The Role of K-ras Signaling in Erythropoiesis In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3136-3136
Author(s):  
Jing Zhang ◽  
Yangang Liu ◽  
Caroline Beard ◽  
Rudolf Jaenisch ◽  
Tyler Jacks ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Ras Signaling ◽  
Erythroid Progenitors ◽  
Akt Activation ◽  
Fetal Liver Cells ◽  
Terminal Erythroid Differentiation

Abstract K-ras plays an important role in hematopoiesis. K-ras-deficient mouse embryos die around E12-E13 with severe anemia. In humans, oncogenic mutations in K-ras gene are identified in ~30% of patients with acute myeloid leukemia. We used mouse primary erythroid progenitors as a model system to study the role of K-ras signaling in vivo. Both Epo- and stem cell factor (SCF) - dependent Akt activation are greatly reduced in K-ras-/- fetal liver cells, whereas other cytokine- induced pathways, including Stat5 and p44/p42 MAP kinase, are activated normally. The reduced Akt activation in erythroid progenitors per se leads to delayed erythroid differentiation. Our data identify K-ras as the major regulator for cytokine-dependent Akt activation, which is important for erythroid differentiation in vivo. Overexpression of oncogenic Ras in primary fetal erythroid progenitors led to their continual proliferation and a block in terminal erythroid differentiation. Similarly, we found that primary fetal liver cells expressing oncogenic K-ras from its endogenous locus undergo abnormal proliferation and terminal erythroid differentiation is partially blocked. We are currently investigating the signal transduction pathways activated by this oncogenic K-ras that underlies these cellular phenotypes.

scholarly journals TAF10 Interacts with GATA1 Transcription Factor and Controls Mouse Erythropoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2912-2912
Author(s):  
Petros Papadopoulos ◽  
Laura Gutierrez ◽  
Jeroen Demmers ◽  
Dimitris Papageorgiou ◽  
Elena Karkoulia ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells ◽  
Fetal Liver Cells ◽  
Gata1 Transcription Factor

Abstract The ordered assembly of a functional preinitiation complex (PIC), composed of general transcription factors (GTFs) is a prerequisite for the transcription of protein coding genes by RNA polymerase II. TFIID, comprised of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs), is the GTF that is thought to recognize the promoter sequences allowing site-specific PIC assembly. Transcriptional cofactors, such as SAGA (Spt-Ada-Gcn5-acetyltransferase), are also necessary to have tightly regulated transcription initiation. However, a new era on the role of the GTFs and specifically on the role of TFIID in tissue specific and promoter specific transcriptional regulation has emerged in the light of novel findings regarding the differentiation programs of different cell types1. TAF10 is a subunit of both the TFIID and the SAGA co-activator HAT complexes2. The role of TAF10 is indispensable for early embryonic transcription and mouse development as knockout (KO) embryos die early in gestation between E3.5 and E5.5, around the stage when the supply of maternal protein becomes insufficient3. However, when analyzing TFIID stability and transcription it was noted that not all cells and tissues were equally affected by the loss of TAF10. The contribution of the two TAF10-containing complexes (TFIID, SAGA) to erythropoiesis remains elusive. Ablation of TAF10 specifically in erythroid cells by crossing the TAF10-Lox with the EpoR-Cre mouse led to a differentiation block at around E13.5 with erythroid progenitor cells accumulating at a higher percentage (26% in the KO embryos vs 16% in the WTs at E12.5) at the double positive stage KIT+CD71+ and giving rise to fewer mature TER119+ cells in the fetal liver. At E13.5 embryos were dead with almost no erythroid cells in the fetal liver. Gene expression analysis of the fetal liver cells of the embryos revealed down-regulation of GATA1 expression and its target genes, bh1&bmaj/min globins and KLF1 transcription factor while expression of other genes known to have a role in mouse hematopoiesis remained unaffected (MYB, GATA2, PU.1). In order to get insight to the role of TAF10 during erythropoiesis we analyzed the composition of both TAF10-containing complexes (TFIID and SAGA) by mass spectrometry. We found that their stoichiometry changes slightly but not fundamentally during erythroid differentiation and development (human fetal liver erythroid progenitors, human blood erythroid progenitors and mouse erythroid progenitor cells) and no major rearrangements were generated in the composition of the TFIID as it was reported in other cell differentiation programs (e.g. skeletal differentiation, hepatogenesis). Additionally, we found GATA1 transcription factor only in the fetal liver and not in the adult erythroid cells in the mass spectrometry data of TAF10 immunoprecipitations (IPs), an interaction that we confirmed by reciprocal IP of TAF10 and GATA1 in MEL and mouse fetal liver cells. Most importantly, we checked whether TAF10 binding is enriched on the GATA1 locus in human erythroid cells during the fetal and the adult stage in erythroid proerythroblasts and we found that there is enriched binding of TAF10 in the palindromic GATA1 site in the fetal stage. Our results support a developmental role for TAF10 in GATA1 regulated genes, including GATA1 itself, during erythroid differentiation emphasizing the crosstalk between the transcriptional machinery and activators in erythropoiesis. References 1. Goodrich JA, Tjian R (2010) Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nature reviews Genetics 11: 549-558 2 .Timmers HT, Tora L (2005) SAGA unveiled. Trends Biochem Sci 30: 7-10 3. Mohan WS, Jr., Scheer E, Wendling O, Metzger D, Tora L (2003) TAF10 (TAF(II)30) is necessary for TFIID stability and early embryogenesis in mice. Mol Cell Biol 23: 4307-4318 Disclosures No relevant conflicts of interest to declare.

