ROS are involved in cytokine signaling in primary hematopoietic progenitor cells. Interestingly, in vivo administration of NAC normalized the number of FoxO3 null-derived CFU-Sd12 in lethally irradiated hosts without any impact on wild type CFU-Sd12 strongly suggesting that ROS mediate specifically enhanced generation of primitive hematopoietic progenitors in FoxO3 null mice. In this context, we were surprised to find similar levels of ROS concentrations in FoxO3 mutant as compared to control hematopoietic progenitors. Thus, we asked whether the increase in FoxO3 null primitive hematopoietic progenitor compartment is due to an increase sensitivity of cytokine signaling to ROS as opposed to increased ROS build up per se in these cells. In search for a mechanism we found the expression of Lnk, a negative regulator of cytokine signaling, to be highly reduced in FoxO3 null primitive hematopoietic progenitor cells. We further demonstrated that retroviral reintroduction of Lnk but not vector control in FoxO3 null primitive bone marrow cells reduced significantly the number of FoxO3 null-derived CFU-Sd12in vivo. Collectively, these results suggest that reduced expression of Lnk hypersensitizes FoxO3-deficient hematopoietic progenitors to ROS generated by cytokine signaling leading to myeloproliferation. These cumulative findings uncover a mechanism by which deregulation of cellular sensitivity to physiological ROS leads to hematopoietic malignancies specifically in disorders in which FoxO play a role.

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Stromal pleiotrophin regulates repopulation behavior of hematopoietic stem cells

Blood ◽

10.1182/blood-2010-05-287235 ◽

2011 ◽

Vol 118 (10) ◽

pp. 2712-2722 ◽

Cited By ~ 26

Author(s):

Rouzanna Istvanffy ◽

Monika Kröger ◽

Christina Eckl ◽

Sylke Gitzelmann ◽

Baiba Vilne ◽

...

Keyword(s):

Stromal Cells ◽

Molecular Level ◽

Donor Cell ◽

Regulatory Mechanisms ◽

Wild Type ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Regulated Expression ◽

Normal Hemostasis

Abstract Pleiotrophin (Ptn) is strongly expressed by stromal cells which maintain HSCs. However, in vivo, Ptn deficiency does not alter steady-state hematopoiesis. However, knockdown of Ptn (PtnKD) in stromal cells increases production of hematopoietic progenitors as well as HSC activity in cocultures, suggesting that Ptn may have a role in HSC activation. Indeed, transplantations of wild-type (Ptn+/+) HSCs into Ptn−/− mice show increased donor cell production in serial transplantations and dominant myeloid regeneration caused by Ptn-dependent regulation of HSC repopulation behavior. This regulation of Lin−Kit+Sca1+ function is associated with increased proliferation and, on a molecular level, with up-regulated expression of cyclin D1 (Ccnd1) and C/EBPα (Cepba), but reduced of PPARγ. The known HSC regulator β-catenin is, however, not altered in the absence of Ptn. In conclusion, our results point to different Ptn-mediated regulatory mechanisms in normal hemostasis and in hematopoietic regeneration and in maintaining the balance of myeloid and lymphoid regeneration. Moreover, our results support the idea that microenvironmental Ptn regulates hematopoietic regeneration through β-catenin–independent regulation of Ccnd1 and Cebpa.

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Relationship between the Hematopoietic Phenotype and the Subtype of Fanconi Anemia Mice.

Blood ◽

10.1182/blood.v110.11.2447.2447 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2447-2447

Author(s):

Susana Navarro ◽

Paula Rio ◽

Nestor W. Meza ◽

Raquel Chinchon ◽

Jose C. Segovia ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Models ◽

Fanconi Anemia ◽

Bone Marrow Cells ◽

Inherited Disease ◽

Wild Type ◽

Hematopoietic Progenitors ◽

Core Complex ◽

Similar Reduction ◽

Hematopoietic Stem

Abstract Fanconi anemia (FA) is a rare inherited disease associated with aplastic anemia and cancer predisposition. Great progress has occurred at a molecular level that allowed the identification of 13 genes involved in the disease. Additionally, several FA mouse models have been developed with the purpose of investigating the biological basis of the disease, and of facilitating the development of new therapies for FA. Because of the genetic heterogeneity of FA, here we have investigated the relevance of different FA genes to preserve the function of the hematopoietic stem cells (HSCs) of the mouse. To this aim, we have comparatively investigated the hematopoietic phenotype of different FA mice: Fanca−/−, Fancc−/−, Fancd1/Brca2Δ27/Δ27and Fancd2−/−. Normal counts of peripheral blood (PB) cells were observed in the different FA mice. Flow cytometry analyses were also incapable of demonstrating significant changes in the hematopoietic subpopulations present in PB, bone marrow (BM) or spleen of the different FA mice. In contrast to these observations, a reduction in the content of the hematopoietic progenitors was observed both in the BM and the spleen of Fancd1/Brca2Δ27/Δ27 and Fancd2−/− mice, either in young or adult. Significantly, a similar reduction in hematopoietic progenitors was only observed in old Fanca−/− and Fancc−/− mice. Moreover, hematopoietic colonies from mice defective in FA proteins downstream the FA core complex were smaller, compared to colonies from wild type, Fanca−/− or Fancc−/− mice. As expected, hematopoietic progenitors from all studied FA mouse models were highly sensitive to the DNA cross-linking agent, mitomycin C (MMC). Concerning the function of the HSCs of these FA mice, reduced competitive repopulation ability was always observed, compared to WT HSCs. However, in animals defective in FA proteins downstream the FA core, CRA values progressively dropped along the transplantation period. Additionally, a spontaneous chromosomal instability was observed in bone marrow cells from Fancd1/Brca2Δ27/Δ27. These animals also showed a marked repopulation defect in their HSCs assessed in their natural physiological environment, when wild type cells were transplanted in unconditioned Brca2Δ27/Δ27 recipients. In summary, our results show a more severe defect in the functionality of the HSC compartment in mice with mutant FA proteins downstream the FA core complex.

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