Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 488-499 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard A. Ashmun ◽  
Donald Orlic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Blood Cell ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Immunoblot Analysis ◽  
Marrow Cells

Abstract The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

Enforced Expression of the GATA-2 Transcription Factor Blocks Normal Hematopoiesis

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 488-499 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard A. Ashmun ◽  
Donald Orlic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Blood Cell ◽  
Fluorescent Protein ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Immunoblot Analysis ◽  
Marrow Cells

The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1+Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1+ Lin−cells. Although Sca-1+ Lin− bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1+Lin− cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.

scholarly journals Development of a Novel Selective Amplifier Gene for Controllable Expansion of Transduced Hematopoietic Cells

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3884-3892 ◽  
Author(s):  
Keiko Ito ◽  
Yasuji Ueda ◽  
Masaki Kokubun ◽  
Masashi Urabe ◽  
Toshiya Inaba ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Selective Amplifier ◽  
Marrow Cells

Abstract To overcome the low efficiency of gene transfer into hematopoietic cells, we developed a novel system for selective expansion of transduced cells. To this end, we constructed a chimeric cDNA (GCRER) encoding the fusion protein between the granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain (HBD) of the estrogen receptor (ER) as a selective amplifier gene. Use of the intracellular signaling pathway of G-CSFR was considered to be appropriate, because G-CSF has the ability not only to stimulate the neutrophil production, but also to expand the hematopoietic stem/progenitor cell pool in vivo. To activate the exogenous G-CSFR signal domain selectively, the estrogen/ER-HBD system was used as a molecular switch in this study. When the GCRER gene was expressed in the interleukin-3 (IL-3)–dependent murine cell line, Ba/F3, the cells showed IL-3–independent growth in response to G-CSF or estrogen. Moreover, the Ba/F3 cells transfected with the Δ(5-195)GCRER, whose product lacks the extracellular G-CSF–binding domain, did not respond to G-CSF, but retained the ability for estrogen-dependent growth. Further, murine bone marrow cells transduced with the GCRER or Δ(5-195)GCRER gene with retroviral vectors formed a significant number of colonies in response to estrogen, as well as G-CSF, whereas estrogen did not stimulate colony formation by untransduced murine bone marrow cells. It is noteworthy that erythroid colonies were apparently formed by the bone marrow cells transduced with the GCRER gene in the presence of estrogen without the addition of erythropoietin, suggesting that the signals from the G-CSFR portion of the chimeric molecules do not preferentially induce neutrophilic differentiation, but just promote the differentiation depending on the nature of the target cells. We speculate that when the selective amplifier genes are expressed in the primitive hematopoietic stem cells, the growth signal predominates and that the population of transduced stem cells expands upon estrogen treatment, even if some of the cells enter the differentiation pathway. The present study suggests that this strategy is applicable to the in vivo selective expansion of transduced hematopoietic stem cells.

Myeloid Transcription Factor C/EBPɛ Is Involved in the Positive Regulation of Lactoferrin Gene Expression in Neutrophils

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3141-3150 ◽  
Author(s):  
Walter Verbeek ◽  
Julie Lekstrom-Himes ◽  
Dorothy J. Park ◽  
Pham My-Chan Dang ◽  
Peter T. Vuong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Bone Marrow Cells ◽  
Granulocytic Differentiation ◽  
Direct Role ◽  
Gram Negative ◽  
Protein Levels ◽  
Factor C ◽  
Marrow Cells

Abstract Targeted mutation of the myeloid transcription factor C/EBPɛ in mice results in gram-negative septic death at 3 to 5 months of age. This study defines the underlying molecular defects in their terminal granulocytic differentiation. The mRNA for the precursor protein of the cathelin-related antimicrobial peptides was almost completely absent in the bone marrow cells of C/EBPɛ−/− mice. This finding may help explain their susceptibility to gram-negative sepsis, because both are bacteriocidal peptides with potent activity against gram-negative bacteria. Superoxide production was found to be reduced in both granulocytes and monocytes of C/EBPɛ−/− mice. While gp91 phox protein levels were normal, p47phox protein levels were considerably reduced in C/EBPɛ −/− granulocytes/monocytes, possibly limiting the assembly of the NADPH oxidase. In addition, expression of mRNA of the secondary and tertiary granule proteins, lactoferrin and gelatinase, were not detected, and levels of neutrophil collagenase mRNA were reduced in bone marrow cells of the knock-out mice. The murine lactoferrin promoter has a putative C/EBP site close to the transcription start site. C/EBPɛ bound to this site in electromobility shift assay studies and mutation of this site abrogated binding to it. A mutation in the C/EBP site reduced the activity of the promoter by 35%. Furthermore, overexpression of C/EBPɛ in U937 cells increased the activity of the wild-type lactoferrin promoter by 3-fold. In summary, our data implicate C/EBPɛ as a critical factor of host antimicrobial defense and suggests that it has a direct role as a positive regulator of expression of lactoferrin in vivo.

