scholarly journals Comprehensive RNA Editome Reveals Azin1 As a Functional Regulator in Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 34-35
Author(s):  
Fengjiao Wang ◽  
Jiahuan He ◽  
Yanni Ma ◽  
Sha Hao ◽  
Siqi Liu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Rna Editing ◽  
Conflicts Of Interest ◽  
Underlying Mechanism ◽  
Vital Role ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Dynamic Landscape

RNA editing, adenosine (A)-to-inosine (I), plays a vital role in many biological processes. Our previous study has demonstrated that the hematopoietic stem and progenitor cells (HSPCs) deficient in adenosine deaminase acting on RNA 1 (Adar1), an RNA-editing enzyme, cannot reconstitute the irradiated recipients in vivo and form colonies in vitro (Xufeng R et al, PNAS 2009). However, the overall profile of RNA editome in hematopoiesis has not been established and the underlying mechanism how RNA editing governs the function of HSPCs is poorly defined. In this study, we sorted 12 murine adult hematopoietic cell populations and performed RNA sequencing. We depicted the landscape of RNA editome in hematopoietic cells and identified 30,796 editing sites in total. The dynamic landscape of RNA editome comprised of stage/group-specific as well as house-keeping editing patterns. Notably, antizyme inhibitor 1 (Azin1) was uncovered to be highly edited in HSPCs. To understand whether edited Azin1 was required for functioning of HSPCs, we transduced c-Kit+ HSPCs with the lentivirus carrying Azin1 cDNAs with distinct editing frequencies. c-Kit+ cells transduced with fully edited Azin1 showed enhanced reconstitution, compared to that transduced with partially edited or non-edited Azin1. Specifically, inability of RNA editing in Azin1 blocked the differentiation of hematopoietic stem cells (HSCs) in vivo. Moreover, a similar finding was obtained when Azin1 was knocked down. In conclusion, RNA editing of Azin1 (i) results in amino acid change to induce AZIN1 translocation to the nucleus, (ii) enhances AZIN1 binding affinity for DEAD box polypeptide 1 (DDX1) to alter the DDX1 chromatin distribution, and (iii) changes the expression of multiple hematopoietic regulators to ultimately promote HSPC differentiation. This work provides a valuable resource for studying RNA editing and delineates an essential role of Azin1 RNA editing in HSPCs. Disclosures No relevant conflicts of interest to declare.

Involvement of the Integrin Alpha 6 Receptor in the Homing and Engraftment of Hematopoietic Stem and Progenitor Cells to Bone Marrow.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1293-1293
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Functional Blocking

Abstract Within the bone marrow environment, adhesive interactions between stromal cells and extracellular matrix molecules are required for stem and progenitor cell survival, proliferation and differentiation as well as their transmigration between bone marrow (BM) and the circulation. This regulation is mediated by cell surface adhesion receptors. In experimental mouse stem cell transplantation models, several classes of cell adhesion receptors have been shown to be involved in the homing and engraftment of stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Using FACS analysis, the integrin a6 chain was now found to be ubiquitously (>95%) expressed in mouse hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, lin−Sca-1+c-Kit+CD34+) both in adult bone marrow and in fetal liver. In vitro, about 70% of mouse BM lin−Sca-1+c-Kit+ cells adhered to laminin-10/11 and 40% adhered to laminin-8. This adhesion was mediated by integrin a6b1 receptor, as shown by functional blocking monoclonal antibodies. We also used a functional blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of hematopoietic stem and progenitor cells. We found that the integrin a6 antibody inhibited the homing of bone marrow progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C was reduced by about 40% as compared to cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells (LTR), antibody treated bone marrow cells were first injected intravenously into lethally irradiated primary recipients. After three hours, bone marrow cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis 16 weeks after transplantation revealed an 80% reduction of stem cell activity of integrin a6 antibody treated cells as compared to cells treated with control antibody. These results suggest that integrin a6 plays an important role for hematopoietic stem and progenitor cell homing in vivo.

