Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P10721 (c-kit)
 6,575 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The "stromal" or adherent cells of long-term murine Dexter explant bone marrow cultures provide the best in vitro model of the bone marrow microenvironment. Colony-stimulating factor-1 (CSF-1) is produced constitutively by these cells and is easily detected, but most investigators have not found constitutive production of the other hemolymphopoietic cytokines. We have previously reported the detection of granulocyte-macrophage-CSF (GM-CSF) in murine stromal cultures and its induction by the lectin Pokeweed mitogen. The present studies analyzing stromal cytokine messenger RNA (mRNA) production by standard Northern blot analysis show constitutive production of mRNAs for CSF-1, GM-CSF, granulocyte-CSF (G-CSF), c-kit ligand (KL), and interleukin-6 (IL-6), but not IL-3, IL-4, or IL-5 by 3-week irradiated or nonirradiated murine Dexter stromal cells. Exposure of stromal cells to Pokeweed mitogen or IL-1 16 hours before RNA harvest induces the messages for GM-CSF, G-CSF, KL, and IL-6, but not IL-3, IL-4, IL-5, or CSF-1. Polymerase chain reaction amplification of cDNA made with reverse transcriptase from stromal RNA using two separate sets of IL-3-specific primers shows the presence of IL-3 message in irradiated stromal cells, which is only detectable with this more sensitive technique. The factor-dependent cell lines FDC-P1 and 32D are supported by the stromal cells without the addition of exogenous growth factors, demonstrating a cytokine activity in these cultures that is inhibited by the addition of anti-IL-3 or anti-GM-CSF antibodies. These data indicate that murine Dexter stromal cells constitutively produce CSF-1, GM-CSF, G-CSF, IL-6, KL, and IL-3. This growth factor production could explain the support of granulocyte, macrophage, and megakaryocyte production and stem cell maintenance in Dexter-type long-term murine bone marrow cultures.
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PMID:Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma. 137 43

FDC-P1 haemopoietic cells were used to select mutations of c-fms that constitutively activate the receptor for macrophage-colony stimulating factor (M-CSF or CSF-1). One mutation changed Ser 929 to Gly within a Ser/Gly rich region of the C-terminal tail and a second changed a nearby, highly conserved Leu 926 for Pro. A third mutation (D802V) changed Asp 802 to Val within the alpha L12/beta 9 region of the tyrosine kinase domain, so supporting the crystallographic evidence that this region triggers kinase activation. A c-kit mutation exactly equivalent to D802V was previously identified in a leukamic cell line and was demonstrated here to be transforming. Surprisingly, although D802V potently transformed FDC-P1 cells, it could not induce Rat-2 fibroblast foci, even in the presence of M-CSF. It is suggested that the accelerated receptor degradation induced by D802V may account for its cell specific effect.
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PMID:Selection of activating mutations of c-fms in FDC-P1 cells. 747 56

Activation of phosphatidylinositol (PI) 3-kinase is a common sequel to tyrosine kinase activation and appears to be essential for tyrosine kinases to induce proliferation. Since multiple hemopoietic growth factors activate tyrosine kinases, we investigated whether these growth factors activate PI 3-kinase. We show that interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), granulocyte-macrophage colony stimulating factor (GM-CSF), and steel factor (SLF) all activate PI 3-kinase. These cytokines increased the amount of PI 3-kinase activity that could be immunoprecipitated with anti-phosphotyrosine antibodies from the MC-9 mast cell line or from the hemopoietic progenitor cell line FDC-P1. Increases in this assay frequently correlate with PI 3-kinase activation in vivo. To determine directly whether these factors activate PI 3-kinase in vivo, we measured the levels of 3-phosphorylated inositol phospholipids in intact 32P-labeled MC-9 cells. IL-3, IL-4, IL-5, GM-CSF, and SLF all caused increased synthesis of the PI 3-kinase products phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate with a relative potency of SLF >> IL-3 > IL-5, GM-CSF > IL-4. In contrast, IL-4 caused the largest increase in the in vitro anti-phosphotyrosine immune complex PI 3-kinase assay. Thus, the in vitro assay does not accurately reflect in vivo activation of PI 3-kinase. Cytokine treatment did not stimulate tyrosine phosphorylation of either the 85-kDa regulatory subunit or the 110-kDa catalytic subunit of PI 3-kinase and is therefore not required for activation of PI 3-kinase by these factors. Cytokine treatment did induce PI 3-kinase to associate with other tyrosine-phosphorylated proteins in a cytokine-specific manner. PI 3-kinase associated with c-kit after SLF stimulation, a 170-kDa protein after IL-4 stimulation, and a 70-kDa protein after treatment with IL-3 or GM-CSF. Thus, multiple hemopoietic growth factors that act through different types of receptors activate PI 3-kinase in vivo and induce factor-specific interactions of PI 3-kinase with other tyrosine-phosphorylated proteins.
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PMID:Multiple cytokines activate phosphatidylinositol 3-kinase in hemopoietic cells. Association of the enzyme with various tyrosine-phosphorylated proteins. 750 38