Nrf2, a Critical Regulator of Oxidative Stress, Is Required for HSC Function and Cytokine Response.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1492-1492
Author(s):  
Akil Merchant ◽  
Anju Singh ◽  
Giselle Joseph ◽  
Qiuju Wang ◽  
Ping Zhang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Increased Sensitivity ◽  
Marrow Cells

Abstract Abstract 1492 Poster Board I-515 Previous studies have established an important role for reactive oxygen species (ROS) in regulating the function and life-span of hematopoietic stem cells (HSC). Nuclear factor erythroid-2–related factor 2 (Nrf2) is a redox-sensitive transcription factor that regulates cellular responses to ROS and detoxification pathways implicated in chemoresistance, however, its role in normal stem cells is unknown. We analyzed Nrf2null mice and found increased total bone marrow cellularity, cKit+Sca1+Lin− (KSL) stem-progenitor cells, and long-term quiescent HSC (CD34−KSL) compared to wild type mice (p<0.05). Transplantation of equal numbers of KSL cells from Nrf2wt and Nrf2null resulted in a five-fold decrease in peripheral blood chimerism from Nrf2null derived cells at 16 weeks (15% wild type vs. 3% null, p<0.05). Unlike other models of deficiencies in genes associated with ROS handling, such as ATM or the FoxO family of transcription factors, basal ROS levels were not elevated in Nrf2null HSC. However, Nrf2null bone marrow cells demonstrated increased sensitivity to induced oxidative stress and in vitro treatment with H2O2 resulted in a 2 fold decrease in colony formation in methylcellulose. We also examined the in vivo sensitivity of Nrf2null cells to oxidative stress by irradiating (400 rads) stably chimeric mice 20 weeks following transplantation with either Nrf2wt or Nrf2null HSC. Mice receiving Nrf2null HSC demonstrated a 50% decrease in peripheral blood chimerism at 4 months following radiation compared to no change in Nrf2wt recipients (p<0.05) confirming that loss of Nrf2 leads to increased sensitivity to oxidative stress. Microarray gene expression analysis from Nrf2wt and Nrf2null mice revealed down regulation of the G-CSF cytokine receptor in Nrf2null HSC and suggested that defective cytokine signaling may contribute to the HSC dysfunction seen in Nrf2null bone marrow cells. To test this hypothesis, we attempted to rescue the function of Nrf2null HSC by treating mice with exogenous G-CSF. Nrf2wt and Nrf2null mice were treated with one week of daily G-CSF and then HSC were harvested and transplanted. In contrast to the defects in engraftment of untreated Nrf2null HSC, there was no significant difference in peripheral blood chimerism following transplantation of G-CSF treated Nrf2wt or Nrf2null HSC, thus demonstrating that G-CSF treatment could rescue the HSC defect in mutant mice. In conclusion, the Nrf2 transcription factor appears to be a novel and essential regulator of normal HSC function through the modulation of oxidative stress response and cytokine signaling. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Serial transplantation of methotrexate-resistant bone marrow: protection of murine recipients from drug toxicity by progeny of transduced stem cells

Blood ◽  
1990 ◽  
Vol 75 (2) ◽  
pp. 337-343 ◽  
Author(s):  
CA Corey ◽  
AD DeSilva ◽  
CA Holland ◽  
DA Williams
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Retroviral Vectors ◽  
Hematopoietic Stem ◽  
Genetic Sequences ◽  
Marrow Cells

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFRr, in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate- induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRr cDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

scholarly journals L3T4 antigen expression by hemopoietic precursor cells.

1989 ◽  
Vol 169 (4) ◽  
pp. 1473-1478 ◽  
Author(s):  
G G Frederickson ◽  
R S Basch
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Antigen Expression ◽  
Precursor Cells ◽  
Hemopoietic Precursor Cells ◽  
Marrow Cells

L3T4 (CD4) is expressed on immature hematopoietic cells. Sorting bone marrow cells on the basis of their expression of this antigen produces populations of cells that are markedly enriched for multipotential stem cells (CFU-s) and for myeloid precursors (CFU-c). We believe that L3T4 is transiently expressed by most, if not all, hematopoietic precursors early in their maturation. We suggest that the expression of CD4 molecules on the surface of immature precursors is required for their interaction with Ia bearing cells within the hemopoietic inductive microenvironment(s) of the marrow and thymus.

