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PubMed2002

Gonadotropins, via cAMP, negatively regulate GATA-1 gene expression in testicular cells.

We and others demonstrated that the mRNAs encoding GATA-binding proteins, GATA-1 and GATA-4, were detected in mouse and rat testis, and in isolated rat Sertoli cells and testicular tumor cell lines derived from Leydig and Sertoli cells. In this study, we investigated the possible effects of gonadotropins and cAMP on the expression of GATA-binding protein genes in testicular cells. Unexpectedly, FSH negatively regulated GATA-1 (but not GATA-4) mRNA in a dose-dependent manner in primary cultures of rat Sertoli cells isolated from 21-d-old animals. GATA-1 mRNA was also negatively regulated by cAMP in a dose- and time-dependent manner in MA-10, a mouse Leydig tumor cell line. When 0.3 mM cAMP was administered to MA-10 cell cultures for 4 h, more than 95% of the GATA-1 mRNA and protein was abolished. The reduction of GATA-1 mRNA by cAMP can be mimicked by treatment with forskolin, which elevates intracellular cAMP levels. The inhibitory effect of cAMP was specific to the GATA-1 gene, given that GATA-4 and alpha-tubulin mRNA levels were not changed by any of the cAMP treatments. Inhibin alpha-subunit mRNA, on the other hand, was evidently increased by cAMP treatment in both MA-10 and Sertoli cells. However, inhibin alpha-subunit mRNA levels were elevated at 60-90 min before the suppression of GATA-1 mRNA detected. The inhibitory effect of cAMP on GATA-1 mRNA and protein was shown to be specific to testicular cells. The GATA-1 mRNA expressed in MEL, a mouse erythroid leukemia cell line, was not affected by cAMP. The reduction of GATA-1 mRNA by cAMP can be prevented when a translational inhibitor, cycloheximide, is added. In summary, we demonstrated that gonadotropins via cAMP negatively regulate the mRNA and protein levels of GATA-1, but not GATA-4, in testicular cells. The inhibitory effect on GATA-1 gene expression was specific to testicular cells and was not observed in erythroid cells.

Animals↗
PubMed2002

The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media.

Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, we have taken the novel approach of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. We first established conditions whereby EBs could form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated beta-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary and sufficient to recover EKLF and GATA1 expression and could be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs were competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevented induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage.

Animals↗
PubMed1992

Erythropoiesis in long term cultures of foetal liver cells is transiently obtained on adult but not on foetal adherent cell layers.

We have previously reported long term erythroid differentiation of adult bone marrow cells seeded onto adherent cells derived from adult bone marrow. In this paper, we show that the adherent cells obtained from foetal liver do not support the erythroid differentiation of either adult bone marrow cells or foetal liver cells. Adherent layers derived from bone marrow of adult W/Wv mice supported differentiation of adult bone marrow precursors, but foetal liver progenitors only produced erythrocytes for a few weeks and the foetal origin of these red cells was confirmed by haemoglobin typing. The duration and extent of erythropoiesis was generally inversely proportional to the cell dose. Foetal progenitors were as sensitive to erythropoietin as adult cells, but were optimally stimulated at a lower plateau concentration. These results suggest that inhibitory cells present in foetal liver may block erythropoiesis and their growing importance with age may provide an explanation for the arrest of erythropoiesis in the liver at late developmental stages.

Anemia↗
PubMed2003

RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation.

