Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.10.1 (ERK)
 95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Inhibition of MAPK by elevated intracellular cAMP has often been correlated with suppression of growth factor-induced proliferation. However, in murine bone marrow-derived macrophages (BMM) we show that the cAMP analogue, 8-bromo cAMP (8BrcAMP) (1mM), despite being a dramatic G1 phase proliferation inhibitor, increased ERK activity both in the absence and presence of CSF-1; these increases were blocked by PD98059 (100 microM) suggesting MEK dependence. In contrast, CSF-1-stimulated p21Ras activity was blocked by 8BrcAMP thus correlating with the inhibition of proliferation. This is the first report to indicate that elevated intracellular cAMP can activate ERK activity while inhibiting proliferation and the data support the concept in CSF-1-treated macrophages of Ras-independent activation of ERK activity. It was also found that the acute but not the sustained elevation of c-fos mRNA expression due to 8BrcAMP was also MEK dependent indicating that there are separate pathways controlling c-fos mRNA expression in BMM.
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PMID:cAMP enhances CSF-1-induced ERK activity and c-fos mRNA expression via a MEK-dependent and Ras-independent mechanism in macrophages. 951 45

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

A point mutation substituting Arg777 by Gln was obtained in a highly conserved region of the human colony-stimulating factor-1 receptor (CSF-1R) sequence. Constitutive expression of wild-type receptors in CHO cells confers susceptibility to CSF-1 for proliferation whereas the mutated receptors exhibited a 90% reduced efficiency in proliferation. We sought to determine the alterations intervening in the CSF-1 signal transduction of the Arg777Gln mutated receptor. We found that ligand binding and ligand-induced CSF-1R internalization were unaffected. CSF-1-induced receptor dimerization and autophosphorylation were impaired to the same extent as mitogen-activated protein kinase activation (90%). However, only phosphatidylinositol 3-kinase activation and ligand-induced receptor ubiquitination were abrogated by the mutation. These features probably reflect the inability of the mutated CSF-1R kinase domain to fold properly and hence to autophosphorylate and/or to associate correctly with transduction proteins. These data may indicate a role for the conserved regions of the RTK kinase domains in the stabilization of the intracellular domain conformation.
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PMID:Arg777 plays a major role in the conformation of the colony-stimulating factor-1 receptor intracellular kinase domain. 961 84

FLT3 ligand (FLT3L) stimulates primitive hematopoietic cells by binding to and activating the FLT3 receptor (FLT3R). We carried out a structure-activity study of human FLT3L in order to define the residues involved in receptor binding. We developed a rapid method to screen randomly mutagenized FLT3L using a FLT3R-Fc fusion protein to probe the relative binding activities of mutated ligand. Approximately 60,000 potential mutants were screened, and the DNA from 59 clones was sequenced. Thirty-one single amino acid substitutions at 24 positions of FLT3L either enhanced or reduced activity in receptor binding and cell proliferation assays. Eleven representative proteins were purified and analyzed for receptor affinity, specific activity, and physical properties. Receptor affinity and bioactivity were highly correlated. FLT3L affinity for receptor improved when four individual mutations that enhance FLT3L receptor affinity were combined in a single molecule. A model of FLT3L three-dimensional structure was generated based on sequence alignment and x-ray structure of macrophage colony-stimulating factor. Most residues implicated in receptor binding are widely dispersed in the primary structure of FLT3L, yet they localize to a surface patch in the tertiary model. A mutation that maps to and is predicted to disrupt the proposed dimerization interface between FLT3L monomers exhibits a Stokes radius that is concentration-dependent, suggesting that this mutation disrupts the FLT3L dimer.
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PMID:Structure-function analysis of FLT3 ligand-FLT3 receptor interactions using a rapid functional screen. 965 58

