scholarly journals Induced cell-autonomous neutropenia systemically perturbs hematopoiesis in Cebpa enhancer-null mice

2021 ◽  
Author(s):  
Roberto Avelino ◽  
Roger Mulet-Lazaro ◽  
Marije Havermans ◽  
Remco Hoogenboezem ◽  
Leonie Smeenk ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Wild Type Mouse ◽  
Mouse Bone Marrow ◽  
Severe Dysplasia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Factor C

The transcription factor C/EBPa initiates the neutrophil gene expression program in the bone marrow. Knockouts of the Cebpa gene or its +37kb enhancer in mice show two major findings: (1) neutropenia in bone marrow and blood; (2) decrease in long-term hematopoietic stem cell (LT-HSC) numbers. Whether the latter finding is cell autonomous (intrinsic) to the LT-HSCs or an extrinsic event exerted on the stem cell compartment remained an open question. Flow cytometric analysis of the Cebpa +37kb enhancer knockout model revealed that the reduction in LT-HSC numbers observed was proportional to the degree of neutropenia. Single cell transcriptomics of wild type mouse bone marrow showed that Cebpa is predominantly expressed in early myeloid-biased progenitors, but not in LT-HSCs. These observations suggest that the negative effect on LT-HSCs is an extrinsic event caused by neutropenia. We transplanted whole bone marrows from +37kb enhancer deleted mice and found that 40% of the recipient mice acquired full blown neutropenia with severe dysplasia and a significant reduction in the total LT-HSC population. The other 60% showed initial signs of myeloid differentiation defects and dysplasia when they were sacrificed, suggesting they were in an early stage of the same pathological process. This phenotype was not seen in mice transplanted with wild type bone marrow cells. Altogether, these results indicate that Cebpa-enhancer deletion causes cell autonomous neutropenia, which reprograms and disturbs the quiescence of HSCs, leading to a systemic impairment of the hematopoietic process.

Understanding the Impact of Inflammation on Hematopoietic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2629-2629
Author(s):  
Ying Zhao ◽  
Flora Ling ◽  
Hong-Cheng Wang ◽  
Xiao-Hong Sun
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Chronic Inflammation ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Inflammatory Conditions ◽  
The Impact ◽  
Lps Treatment ◽  
Marrow Cells

