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Query: UNIPROT:P06889 (Mol)
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Stem cell factor (SCF) plays a crucial role in hematopoiesis through its interaction with the receptor tyrosine kinase c-kit. However, the signaling events that are activated by this interaction and involved in the control of growth or differentiation are not completely understood. We demonstrate here that Tec, a cytoplasmic, src-related kinase, physically associates with c-kit through a region that contains a proline-rich motif, amino terminal of the SH3 domain. Following SCF binding, Tec is tyrosine phosphorylated and its in vitro kinase activity is increased. Tyrosine phosphorylation of Tec is not detected in the response to other cytokines controlling hematopoiesis, including colony-stimulating factor-1 (CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). Conversely, the cytoplasmic kinase JAK2 is activated by IL-3 but not by SCF stimulation. The activation of distinct cytoplasmic kinases may account for the synergy seen in the actions of SCF and IL-3 on hematopoietic stem cells.
Mol Cell Biol 1994 Dec
PMID:Tec kinase associates with c-kit and is tyrosine phosphorylated and activated following stem cell factor binding. 752 58

The different 3' noncoding AU-rich elements (ARE) that mediate the degradation of many short-lived mRNAs may function through distinct decay pathways; translation-dependent and -independent mechanisms have been proposed. To investigate the cotranslational model, we designed an expression system that exploits the properties of the ferritin iron-responsive element to shuttle chimeric mRNAs from ribonucleoproteins to polyribosomes. The iron-responsive element was introduced in the 5' untranslated regions of alpha-globin mRNAs that harbored in their 3' untranslated regions either the c-fos ARE or the granulocyte-macrophage colony-stimulating factor ARE as prototypes of the different ARE subsets. The cytoplasmic location of the transcripts was controlled by intracellular iron availability and monitored by polysomal profile analysis. We report that these two mRNA subsets behaved identically in this system. Iron deprivation by desferrioxamine treatment stabilized both transcripts by sequestering them away from polyribosomes. Sequential treatments with desferrioxamine, followed by hemin to concentrate the mRNAs in the ribonucleoprotein pool prior to translation, showed that rapid degradation occurred only upon redistribution of the transcripts to polyribosomes. Deletion of a critical cytosine in the iron-responsive element abolished targeted sequestration and restored high-level constitutive mRNA instability. These observations demonstrate that the c-fos and granulocyte-macrophage colony-stimulating factor ARE subsets mediate selective mRNA degradation through similar polysome-associated mechanisms coupled with ongoing translation.
Mol Cell Biol 1995 Jul
PMID:Rapid mRNA degradation mediated by the c-fos 3' AU-rich element and that mediated by the granulocyte-macrophage colony-stimulating factor 3' AU-rich element occur through similar polysome-associated mechanisms. 754 Jul 19

Granulocyte-macrophage colony-stimulating factor (GM-CSF) mainly stimulates proliferation and maturation of myeloid progenitor cells. Although the signal transduction pathways triggered by GM-CSF receptor (GMR) have been extensively characterized, the roles of GMR signals in differentiation have remained to be elucidated. To examine the relationship between receptor expression and differentiation of hemopoietic cells, we used transgenic mice (Tg-mice) that constitutively express human (h) GMR at almost all stages of hemopoietic cell development. Proliferation and differentiation of hemopoietic progenitors in bone marrow cells from these Tg-mice were analyzed by methylcellulose colony formation assay. High affinity GMR interacts with GM-CSF in a species-specific manner, therefore one can analyze the effects of hGMR signals on differentiation of mouse hemopoietic progenitors using hGM-CSF. Although mouse (m) GM-CSF yielded only GM colonies, hGM-CSF supported various types of colonies including GM, eosinophil, mast cell, erythrocyte, megakaryocyte, blast cell, and mixed hemopoietic colonies. Thus, the effects of hGM-CSF on colony formation more closely resembled mIL-3 than those of mGM-CSF. In addition, hGM-CSF generated a much larger number of blast cell colonies and mixed cell colonies than did mIL-3. hGM-CSF also generated erythrocyte colonies in the absence of erythropoietin. Therefore, GM-CSF apparently has the capacity to promote growth of cells of almost all hemopoietic cell lineages, if functional hGMR is present.
Mol Biol Cell 1995 May
PMID:A human GM-CSF receptor expressed in transgenic mice stimulates proliferation and differentiation of hemopoietic progenitors to all lineages in response to human GM-CSF. 754 29