scholarly journals Expression of oncogenic K-ras from its endogenous promoter leads to a partial block of erythroid differentiation and hyperactivation of cytokine-dependent signaling pathways

Blood ◽  
2007 ◽  
Vol 109 (12) ◽  
pp. 5238-5241 ◽  
Author(s):  
Jing Zhang ◽  
Yangang Liu ◽  
Caroline Beard ◽  
David A. Tuveson ◽  
Rudolf Jaenisch ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Erythroid Differentiation ◽  
Retroviral Vectors ◽  
Ras Signaling ◽  
Erythroid Progenitors ◽  
Oncogenic Ras ◽  
Endogenous Promoter ◽  
High Level ◽  
Terminal Erythroid Differentiation

Abstract When overexpressed in primary erythroid progenitors, oncogenic Ras leads to the constitutive activation of its downstream signaling pathways, severe block of terminal erythroid differentiation, and cytokine-independent growth of primary erythroid progenitors. However, whether high-level expression of oncogenic Ras is required for these phenotypes is unknown. To address this issue, we expressed oncogenic K-ras (K-rasG12D) from its endogenous promoter using a tetracycline-inducible system. We show that endogenous K-rasG12D leads to a partial block of terminal erythroid differentiation in vivo. In contrast to results obtained when oncogenic Ras was overexpressed from retroviral vectors, endogenous levels of K-rasG12D fail to constitutively activate but rather hyperactivate cytokine-dependent signaling pathways, including Stat5, Akt, and p44/42 MAPK, in primary erythroid progenitors. This explains previous observations that hematopoietic progenitors expressing endogenous K-rasG12D display hypersensitivity to cytokine stimulation in various colony assays. Our results support efforts to modulate Ras signaling for treating hematopoietic malignancies.

scholarly journals The Anemic Friend Virus gp55 Envelope Protein Induces Erythroid Differentiation in Fetal Liver Colony-Forming Units-Erythroid

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1163-1172 ◽  
Author(s):  
Stefan N. Constantinescu ◽  
Hong Wu ◽  
Xuedong Liu ◽  
Wendy Beyer ◽  
Amy Fallon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Erythropoietin Receptor ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Fetal Liver Cells

Abstract The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR−/− fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell lines. Using chimeric gp55 P/A proteins, we extend earlier work showing that the transmembrane sequence determines the capacity of gp55 proteins to differentially activate EpoR signaling. We discuss the possibilities for different signaling capacities of gp55-A and -P in fetal liver and bone marrow-derived erythroid progenitor cells.