Megakaryocytic and erythroid lineages derive from a common bipotential progenitor and share many transcription factors, most prominently factors of the GATA zinc-finger family. Little is known about transcription factors unique to the megakaryocytic lineage that might program divergence from the erythroid pathway. To identify such factors, we used the K562 system in which megakaryocyte lineage commitment is dependent on sustained extracellular regulatory kinase (ERK) activation and is inhibited by stromal cell contact. During megakaryocytic induction in this system, the myeloid transcription factor RUNX1 underwent up-regulation, dependent on ERK signaling and inhibitable by stromal cell contact. Immunostaining of healthy human bone marrow confirmed a strong expression of RUNX1 and its cofactor, core-binding factor beta (CBFbeta), in megakaryocytes and a minimal expression in erythroblasts. In primary human hematopoietic progenitor cultures, RUNX1 and CBFbeta up-regulation preceded megakaryocytic differentiation, and down-regulation of these factors preceded erythroid differentiation. Functional studies showed cooperation among RUNX1, CBFbeta, and GATA-1 in the activation of a megakaryocytic promoter. By contrast, the RUNX1-ETO leukemic fusion protein potently repressed GATA-1-mediated transactivation. These functional interactions correlated with physical interactions observed between GATA-1 and RUNX1 factors. Enforced RUNX1 expression in K562 cells enhanced the induction of the megakaryocytic integrin proteins alphaIIb and alpha2. These results suggest that RUNX1 may participate in the programming of megakaryocytic lineage commitment through functional and physical interactions with GATA transcription factors. By contrast, RUNX1-ETO inhibition of GATA function may constitute a potential mechanism for the blockade of erythroid and megakaryocytic differentiation seen in leukemias with t(8;21).

Cell Differentiation↗
PubMed2002

Nuclear transcription factor GATA-1 is activated during aclacinomycin-induced erythroid differentiation.

Anthracycline antitumor drugs induce erythroid differentiation of the K562 erythroleukemic cell line at subtoxic concentrations. Aclacinomycin (ACM) stimulates this process by activating the erythroid transcription factor GATA-1, that controls genes involved in hemoglobin biosynthesis. To investigate the implication of GATA-1 in this process, we used a specific anti-GATA-1 polyclonal antibody that we produced in our laboratory. The GATA-1 transcription factor was then monitored during erythroid differentiation induced by aclacinomycin. Here we show that a cellular redistribution and a modification of the phosphorylation state of this transcription factor occurred during ACM-mediated cell differentiation. It suggests that anthracyclines can induce the erythroid differentiation of neoplastic cells by activating the transcription factor GATA-1, probably via its clustering into nuclear foci.

Aclarubicin↗
PubMed2001

An optimized system for studies of EPO-dependent murine pro-erythroblast development.

OBJECTIVE: Objectives were to develop new means to isolate useful numbers of primary progenitor cells and to quantitatively assay the stepwise maturation of erythroblasts. METHODS: Approaches involved dosing mice with thiamphenicol (TAP) to yield staged cohorts of pro-erythroid cells; optimizing conditions for their EPO-dependent in vitro growth and survival; developing assays for CFU-E maturation; analyzing stage-specific transcript expression; and expressing a heterologous, erythroid-specific tag (EE372) in transgenic mice. RESULTS: Per TAP-treated mouse, 3 x 10(7) highly EPO-responsive erythroid progenitor cells were generated that represented up to 30% of total splenocytes and showed strict dependence on EPO for survival, growth, and immediate response gene expression. In this developing cohort, a tightly programmed sequence of gene expression was observed, and maximal expression of c-kit, EPO receptor, and beta-globin transcripts occurred at 72, 96, and 120 hours post-TAP withdrawal, respectively. Also, the newly discovered erythroid-specific dual-specificity kinase, DYRK3, was revealed to be expressed at a late CFU-E stage. In vitro, these progenitor cells matured stepwise from high FALS Ter119- cells (24-hour culture) to high FALS Ter119+ cells (24-36 hours) to low FALS Ter119+ maturing erythroblasts (40-48 hours) and sharp differences in their morphologies were observed. Finally, a MACS-based procedure for the purification of erythroid progenitor cells from TAP-treated EE372 transgenic mice also was developed. CONCLUSIONS: A comprehensive new system for isolating large numbers of primary murine erythroid progenitor cells and quantitatively monitoring their development is established that should serve well in investigations of endogenous and pharmacological regulators of red blood cell development.

Animals↗
PubMed1991

Murine erythroid cell lines derived with c-myc retroviruses respond to leukemia-inhibitory factor, erythropoietin, and interleukin 3.