Cytokine-suppressing anti-inflammatory drugs (CSAIDs) are reported to inhibit production of proinflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) by affecting a stress-induced kinase. To gain a better understanding of the selectivity and cellular dynamics of this type of inhibitor, we studied in vitro the prototype member of this class of agents, SKF86002. Lipopolysaccharide (LPS)-activated human monocytes treated with SKF86002 produced less proIL-1 beta but normal amounts of the noncytokine lysozyme. Two-dimensional gel analysis indicated that only eight polypeptides produced by monocytes were decreased by SKF86002. Inhibition of IL-1 beta production was achieved by affecting two separate steps in this cytokine's biogenesis. First, SKF86002 lowered proIL-1 beta synthesis. By pulse-chase analysis, this effect was localized to a posttranscriptional site of action; maximal inhibition was observed when SKF86002 was added at the time of cytokine translation. Exposure of monocytes to SKF86002 for > 2 hr led to a loss of IL-1 beta inhibitory activity, suggesting that these cells adapted to this agent. Moreover, LPS-activated monocytes that were pretreated with granulocyte-macrophage colony-stimulating factor were less sensitive to the proIL-1 beta inhibitory effect of SKF86002, and production of proIL-1 beta by cytokine-stimulated human fibroblasts was impaired only modestly by the CSAID. A second effect of SKF86002 was to inhibit release of IL-1 beta into the medium in response to high concentrations of LPS; this effect is observed only with freshly isolated human monocytes as other IL-1 beta-producing cells do not release significant cytokine in response to LPS. The ability of SKF86002 to inhibit this posttranslational mechanism was mimicked by lysosomotrophic agents such as chloroquine, quinacrine, and methylamine. In contrast, chloroquine, and quinacrine were not effective inhibitors of monocyte proIL-1 beta translation. Thus, SKF86002 inhibits IL-1 beta production by affecting at least two distinct steps in the biosynthesis of this cytokine. Manifestation of these two effects, however, is dependent on the length of time for which cells are exposed to this agent and the nature of the cytokine-producing cellular system.
Mol Pharmacol 1995 Sep
PMID:Inhibition of interleukin-1 beta production by SKF86002: evidence of two sites of in vitro activity and of a time and system dependence. 756 23

Previously we have shown that the zinc finger transcription factor Egr-1 is essential for and restricts differentiation of hematopoietic cells along the macrophage lineage, raising the possibility that Egr-1 actually plays a deterministic role in governing the development of hematopoietic precursor cells along the monocytic lineage. To test this hypothesis, we have taken advantage of interleukin-3-dependent 32Dcl3 hematopoietic precursor cells which, in addition to undergoing granulocytic differentiation in response to granulocyte colony-stimulating factor, were found to be induced for limited proliferation, but not differentiation, by granulocyte-macrophage colony-stimulating factor. It was shown that ectopic expression of Egr-1 blocked granulocyte colony-stimulating factor-induced terminal granulocytic differentiation, consistent with previous findings. In addition, ectopic expression of Egr-1 endowed 32Dcl3 cells with ability to be induced by granulocyte-macrophage colony-stimulating factor for terminal differentiation exclusively along the macrophage lineage. Thus, evidence that Egr-1 potentiates terminal macrophage differentiation has been obtained, suggesting that Egr-1 plays a deterministic role in governing the development of hematopoietic cells along the macrophage lineage.
Mol Cell Biol 1995 Oct
PMID:The zinc finger transcription factor Egr-1 potentiates macrophage differentiation of hematopoietic cells. 756 1