Role of Ras signaling in erythroid differentiation of mouse fetal liver cells: functional analysis by a flow cytometry–based novel culture system

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 3938-3946 ◽  
Author(s):  
Jing Zhang ◽  
Merav Socolovsky ◽  
Alec W. Gross ◽  
Harvey F. Lodish
Keyword(s):  
Flow Cytometry ◽  
Colony Formation ◽  
Culture System ◽  
Fetal Liver ◽  
Erythroid Differentiation ◽  
Dominant Negative ◽  
Ras Signaling ◽  
Proliferation And Differentiation

Abstract Ras signaling plays an important role in erythropoiesis. Its function has been extensively studied in erythroid and nonerythroid cell lines as well as in primary erythroblasts, but inconclusive results using conventional erythroid colony-forming unit (CFU-E) assays have been obtained concerning the role of Ras signaling in erythroid differentiation. Here we describe a novel culture system that supports terminal fetal liver erythroblast proliferation and differentiation and that closely recapitulates erythroid development in vivo. Erythroid differentiation is monitored step by step and quantitatively by a flow cytometry analysis; this analysis distinguishes CD71 and TER119 double-stained erythroblasts into different stages of differentiation. To study the role of Ras signaling in erythroid differentiation, different H-ras proteins were expressed in CFU-E progenitors and early erythroblasts with the use of a bicistronic retroviral system, and their effects on CFU-E colony formation and erythroid differentiation were analyzed. Only oncogenic H-ras, not dominant-negative H-ras, reduced CFU-E colony formation. Analysis of infected erythroblasts in our newly developed system showed that oncogenic H-ras blocks terminal erythroid differentiation, but not through promoting apoptosis of terminally differentiated erythroid cells. Rather, oncogenic H-ras promotes abnormal proliferation of CFU-E progenitors and early erythroblasts and supports their erythropoietin (Epo)–independent growth.

Distinct Roles of Integrins α6 and α4 in Fetal Liver Hematopoietic Stem and Progenitor Cell Homing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 353-353
Author(s):  
Hong Qian ◽  
Elisabeth Georges-Labouesse ◽  
Alexander Nyström ◽  
Anna Domogatskaya ◽  
Karl Tryggvason ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Critical Role ◽  
Liver Cells ◽  
Hematopoietic Stem ◽  
Knockout Mouse Model ◽  
Genetic Ablation ◽  
Integrin Α4 ◽  
Fetal Liver Cells

Abstract During development and after transplantation, intravenously injected hematopoietic stem and progenitor cells (HSPCs) selectively transmigrate through the sinusoidal walls into the bone marrow (BM) niches to engraft and reconstitute hematopoiesis. The rate of reconstitution following transplantation varies depending on the source of hematopoietic stem cells (HSCs) (To, et al 1992). However, the molecular pathways that control the homing of HSCs, in particular, of fetal HSCs are still not well understood. In the present study we studied the contribution of α6 and α4 integrins in homing of fetal liver HSPCs into adult BM by using function-blocking antibodies and an integrin α6 knockout mouse model. We found an ubiquitous expression of both integrin α6 and α4 receptors on fetal liver Lin−Sca-1+c-kit+ (LSK) HSPCs. Genetic ablation of integrin α6 resulted in reduced homing of fetal liver progenitors (HPCs) to BM of lethally irradiated adult recipients. In agreement with this, the integrin α6 antibody inhibited homing of fetal liver HPCs into BM and spleen. The role of integrin a6 in homing and engraftment of fetal liver HSCs was studied by a competitive repopulation assay by using integrin α6−/− or α6+/+ fetal liver cells. Absence of α6 integrin in fetal liver cells did not cause any engraftment defect or mobilization hypersensitivity as compared to wild-type cells. In agreement with this, anti-integrin α6 antibody did not either inhibit BM homing of short-term or long-term HSCs. In contrast, homing of fetal liver HSCs and HPCs to BM was virtually abrogated after treatment with integrin α4 antibody. Our results show that the α6 integrin receptors are functional during homing of fetal liver HPCs, but not multilineage repopulating HSC in vivo. Furthermore, we show the critical role of integrin α4 receptor for homing of both fetal liver HPCs and multilineage repopulating HSCs to BM, indicating distinct developmentally regulated functions for integrin α6 and α4 receptors during fetal hematopoiesis

scholarly journals A critical role of VLA-4 in erythropoiesis in vivo

Blood ◽  
1996 ◽  
Vol 87 (6) ◽  
pp. 2513-2517 ◽  
Author(s):  
K Hamamura ◽  
H Matsuda ◽  
Y Takeuchi ◽  
S Habu ◽  
H Yagita ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Fetal Liver ◽  
Critical Role ◽  
In Utero ◽  
In Vitro Studies ◽  
Specific Interactions ◽  
Erythroid Progenitors