The transforming potential of the c-myc gene is shown here, for the first time, to include murine erythroid cells. Continuously growing cell lines were reproducibly generated by infection of day 13 CBA fetal liver cells with novel recombinant c-myc retroviruses. By cytostaining, most cells resembled early erythroblasts, but certain lines also contained significant numbers of hemoglobinized cells. RNA analysis revealed substantial expression of the genes encoding beta-globin and the erythroid-specific transcription factor GF-1. Although apparently immortal, the lines were not initially transplantable. Thus, constitutive myc expression in early erythroid cells can enhance their self-renewal capacity but is insufficient to fully transform them. The cell lines proliferated without the addition of exogenous factors, but their clonogenicity in semisolid medium was enhanced in the presence of erythropoietin, interleukin 3, and/or leukemia-inhibitory factor. In combination with either interleukin 3 or erythropoietin, leukemia-inhibitory factor also facilitated differentiation of certain lines. These results suggest that leukemia-inhibitory factor may have a previously unsuspected role in the regulation of erythropoiesis and could be considered as a possible therapeutic agent for the clinical management of erythroleukemia.

Animals↗
PubMed1997

GATA-1 DNA binding activity is down-regulated in late S phase in erythroid cells.

We have set out to test a model for tissue-specific gene expression that relies on the early replication of expressed genes to sequester limiting activating transcription factors. Using an erythroid cell line, we have tested the changes in the DNA binding activity of the lineage-restricted transcription factor GATA-1 through the cell cycle. We find that GATA-1 activity is low in G1, peaks in mid-S phase, and then decreases in G2/M. In contrast, the binding activities of two ubiquitous transcription factors, Oct1 and Sp1, remain high in G2/M. GATA-1 protein and mRNA vary in a similar manner through the cell cycle, suggesting that the expression of the gene or the stability of its message is regulated. Although a number of transcription factors involved in the control of the cell cycle or DNA replication have been shown to peak in S phase, this is the first example of a lineage-restricted transcription factor displaying S phase-specific DNA binding activity. One interpretation of these data leads to a model in which the peak in GATA-1 DNA binding amplifies the effect of early replication on the activation of erythroid-specific genes at the same time as preventing activation of non-erythroid genes containing GATA-responsive elements. These results may also relate to recent data implicating GATA-1 function in apoptosis and cell cycle progression.

Cell Cycle↗
PubMed1994

Retinoic acid downmodulates erythroid differentiation and GATA1 expression in purified adult-progenitor culture.

All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of interleukin-3 (IL-3) granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (Ep) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS- liquid-suspension culture supplemented with saturating Ep level and low-dose IL-3/GM-CSF, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master GATA1 gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of GATA1 mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of GATA1 induction in the early stages of erythropoietic differentiation.

Adult↗
PubMed2005

Graded levels of GATA-1 expression modulate survival, proliferation, and differentiation of erythroid progenitors.

Transcription factor GATA-1 plays an important role in gene regulation during the development of erythroid cells. Several reports suggest that GATA-1 plays multiple roles in survival, proliferation, and differentiation of erythroid cells. However, little is known about the relationship between the level of GATA-1 expression and its nature of multifunction to affect erythroid cell fate. To address this issue, we developed in vitro embryonic stem (ES) culture system by using OP9 stromal cells (OP9/ES cell co-culture system), and cultured the mutant (GATA-1.05 and GATA-1-null) and wild type (WT)ES cells, respectively. By using this OP9/ES cell co-culture system, primitive and definitive erythroid cells were developed individually, and we examined how expression level of GATA-1 affects the development of erythroid cells. GATA-1.05 ES-derived definitive erythroid cells were immature with the appearance of proerythroblasts, and highly proliferated, compared with WT and GATA-1-null ES-derived erythroid cells. Extensive studies of cell cycle kinetics revealed that the GATA-1.05 proerythroblasts accumulated in S phase and expressed lower levels of p16(INK4A) than WT ES cell-derived proerythroblasts. We concluded that GATA-1 must achieve a critical threshold activity to achieve selective activation of specific target genes, thereby influencing the developmental decision of an erythroid progenitor cell to undergo apoptosis, proliferation, or terminal differentiation.

Animals↗
PubMed1991

Deficiency of alpha-spectrin synthesis in burst-forming units-erythroid in lethal hereditary spherocytosis.