High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1758-1758
Author(s):  
Axel Schambach ◽  
Bernhard Schiedlmeier ◽  
Jens Bohne ◽  
Dorothee von Laer ◽  
Geoff Margison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

Implication of AML1/RUNX1 Function in the Homeostasis and Leukemic Transformation of Hematopoietic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4117-4117
Author(s):  
Motoshi Ichikawa ◽  
Masataka Takeshita ◽  
Susumu Goyama ◽  
Takashi Asai ◽  
Eriko Nitta ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Chimeric Protein ◽  
Chromosomal Translocation ◽  
Hematopoietic Stem ◽  
Quiescent Hscs ◽  
Marrow Cells

Abstract Transcription factor AML1/RUNX1, initially isolated from the t(8;21) chromosomal translocation in human leukemia, is essential for the development of multilineage hematopoiesis in mouse embryos. AML1 negatively regulates the number of immature hematopoietic cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. However, it remains yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population fraction and c-Kit-positive cells in the G0 cell cycle status were increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We also found an increase in HSC number within the AML1-deficient bone marrow using limiting dilution bone marrow transplantation assays. Thus, the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia. To identify mechanisms through which functional loss of AML1 exerts leukemogenic potential, we focused on the AML1-Evi-1 chimeric protein, which is generated by the t(3;21) chromosomal translocation and disturbs the normal function of AML1. We introduced AML1-Evi-1 and its mutants into murine bone marrow cells, and evaluated hematopoietic cell transformation by colony replating assays. The transforming activity of AML1-Evi-1 was impaired when any of the major functional domains of AML1-Evi-1 was lost. Moreover, overexpression of Evi-1 could not transform AML1-deleted bone marrow cells, suggesting that fusion of AML1 and Evi-1, rather than AML1 suppression and Evi-1 overexpression, is essential for AML1-Evi-1 leukemogenesis. Intriguingly, among the hematopoietic progenitor cell fractions, AML1-Evi-1 could transform only the uncommitted, immature hematopoietic cells, which contrasts with MLL-ENL, a chimeric protein generated in t(11;19) leukemia. AML1-Evi-1 transformed cells show a surface marker profile different from that of the cells transformed by AML1-MTG8/ETO, another leukemic gene product that also perturbs AML1 function. These results provide a valuable clue to a distinct mechanism determined by the Evi-1 moiety in the AML1-Evi-1 leukemogenesis and to a role of AML1 loss in the self-renewal of leukemic stem cells.

Balanced Wnt5a-Mediated Signaling Is Necessary for Normal Proliferation of Primitive Hematopoietic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2533-2533
Author(s):  
Benjamin Povinelli ◽  
Michael Baranello ◽  
Kathleen Kokolus ◽  
Michael Nemeth
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transcription Factors ◽  
Developmental Stage ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Cells Cultured ◽  
Marrow Cells