Abstract Abstract 2629 The overall objectives of this study are to investigate the impact of inflammatory conditions on hematopoietic stem cell (HSC) maintenance and to elucidate the underlying mechanisms. HSCs are exposed to a variety of inflammatory conditions through life. How these conditions influence the integrity of HSCs is a fundamental issue of clinical importance but it is poorly understood. Equally unknown is the molecular regulation of HSC maintenance during inflammatory. In this context, our focus is on the role of basic helix-loop-helix (bHLH) proteins, which include transcription activators such as E2A proteins and their inhibitors including Id proteins. We and others have shown that these regulators are involved in normal hematopoiesis such as stem cell function and lineage specific differentiation. Recently, we have obtained evidence to suggest that signaling through Toll-like receptors (TLRs), which is closely linked to inflammation, causes down-regulation of E2A function by stimulating Id1 expression. Therefore, we hypothesize that inflammatory conditions causes down-regulation of E protein function, which disturbs the quiescence of long-term (LT)-HSC, leading to stem cell exhaustion over time. To test this hypothesis, we induced chronic inflammation in wild type and Id1-/- mice by daily injection of 1 mg of LPS, i.p. for 30 days. Peripheral blood was collected on days 15 and 30 and levels of a panel of inflammatory cytokines were assayed using a Luminex multiplex kit. On day 15, dramatic increases were found in the levels of IL-10, IL-6, KC and TNFα but not IFN-γ, IL12-p70 and IL-1β. Interestingly, levels of IL-6 and TNFα were significantly lower in Id1-/- mice compared to wild type mice. By day 30 of LPS treatment, levels of these cytokines returned to the levels in animals without LPS injection. These results suggest that this chronic LPS treatment indeed elicited an inflammatory response that included transient elevation of inflammatory cytokines. Whether secretion of these cytokines has any direct effects on HSCs remains to be determined. To measure HSC activity in these LPS-treated mice, we performed serial bone marrow transplant assays. Lin−Sca-1+c-kit+ (LSK) stem/progenitor cells were isolated from wild type or Id1-/- mice treated with or without LPS. These cells were transplanted into lethally irradiated CD45.1+ recipients along with equal numbers of YFP-expressing LSK as competitors. Six weeks later, cohorts of mice were sacrificed and bone marrow cells were collected. Pooled whole bone marrow cells within each cohort were injected into lethally irradiated secondary recipients. Secondary recipients were sacrificed 8 and 16 weeks post transplant. For assessment of primary and secondary engraftment, bone marrow cells were examined for expression of donor and lineage specific markers. Robust engraftment was observed in primary or secondary recipients. Donor derived cells were then gated for YFP− and YFP+ cells, which separate cells originated from tester and competitor LSK, respectively. While YFP− and YFP+ cells engrafted equivalently in primary recipients transplanted with cells treated with or without LPS, LPS treatment of wild type mice caused a great disparity in secondary recipients. In contrast, HSC in Id1-/- mice did not appear to be affected by the same treatment even though HSCs in Id1 deficient mice are normally lower in numbers and activities as we previously reported. These results suggest that chronic inflammation diminishes the LT-stem cell activity and this may involve the up-regulation of Id1 expression. To investigate the underlying mechanism, we performed label retaining assays to examine the quiescence of LT-HSCs. We found that BrdU-labeling in HSCs was 2-fold lower in mice treated with LPS compared to the untreated controls, suggesting that treatment with LPS promoted the cycling of HSCs, thus impairing their stem cell function. Taken together, our study illustrates that chronic inflammation has a detrimental effect on LT-stem cell activity. Although HSCs have an enormous capability to repopulate the bone marrow by compensatory proliferation, pro-longed inflammation could eventually lead to stem cell exhaustion and seriously compromise hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

The F-Box Protein NIPA Regulates the Hematopoietic Stem Cell Pool

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2330-2330
Author(s):  
Stefanie Kreutmair ◽  
Anna Lena Illert ◽  
Rouzanna Istvanffy ◽  
Melanie Sickinger ◽  
Christina Eckl ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Wild Type ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Pool ◽  
F Box Protein ◽  
Stem Cell Pool

