Stimulation of FSH release by erythroid differentiation factor (EDF)

The action of erythroid differentiation factor (EDF) on primary culture of rat anterior pituitary cells was examined. EDF stimulates FSH secretion in a dose dependent manner but not of LH secretion. ED50 of EDF for FSH secretion was 5 X 10(-10) M, while ED50 of LHRH for FSH secretion was 5 X 10(-9) M. These data indicate that EDF is a potent agonist for FSH secretion and the biological activity of EDF on anterior pituitary seems to be identical as that of FSH releasing protein (FRP).     

Expression of erythroid differentiation factor (EDF) in Chinese hamster ovary cells

Plasmid DNA containing EDF subunit cDNA and mouse dihydrofolate reductase (DHFR) cDNA was transfected into CHO DHFR- cells by the calcium-phosphate method. DHFR positive transformants secreted recombinant EDF (r-EDF) constitutively in an active form and accumulated it in the conditioned medium. Furthermore, cells which were resistant to methotrexate (MTX : 0.5 microM) secreted r-EDF up to 1 microgram/ml. r-EDF was identical to natural EDF (n-EDF) produced by human acute monocytic leukemia cell line, THP-1, as regards its dimeric structure and a biological activity.     

Purification and characterization of erythroid differentiation factor (EDF) isolated from human leukemia cell line THP-1

We isolated a protein, from a cell line of human origin, which exhibits extensive differentiation inducing activity toward Friend leukemia cells. The protein, called Erythroid Differentiation Factor (EDF), was found in a 4 day culture of THP-1 cells performed in the presence of 4 beta-phorbol 12-myristate 13-acetate(PMA). EDF is a homodimer of a molecular weight of 25,000, with an NH2-terminal sequence identical to that of the beta A-chain of porcine Inhibin. It was suggested that a single protein species is responsible for the activities of both EDF and FRP, a FSH releasing protein isolated from porcine ovarian follicular fluid.     

Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor)

Summary Recently the mesoderm-inducing effects of the transforming growth factor (TGF-) family of proteins have been widely examined. In an attemt to elucidate the functions of these proteins, porcine inhibin A and activin A (erythroid differentiation factor; EDF) were examined. Treatment of explants with activin A led to differentiation of mesodermal derivatives such as mesenchyme, notochord, blood cells and muscle, but inhibin A had a much lesser effect. The mesodermal differentiation induced by activin A was also comfirmed by analyses using a polyclonal antibody against muscle myosin. By indirect immunofluorescence analysis, the differentiation of muscle blocks was observed in the activin-A-treated explants, whereas no differentiation was observed in inhibin-A-treated and control explants. These findings confirm that this protein of the TGF- family has mesoderm-inducing ability.     

An intracellular factor that induces erythroid differentiation in mouse erythroleukemia (Friend) cells

We have previously shown that in vitro erythroid differentiation of mouse Friend cells is a result of a synergistic action of two distinctive intracellular reactions. We now have evidence that a factor in the cell free extract is involved in one of the reactions. This factor triggers erythroid differentiation when introduced into undifferentiated mouse Friend cells, provided the cells have been briefly exposed to dimethyl sulfoxide. The factor is induced in nonerythroid cells as well following treatment of the cells by agents that affect DNA replication. Cycloheximide inhibited the induction of the factor. The factor, which is in the cytoplasm, was partially purified and proteinaceous. When introduced into the cells the partially purified factor converts 60% to 70% of undifferentiated Friend cells to erythroid cells, at an efficiency almost equivalent to the efficiencies achieved by typical inducing agents. The factor's biochemical characteristics and possible role in erythroid differentiation are also discussed.     

The vegetalizing factor from chicken embryos: its EDF (activin A)-like activity

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng/ml), notochord- and endoderm-like tissues are induced in a high percentage.     