Poly(A) tail removal is a critical first step in the decay pathway for many yeast and mammalian mRNAs. Poly(A) shortening rates can be regulated by cis-acting sequences within the transcribed portion of mRNA, which in turn control mRNA turnover rates. The AU-rich element (ARE), found in the 3' untranslated regions of many highly labile mammalian mRNAs, is a well-established example of this type of control. It represents the most widespread RNA stability determinant among those characterized in mammalian cells. Here, we report that two structurally different AREs, the c-fos ARE and the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE, both direct rapid deadenylation as the first step in mRNA degradation, but by different kinetics. For c-fos-ARE-mediated decay, the mRNA population undergoes synchronous poly(A) shortening and is deadenylated at the same rate, implying the action of distributive or nonprocessive ribonucleolytic digestion of poly(A) tails. In contrast, the population of granulocyte-macrophage colony-stimulating factor ARE-containing mRNAs is deadenylated asynchronously, with the formation of fully deadenylated intermediates, consistent with the action of processive ribonucleolytic digestion of poly(A) tails. An important general implication of this finding is that different RNA-destabilizing elements direct deadenylation either by modulating the processivity at which a single RNase functions or by recruiting kinetically distinct RNases. We have also employed targeted inhibition of translation initiation to demonstrate that the RNA-destabilizing function of both AREs can be uncoupled from translation by ribosomes. In addition, a blockade of ongoing transcription has been used to further probe the functional similarities and distinctions of these two AREs. Our data suggest that the two AREs are targets of two distinct mRNA decay pathways. A general model for ARE-mediated mRNA degradation involving a potential role for certain heterogeneous nuclear ribonucleoproteins and ARE-binding proteins is proposed.
Mol Cell Biol 1995 Oct
PMID:mRNA decay mediated by two distinct AU-rich elements from c-fos and granulocyte-macrophage colony-stimulating factor transcripts: different deadenylation kinetics and uncoupling from translation. 756 31

T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte-macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3 receptor (VDR). We therefore examined vitamin D3-mediated repression of activated IL-2 expression by cotransfecting Jurkat cells with IL-2 promoter/reporter constructs and a VDR overexpression vector and by DNA binding. We delineated an element conferring both DNA binding by the receptor in vitro and 1,25(OH)2D3-mediated repression in vivo to a short 40-bp region encompassing an important positive regulatory element, NF-AT-1, which is bound by a T-cell-specific transcription factor, NFATp, as well as by AP-1. VDR DNA-binding mutants were unable to either bind to this element in vitro or repress in vivo; the VDR DNA-binding domain alone, however, bound the element but also could not repress IL-2 expression. These results indicate that DNA binding by VDR is necessary but not sufficient to mediate IL-2 repression. By combining partially purified proteins in vitro, we observed the loss of the bound NFATp/AP-1-DNA complex upon inclusion of VDR or VDR-retinoid X receptor. Order of addition and off-rate experiments indicate that the VDR-retinoid X receptor heterodimer blocks NFATp/AP-1 complex formation and then stably associates with the NF-AT-1 element. This direct inhibition by a nuclear hormone receptor of transcriptional activators of the IL-2 gene may provide a mechanistic explanation of how vitamin derivatives can act as potent immunosuppressive agents.
Mol Cell Biol 1995 Oct
PMID:Transcriptional repression of the interleukin-2 gene by vitamin D3: direct inhibition of NFATp/AP-1 complex formation by a nuclear hormone receptor. 756 32

Growth factor receptors play an important role in hematopoiesis. In order to further understand the mechanisms directing the expression of these key regulators of hematopoiesis, we initiated a study investigating the transcription factors activating the expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor alpha gene. Here, we demonstrate that the human GM-CSF receptor alpha promoter directs reporter gene activity in a tissue-specific fashion in myelomonocytic cells, which correlates with its expression pattern as analyzed by reverse transcription PCR. The GM-CSF receptor alpha promoter contains an important functional site between positions -53 and -41 as identified by deletion analysis of reporter constructs. We show that the myeloid and B cell transcription factor PU.1 binds specifically to this site. Furthermore, we demonstrate that a CCAAT site located upstream of the PU.1 site between positions -70 and -54 is involved in positive-negative regulation of the GM-CSF receptor alpha promoter activity. C/EBP alpha is the major CCAAT/enhancer-binding protein (C/EBP) form binding to this site in nuclear extracts of U937 cells. Point mutations of either the PU.1 site or the C/EBP site that abolish the binding of the respective factors result in a significant decrease of GM-CSF receptor alpha promoter activity in myelomonocytic cells only. Furthermore, we demonstrate that in myeloid and B cell extracts, PU.1 forms a novel, specific, more slowly migrating complex (PU-SF) when binding the GM-CSF receptor alpha promoter PU.1 site. This is the first demonstration of a specific interaction with PU.1 on a myeloid PU.1 binding site. The novel complex is distinct from that described previously as binding to B cell enhancer sites and can be formed by addition of PU.1 to extracts from certain nonmyeloid cell types which do not express PU.1, including T cells and epithelial cells, but not from erythroid cells. Furthermore, we demonstrate that the PU-SF complex binds to PU.1 sites found on a number of myeloid promoters, and its formation requires an intact PU.1 site adjacent to a single-stranded region. Expression of PU.1 in nonmyeloid cells can activate the GM-CSF receptor alpha promoter. Deletion of the amino-terminal region of PU.1 results in a failure to form the PU-SF complex and in a concomitant loss of transactivation, suggesting that formation of the PU-SF complex is of functional importance for the activity of the GM-CSF receptor alpha promoter. Finally, we demonstrate that C/EBP alpha can also active the GM-CSF receptor alpha promoter in nonmyeloid cells. These results suggest that PU.1 and C/EBP alpha direct the cell-type-specific expression of GM-CSF receptor alpha, further establish the role of PU.1 as a key regulator of hematopoiesis, and point to C/EBP alpha as an additional important factor in this process.
Mol Cell Biol 1995 Oct
PMID:PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene. 756 36