Abstract Abstract 2330 Hematopoietic stem cells (HSCs) are characterized by their ability to self-renewal and multilineage differentiation. Since mostly HSCs exist in a quiescent state re-entry into cell cycle is essential for their regeneration and differentiation and the expression of numerous cell cycle regulators must be tightly controlled. We previously characterized NIPA (Nuclear Interaction Partner of ALK) as a F-Box protein that defines an oscillating ubiquitin E3 ligase targeting nuclear cyclin B1 in interphase thus contributing to the timing of mitotic entry. To examine the function of NIPA on vivo, we generated NIPA deficient animals, which are viable but sterile due to a defect in recombination and testis stem cell maintenance. To further characterize the role of NIPA in stem cell maintenance and self-renewal we investigated hematopoiesis in NIPA deficient animals. Peripheral blood counts taken at different ages revealed no apparent difference between NIPA knockout and wild type mice in numbers and differentiation. In contrast, looking at the hematopoietic stem cell pool, FACS analyses of bone marrow showed significantly decreased numbers of Lin-Sca1+cKit+ (LSK) cells in NIPA deficient animals, where LSKs were reduced to 40% of wild type littermates (p=0,0171). This effect was only apparent in older animals, where physiologically higher LSK numbers have to compensate for the exhaustion of the stem cell pool. Additionally, older NIPA deficient mice have only half the amount of multi myeloid progenitors (MMPs) in contrast to wild type animals. To examine efficient activation of stem cells to self-renew in response to myeloid depression, we treated young and old mice with the cytotoxic drug (5-FU) four days before bone marrow harvest. As expected, 5-FU activated hematopoietic progenitors in wild type animals, whereas NIPA deficient progenitors failed to compensate to 5-FU depression, e.g. LSKs of NIPA knockout mice were reduced to 50% of wild type levels (p<0.001), CD150+CD34+ Nipa deficient cells to 20% of wild type levels (p<0.0001). Interestingly, these effects were seen in all NIPA deficient animals independent of age, allowing us to trigger the self-renewal phenotype by activating the hematopoietic stem cell pool. Using competitive bone marrow transplantation assays, CD45.2 positive NIPA deficient or NIPA wild type bone marrow cells were mixed with CD45.1 positive wild type bone marrow cells and transplanted into lethally irradiated CD45.2 positive recipient mice. Thirty days after transplantation, FACS analysis of peripheral blood and bone marrow showed reduced numbers of NIPA knockout cells in comparison to NIPA wild type bone marrow recipient mice. This result was even more severe with aging of transplanted mice, where NIPA deficient cells were reduced to less than 10% of the level of wild type cells in bone marrow of sacrificed mice 6 months after transplantation, pointing to a profound defect in repopulation capacity of NIPA deficient HSCs. Taken together our results demonstrate a unique and critical role of NIPA in regulating the primitive hematopoietic compartment as a regulator of self-renewal, cycle capacity and HSC expansion. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic Stem Cell Engraftment in Bone Marrow Is Dependent upon Gsα.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 857-857
Author(s):  
Gregor B. Adams ◽  
Ian R. Alley ◽  
Karissa T. Chabner ◽  
Ung-il Chung ◽  
Emily S. Marsters ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Conditional Knockout ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract During development, hematopoietic stem cells (HSCs) translocate from the fetal liver to the bone marrow, which remains the site of hematopoiesis throughout adulthood. In the bone marrow the HSCs are located at the endosteal surface, where the osteoblasts are a key component of the stem cell niche. The exogenous signals that specifically direct HSCs to the bone marrow have been thought to include stimulation of the chemokine receptor CXCR4 by its cognate ligand stromal derived factor-1α (SDF-1α or CXCL12). However, experiments in which CXCR4−/− fetal liver hematopoietic cells were transplanted into wild-type hosts demonstrated efficient engraftment of the HSCs in the bone marrow. In addition, treatment of HSCs with inhibitors of Gαi-coupled signaling, which blocks transmigration towards SDF-1αin vitro, does not affect bone marrow homing and engraftment in vivo. Therefore, we examined whether Gsα-coupled mechanisms play a key role in the engraftment of the HSCs in the bone marrow environment. Utilizing an inducible-conditional knockout of Gsα, we found that deletion of the gene in hematopoietic bone marrow cells did not affect their ability to perform in the in vitro primitive CFU-C or LTC-IC assay systems. However, Gsα−/− cells were unable to establish effective hematopoiesis in the bone marrow microenvironment in vivo in a competitive repopulation assay (41.1% contribution from wild-type cells versus 1.4% from knockout cells). These effects were not due to an inability of the cells to function in the bone marrow in vivo as deletion of Gsα following establishment of hematopoiesis had no effects on the HSCs. Examining the ability of the HSCs to home to the bone marrow, though, demonstrated that deletion of Gsα resulted in a marked impairment of the ability of the stem cells to localize to the marrow space (approximately 9-fold reduction in the level of primitive cell homing). Furthermore, treatment of BM MNCs with an activator of Gsα augmented the cells homing and thus engraftment potential. These studies demonstrate that Gsα is critical to the localization of HSCs to the bone marrow. Which receptors utilize this pathway in this context remains unknown. However, Gsα represents a previously unrecognized signaling pathway for homing and engraftment of HSCs to bone marrow. Pharmacologic activation of Gsα in HSC ex vivo prior to transplantation offers a potential method for enhancing stem cell engraftment efficiency.