Regulation of erythroid differentiation by miR-376a and its targets

Lineage differentiation is a continuous process during which fated progenitor cells execute specific programs to produce mature counterparts. This lineage-restricted pathway can be controlled by particular regulators, which are usually exclusively expressed in certain cell types or at specific differentiation stages. Here we report that miR-376a participates in the regulation of the early stages of human erythropoiesis by targeting cyclin-dependent kinase 2 (CDK2) and Argonaute 2 (Ago2). Among various human leukemia cell lines, miR-376a was only detected in K562 cells which originated from a progenitor common to the erythroid and megakaryotic lineages. Enforced expression of miR-376a or silencing of CDK2 and Ago2 by RNAi inhibits erythroid differentiation of K562 cells. Hematopoietic progenitor cells transduced with miR-376a showed a significant reduction of their erythroid clonogenic capacity. MiR-376a is relatively abundant in erythroid progenitor cells, where it reduces expression of CDK2 and maintains a low level of differentiation due to cell cycle arrest and decreased cell growth. Following erythroid induction, miR-376a is significantly down-regulated and CDK2 is released from miR-376a inhibition, thereby facilitating the escape of progenitor cells from the quiescent state into erythroid differentiation. Moreover, our results establish a functional link between miR-376a and Ago2, a key factor in miRNA biogenesis and silencing pathways with novel roles in human hematopoiesis.     

Expression of c-myc oncogene during differentiation of human burst-forming unit, erythroid (BFU-E)

We investigated the serial expression of c-myc oncogene during differentiation of normal human burst-forming unit, erythroid (BFU-E), in vitro, together with the replating efficiency and the expression of transferrin receptors. We found that the c-myc oncogene was expressed in the early progeny of BFU-E and that this expression decreased during erythroid differentiation. This change of the c-myc expression was well correlated to changes in the replating efficiency and the expression of transferrin receptors. These observations indicate that the c-myc oncogene probably has a physiological role in normal human hematopoiesis as well as leukemic hematopoiesis.     

Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions

Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions.     

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells

Background

We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β_B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.

Methodology

hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β_B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey''s test.

Principal Findings

GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β_B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β_B-subunit mRNA and inhibin B production were attenuated by adding FST.

Conclusion

GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β_B-subunit mRNA expression and inhibin B production in hGL cells.     

Identification of a specific receptor for erythroid differentiation factor on follicular granulosa cell

A cellular receptor for erythroid differentiation factor (EDF) was demonstrated by incubation of 125I-labeled EDF with rat follicular granulosa cell cultures. The specific binding of labeled EDF to the cells showed saturation; Scatchard analysis of binding data indicated a single class of receptors having Kd = 3.4 x 10(-10) M. A large excess of unlabeled EDF reduced labeled EDF binding almost completely, whereas similar doses of inhibin and transforming growth factor type beta, which are quite similar to EDF in protein structure and subunit organization, had no effect; EDF did not share receptors with those factors. EDF receptor (activin A receptor) expression was enhanced in granulosa cells cultured in the presence of follicle-stimulating hormone; follicle-stimulating hormone raised the number of EDF binding sites/cell from 13,000 to 96,000 without altering the binding affinity.     

Induction of differentiation of human stem cells ex vivo: Toward large-scale platelet production

Platelet transfusion is one of the most reliable strategies to cure patients suffering from thrombocytopenia or platelet dysfunction. With the increasing demand for transfusion, however, there is an undersupply of donors to provide the platelet source. Thus, scientists have sought to design methods for deriving clinical-scale platelets ex vivo. Although there has been considerable success ex vivo in the generation of transformative platelets produced by human stem cells (SCs), the platelet yields achieved using these strategies have not been adequate for clinical application. In this review, we provide an overview of the developmental process of megakaryocytes and the production of platelets in vivo and ex vivo, recapitulate the key advances in the production of SC-derived platelets using several SC sources, and discuss some strategies that apply three-dimensional bioreactor devices and biochemical factors synergistically to improve the generation of large-scale platelets for use in future biomedical and clinical settings.     