Integrin alpha 6 Is Essential for Fetal Hematopoietic Progenitor, but Not Fetal Stem Cell Homing to Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1387-1387
Author(s):  
Hong Qian ◽  
Sten Eirik W. Jacobsen ◽  
Marja Ekblom
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Progenitor Cell ◽  
Fetal Liver ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Integrin Alpha 6

Abstract Homing of transplanted hematopoietic stem cells (HSC) in the bone marrow (BM) is a prerequisite for establishment of hematopoiesis following transplantation. However, although multiple adhesive interactions of HSCs with BM microenviroment are thought to critically influence their homing and subsequently their engraftment, the molecular pathways that control the homing of transplanted HSCs, in particular, of fetal HSCs are still not well understood. In experimental mouse stem cell transplantation models, several integrins have been shown to be involved in the homing and engraftment of both adult and fetal stem and progenitor cells in BM. We have previously found that integrin a6 mediates human hematopoietic stem and progenitor cell adhesion to and migration on its specific ligands, laminin-8 and laminin-10/11 in vitro (Gu et al, Blood, 2003; 101:877). Furthermore, integrin a6 is required for adult mouse HSC homing to BM in vivo (Qian et al., Abstract American Society of Hematology, Blood 2004 ). We have now found that the integrin a6 chain like in adult HSC is ubiquitously (>99%) expressed also in fetal liver hematopoietic stem and progenitor cells (lin−Sca-1+c-Kit+, LSK ). In vitro, fetal liver LSK cells adhere to laminin-10/11 and laminin-8 in an integrin a6b1 receptor-dependent manner, as shown by function blocking monoclonal antibodies. We have now used a function blocking monoclonal antibody (GoH3) against integrin a6 to analyse the role of the integrin a6 receptor for the in vivo homing of fetal liver hematopoietic stem and progenitor cells to BM. The integrin a6 antibody inhibited homing of fetal liver progenitors (CFU-C) into BM of lethally irradiated recipients. The number of homed CFU-C in BM was reduced by about 40% as compared to the cells incubated with an isotype matched control antibody. To study homing of long-term repopulating stem cells, BM cells were first incubated with anti-integrin alpha 6 or anti-integrin alpha 4 or control antibody, and then injected intravenously into lethally irradiated primary recipients. After three hours, BM cells of the primary recipients were analysed by competitive repopulation assay in secondary recipients. Blood analysis up to 16 weeks after transplantation showed that no reduction of stem cell reconstitution from integrin a6 antibody treated cells as compared to cells treated with control antibody. In accordance with this, fetal liver HSC from integrin a6 gene deleted embryos did not show any impairment of homing and engraftment in BM as compared to normal littermates. These results suggest that integrin a6 plays an important developmentally regulated role for homing of distinct hematopoietic stem and progenitor cell populations in vivo.

Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 83-83
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Ann Atzberger ◽  
Dengli Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

scholarly journals In vitro and in vivo evaluation of cord blood hematopoietic stem and progenitor cells amplified with glycosaminoglycan mimetic

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Lionel Faivre ◽  
Véronique Parietti ◽  
Fernando Siñeriz ◽  
Sandrine Chantepie ◽  
Marie Gilbert-Sirieix ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
In Vivo Evaluation ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

scholarly journals IL-33 promotes anemia during chronic inflammation by inhibiting differentiation of erythroid progenitors

2020 ◽  
Vol 217 (9) ◽  
Author(s):  
James W. Swann ◽  
Lada A. Koneva ◽  
Daniel Regan-Komito ◽  
Stephen N. Sansom ◽  
Fiona Powrie ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Chronic Inflammation ◽  
Inflammatory Disease ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

An important comorbidity of chronic inflammation is anemia, which may be related to dysregulated activity of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Among HSPCs, we found that the receptor for IL-33, ST2, is expressed preferentially and highly on erythroid progenitors. Induction of inflammatory spondyloarthritis in mice increased IL-33 in BM plasma, and IL-33 was required for inflammation-dependent suppression of erythropoiesis in BM. Conversely, administration of IL-33 in healthy mice suppressed erythropoiesis, decreased hemoglobin expression, and caused anemia. Using purified erythroid progenitors in vitro, we show that IL-33 directly inhibited terminal maturation. This effect was dependent on NF-κB activation and associated with altered signaling events downstream of the erythropoietin receptor. Accordingly, IL-33 also suppressed erythropoietin-accelerated erythropoiesis in vivo. These results reveal a role for IL-33 in pathogenesis of anemia during inflammatory disease and define a new target for its treatment.