Deregulation of MicroRNA Underlies Severe Congenital Neutropenia Pathogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 313-313
Author(s):  
ChinavenMeni S. Velu ◽  
Avinash M Baktula ◽  
Tristan Bourdeau ◽  
H. Leighton Grimes
Keyword(s):  
Bone Marrow ◽  
Zinc Finger Protein ◽  
Bone Marrow Cells ◽  
Flow Cytometric Analysis ◽  
Leukemia Cell ◽  
Severe Congenital Neutropenia ◽  
Wild Type ◽  
Congenital Neutropenia ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract The zinc finger protein Growth factor independent-1 (Gfi1) is a transcriptional repressor that regulates hematopoietic stem cell (HSC) maintenance and granulocytic lineage differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 (encoding GFI1N382S) which generate dominant negative acting proteins. GFI1N382S proteins sequester limiting cofactors to deregulate a subset of GFI1 target genes. Here we show that Gfi1 is a master regulator of microRNAs and suggest that that transcriptional control of microRNA genes is critical for GFI1N382S-associated SCN phenotypes. First, the expression of Gfi1 and miR21 and miR-196 is reciprocal: 1) in wild type and Gfi1−/− marrow cells, 2) during normal differentiation from common myeloid progenitors (CMP) to granulocyte monocyte progenitors (GMP), 3) during treatment-induced differentiation of human myeloid leukemia cell lines, and 4) upon conditional deletion of Gfi1 in primary sorted murine CMP and GMP. Biochemical analyses reveal that miR21 and miR-196 are direct transcriptional targets of Gfi1. Subsequently, forced expression of wild type Gfi1 rescues expression of microRNA in Gfi1−/− Lin- bone marrow cells, while forced expression of Gfi1N382S in wild type Lin- bone marrow cells significantly deregulates miR-21 and miR-196 expression. Similarly, we demonstrate elevated miR21 and miR196b levels in CD34+ cells from a GFI1N382S SCN patient. Flow cytometric analysis and colony assays reveal that the overexpression or knockdown of either miR induces changes in myeloid development, but that co-expression of both miR (as seen in Gfi1−/− mice and GFI1N382S SCN patients) completely blocks G-CSF-induced granulopoiesis. These data provide a molecular understanding of SCN disease pathogenesis.

Chronic RPS19 Deficiency Leads to Bone Marrow Failure In a Mouse Model for Diamond-Blackfan Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 193-193
Author(s):  
Pekka Jaako ◽  
Johan Flygare ◽  
Karin Olsson ◽  
Ronan Quere ◽  
Jonas Larsson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Bone Marrow Failure ◽  
Flow Cytometric Analysis ◽  
Wild Type ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem ◽  
Flow Cytometric

Abstract Abstract 193 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Of the many different DBA disease genes known, all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). The generation of animal models for DBA is pivotal in order to understand the disease mechanisms and to evaluate novel therapies. We have generated two mouse models for RPS19-deficient DBA by taking advantage of RNA interference (Jaako et al, 2009 ASH meeting abstract). These models contain RPS19-targeting shRNAs expressed by a doxycycline-responsive promoter downstream of the Collagen A1 locus allowing an inducible and dose-dependent regulation of shRNA. As we have previously reported, the induction of RPS19 deficiency results in a reduction in the number of erythrocytes, platelets and white blood cells, and flow cytometric analysis of bone marrow after a short-term induction reveals increased frequencies of hematopoietic stem and progenitor cells reflecting the onset of stress hematopoiesis. In the current study we have analyzed the long-term effect of RPS19 deficiency in bone marrow. In contrast to a short-term induction, flow cytometric analysis of bone marrow after 51 days revealed decreased frequencies of hematopoietic stem and progenitor cells that correlate with a severe peripheral blood phenotype. In addition, we observed a 3–6 fold increase in apoptosis in RPS19-deficient bone marrow compared to controls based on TUNEL assay. Furthermore, transplantation of whole bone marrow cells from transgenic donors into wild type lethally irradiated recipients confirms that the observed phenotype is autonomous to the blood system. To study whether long-term RPS19 deficiency functionally impairs hematopoietic stem cells, we pre-induced mice for 30 days followed by 15 days without doxycycline to restore the RPS19 expression. Mice were sacrificed and total bone marrow cells were transplanted together with wild-type competitor cells (1:1) into wild type lethally irradiated recipients without doxycycline. This experimental setting allows us to assess the functionality of pre-induced hematopoietic stem cells in absence of ribosomal stress. Flow cytometric analysis of peripheral blood one month after transplantation clearly demonstrates decreased reconstitution from pre-induced donors compared to the wild-type competitor. While this time point reflects mainly the function of transplanted progenitors, long-term analysis of hematopoietic stem cell function in these recipients is ongoing. To study the molecular mechanisms underlying the hematopoietic defect we performed comparative microarray analysis. We chose to analyze preCFU-E/CFU-E erythroid progenitors since we have previously located the erythroid defect at the CFU-E – proerythroblast transition based on flow cytometry and clonogenic proliferation cultures of prospectively isolated erythroid progenitors. Microarray analysis of preCFU-E/CFU-E progenitors reveals deregulation of several genetic pathways, including a robust upregulation of p53 pathway genes, and these targets have been confirmed by real-time PCR. Furthermore, many of p53 target genes are also upregulated in the Lineage− Sca-1+ c-Kit+ (LSK) population that contains immature hematopoietic progenitors and stem cells suggesting that the activation of p53 is not restricted to the erythroid lineage. To ask whether increased activity of p53 can solely explain the hematopoietic phenotype, we have crossed our mouse model into a p53-null background. In summary, our data suggest that RPS19-deficient mice fail to uphold stress hematopoiesis for extended periods of time, with chronic RPS19 deficiency causing bone marrow failure. Disclosures: No relevant conflicts of interest to declare.

Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A–null mice

Blood ◽  
2003 ◽  
Vol 101 (2) ◽  
pp. 517-523 ◽  
Author(s):  
Caryn Y. Ito ◽  
Carol Y. J. Li ◽  
Alan Bernstein ◽  
John E. Dick ◽  
William L. Stanford
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Bone Marrow Cells ◽  
Lymphocyte Activation ◽  
Wild Type ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Competitive Disadvantage ◽  
Colony Forming Units

Despite its wide use as a marker for hematopoietic stem cells (HSCs), the function of stem cell antigen–1 (Sca-1) (also known as lymphocyte activation protein–6A [Ly-6A]) in hematopoiesis remains poorly defined. We have previously established that Sca-1−/−T cells develop normally, although they are hyperresponsive to antigen. Here, we report detailed analysis of hematopoiesis in Sca-1–deficient animals. The differentiation potential of Sca-1–null bone marrow was determined from examination of the most mature precursors (culture colony-forming units [CFU-Cs]) to less committed progenitors (spleen CFUs [CFU-Ss]) to long-term repopulating HSCs. Sca-1–null mice are mildly thrombocytopenic with a concomitant decrease in megakaryocytes and their precursors. Bone marrow cells derived fromSca-1−/− mice also have decreased multipotential granulocyte, erythroid, macrophage, and megakaryocyte CFU (GEMM-CFU) and CFU-S progenitor activity. Competitive repopulation assays demonstrated that Sca-1−/−HSCs are at a competitive disadvantage compared with wild-type HSCs. To further analyze the potential of Sca-1−/−HSCs, serial transplantations were performed. While secondary repopulations using wild-type bone marrow completely repopulatedSca-1−/−mice, Sca-1−/−bone marrow failed to rescue one third of lethally irradiated wild-type mice receiving secondary bone marrow transplants from irradiation-induced anemia and contributed poorly to the surviving transplant recipients. These data strongly suggest that Sca-1 is required for regulating HSC self-renewal and the development of committed progenitor cells, megakaryocytes, and platelets. Thus, our studies conclusively demonstrate that Sca-1, in addition to being a marker of HSCs, regulates the developmental program of HSCs and specific progenitor populations.