Hematopoiesis requires specific interactions with the microenvironments, and VLA-4 has been implicated in these interactions based on in vitro studies. To study the role of VLA-4 in hematopoiesis in vivo, we performed in utero treatment of mice with an anti-VLA-4 monoclonal antibody. Although all hematopoietic cells in fetal liver expressed VLA-4, the treatment specifically induced anemia. It had no effect on the development of nonerythroid lineage cells, including lymphoids and myeloids. In the treated liver almost no erythroblast was detected, whereas the erythroid progenitors, which give rise to erythroid colonies in vitro, were present. These results indicate that VLA-4 plays a critical role in erythropoiesis, while it is not critical in lymphopoiesis in vivo.

Clathrin Assembly Protein CALM Plays a Key Role In Erythroid Differentiation Via Regulating Transferrin Receptor Endocytosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4256-4256
Author(s):  
Yuichi Ishikawa ◽  
Manami Maeda ◽  
Min Li ◽  
Sung-Uk Lee ◽  
Julie Teruya Feldstein ◽  
...  
Keyword(s):  
Transferrin Receptor ◽  
Mammalian Cells ◽  
Research Funding ◽  
Molecular Mechanisms ◽  
Erythroid Differentiation ◽  
Global Changes ◽  
Erythroid Progenitors ◽  
Erythroid Development ◽  
Terminal Erythroid Differentiation

Abstract Abstract 4256 Clathrin assembly lymphoid myeloid leukemia protein (CALM, also known as PICALM) is ubiquitously expressed in mammalian cells and implicated in clathrin dependent endocytosis (CDE). The CALM gene is the target of the t(10;11)(p13;q14-21) translocation, which is rare, but recurrently observed mutation in multiple types of acute leukemia. While the resultant CALM/AF10 fusion gene could act as an oncogene in vitro and in vivo in animal models, molecular mechanisms by which the fusion protein exerts its oncogenic activity remains elusive. Since CDE is implicated in the regulation of growth factor/cytokine signals, we hypothesized that the CALM/AF10 fusion oncoprotein could affect normal Calm function, leading to leukemogenesis. To determine the role of CALM and CDE in normal hematopoiesis, we generated and characterized both conventional (Calm+/−) and conditional (CalmF/F Mx1Cre+) Calm knockout mutants. While we didn't observe a gross defect in the heterozygous mutant (Calm+/−), homozygous deletion of the Calm gene (Calm-/-) resulted in late embryonic lethality. Total numbers of fetal liver (FL) cells were significantly reduced in Calm-/-embryos compared to that of control due to inefficient erythropoiesis. Proportions of mature erythroblasts (CD71-Ter119+) in FL were significantly reduced in the absence of the Calm gene. Furthermore, Calm deficient Megakaryocyte-Erythroid Progenitors (MEPs) gave rise to less CFU-E colonies when seeded in methyl cellulose plates, suggesting that Calm is required for terminal erythroid differentiation in a cell autonomous manner. To determine the role of Calm in adult hematopoiesis, we analyzed peripheral blood (PB), bone marrow (BM) and spleen of CalmF/F Mx1Cre+ mice after pIpC injection. CalmF/F Mx1Cre+ mice demonstrated hypochromic anemia, T-lymphocytopenia and thrombocytosis one month after pIpC injection. Levels of plasma transferrin and ferritin were intact in CalmF/F Mx1Cre+ mice, while plasma iron levels were increased, indicating that iron uptake is impaired in Calm deficient erythroblasts. We observed significant reduction of mature erythroblasts and erythrocytes in both BM and spleen with concomitant increase of immature erythroblasts (CD71+Ter119+) in CalmF/F Mx1Cre+ mice. The increased population mainly consists of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts, and Benzidine staining of PB and splenic erythroblasts revealed reduced hemoglobinization in Calm deficient erythroblasts. To examine the global changes in transcriptome of CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts with or without the Calm gene, we compared mRNA expression profile by gene chip microarray analysis. Over 400 genes, including genes associated with iron metabolism and CDE pathway, were up- or down-regulated more than 1.5-fold in Calm deficient polychromatophilic erythroblasts as compared to control. Genes Set Enrichment Analysis (GSEA) revealed that multiple metabolic pathways were downregulated in Calm deficient polychromatophilic erythroblasts. Calm deficient CD71+Ter119+CD44+FSCdim polychromatophilic erythroblasts demonstrated a defect in cellular proliferation revealed by cell cycle analysis. Transferrin receptor 1 (TFR1, CD71) is highly expressed in rapidly dividing cells and erythroblasts, and uptake of iron-bound transferrin through TFR1 is the main pathway of iron intake to erythroid precursors. Since CDE is implicated in TFR1 endocytosis, we next examined surface expression levels of CD71 in Calm deficient erythroid progenitors and erythroblasts. While CD71 is normally expressed at low level in early stage of megakaryo/erythroid progenitors and highly expressed in CFU-E through polychromatophilic erythroblasts, its expression was dramatically up-regulated throughout the erythroid development in CalmF/F Mx1Cre+ mice. Up-regulation of surface CD71 expression was also evident in K562 erythroid leukemia cell lines upon ShRNA-mediated CALM knockdown. Taken together, our data indicate that CALM plays an essential role in terminal erythroid differentiation via regulating TFR1 endocytosis. Since iron is required for both erythroblast proliferation and hemoglobinization, Calm deficiency significantly impacts erythroid development at multiple levels. Disclosures: Naoe: Chugai Pharm. Co.: Research Funding; Zenyaku-Kogyo Co.: Research Funding; Kyowa-Kirin Co.: Research Funding; Dainippon-Sumitomo Pharm. Co.: Research Funding; Novartis Pharm. Co.: Research Funding; Janssen Pharm. Co.: Research Funding.

