Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P04141 (granulocyte-macrophage colony-stimulating factor)
 6,790 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

AIC2A and AIC2B are closely related genes encoding components of the receptors for murine interleukin-3 (IL-3) (AIC2A) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5 (AIC2B). We have studied the parallel regulation of expression of these genes in erythroid and myeloid progenitor cell lines. AIC2A and AIC2B transcription was transiently induced in these cells in response to a variety of hematopoietic growth factors, including erythropoietin (EPO), monocyte-CSF, IL-3, GM-CSF, and stem cell factor (SCF or kit ligand). Run-on assays established that the increase occurred mainly at the transcriptional level. Immunoprecipitation experiments confirmed that the increase in messenger RNA expression resulted in augmented synthesis of both AIC2A and AIC2B proteins, and binding studies further showed these proteins to be functional. We observed a fourfold increase in low-affinity IL-3 sites in an erythroid precursor cell line stimulated with EPO, and a threefold increase in GM-CSF high-affinity sites in a myeloid cell line stimulated with IL-3. In addition, we showed that the increase in the IL-3 receptor chain AIC2A in the erythroid precursor cell line correlated with the ability of IL-3 to exert a cooperative effect with EPO in the induction of beta-globin in these cells.
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PMID:Enhanced expression of interleukin-3 and granulocyte-macrophage colony-stimulating factor receptor subunits in murine hematopoietic cells stimulated with hematopoietic growth factors. 138 62

Hematopoietic growth factors are produced by a number of human tumors. We extracted RNA from selected human tumor cells known to produce at least one hematopoietic growth factor and found high levels of abnormally stable cytokine messenger (m)RNA. Half-life experiments performed after preventing RNA synthesis by exposing cells to actinomycin D before RNA extraction showed stabilization of cytokine messages in tumor cells in liquid culture as well as in human tumor xenografts grown in mice. Exposure to the phorbol ester phorbol 12-myristate 13-acetate (TPA) caused enhancement of granulocyte-macrophage colony-stimulating factor (GM-CSF) message level in lung cancer cells and in control fibroblasts but elevated levels persisted far longer in the tumor cells. In normal cells, an AU-rich sequence in the 3' untranslated region of cytokine mRNAs confers lability to the message. Although a beta-globin gene expression vector containing this region appears to produce unstable mRNA in lung cancer cells, cytokine mRNAs, which also contain this sequence, are very stable in the tumors we studied. This may indicate that another region of the cytokine mRNA molecule is of greater importance than the AU-rich region in determining mRNA stability in tumor cells.
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PMID:Cytokine messenger RNA stability is enhanced in tumor cells. 184 61

An AU-rich sequence present within the 3' untranslated region has been shown to mark some short-lived mRNAs for rapid degradation. We demonstrate by label transfer and gel shift experiments that a 32-kDa polypeptide, present in nuclear extracts, specifically interacts with the AU-rich domains present within the 3' untranslated region of human granulocyte-macrophage colony-stimulating factor, c-fos, and c-myc mRNAs and a similar domain downstream of the poly(A) addition site of the adenovirus IVa2 mRNA. Competition experiments and partial protease analysis indicated that the same polypeptide interacts with all four RNAs. A single AUUUA sequence in a U-rich context was sufficient to signal binding of the 32-kDa polypeptide. Insertion of three copies of this minimal recognition site led to markedly reduced accumulation of beta-globin RNA, while the same insert carrying a series of U-to-G changes had little effect on RNA levels. Steady-state levels of beta-globin-specific nuclear RNA, including incompletely processed RNA, and cytoplasmic mRNA were reduced. Cytoplasmic mRNA containing the AU-rich recognition sites for the 32-kDa polypeptide exhibited a half-life shorter than that of mRNA with a mutated insert. We suggest that binding of the 32-kDa polypeptide may be involved in the regulation of mRNA half-life.
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PMID:A 32-kilodalton protein binds to AU-rich domains in the 3' untranslated regions of rapidly degraded mRNAs. 190 42