SIRT1 Deacetylase Is Essential for Hematopoietic Stem Cell Activity Via Regulation of Foxo3.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2315-2315 ◽  
Author(s):  
Pauline Rimmele ◽  
Carolina L. Bigarella ◽  
Valentina d'Escamard ◽  
Brigitte Izac ◽  
David Sinclair ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Leukemic Stem Cells ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract Abstract 2315 SIRT1 is a member of the NAD-dependent family of sirtuin deacetylases with critical functions in cellular metabolism, response to stress and aging. Although SIRT1 is clearly a regulator of embryonic stem cells, reports on the function of SIRT1 in adult hematopoietic stem cell (HSC) have been conflicting. While SIRT1 was positively associated with HSC activity on a genetic screen, using a germline deletion of SIRT1 three groups found SIRT1 to be dispensable for adult HSC. Here, we first showed that nuclear SIRT1 expression is enriched in bone marrow-derived Lin−Sca1+cKit+ (LSK) cells, as compared to total bone marrow cells. Germline deletion of SIRT1 is associated with developmental defects and high perinatal mortality resulting in only 10% of mice reaching adulthood. To circumvent the potential developmental adaptation of these mice, we used an adult-tamoxifen inducible SIRT1 knockout mouse model. Full-length SIRT1 protein was nearly undetectable in the bone marrow and spleen of SIRT1−/− mice. Analysis of wild type and SIRT1−/− bone marrow cells, 4 weeks after tamoxifen treatment, showed that loss of SIRT1 increased the size and frequency of the LSK compartment. Interestingly, this was associated with a significant decrease in the frequency of long-term repopulating HSC as determined by SLAM markers (CD48−CD150+LSK) within LSK cells. This decrease was even more pronounced with time. In agreement with these results, the long-term repopulation ability of CD48−CD150+LSK cells is severely compromised in SIRT1−/− mice as measured 16 weeks after transplantation, strongly suggesting that SIRT1 is essential for long-term HSC function. Thus, loss of SIRT1 results in loss of long-term repopulating stem cells in favor of total LSK cells that is a more heterogeneous population of stem cells. SIRT1 has several substrates with a potential function in HSC. Among these, we focused on Foxo3 Forkhead transcription factor which is essential for the maintenance of hematopoietic and leukemic stem cell pool. Despite the importance of Foxo3 to the control of HSC function, mechanisms that regulate Foxo3 activity in HSC remain unknown. Negative regulation of FoxOs by AKT phosphorylation promotes their cytosolic localization in response to growth factors stimulation. Interestingly, Foxo3 is constitutively nuclear in bone marrow LSK and in leukemic stem cells, strongly suggesting that negative phosphorylation may not be the sole Foxo3 regulatory mechanism in these stem cells. FoxO proteins are regulated by several post-translational modifications including acetylation in addition to phosphorylation, although the impact of acetylation on Foxo3 function remains unresolved. Therefore, we asked whether regulation of adult HSC activity by SIRT1 deacetylase is mediated by Foxo3. The in vivo injection of sirtinol, a SIRT1 inhibitor, for 3 weeks compromised significantly the long-term repopulation capacity of wild type but not Foxo3−/− HSC as measured by the repopulation ability of CD48−CD150+LSK cells in lethally irradiated mice after 16 weeks. These results suggest that Foxo3 is likely to be required for SIRT1 regulation of HSC activity. In agreement with this, we showed that in contrast to wild type LSK cells, Foxo3 is mostly cytoplasmic in SIRT1−/− LSK cells, indicating that loss of SIRT1 is sufficient to translocate Foxo3 to the cytosol and presumably inhibit its activity. We further showed that ectopically expressed acetylation-mimetic mutant of Foxo3 where all putative acetyl-lysine residues are mutated to glutamine, in bone marrow mononuclear cells, is mostly localized in the cytosol in contrast to wild type Foxo3 protein and results in significant decrease of colony-forming unit-spleen (CFU-S) activity. Using pharmacological antagonism as well as conditional deletion of SIRT1 in adult HSC, we identified a critical function for SIRT1 in the regulation of long-term HSC activity. Our results contrast with previously published data obtained from germline deleted SIRT1 mice, and suggest that the use of a conditional approach is essential for unraveling SIRT1 function in adult tissues. Our data also suggest that SIRT1 regulation of HSC activity is through activation of Foxo3. These findings are likely to have an important impact on our understanding of the regulation of hematopoietic and leukemic stem cells and may be of major therapeutic value for hematological malignancies and disorders of stem cells and aging. Disclosures: No relevant conflicts of interest to declare.