Leptin Receptor As a Marker for a Subset of Long-Term Functional Hematopoietic Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-32
Author(s):  
Thao Trinh ◽  
James Ropa ◽  
Arafat Aljoufi ◽  
Scott Cooper ◽  
Edward F. Srour ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Leptin Receptor ◽  
Conflicts Of Interest ◽  
Gene Set Enrichment Analysis ◽  
Type I ◽  
Hematopoietic Stem ◽  
Therapeutic Implications

The hematopoietic system is maintained by the hematopoetic stem and progenitor cells (HSCs/HPCs), a group of rare cells that reside in a hypoxic bone marrow (BM) microenvironment. Leptin (Lep) is well-known for its neuroendocrine and immunological functions, and its receptor (Lepr) has been studied extensively in the BM niche cells. Yet, its biological implications in HSC/HPC biology remained largely unknown. In this study, we hypothesized that Lepr-expressing HSCs/HPCs are functionally and transcriptomically distinct from their negative counterparts. To test our hypothesis, we utilized both in vitro and in vivo approaches. We first employed Fluorescence-activated cell sorting (FACS) analysis to confirm expression of Lepr on HSCs/HPCs in adult mouse BM. We then isolated equal numbers of Lepr+Lineage-Sca1+cKit+ (LSK cells - a heterogenous population of long-term, short-term HSCs and multipotent HPCs) and Lepr-LSK cells from C57BL/6 (CD45.2+) mouse BM to perform colony-forming unit (CFU) assay and competitive transplantation assay, which also included using competitor cells from BoyJ (CD45.1+) unseparated BM and lethally-irradiated F1 (CD45.1+CD45.2+) as hosts. To determine whether Lepr can further hierarchize HSCs into two distinct populations, we repeated the competitive transplants using freshly isolated C57BL/6 Lepr+HSCs or Lepr-HSCs cells instead. At the end of primary transplants, whole BM were analyzed for donor chimerisms in the peripheral blood (PB) and BM as well as transplanted in a non-competitive fashion into lethally-irradiated secondary recipients. To gain mechanistic insights, we assessed homing potential as homing plays a role in increased engraftment. We also performed bulk RNA-seq using freshly sorted BM Lepr+HSCs or Lepr-HSCs to elucidate potential molecular pathways that are responsible for the differences in their functional capacity. By phenotypic studies, our FACS analyses showed that Lepr+ cells represented a smaller population within the hematopoietic compartment in the BM. However, HSCs contained a higher percentage of Lepr+ cells than other HPC populations. By functional assessments, Lepr+LSK cells were more highly enriched for colony-forming progenitor cells in CFU assay as compared to Lepr-LSK cells. Interestingly, Lepr+LSK cells exhibited more robust engraftment capability in primary transplants and substantial self-renewal capacity in secondary transplants throughout different time points in both PB and BM. In addition, Lepr+HSCs showed significantly higher donor chimerisms in PB month 1, 2, 4 and BM month 4 with similar lineage output compared to Lepr-HSCs. Higher engraftment could be due to increased homing of HSCs to the BM; however, Lepr+HSCs and Lepr-HSCs showed similar homing capacity as well as levels of surface CXCR4 expression. Molecularly, Fast Preranked Gene Set Enrichment Analysis (FGSEA) showed that Lepr+HSCs were enriched for Type-I Interferon and Interferon-gamma response pathways with Normalized Enrichment Scores of 2 or higher. Lepr+HSC transcriptomic study also revealed that these cells as compared to Lepr-HSCs expressed significantly higher levels of genes involved in megakaryopoiesis and proinflammatory immune responses including the NF-κB subunits (Rel and Relb). Interestingly, both IFN-γ and NF-κB signalings have been demonstrated to be critical for the emergence of HSCs from the hemogentic endothelium during embryonic development. In summary, although Lepr+LSK cells occupied a minor fraction compared to their negative counterparts in the BM, they possessed higher colony-forming capacity and were more highly enriched for long-term functional HSCs. In line with this, Lepr+HSCs engrafted significantly higher and self-renewed more extensively than Lepr-HSCs, suggesting that Lepr not only can be used as a marker for functional HSCs but also further differentiate HSCs into two functionally distinguishable populations. Intriguingly, Lepr+HSCs were characterized with a proinflammatory transcriptomic profile that was previously suggested to be critical for the development of HSCs in the embryo. All together, our work demonstrated that Lepr+HSCs represent a subset of highly engrafting adult BM HSCs with an embryonic-like transcriptomic signature. This can have potential therapeutic implications in the field of hematopoietic transplantation as Lepr is highly conserved between mice and human. Disclosures No relevant conflicts of interest to declare.