scholarly journals Support of early B-cell differentiation in mouse fetal liver by stromal cells and interleukin-7

Blood ◽  
1991 ◽  
Vol 77 (12) ◽  
pp. 2612-2617 ◽  
Author(s):  
Y Gunji ◽  
T Sudo ◽  
J Suda ◽  
Y Yamaguchi ◽  
H Nakauchi ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Liver Cells ◽  
B Cell Differentiation ◽  
Stromal Cell Line ◽  
Fetal Liver Cells

We compared the development of B-cell progenitors with that of myeloid progenitors in fetal liver cells at various gestational ages. Day 12 to 14 fetal liver cells did not form pre-B-cell colonies. Pre-B-cell colonies were developed from day 15 fetal liver cells. The incidence of colonies increased with increases in gestational age and reached a maximum on days 18 to 19. In contrast, the incidence of myeloid colonies formed in the presence of interleukin-3 (IL-3) and erythropoietin did not change significantly during days 13 to 21 of gestation. After coculturing day 13 fetal liver cells with IL-7- producing stromal cell line ST-2, they could respond to IL-7 and proliferate. Analysis of the phenotypes showed that day 13 fetal liver cells were B220-, IgM-, while culturing day 13 fetal liver cells with ST-2 and untreated day 18 fetal liver cells contained the population of B220+ cells. Even in the presence of IL-7-defective stromal cell line FLS-3, IL-7-responsive cells could be induced from day 13 fetal liver cells. IL-7 acted on B220+ cells and induced pre-B-cell colonies that contained IgM+ cells in the methylcellulose culture. IL-7 mRNA was expressed in days 13 and 18 fetal liver cells but not in pre-B cells or adult liver cells. From these findings, it is suggested that stromal cells or stromal-derived factors but not IL-7 were required for the differentiation from B220- cells to B220+ cells. In the second stage, B220+, IgM- cells proliferated and some of them differentiated to IgM+ cells in the presence of IL-7 alone. The two-step model can apply to in vivo early B lymphopoiesis.

scholarly journals The mouse Klf1 Nan variant impairs nuclear condensation and erythroid maturation

2018 ◽  
Author(s):  
Ileana Cantú ◽  
Harmen J.G. van de Werken ◽  
Nynke Gillemans ◽  
Ralph Stadhouders ◽  
Steven Heshusius ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Chromatin Condensation ◽  
Erythroid Differentiation ◽  
Liver Cells ◽  
Single Amino Acid ◽  
Erythroid Cells ◽  
Nuclear Condensation ◽  
Similar Phenotype ◽  
Fetal Liver Cells ◽  
Erythroid Maturation

ABSTRACTKrüppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >65 KLF1 variants, causing different erythroid phenotypes, have been described. The Klf1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the ~780 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We conclude that reduced expression of XPO7 is partially responsible for the erythroid defects observed in Nan erythroid cells.

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