Peripheral blood blasts from a patient with acute megakaryoblastic leukemia were placed into liquid cultures with recombinant growth factors. Growth, but not differentiation, was supported by interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for the first 30 days of culture. Sustained growth occurred only with GM-CSF and gave rise to the cell line MB-02, which has been in continuous culture for over 1 year. The cell line retained the surface phenotype of the leukemic megakaryoblasts except for the loss of glycoproteins Ib and IIb/IIIa, which were induced after exposure to phorbol esters. The induction of erythropoiesis occurred when GM-CSF-deprived cells were cultured with erythropoietin (Epo). Well-defined morphologic stages of differentiation ranging from primitive erythroblasts to nuclei-extruding normoblasts were seen. Transforming growth factor-beta inhibited GM-CSF- and Epo-dependent growth, but not erythroid maturation. Indirect immunofluorescence using globin chain-specific monoclonal antibodies detected fetal, but not adult hemoglobin in the uninduced cells. beta-globin was induced and gamma-globin was increased after Epo exposure. Both globin species accumulated in the developing erythrocytes until terminal differentiation. Quantitative S1 analysis of beta-like globin transcripts showed very low levels of epsilon- and beta-globin expression and high levels of gamma-globin expression in cells maintained in GM-CSF. Five days after induction with Epo, epsilon message decreased to barely detectable levels while gamma and beta transcripts increased threefold and 20-fold, respectively. This novel cell line not only retains many characteristics of the leukemic megakaryoblasts from which it was derived, but also can be induced to recapitulate apparent normal erythropoiesis.
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PMID:Granulocyte-macrophage colony-stimulating factor-dependent growth and erythropoietin-induced differentiation of a human cell line MB-02. 195 74

Many RNAs coding for either cytokines or oncogenes are unstable and have a short half-life (t1/2). The AUUUA motif is a highly conserved sequence and is repeated three or more times in the 3' untranslated region (3'UTR) of RNAs encoding many of these short-lived cytokines and oncogenes. These sequences can confer instability. In this study, we investigated the role of number and location of AUUUA motifs in stabilization of RNA. We introduced 1xATTTA, 2xATTTA, ATTTTTTTA (second adenosine of 2xATTTA was substituted with a thymidine), 3xATTTA, 5xATTTA, 7xATTTA [AT-rich sequence from granulocyte-macrophage colony-stimulating factor [GM-CSF] gene (AT-62)], and GC-62 (GC sequences were substituted for ATTTA sequences in the 7xATTTA) into the 3'UTR of rabbit beta-globin (R beta G) gene. This construct also contained the neomycin-resistance gene. These expression vectors were transfected into human lung fibroblasts (W138), which constitutively expressed low levels of GM-CSF mRNA. Stable transfectants were selected by growth in G418. Northern blot analysis of actinomycin D-treated, stably transfected cells demonstrated that the number of AUUUA sequences correlated with rapidity of turnover of the chimeric R beta G mRNA. The rank order of stability was GC-62 = 1xATTTA = 2xATTTA (no RNA decay at 4 hours) > 3xATTTA = 5xATTTA (t1/2, 4 hours) > 7xATTTA (t1/2, 2 hours). Stability of mRNA of R beta G also was reduced (t1/2, 2 to 4 hours) when AT-62 was introduced into the second exon of R beta G gene. In these same cells, the t1/2 of GM-CSF RNA was approximately 10 to 15 minutes, suggesting that the AUUUA motifs cannot alone account for the rapid degradation of this cytokine mRNA. Phorbol diesters, including 12-0-tetradecanoyl phorbol 13-acetate (TPA), stabilize a variety of transiently expressed RNAs, including GM-CSF RNA. We found that TPA markedly increased (> 30-fold) the accumulation of GM-CSF RNA. In contrast, TPA was unable to stimulate the levels of the chimeric R beta G when either 1x, 2x, 3x, or 5xATTTA motifs were fused to 3'UTR, or when either AT-62 or GC-62 control sequences were fused to the second exon. The chimeric beta-globin construct with either AT-62 or ATTTTTTTA in the 3'UTR had only an approximately twofold to threefold increase in accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Number and location of AUUUA motifs: role in regulating transiently expressed RNAs. 819 53

Delineating the molecular basis for agonist-induced destabilization of mRNA of G-protein-linked receptors that contributes to receptor down-regulation is fundamental to our understanding of long-term regulation of receptors by agonist. Previously we identified a prominent, M(r) 35,000 cytosolic RNA-binding protein that (i) binds selectively to beta 1 and beta 2-adrenergic receptor mRNAs, both of which undergo agonist-induced down-regulation; (ii) does not bind either to alpha 1b-adrenergic receptor mRNA, which does not undergo agonist-induced down-regulation, or to beta-globin mRNA; (iii) displays binding to beta 2-adrenergic receptor mRNA that is selectively competed by poly(U) RNA, but not poly(A),-(C), or -(G) RNA; and (iv) its abundance varies inversely with the level of receptor mRNA, being induced by agonists that down-regulate receptor mRNA (Port, J. D., Huang, L.-y., and Malbon (1992) J. Biol. Chem. 267, 24103-24108). We demonstrate here that the binding of beta-adrenergic receptor mRNA by this protein, termed beta-ARB protein, is sensitive to competition by AU-rich domains of the 3'-untranslated regions of c-fos, c-myc, and human granulocyte-macrophage colony-stimulating factor. Using the AU-rich 3'-untranslated regions of wild-type adenovirus IVa2 mRNA and variants with defined mutations in the AUUUApentamer, AU-rich, and U-rich domains, we were able to define sequences critical to the binding of the beta 2-receptor mRNA by the beta-ARB protein. Recognition of beta-ARB protein requires not only an AUUUA destabilization pentamer, but also a flanking U-rich domain(s). Using radiolabeled 3'-untranslated regions of short-lived mRNA, we were able to identify this same M(r) 35,000 cytosolic RNA-binding protein(s), beta-ARB protein, as selective for beta 2-adrenergic receptor mRNA.
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PMID:The M(r) 35,000 beta-adrenergic receptor mRNA-binding protein induced by agonists requires both an AUUUA pentamer and U-rich domains for RNA recognition. 824 13