GSK3β Signaling Regulates the Motility of Hematopoietic Progenitors Via Prune.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1553-1553
Author(s):  
Kfir Lapid ◽  
Gabriele D‘Uva ◽  
Alexander Kalinkovich ◽  
Aya Ludin ◽  
Karin Golan ◽  
...  
Keyword(s):  
Steady State ◽  
Defense Mechanisms ◽  
Conflicts Of Interest ◽  
Glycogen Synthase ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Stem And Progenitor Cells ◽  
Migration Capacity

Abstract Abstract 1553 One of the hallmarks of hematopoietic stem and progenitor cells (HSPC) is their motility. In steady state, HSPC are mostly retained in the bone marrow (BM), allowing ongoing hematopoiesis, concomitantly with slow release to the circulation as part of homeostasis and host defense mechanisms. While stress-induced recruitment and clinical mobilization processes are extensively studied, steady state egress mechanisms are poorly understood. In this study, we demonstrate that inhibition of Glycogen Synthase Kinase 3b (GSK3β) directly or via upstream Insulin-like Growth Factor-1 (IGF-1) signaling limited murine HSPC egress to the circulation. Indeed, inhibition of GSK3β resulted in reduced HSPC migration capacity towards a gradient of the chemokine stromal derived factor-1 (SDF-1, also termed CXCL12) in vitro and was found to reduce HSPC mobilization by IGF-1 receptor antagonist treatment. Interestingly, GSK3β signaling also regulated SDF-1 transcription by BM stromal cells in vitro and in vivo, probably as part of HSPC maintenance, since murine CXCR4 signaling is essential for hematopoietic stem cell quiescence. We revealed that the involvement of GSK3β in directional HSPC motility is mediated by the downstream phosphodiesterase Prune. Prune, which is over-expressed in several human cancers, was recently found to localize in focal adhesion sites, promoting the motility of malignant cells. Herein, we show that Prune is also expressed in normal leukocytes, including HSPC. Accordingly, inhibition of Prune resulted in reduced SDF-1 induced migration of murine HSPC in vitro as well as reduced steady state egress in vivo. Prune activity was also shown to regulate the actin cytoskeleton by contributing to its polymerization. In general, highly regulated actin turnover is essential for spontaneous and directional motility mechanisms. Altogether, we present GSK3β and Prune as novel players in physiological HSPC motility, dictating an active rather than passive nature for steady state egress from the BM reservoir to the blood circulation as part of homeostasis. Disclosures: No relevant conflicts of interest to declare.

IFNs Upregulate Sca-1 and Block Proliferation in Murine Hematopoietic Stem and Progenitor Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3394-3394
Author(s):  
Kaitlyn Shank ◽  
Yusup Shin ◽  
Carson Wills ◽  
Nicole Cunningham ◽  
Alevtina Domashenko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Caspase 3 ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Brdu Incorporation ◽  
Apparent Paradox ◽  
Hematopoietic Stem