Natural Ligands for Rxra, but Not Rara, Are Observed in Mouse Bone Marrow Cells, and Are Augmented in Response to 5FU and GCSF

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4339-4339
Author(s):  
Haixa Niu ◽  
John Welch
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Transcription Factors ◽  
Retinoic Acid ◽  
Bone Marrow Cells ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Natural Ligand ◽  
Mouse Bone Marrow Cells ◽  
Marrow Cells

Abstract The retinoid receptors RARA and RXRA are both important transcription factors that influence hematopoietic cell growth and differentiation. RARA and RXRA are both ligand-dependent transcription factors. The natural ligand for RARA is thought to be all-trans retinoic acid (ATRA), the natural ligand for RXRA is unclear, although 9-cis retinoic acid is an active ligand in vitro. Aldehyde dehydrogenase (ALDH) metabolism is the rate-limiting step in ATRA synthesis, and ALDH activity is associated with stem cell self-renewal in hematopoietic stem cell (HSCs) and in cancer stem cells. It is unknown whether these two functions are related. In order to measure the presence and regulation of natural retinoids in vivo, we developed a UAS-GFP reporter mouse. We found this system to be highly sensitive and specific. We transplanted UAS-GFP mouse bone marrow cells with virus expressing either Gal4-RARA-IRES-mCherry (Gal4-RARA-IC) or Gal4-RXRA-IRES-mCherry (Gal4-RXRA-IC). Because PML-RARA is proposed to act as a dominant-negative, and ATRA induces significant differentiation of myeloid cells, we were surprised to observe no GFP expression in mice transplanted with Gal4-RARA-IC. Instead, we observed GFP in mice transplanted with Gal4-RXRA-IC, consistent with natural RXRA ligands, but not RARA ligands, in bone marrow cells. When we treated mice with either ATRA or bexarotene, we observed that most hematopoietic cell types can respond to an active retinoid, with the exception of Kit+Lin-Sca+ HSCs, which had a significantly attenuated response. Ex vivo, we found that the P450 inhibitors liarozole and talarozole augmented response to retinoids in Kit+ hematopoietic stem/progenitor cells, suggesting that HSCs have high rates of retinoid degradation via active P450 pathways, and thus maintain a retinoid deplete environment. We further tested whether hematopoietic cells might respond to hematopoietic stress through retinoid receptor signaling. We observed a significant increase in the number of GFP+ cells when Gal4-RXRA-IC transplanted mice were treated with either 5FU or GCSF, but no response in Gal4-RARA-IC transplanted mice. As a control, we repeated the studies using a Gal4-RXRA vector with the AF2 domain deleted. The RXRAdeltaAF2 mutation can bind to ligand, but does not respond to it, although it still can be activated through a heterodimeric partner. We observed no GFP induction by 5FU or GCSF with the RXRAdeltaAF2 mutation, suggesting that the GFP response is to natural RXRA ligands, and not to alternative signaling through a heterodimeric partner (e.g. Lxra or Pparg). These data suggest that HSCs maintain low levels of natural retinoids, and that the mechanism of stem-cell associated ALDH is therefore not through ATRA production and RARA activation. In addition, bone marrow cells are exposed to natural RXRA ligands, but not RARA ligands, under homeostatic conditions, and this increases during response to 5FU and GCSF, suggesting that ligand-dependent RXRA activation may play a critical role in hematopoietic response to 5FU and GCSF. Disclosures No relevant conflicts of interest to declare.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal

Blood ◽  
2021 ◽  
Author(s):  
Yuqing Yang ◽  
Andrew J Kueh ◽  
Zoe Grant ◽  
Waruni Abeysekera ◽  
Alexandra L Garnham ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
High Rate ◽  
Embryonic Patterning ◽  
Self Renewal ◽  
Hematopoietic Stem

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs). The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

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