Erythroid differentiation involves the activation of a number of erythroid-specific genes, most of which, including the globin genes and the erythropoietin receptor (Epo-R) gene, are, at least in part, regulated by the transcription factor GATA-1. In order to understand the relationship, if any, between expression of GATA-1, response to Epo and erythroid differentiation, we analyzed the expression of GATA-1, Epo-R and globin genes in an Epo-dependent human cell line, UT-7 Epo. The results were compared to those obtained with the parental granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent cell line, UT-7, which has a predominantly megakaryoblastic phenotype and is unable to proliferate continuously in the presence of Epo. UT-7 Epo and UT-7 expressed similar levels of GATA-1 mRNA and binding activity. The two lines also expressed comparable levels of Epo-R mRNA while the number of Epo-binding sites on UT-7 Epo cells was one-sixth the number of UT-7 cells (2400 +/- 3 vs. 13,800 +/- 300). This difference in the number of binding sites could be due to differences in cell surface (UT-7 cells are 20% smaller than the parental UT-7 cells) or in receptor turnover. By Northern analysis, UT-7 cells expressed detectable levels of beta- and gamma-globin but not alpha-globin. In comparison, UT-7 Epo cells expressed alpha-globin and higher levels of gamma-globin (5-fold) and beta-globin (from barely to clearly detectable). Globin chains (alpha, beta and gamma) were clearly detectable by affinity chromatography in UT-7 Epo but not in UT-7 cells. The frequency of the cells which expressed beta- and gamma-globin genes in the two cell populations was measured by immunofluorescence with beta- and gamma-specific antibodies. The number of gamma-positive cells and their fluorescence intensity were higher in UT-7 Epo than in UT-7 cells (0 to 17% barely positive cells and 23 to 40% clearly positive cells, respectively), indicating that the increase in globin mRNA observed in UT-7 Epo is due to both an increase of gene expression per cell and an increase in numbers of cells containing gamma-globin. The levels of GATA-1, Epo-R and globin mRNA expressed were not affected by a 24-hour incubation of either cell line with Epo, GM-CSF or interleukin-3 (IL-3).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Dependence for the proliferative response to erythropoietin on an established erythroid differentiation program in a human hematopoietic cell line, UT-7. 851 68

We have investigated, by semiquantitative RT-PCR, the kinetics of activation of hematopoietic receptors and differentiation markers in partially purified murine hematopoietic stem cells (HSC) induced to differentiate in serum-free culture with combinations of growth factor (GF). The combinations of GF used sustained either multilineage [stem cell factor (SCF) + interleukin 3 (IL-3), or erythroid [SCF + IL-3 + erythropoietin (Epo)] or myeloid [SCF + IL-3 + granulocyte colony-stimulating factor (G-CSF)] differentiation. The GF receptor genes investigated were the alpha and beta subunits of the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin receptor, the G-CSF receptor, and c-Fms, the receptor for macrophage colony-stimulating factor (M-CSF). The expression of Gata1 and alpha- and beta-globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, which include partitioning of lineage-negative bone marrow cells with the mitochondrial dye Rhodamine 123 (Rho) into Rho-dull (> or = 17% of which reconstitute long-term hematopoiesis in recipient mice) and into Rho-bright (which are as capable as Rho-dull of multilineage differentiation but do not permanently reconstitute the host). The following pattern of expression was observed: the alpha subunit of the IL-3 receptor clearly was expressed in both Rho-bright and Rho-dull cells at the outset, and its expression did not change over time in culture. The beta subunits of the IL-3 and GM-CSF receptor, the alpha subunit of the GM-CSF receptor, the Epo and G-CSF receptors and Fms barely were expressed in purified Rho-bright and Rho-dull cells, but their expression increased in cells cultured both in erythroid and in myeloid GF combinations. Gata1 was expressed maximally in Rho-bright cells but was below the level of detection in Rho-dull cells. Rho-dull cells expressed Gata1 when cultured both in erythroid and in myeloid GF combinations. In contrast, alpha- and beta-globin, which also were not expressed in the purified cells, were induced only in cells stimulated with Epo. These results indicate that the genes for all the GF receptors investigated (with the exception of the alpha subunit of the IL-3 receptor) are expressed at low levels, if any, in purified Rho-bright or Rho-dull cells, but are expressed in their progeny cultured either in erythroid or myeloid GF combinations. The expression of the Epo receptor, in particular, is activated both in erythroid (alpha- and beta-globin positive and in myeloid (alpha- and beta-globin negative) cells. Therefore, activation of the expression of the Epo receptor gene and activation of the erythroid differentiation program are two independent events in normal hematopoiesis.
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PMID:Growth factor receptor expression during in vitro differentiation of partially purified populations containing murine stem cells. 918 Sep 4