The cell-surface receptor c-kit and its cognate ligand stem-cell factor (SCF) or steel factor (SLF) are important for the maintenance of hematopoiesis both in vitro and in vivo. Transforming growth factor-beta (TGF-beta) has been shown to be a potent inhibitor of SLF-mediated synergistic growth of murine Lin-Sca-1+ progenitor cells, as well as more committed progenitors. In the present study, we examined the regulation of c-kit mRNA and cell-surface expression by TGF-beta. Among the murine hematopoietic progenitor cells tested, the myeloid cell line FDC-P1 and the mast-cell line MC-6, as well as progenitor-enriched bone marrow cells, constitutively expressed functional cell-surface c-kit. Treatment of these progenitor cell lines and primary progenitor cells with TGF-beta resulted in downregulation of cell-surface c-kit expression. This effect was not a secondary event of cell-cycle status. TGF-beta inhibition was dose- and time-dependent, with 50% inhibition seen between 0.3 to 3 ng/mL TGF-beta and maximal inhibition at 30 ng/mL. Using the FDC-P1 cell line, we observed that the inhibition of cell-surface c-kit expression by TGF-beta is preceded by a marked reduction in c-kit mRNA levels starting 2 hours after TGF-beta treatment, and reaches a maximum by 6 hours. The inhibition in steady-state c-kit mRNA levels is explained, in part, by a decrease in the half-life of c-kit transcripts (2 to 4 hours for control cells v 0.5 to 1.5 hours for TGF-beta-treated cells). These findings suggest that TGF-beta regulates the responsiveness of murine hematopoietic progenitors to SLF through a decrease in c-kit message stability leading to decreased cell-surface expression.
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PMID:Transforming growth factor-beta regulates c-kit message stability and cell-surface protein expression in hematopoietic progenitors. 751

The c-kit receptor tyrosine kinase (KIT) is activated upon ligand binding, thereby leading to a variety of signaling events that play a fundamental role in hematopoiesis. In addition to ligand-dependent activation, we have previously shown that KIT is constitutively activated in a ligand-independent manner by two point mutations, Val-559-->Gly (G559) mutation in the juxtamembrane domain and Asp-814-->Val (V814) mutation in the phosphotransferase domain. To investigate the biochemical consequence and biologic significance of these mutations, retroviral vectors encoding KITG559 or KITV814 were introduced into murine pro-B-type Ba/F3 cells and myeloid FDC-P1 cells, both of which require interleukin-3 (IL-3) for their growth and survival. In the cells, KITG559 or KITV814 were found to be constitutively phophorylated on tyrosine in the absence of stem cell factor (SCF) that is a ligand for KIT. Chemical cross-linking analysis showed that a substantial fraction of the phosphorylated KITG559 underwent dimerization even in the absence of SCF, whereas the phosphorylated KITV814 did not, suggesting the distinct mechanisms underlying constitutive activation of KIT by G559 and V814 mutations. Furthermore, the cells expressing either KITG559 or KITV814 were found to show a factor-independent growth, whereas the cells expressing wild-type KIT (KITWT) proliferated in response to SCF as well as IL-3. Moreover, subcutaneous injection of Ba/F3 cells expressing KITG559 or KITV814 into nude mice resulted in production of large tumors at all sites of the injection within 2 weeks, and all nude mice quickly succumbed to leukemia and died. These results suggest that, although the mechanisms underlying constitutive activation of KITG559 or KITV814 may be different, both of the activating mutations have a function to induce a factor-independent and tumorigenic phenotype. Also, the data of this study raise the possibility that the constitutively activating mutations of c-kit may play a causal role in development of hematologic malignancies.
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PMID:Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. 753 May 9