The 3' noncoding region (NCR) AU-rich element (ARE) selectively confers rapid degradation on many mRNAs via a process requiring translation of the message. The role of cotranslation in destabilization of ARE mRNAs was examined by insertion of translation-blocking stable secondary structure at different sites in test mRNAs containing either the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE or a control sequence. A strong (-80 kcal/mol [1 kcal = 4.184 kJ]) but not a moderate (-30 kcal/mol) secondary structure prevented destabilization of mRNAs when inserted at any position upstream of the ARE, including in the 3' NCR. Surprisingly, a strong secondary structure did not block rapid mRNA decay when placed immediately downstream of the ARE. Studies are also presented showing that the turnover of mRNAs containing control or ARE sequences is not altered by insertion of long (1,000-nucleotide) intervening segments between the stop codon and the ARE or between the ARE and poly(A) tail. Characterization of ARE-containing mRNAs in polyadenylated and whole cytoplasmic RNA fractions failed to find evidence for decay intermediates degraded to the site of strong secondary structure from either the 5' or 3' end. From these and other data presented, this study demonstrates that complete translation of the coding region is essential for activation of rapid mRNA decay controlled by the GM-CSF ARE and that the structure of the 3' NCR can strongly influence activation. The results are consistent with activation of ARE-mediated decay by possible entry of translation-linked decay factors into the 3' NCR or translation-coupled changes in 3' NCR ribonucleoprotein structure or composition.
Mol Cell Biol 1995 Nov
PMID:Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5' to the ARE. 756 86

Although human eosinophils express low concentrations of CD4, the capacity of mature, non-replicating eosinophils to be infected with human immunodeficiency virus-1 (HIV-1) has not been established. Using peripheral blood eosinophils isolated free of contaminating lymphocytes and mononuclear leukocytes, we evaluated eosinophil infection with HIV-1. Eosinophils could be infected with strains of HIV-1 as evidenced by HIV-induced cytolytic effects, progressive release of p24 antigen in cultures of infected eosinophils, recovery of HIV from infected eosinophils by co-cultivation, and detection of HIV-1 gag viral DNA from infected eosinophils by polymerase chain reaction (PCR) amplification. Greater p24 antigen release from infected eosinophils was elicited by the phorbol ester, PMA; and eosinophil killing by HIV-1 was enhanced by the cytokine GM-CSF. By light and electron microscopy, HIV-infected eosinophils demonstrated apoptosis and necrosis. Apoptotic subdiploid nuclear staining was detected by flow cytometric analyses of propidium iodide-stained nuclei from HIV-infected eosinophils, and DNA isolated from HIV-infected eosinophils showed both nucleosomal fragmentation and diffuse degradation. Thus, mature eosinophils, non-replicating terminally differentiated leukocytes, can be infected with HIV-1. HIV-1 expression in eosinophils is promoted by increased granulocyte-macrophage colony-stimulating factor (GM-CSF) and can cause eosinophils to undergo death due to apoptosis and necrosis.
Am J Respir Cell Mol Biol 1995 Nov
PMID:Infection, apoptosis, and killing of mature human eosinophils by human immunodeficiency virus-1. 757 98


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