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors share a common beta chain (beta(c)), and both cytokines enhance erythropoietin (Epo)-dependent in vitro erythropoiesis by primary hematopoietic progenitors and factor-dependent cells. These data suggest that the Epo receptor and beta(c) may functionally interact. To determine whether such interactions can be documented, we studied a murine factor-dependent cell line (Ba/F3), which endogenously expresses IL-3R. First, Ba/F3 cells were transfected with murine EpoR, making them responsive to both IL-3 and Epo. Next, the EpoR expressing cells were transfected with murine beta(c). This resulted in an enhanced sensitivity of these cells to Epo, which was especially pronounced at low Epo concentrations. Ba/F3-EpoR were then treated with antisense oligodeoxynucleotides to the murine beta. Control sense and nonsense had no effect on Epo-dependent growth, but the antisense markedly and specifically inhibited Epo-dependent growth. In contrast, the antisense did not affect beta-globin message levels (another Epo-responsive effect in these cells) detectable by Northern blot. Finally, Western blot analysis of proteins immunoprecipitated from cells expressing both receptors with antibody against beta and blotted with antibody against EpoR, or immunoprecipitated with antibody against EpoR and blotted with antibody against beta, showed that EpoR and beta coimmunoprecipitate. These data show that the beta chain functionally and physically associates with the EpoR. This suggests that these cytokine receptors exist as a large supercomplex and offers the first molecular explanation for the synergistic effects of IL-3 and GM-CSF with Epo during erythropoiesis.
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PMID:The beta chain of the interleukin-3 receptor functionally associates with the erythropoietin receptor. 929 19

Mechanisms underlying fetal hemoglobin (HbF) reactivation in adult life have not been elucidated; particularly, the role of growth factors (GFs) is controversial. Interestingly, histone deacetylase (HD) inhibitors (sodium butyrate, NaB, trichostatin A, TSA) reactivate HbF. We developed a novel model system to investigate HbF reactivation: (1) single hematopoietic progenitor cells (HPCs) were seeded in serum-free unilineage erythroid culture; (2) the 4 daughter cells (erythroid burst-forming units, [BFU-Es]), endowed with equivalent proliferation/differentiation and HbF synthesis potential, were seeded in 4 unicellular erythroid cultures differentially treated with graded dosages of GFs and/or HD inhibitors; and (3) HbF levels were evaluated in terminal erythroblasts by assay of F cells and gamma-globin content (control levels, 2.4% and 1.8%, respectively, were close to physiologic values). HbF was moderately enhanced by interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor treatment (up to 5%-8% gamma-globin content), while sharply reactivated in a dose-dependent fashion by c-kit ligand (KL) and NaB (20%-23%). The stimulatory effects of KL on HbF production and erythroid cell proliferation were strictly correlated. A striking increase of HbF was induced by combined addition of KL and NaB or TSA (40%-43%). This positive interaction is seemingly mediated via different mechanisms: NaB and TSA may modify the chromatin structure of the beta-globin gene cluster; KL may activate the gamma-globin promoter via up-modulation of tal-1 and possibly FLKF transcription factors. These studies indicate that KL plays a key role in HbF reactivation in adult life. Furthermore, combined KL and NaB administration may be considered for sickle cell anemia and beta-thalassemia therapy.
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PMID:Hemoglobin switching in unicellular erythroid culture of sibling erythroid burst-forming units: kit ligand induces a dose-dependent fetal hemoglobin reactivation potentiated by sodium butyrate. 1082 43


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