Abstract Abstract 3394 Hematopoietic stem cells (HSC) replenish the cellular components of the blood throughout life by a homeostatic process in which the majority of HSCs remain quiescent while a small percentage enter the cell cycle to either self-review or differentiate. During inflammatory responses to infections, Interferons (IFNa, IFNg) perturb HSC homeostasis, presumably in response to the demand for increased numbers of inflammatory cells. Previous studies have highlighted an apparent paradox, i.e. IFNs suppress the proliferation of normally cycling murine hematopoietic progenitor cells (HPCs), yet increase the fraction of normally quiescent Sca+ HSCs that proliferate. To investigate the mechanisms underlying this paradox, we dissected the dynamics of cell surface phenotypes, cell cycle kinetics, pro- and anti-apoptotic pathways within the HSC and HPC compartments in response to pIpC and IFNs both in vivo and in vitro. Forty-eight hours after pIpC injection, bone marrow (BM) cellularity declined by 60%, the proportion of Sca- kit+ HPCs fell from 0.45% to 0.05%, while the proportion of BM cells with the Sca+ kit+ HSC phenotype increased from 0.17 to 0.26%. To determine whether the increase in Sca+kit+ cells was due to proliferation of HSCs or upregulation of Sca-1 on HPCs, we cultured purified CD150+ Sca-Kit+ HPCs and CD150+Sca+kit+ HSCs in vitro with IFNa, IFNg, or PBS. Sca expression was induced on previously Sca- HPCs, and the level of Sca expression on HSCs was also increased. This induction was detectable as early as 6 hours after treatment and accompanied by an increase in Sca mRNA. BrdU incorporation into both HPC and HSC populations decreased from pre-treatment baselines, further indicating that the increase in cells with the HSC phenotype was not due to HSC proliferation, but rather the appearance of cycling HPCs within the HSC staining gate following IFN-induced upregulation of Sca. Staining with FITC-DEVD-FMK identified active cleaved capase-3 in pIpC- or IFN-treated cells, suggesting that the reduced cellularity following IFN reflected a cellular stress that killed Lin+ precursors cells and some HPCs, but spared HSCs. In contrast to lin+kit- precursors, all kit + HPCs and HSCs expressed bcl-2, suggesting that expression of anti-apoptotic proteins may prevent IFN-induced stress from resulting in HSC/HPC apoptosis despite the initial triggering of caspase-3 cleavage. In summary, acute treatment with IFNs has anti-proliferative effects on all hematopoietic cells, including precursors, HPCs and HSCs, with the apparent increase in HSC proliferation the result of HPCs masquerading as Sca+HSCs after exposure to IFN. Unlike precursors, HSCs and some HPCs survive treatment to IFNs despite activation of cleaved caspase-3, possibly due to their expression of bcl-2, and likely related anti-apoptotic regulators. The previously observed increase in HSC proliferation days and weeks following IFN treatment is most likely due to the homeostatic response of HSCs to the depopulation of the precursor and HPCs caused by acute IFN exposure. Disclosures: No relevant conflicts of interest to declare.

Differential Response of Fanconi Anemia Hematopoietic Stem and Progenitor Cells to Oxidative and Oncogenic Stresses

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3593-3593
Author(s):  
Wei Du ◽  
Surya Amarachintha ◽  
Erden Ozlem ◽  
Qishen Pang
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Progenitor Cells ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Arginine Methylation ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Oncogenic Stress

Abstract Members of the Fanconi anemia (FA) protein family are involved in DNA damage response. A common damage to DNA in vivo is oxidative stress, and compelling evidence suggests that FA cells are in an in vivo pro-oxidant state. In response to oncogenic activation, normal cells induce genetically encoding programs that prevent deregulated proliferation and thus protect multicellular organisms from cancer progression. How FA cells respond to oxidative DNA damage and oncogenic stress is largely unknown. By employing an in vivo stress-response model expressing the Gadd45b-luciferase transgene, we show here that hematopoietic stem and progenitor cells (HSPCs) from mice deficient for the FA gene Fanca or Fancc differentially responded to oxidative and oncogenic stresses. Compared to wild-type controls, Fanca-/- or Fancc-/- HSPCs exhibited a persistent response to oxidative stress. Mechanistically, we demonstrated that accumulation of unrepaired DNA damage, particularly in oxidative damage-sensitive genes, was responsible for the long-lasting response in FA HSPCs. In contrast, using two inducible models of oncogenic activation (LSL-K-rasG12D and MycER), we identify a short-lived response of FA HSPCs to oncogenic insults both in vitro and in vivo. Mechanistic studies revealed that loss of Fanca or Fancc impaired oncogenic stress-induced senescence (OIS), and genetic correction of Fanca or Fancc deficiency restored OIS in HSPCs. Furthermore, FA deficiency compromised K-rasG12D-induced arginine methylation of p53 mediated by the protein arginine methyltransferase 5 (PRMT5). Finally, forced expression of PRMT5 in HSPCs from LSL-K-rasG12D/CreER-Fanca-/- mice prolonged oncogenic response and delayed leukemia development in recipient mice. Taken together, our study demonstrates differential responses of HSPCs to oxidative and oncogenic stresses and identifies the FA pathway as an integral part of this versatile cellular mechanism. Disclosures No relevant conflicts of interest to declare.

scholarly journals Megakaryocytic TGFβ1 Partitions Hematopoiesis into Amplifying Stem and Progenitor Cells and Maturing Effector Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 81-81
Author(s):  
Silvana Di Giandomenico ◽  
Pouneh Kermani ◽  
Nicole Molle ◽  
Mia Yabut ◽  
Fabienne Brenet ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Progenitor Cells ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Erythroid Precursors ◽  
Stem And Progenitor Cells