Translation initiation factor 4E inhibits differentiation of erythroid progenitors

Stem cell factor (SCF) delays differentiation and enhances the expansion of erythroid progenitors. Previously, we performed expression-profiling experiments to link signaling pathways to target genes using polysome-bound mRNA. SCF-induced phosphoinositide-3-kinase (PI3K) appeared to control polysome recruitment of specific mRNAs associated with neoplastic transformation. To evaluate the role of mRNA translation in the regulation of expansion versus differentiation of erythroid progenitors, we examined the function of the eukaryote initiation factor 4E (eIF4E) in these cells. SCF induced a rapid and complete phosphorylation of eIF4E-binding protein (4E-BP). Overexpression of eIF4E did not induce factor-independent growth but specifically impaired differentiation into mature erythrocytes. Overexpression of eIF4E rendered polysome recruitment of mRNAs with structured 5' untranslated regions largely independent of growth factor and resistant to the PI3K inhibitor LY294002. In addition, overexpression of eIF4E rendered progenitors insensitive to the differentiation-inducing effect of LY294002, indicating that control of mRNA translation is a major pathway downstream of PI3K in the regulation of progenitor expansion.     

Fluctuation of gene expression for poly(ADP-ribose) synthetase during hemin-induced erythroid differentiation of human leukemia K562 cells and its reversion process

We have studied the regulation of gene expression for poly(ADP-ribose) synthetase during erythroid differentiation and its reversion process. When human leukemia K562 cells were incubated in the presence of 80 microM hemin, benzidine-positive cells appeared at day 2 and 90% of the cells became positive at day 6. However, RNA blot analysis reveals that mRNA for gamma-globin was already abundant in untreated K562 cells and the level of the message was slightly increased by hemin-treatment. Spectroscopic analysis and polyacrylamide gel electrophoresis of the induced cell extracts indicate that hemoglobin molecules were not detected in untreated cells, and increased successively up to day 6. The hemin-induced cells were thoroughly washed, and then recultured in the absence of hemin. The benzidine-positive cells mostly disappeared 3 days after the elimination of the inducer. During the hemin-induced erythroid differentiation, the activity and mRNA for poly(ADP-ribose) synthetase decreased to 50% and 20% of the initial level at day 3 and a low level of the gene expression was maintained afterwards, whereas the activity and mRNA returned to the initial value 1 day after hemin elimination. The results indicate that the hemin-induced erythroid differentiation of K562 cells is a reversible process and depression of the synthetase may be involved in the progress of differentiation.     

Inhibition of erythroid differentiation of mouse erythroleukemia cells by a macrophage product(s)

Conditioned media from established murine macrophage cell lines (RAW264.7, P388D1, and WEHI-3) incubated with endotoxin in a serum-free medium contain an erythroid inhibitory activity (EIA) that inhibited dimethylsulfoxide-induced erythroid differentiation of mouse Friend virus-transformed erythroleukemia cells. Endotoxin itself has no EIA activity. Partial purification of EIA demonstrated that it is distinct from other macrophage products such as IL-1, TGF beta, ECGF, FGF, G-CSF, hepatocyte stimulating factor, interferon, PDGF, and cachectin/TNF. These findings indicate that EIA is a macrophage product distinct from other monokines.     

K-Cl cotransporter gene expression during human and murine erythroid differentiation

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.     

Nerve growth factor (NGF) induced differentiation of human neuroblastoma cells

1. Nerve growth factor (NGF) induced differentiation of human neuroblastoma cell line. 2. The differentiated cells had a relatively high activity of adenylate cyclase and cyclic AMP phosphodiesterase, and a high intracellular level of cyclic AMP. 3. These cells synthesized a higher amount of met5-o-enkephalin than undifferentiated cells. 4. Undifferentiated cells bound less met5-enkephalin than differentiated cells. The maximum number of [3H]met5-enkephalin receptor sites per mg of membrane protein increased more in differentiated cells. 5. Previous observations taken together with our results suggests that increased intracellular levels of cyclic AMP after treatment with NGF induced differentiation of human neuroblastoma cells. Reversal of undifferentiated tumor cells into the differentiated changes the capacity of synthesis of met5-enkephalin and its interaction with receptors.