A child diagnosed in utero with hydrops fetalis and a hematocrit of 6.4% was studied to determine the etiology of the anemia. Fetal red blood cells (RBCs) obtained during in utero transfusion had extremely abnormal osmotic fragility. A maternal history of mild autosomal dominant hereditary spherocytosis was present, and the father, who was hematologically normal, had a slightly abnormal osmotic fragility test. The patient was transfusion dependent after birth, with circulating nucleated RBCs but less than 1% reticulocytes. The patient's anemia failed to respond to splenectomy. Because mature RBCs of the patient were not available for study, progenitor-derived erythroblasts grown in culture were investigated. Immunodot assays of the patient's progenitor-derived cells showed a total cell spectrin content 26% of normal. Immunoprecipitation of whole burst-forming units-erythroid-derived cells and solubilized membranes from cells pulse-labeled with 35S-methionine showed a severe deficiency in alpha-spectrin synthesis and a markedly reduced amount of alpha- and beta-spectrin on cell membranes. No alpha-spectrin degradation products were found within the cells or were produced during membrane preparation. Ankyrin content and band 3 synthesis were not different from control. Inheritance of two genetic defects causing severely reduced alpha-spectrin synthesis is proposed as the cause of the lethal anemia, resulting in cell fragmentation during precursor enucleation or during egress from bone marrow.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1997

FOG, a multitype zinc finger protein, acts as a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic differentiation.

The hematopoietic transcription factor GATA-1 is essential for development of the erythroid and megakaryocytic lineages. Using the conserved zinc finger DNA-binding domain of GATA-1 in the yeast two-hybrid system, we have identified a novel, multitype zinc finger protein, Friend of GATA-1 (FOG), which binds GATA-1 but not a functionally inactive mutant lacking the amino (N) finger. FOG is coexpressed with GATA-1 during embryonic development and in erythroid and megakaryocytic cells. Furthermore, FOG and GATA-1 synergistically activate transcription from a hematopoietic-specific regulatory region and cooperate during both erythroid and megakaryocytic cell differentiation. These findings indicate that FOG acts as a cofactor for GATA-1 and provide a paradigm for the regulation of cell type-specific gene expression by GATA transcription factors.

3T3 Cells↗
PubMed2000

Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients.

Mononuclear cells from peripheral blood of thalassemic patients were treated with morpholino oligonucleotides antisense to aberrant splice sites in mutant beta-globin precursor mRNAs (pre-mRNAs). The oligonucleotides restored correct splicing and translation of beta-globin mRNA, increasing the hemoglobin (Hb) A synthesis in erythroid cells from patients with IVS2-654/beta(E), IVS2-745/IVS2-745, and IVS2-745/IVS2-1 genotypes. The maximal Hb A level for repaired IVS2-745 mutation was approximately 30% of normal; Hb A was still detectable 9 days after a single treatment with oligonucleotide. Thus, expression of defective beta-globin genes was repaired and significant level of Hb A was restored in a cell population that would be targeted in clinical applications of this approach.

Cell Nucleus↗
PubMed1999

In vitro evidence for the presence of hematopoietic stem cells in the adult human liver.

Previous studies have identified novel lymphoid phenotypes in the adult human liver and provided evidence to suggest that lymphoid differentiation can occur locally in this organ. The aim of this study was to examine the adult human liver for the presence of hematopoietic stem cells that may provide the necessary precursor population for local hematopoietic and lymphoid differentiation. Hepatic mononuclear cells (HMNC) were extracted from normal adult liver biopsy specimens using a combination of mechanical disruption and enzymatic digestion. The stem cell marker CD34 was found on 0.81% to 2.35% of isolated HMNCs by flow cytometry. CD34(+) HMNCs were positively selected using magnetically labeled beads, and the enriched population was further examined for surface markers characteristically expressed by immature hematopoietic cells and early progenitors. CD45 was expressed by 49% (+/-23%) of CD34(+) HMNCs, indicating their hematopoietic origin. CD38, one of the first markers to be expressed by developing progenitor cells was found on 50% (+/-22%) of CD34(+) HMNCs indicating the presence of both pluripotent stem cells and committed precursors. The majority (90%) of CD34(+) HMNCs coexpressed the activation marker human leukocyte antigen DR, consistent with actively cycling cells. Functional maturation of these hepatic progenitors was shown by the detection of multilineage hematopoietic colony formation after tissue culture. Erythroid (BFU-E), granulocyte-monocyte (CFU-GM), and mixed colonies (CFU-GEMM) were detected after culture of unseparated HMNCs and the enriched CD34(+) HMNC population; 14.3 +/- 13.2 (mean +/- SD) BFU-E, 3.1 +/- 3.1 CFU-GM, and 0.4 +/- 0.9 CFU-GEMM per 1 x 10(5) unseparated HMNCs and 16.0 +/- 9.5 BFU-E and 1.7 +/- 0.9 CFU-GM were identified per 2.4 x 10(3) CD34(+) HMNCs plated. The detection of surface markers characteristic of immature hematopoietic cells and colony formation in tissue culture provides evidence for the presence of hematopoietic stem cells and early progenitor cells in the adult human liver. This would suggest that the adult human liver continues to contribute to hematopoiesis and may be an important site for the differentiation of lymphohematopoietic cells involved in disease states, such as autoimmune hepatitis and graft rejection after liver transplantation.