Abstract Background: Chronic anemia is a significant problem affecting over 3 million Americans annually. Therapies are restricted to transfusion and Erythropoietin Stimulating Agents (ESA). There is a need for new approaches to treat chronic anemia. Immature erythroid progenitors are thought to be continuously produced and then permitted to survive and mature if there is sufficient erythropoietin (Epo) available. This model is elegant in that oxygen sensing within the kidney triggers Epo production so anemia can increase Epo and promote erythroid output. However, during homeostasis this model suggests that considerable energy is used to produce unneeded erythroid progenitors. We searched for independent control and compartmentalization of erythropoiesis that could couple early hematopoiesis to terminal erythroid commitment and maturation. Methods: We previously found the proportion of bone marrow megakaryocytes (MKs) staining for active, signaling-competent TGFβ transiently increases during bone marrow regeneration after chemotherapy. To assess the functional role of Mk-TGFβ, we crossed murine strains harboring a floxed allele of TGFβ1 (TGFβ1Flox/Flox) littermate with a Mk-specific Cre deleter to generate mice with Mk-specific deletion of TGFβ1 (TGFβ1ΔMk/ΔMk). We analyzed hematopoiesis of these mice using high-dimensional flow cytometry, confocal immunofluorescent microscopy and in vitro and in vivo assays of hematopoietic function (Colony forming assays, and in vivo transplantation). Results: Using validated, 9-color flow cytometry panels capable of quantifying hematopoietic stem cells (HSCs) and six other hematopoietic progenitor populations, we found that Mk-specific deletion of TGFβ1 leads to expansion of immature hematopoietic stem and progenitor cells (HSPCs) (Fig1A&B). Functional assays confirmed a more than three-fold increase in hematopoietic stem cells (HSCs) capable of serially-transplanting syngeneic recipients in the bone marrow (BM) of TGFβ1ΔMk/ΔMk mice compared to their TGFβ1Flox/Flox littermates. Expansion was associated with less quiescent (Go) HSCs implicating Mk-TGFβ in the control of HSC cell cycle entry. Similarly, in vitro colony forming cell assays and in vivo spleen colony forming assays confirmed expansion of functional progenitor cells in TGFβ1ΔMk/ΔMk mice. These results place Mk-TGFβ as a critical regulator of the size of the pool of immature HSPCs. We found that the blood counts and total BM cellularity of TGFβ1ΔMk/ΔMk mice was normal despite the dramatic expansion of immature HSPCs. Using a combination of confocal immunofluorescence microscopy (cleaved caspase 3) (Fig1C) and flow cytometry (Annexin V and cleaved caspase 3) (Fig1D), we found ~10-fold greater apoptosis of mature precursor cells in TGFβ1ΔMk/ΔMk BM and spleens. Coincident with this, we found the number of Epo receptor (EpoR) expressing erythroid precursors to be dramatically increased. Indeed, apoptosis of erythroid precursors peaked as they transitioned from dual positive Kit+EpoR+ precursors to single positive cells expressing EpoR alone. Epo levels were normal in the serum of these mice. We reasoned that the excess, unneeded EpoR+ cells were not supported physiologic Epo levels but might respond to even small doses of exogenous Epo. Indeed, we found that the excess erythroid apoptosis could be rescued by administration of very low doses of Epo (Fig1E). Whereas TGFβ1Flox/Flox mice showed minimal reticulocytosis and no change in blood counts, TGFβ1ΔMk/ΔMk mice responded with exuberant reticulocytosis and raised RBC counts almost 10% within 6 days (Fig. 1F). Low dose Epo also rescued survival of Epo receptor positive erythroid precursors in the bone marrow, spleen and blood of TGFβ1ΔMk/ΔMk mice. TGFβ1ΔMk/ΔMk mice showed a similarly brisk and robust erythropoietic response during recovery from phenylhydrazine-induced hemolysis (Fig.1G). Exogenous TGFβ worsened BM apoptosis and caused anemia in treated mice. Pre-treatment of wild-type mice with a TGFβ signaling inhibitor sensitized mice to low dose Epo. Conclusion: These results place megakaryocytic TGFβ1 as a gate-keeper that restricts the pool of immature HSPCs and couples immature hematopoiesis to the production of mature effector cells. This work promises new therapies for chronic anemias by combining TGFβ inhibitors to increase the outflow of immature progenitors with ESAs to support erythroid maturation. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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