TNF-alpha is a pleiotropic cytokine with stimulatory as well as inhibitory effects on hematopoiesis. We have previously demonstrated that TNF-alpha directly inhibits CSF-induced proliferation of primitive murine lineage-negative bone marrow progenitors (Lin-) and stem cell antigen-1 hematopoietic progenitors through the 75-kDa TNF receptor (TNF-R2), whereas TNF-alpha-induced inhibition of more committed Lin- progenitors is mediated through the 55-kDa TNF-R (TNF-R1), indicating a differential role of the two TNF-Rs in hematopoiesis. Numerous studies have demonstrated the ability of stem cell factor (SCF), a key regulator of hematopoiesis signaling through c-kit, to synergize with other hematopoietic growth factors, but little is known about cytokines capable of inhibiting hematopoiesis induced by SCF. While TNF-alpha has been demonstrated to enhance SCF-induced proliferation of myeloid leukemia blasts, the present report demonstrates that TNF-alpha, by signaling through TNF-R2, inhibits SCF-induced proliferation of normal murine Lin- and stem cell antigen-1 hematopoietic progenitors. SCF-stimulated proliferation of the hematopoietic cell line FDC-P1 was also potently inhibited by TNF-alpha and was accompanied by down-regulation of c-kit cell surface expression as well as c-kit mRNA levels. Finally, treatment of the FDC-P1 cell line with TNF-alpha resulted in increased levels of the tumor suppressor p53 mRNA, suggesting another mechanism by which hematopoietic effects of TNF-alpha may be mediated.
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PMID:Inhibition of stem cell factor-induced proliferation of primitive murine hematopoietic progenitor cells signaled through the 75-kilodalton tumor necrosis factor receptor. Regulation of c-kit and p53 expression. 753 12

Monocytes and macrophages show marked phenotypic variation dependent on their tissue of origin. Peripheral blood monocytes have been found to be sources of a variety of cytokines, but isolated marrow macrophages have not been characterized in this regard. Marrow macrophages form a predominant component of murine adherent Dexter stromal cells and can be isolated by sequential explant culture in colony-stimulating factor-1 (CSF-1). We have studied murine (Balb/c) bone marrow macrophage (BMM) cytokine production in the presence or absence of CSF-1, the lectin pokeweed mitogen (PWM) or interleukin-3 (IL-3). Biologic activity in conditioned media (cm) from control and induced BMM was assessed using the factor-dependent cell lines 32D, NFS-60, T1165, MC-6 and FDC-P1. Cell line stimulation and antibody blocking indicated the presence of c-kit ligand, interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF). This stimulatory activity was increased by exposure to PWM or the combination of CSF-1 and PWM or CSF-1 and IL-3. CSF-1, as determined by radioimmunoassay (RIA), was essentially undetectable in baseline cm and induction was not seen with PWM or CSF-1. Baseline or "constitutive" expression of BMM and mRNA for CSF-1 and c-kit ligand was seen. Uninduced BMM did not express mRNA for G-CSF, granulocyte-macrophage CSF (GM-CSF), IL-6 or IL-3. CSF-1 induced increased expression of IL-6 mRNA, PWM induced increased expression of G-CSF and IL-6 mRNA and the combination of PWM and CSF-1 induced expression of CSF-1, G-CSF and IL-6 mRNA. Varying levels of CSF-1 had differential effects on cytokine production. Increasing levels of CSF-1 increased IL-6 mRNA and downmodulated CSF-1 mRNA expression. There was a biphasic response of c-kit ligand mRNA expression to CSF-1 exposure; low levels of CSF-1 (50 U/mL) induced, while higher levels (2000 U/mL) inhibited, expression. These data indicate that BMM (and by analogy the macrophage component of Dexter culture stroma), are important sources of CSF-1 and c-kit ligand but not GM-CSF or IL-3. BMM can also be induced to express IL-6 and/or G-CSF. Lastly, CSF-1, by differentially modulating BMM cytokine production in a holocrine or autocrine manner, may function as a central regulator of stromal based hematopoiesis.
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PMID:Cytokine expression from bone marrow derived macrophages. 767 17