Abstract Abstract 2533 Poster Board II-510 Multiple members of the Wnt family of ligands have been implicated in the regulation of self-renewal and proliferation of hematopoietic stem cells (HSCs). Previously, we have observed that ex vivo expansion of HSCs in the presence of recombinant murine Wnt5a (rmWnt5a) resulted in increased hematopoietic repopulation. Based on these data, we hypothesized that Wnt5a is necessary for normal function of HSCs and hematopoietic progenitors (HPCs). Since Wnt5a deficiency (Wnt5a−/−) is perinatal lethal in vivo, we tested this hypothesis using in vitro Dexter stroma cultures established using whole bone marrow. To determine the ability of Wnt5a to support hematopoiesis in the context of the adult hematopoietic microenvironment, we cultured lineage-negative (lin−) HPCs on irradiated bone marrow stroma in the presence of 5 μg/ml Wnt5a-neutralizing antibody (Wnt5a-Ab). After two weeks, we observed that hematopoietic cells cultured on untreated stroma contained 4.8-fold more myeloid CFU (33.1 ± 12.3 CFU/104 cells) than cells cultured on Wnt5a-Ab stroma (6.9 ± 0.7 CFU; n = 3, p < .01). A similar difference was observed after 4 weeks (control: 16.0 ± 7.2 CFU/104 cells; Wnt5a-Ab: 1.3 ± 2.3 CFU; n = 3; p = .03). In the converse experiment, lin− HPCs were cultured on stroma in the presence of Wnt5a conditioned medium (Wnt5a-CM). We observed after two weeks that hematopoietic cells cultured on stroma with control-CM contained 4.4-fold more myeloid CFU (29.8 ± 13.5 CFU/104 cells) than cells cultured on Wnt5a-CM stroma (6.8 ± 2.2 CFU; n = 4, p = .02). Together, these data indicate that Wnt5a-mediated signaling must be balanced in order for normal hematopoiesis to occur. To determine if the effects of Wnt5a required the presence of developmental stage-specific factors, we established stroma cultures from fetal spleens harvested from E17.5 Wnt5a−/− mice and littermate controls and seeded them with lin− HPCs. We observed that hematopoietic cells cultured on control fetal spleen stroma contained 12.5-fold more myeloid CFU (72.6 ± 21.6 CFU/104 cells) than cells cultured on Wnt5a−/− fetal spleen stroma (5.8 ± 5.8; n = 6, p < .001) after two weeks. These data indicate that the effect of Wnt5a on hematopoietic cells is independent of the developmental stage of the surrounding microenvironment. To determine if the effects of Wnt5a were due to regulation of proliferation or differentiation of hematopoietic cells, we cultured bone marrow cells in cytokine-supplemented methylcellulose in the presence of 300 ng/ml rmWnt5a. We observed a 5.5-fold decrease in the number of myeloid CFU formed in cultures with rmWnt5a (14.1 ± 3.8/104 cells) compared to control (77.6 ± 5.1 CFU; n = 3; p < .001), suggesting that Wnt5a could be regulating both processes. In the converse experiment, we cultured bone marrow cells for 4 days in cytokine-supplemented serum-free media with the same dose of rmWnt5a after which equal numbers of cells were plated in rmWnt5a-free methylcellulose. We did not observe any difference in CFU frequency between control (19.3 ± 4.2 CFU/104 cells) and rmWnt5a (24.0 ± 2.6 CFU; n = 3) cultures, indicating that treatment with rmWnt5a inhibited hematopoietic proliferation but not differentiation. To identify the mechanism by which Wnt5a regulates HSC and HPC proliferation, we analyzed potential Wnt5a-mediated signaling pathways. We observed that Wnt5a induced intracellular Ca2+ (iCa2+) flux in HSCs (defined as lin−, Sca-1HI, c-kitHI; LSK). Previous studies have shown that Wnt5a-mediated induction of iCa2+ can result in activation of the NFAT family of transcription factors. Since NFATc1 promotes quiescence of hair follicle stem cells and is expressed in HSCs, we hypothesized that the effects of Wnt5a required activation of NFAT family members. We cultured bone marrow cells with Wnt5a-CM in the presence of cyclosporine A (CsA), which inhibits activation of NFAT factors. In agreement with our earlier findings, we observed that culturing bone marrow cells in Wnt5a-CM increased the percentage of quiescent (defined as Ki-67-) LSKCD34− HSCs (79.7 ± 3.3%) compared to control-CM (55.0 ± 1.6%; n = 3; p < .001). This increase was inhibited by CsA (69.4 ± 2.6%; n = 3; p = .01 compared to Wnt5a-CM alone). In conclusion, our data point to a role for Wnt5a in regulating HSC and HPC proliferation and that this function may require the activation of NFAT transcription factors. Disclosures: No relevant conflicts of interest to declare.

Myeloid Transcription Factor C/EBPɛ Is Involved in the Positive Regulation of Lactoferrin Gene Expression in Neutrophils

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3141-3150 ◽  
Author(s):  
Walter Verbeek ◽  
Julie Lekstrom-Himes ◽  
Dorothy J. Park ◽  
Pham My-Chan Dang ◽  
Peter T. Vuong ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Bone Marrow Cells ◽  
Granulocytic Differentiation ◽  
Direct Role ◽  
Gram Negative ◽  
Protein Levels ◽  
Factor C ◽  
Marrow Cells

Targeted mutation of the myeloid transcription factor C/EBPɛ in mice results in gram-negative septic death at 3 to 5 months of age. This study defines the underlying molecular defects in their terminal granulocytic differentiation. The mRNA for the precursor protein of the cathelin-related antimicrobial peptides was almost completely absent in the bone marrow cells of C/EBPɛ−/− mice. This finding may help explain their susceptibility to gram-negative sepsis, because both are bacteriocidal peptides with potent activity against gram-negative bacteria. Superoxide production was found to be reduced in both granulocytes and monocytes of C/EBPɛ−/− mice. While gp91 phox protein levels were normal, p47phox protein levels were considerably reduced in C/EBPɛ −/− granulocytes/monocytes, possibly limiting the assembly of the NADPH oxidase. In addition, expression of mRNA of the secondary and tertiary granule proteins, lactoferrin and gelatinase, were not detected, and levels of neutrophil collagenase mRNA were reduced in bone marrow cells of the knock-out mice. The murine lactoferrin promoter has a putative C/EBP site close to the transcription start site. C/EBPɛ bound to this site in electromobility shift assay studies and mutation of this site abrogated binding to it. A mutation in the C/EBP site reduced the activity of the promoter by 35%. Furthermore, overexpression of C/EBPɛ in U937 cells increased the activity of the wild-type lactoferrin promoter by 3-fold. In summary, our data implicate C/EBPɛ as a critical factor of host antimicrobial defense and suggests that it has a direct role as a positive regulator of expression of lactoferrin in vivo.

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