Antigens, CD↗
PubMed2002

Hydroxyurea exerts bi-modal dose-dependent effects on erythropoiesis in human cultured erythroid cells via distinct pathways.

Hydroxyurea (HU) has been shown to increase the proportion of fetal haemoglobin (HbF) in most sickle cell patients. A low-dosage regimen increased total haemoglobin (Hb) levels in some thalassaemia intermedia patients by preferentially increasing beta-globin biosynthesis. To further characterize these apparent dose-dependent effects of HU, we examined erythroid cells exposed to HU (5-100 micro mol/l) in two-phase liquid culture. Low doses (from 5 to 25 micro mol/l) increased Hb levels by up to 2.7-fold, and a high dose (100 micro mol/l) increased Hb levels when added at d 3-6 of phase II, with no significant changes in response to HU during the late stage of phase II culture (> or = 9 d). HU exposure during d 0-3 of phase II culture increased the number of erythroid colonies to a maximum of fivefold at 5 micro mol/l HU. GATA-1 mRNA was downregulated at a high dose and GATA-2 was dose dependently upregulated over a lower dosage range. Treatment with 100 micro mol/l HU dramatically upregulated the death receptor DR-5, caspase 3, as determined by cDNA microarray analysis. In contrast, 10 micro mol/l HU modestly upregulated mRNA levels of the early growth response gene. Our results suggest that HU exerts concentration-dependent effects on HbF production and erythropoiesis and that these two effects are mediated by distinct molecular mechanisms.

Adult↗
PubMed1997

Arrest in primitive erythroid cell development caused by promoter-specific disruption of the GATA-1 gene.

To elucidate the in vivo function of GATA-1 during hematopoiesis, we specifically disrupted the erythroid promoter of the GATA-1 gene in embryonic stem cells and generated germ line chimeras. Male offspring of chimeras bearing the targeted mutation were found to die by 12.5 days post coitus due to severe anemia while heterozygous females displayed characteristics ranging from severe anemia to normal erythropoiesis. When female heterozygotes were crossed with transgenic males carrying a reporter gene, which specifically marks primitive erythroid progenitors, massive accumulation of undifferentiated erythroid cells were observed in the yolk sacs of the GATA-1-mutant embryos, demonstrating that GATA-1 is required for the terminal differentiation of primitive erythroid cells in vivo.

Animals↗
PubMed2003

GATA-1 and c-myb crosstalk during red blood cell differentiation through GATA-1 binding sites in the c-myb promoter.

GATA-1 and c-Myb transcription factors represent key regulators of red blood cell development. GATA-1 is upregulated and c-myb proto-oncogene expression is downregulated when red cell progenitors differentiate into erythrocytes. Here we have employed a culture system, that faithfully recapitulates red blood cell differentiation in vitro, to follow the kinetics of GATA-1 and c-myb expression. We show that c-myb proto-oncogene expression is high in progenitors and effectively downregulated at the time when nuclear GATA-1 accumulates and cells differentiate into erythrocytes. Additionally, we identified two GATA-1 binding sites within the c-myb promoter and demonstrate that GATA-1 protein binds to these sites in vitro. Furthermore, GATA-1 represses c-myb expression through one of the GATA-1 binding sites in transient transfection experiments and this requires FOG-1. Thus, our study provides evidence for a direct molecular link between GATA-1 activity and c-myb proto-oncogene expression during terminal red cell differentiation.

Animals↗