Cells of the murine interleukin-3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) factor-dependent line, FDC-P1, express the tyrosine kinase receptor, c-kit. The ligand for c-kit, steel factor (SLF), encoded by the steel (Sl) locus, is produced as both membrane-bound and soluble forms by fibroblastoid cells. Fibroblasts derived from normal (+/+) WCB6F1 mice are known to produce both forms of SLF and were able to support FDC-P1 cells in a contact-dependent manner in the presence of neutralizing anti-GM-CSF antiserum. In contrast, Sl/Sld mutant fibroblasts, which produce only a soluble form of SLF, were incapable of supporting FDC-P1 cells in the presence of GM-CSF antiserum. These results suggested that FDC-P1 cells were being supported on fibroblast layers by membrane-bound SLF. Attempts to grow FDC-P1 cells in high levels of soluble recombinant SLF to mimic the SLF-dependent response seen in co-culture experiments showed that cells which had been previously grown in GM-CSF or IL-3 were minimally responsive to SLF at concentrations up to 100 ng/mL. Although these cultures were not supported by SLF alone, the cells showed synergistic proliferative responses to SLF combined with suboptimal levels of GM-CSF or IL-3. FDC-P1 cells could, however, be adapted to grow in SLF alone by gradual substitution of SLF for GM-CSF over a period of 3 weeks. These cells showed 5.6- to 8.4-fold and 2.5-fold higher levels of c-kit mRNA than cells grown in GM-CSF or IL-3, respectively. Downregulation of surface c-kit protein was also seen in FDC-P1 cells grown in GM-CSF or IL-3 compared with cells grown in SLF. Although FDC-P1 cells propagated in SLF were more responsive to SLF, they were still able to proliferate as well in GM-CSF and IL-3 as the cells originally grown in the latter factors. Thus, functional downregulation of c-kit by GM-CSF and IL-3 was unidirectional.
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PMID:Responses of the murine myeloid cell line FDC-P1 to soluble and membrane-bound forms of steel factor (SLF). 768

Signaling molecules that are responsible for proliferation and differentiation of hematopoietic cells following ectopic expression of receptor tyrosine kinases (RTKs) were investigated in the interleukin 3 (IL-3)-dependent hematopoietic cell line, FDC-P1. Cells were transfected with human platelet-derived growth factor receptor (PDGF-R), macrophage colony stimulating factor-1 receptor (CSF-1R), epidermal growth factor receptor (EGF-R), and chimeras consisting of the extracellular domain of EGF-R and the transmembrane and cytoplasmic domains of either HER2 (HER1-2) or c-kit (EK-R). All FDC-P1 transfectants proliferated in response to the corresponding growth factor in the absence of IL-3. However, only cells expressing PDGF-R, CSF-1R, and EK-R (type III RTKs) differentiated along the monocyte-macrophage lineage after treatment with their activating ligands. Analysis of proteins from these RTK-expressing cells revealed that a Mr 85,000 protein showed in vitro phosphorylation, and V8 protease peptide mapping showed that this protein was p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase). Accordingly, activation of PDGF-R-, CSF-1R-, and EK-R-expressing cells led to an increase in PI3-kinase activity. Expression of EK-R mutant Y721F, which lacked the known p85 binding site, blocked differentiation and activation of PI3-kinase, without affecting proliferation. Last, addition of wortmannin to cells expressing PDGF-R, CSF-1R, and EK-R blocked ligand-induced differentiation in a concentration-dependent manner, and this effect correlated with wortmannin's ability to inhibit PI3-kinase. Thus, ectopic expression of both type I and III RTKs could stimulate FDC-P1 proliferation in the absence of IL-3; however, only activation of type III RTKs led to differentiation via selective coupling to p85 and PI3-kinase activation.
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PMID:Activation of phosphatidylinositol 3-kinase is necessary for differentiation of FDC-P1 cells following stimulation of type III receptor tyrosine kinases. 954 91

Hematopoietic cell growth, differentiation, and commitment to a restricted lineage are guided by a set of cytokines acting exclusively on cells expressing the corresponding cytokine receptor. The macrophage colony stimulating factor (M-CSF, also termed CSF-1) and its cognate receptor, the tyrosine kinase c-Fms, are essential for monocyte and macrophage development. The underlying molecular mechanism, however, is poorly understood. Here we identified a novel Fms-interacting protein (FMIP, MW 78 kDa) which binds transiently via its N-terminal 144 residues to the cytoplasmic domain of activated Fms-molecules. Binding of FMIP was paralleled by rapid tyrosine phosphorylation within the binding domain which drastically reduced its ability to associate with Fms. Binding was specific as evidenced by co-immunoprecipitation and association with recombinant GST-Fms fusion proteins. No binding was observed with the tyrosine phosphorylated cytoplasmic domains of c-Kit, TrkA, c-Met, and the insulin receptor. The role of FMIP in hematopoietic differentiation was studied in the bipotential myeloid progenitor cell line, FDC-P1Mac11. Overexpression of FMIP prevented M-CSF induced macrophage differentiation. Instead, cells differentiated into granulocytes. Our data suggest that the level of FMIP expression could form a threshold that decides about differentiation either into macrophages or into granulocytes.
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PMID:FMIP, a novel Fms-interacting protein, affects granulocyte/macrophage differentiation. 1059 51


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