The fate of hematopoietic progenitor cells (HPCs) in the bone marrow (BM) microenvironment is determined by two different interactions: 1) they adhere (via integrins) to both extracellular matrix molecules and BM stromal cells; and 2) stromal cells produce cytokines that influence their survival, proliferation, differentiation, and mobilization. The ligands for the protein tyrosine kinase receptors c-KIT and FLT3/FLK2, stem cell factor (SCF), and FL are produced by BM stromal cells and are known to affect several facets of hematopoiesis. We studied another protein tyrosine kinase receptor, c-MET, and its ligand hepatocyte growth factor (HGF), also known as scatter factor (SF), which play a similar role in hematopoiesis. c-MET mRNA is expressed in immature human BM HPCs (CD34+CD33- or CD34+CD38-), but not in more mature HPCs (CD34+CD33+ or CD34+CD38+). The ligand HGF/SF is predominantly produced by BM stromal cells at both the mRNA and protein levels. We confirmed functionally that HGF/SF alone has no effect on proliferation of HPCs, but that when combined with granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin-3 it acts as a synergistic proliferative factor, although not as potently as kit-ligand or FLT-3/FLK-2 ligand. Furthermore, HGF/SF promotes adhesion of HPCs to immobilized fibronectin. HGF/SF-induced adhesion to fibronectin is probably caused by activation of the integrins alpha4beta1 and alpha5beta1, insofar as we were able to block this interaction by using monoclonal blocking antibodies directed against these integrin subunits. Addition of the tyrosine-phosphorylation inhibitor genistein inhibited HGF/SF-induced adhesion, supporting the idea that HGF/SF-induced effects are the result of signaling via the receptor c-MET after ligand binding. The enhanced adhesion of HGF/SF to fibronectin proved to be beneficial for the maintenance of the colony-forming potential of HPCs. HGF/SF alone and especially in combination with fibronectin prolongs survival of GM colony-forming cells in liquid culture. Our data indicate that HGF/SF is a polyfunctional cytokine in the BM microenvironment. It is produced by human BM stromal cells and directly or indirectly promotes proliferation, adhesion, and survival of human HPCs.
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PMID:Hepatocyte growth factor/scatter factor (HGF/SF) is produced by human bone marrow stromal cells and promotes proliferation, adhesion and survival of human hematopoietic progenitor cells (CD34+). 969 10

It was recently reported that transgenic expression in the liver of truncated human Met renders hepatocytes constitutively resistant to apoptosis and reproducibly permits their immortalization. The derived stable cell lines (MMH from Met murine hepatocyte) are highly differentiated and nontransformed. In this report, the capacity of MMHs to support in vitro hematopoiesis is characterized. By reverse-transcription polymerase chain reaction, the expression by MMHs of cytokines involved in the survival and self-renewal of early progenitor cells (stem cell factor and FLT3 ligand) as well as those acting at different stages of progenitor differentiation (interleukin [IL] 1beta, IL-3, leukemia inhibitory factor, IL-6, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and thrombopoietin) was shown. A ribonuclease protection assay further substantiated the presence of at least six cytokine transcripts in MMH lines. Cocultures between MMH layers and progenitor-enriched fetal liver hematopoietic cells resulted in a 40-fold to 80-fold expansion of total hematopoietic cells and in a 2.5-fold expansion of clonogenic progenitors after 1 to 2 weeks. Hematopoiesis was maintained for up to 6 weeks with formation of typical cobblestone cell areas and continuous differentiation of precursor into cells at various degrees of maturation. At 5 weeks of coculture, clonogenic progenitors were maintained at 20% of the input level in coculture with embryonic-derived hepatocytes, showing the ability of hepatocyte feeder layer to support survival and possibly self-renewal of clonogenic progenitors. Therefore, the data emphasize a direct role of the hepatocyte in sustaining hematopoietic cell proliferation and differentiation.
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PMID:Hematopoietic support and cytokine expression of murine-stable hepatocyte cell lines (MMH). 982 30

The escape of malignant cells from the immune response against the tumor may result from a defective differentiation or function of professional antigen-presenting cells (APC), ie, dendritic cells (DC). To test this hypothesis, the effect of human renal cell carcinoma cell lines (RCC) on the development of DC from CD34(+) progenitors was investigated in vitro. RCC cell lines were found to release soluble factors that inhibit the differentiation of CD34(+) cells into DC and trigger their commitment towards monocytic cells (CD14(+)CD64(+)CD1a-CD86(-)CD80(-)HLA-D Rlow) with a potent phagocytic capacity but lacking APC function. RCC CM were found to act on the two distinct subpopulations emerging in the culture at day 6 ([CD14(+)CD1a-] and [CD14(-)CD1a+]) by inhibiting the differentiation into DC of [CD14(+)CD1a-] precursors and blocking the acquisition of APC function of the [CD14(-)CD1a+] derived DC. Interleukin-6 (IL-6) and macrophage colony-stimulating factor (M-CSF) were found to be responsible for this phenomenon: antibodies against IL-6 and M-CSF abrogated the inhibitory effects of RCC CM; and recombinant IL-6 and/or M-CSF inhibited the differentiation of DC similarly to RCC CM. The inhibition of DC differentiation by RCC CM was preceeded by an induction of M-CSF receptor (M-CSFR; CD115) and a loss of granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR; CD116) expression at the surface of CD34(+) cells, two phenomenon reversed by anti-IL-6/IL-6R and anti-M-CSF antibodies, respectively. Finally, a panel of tumor cell lines producing IL-6 and M-CSF induced similar effects. Taken together, the results suggest that the inhibition of DC development could represent a frequent mechanism by which tumor cells will escape immune recognition.
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PMID:Inhibition of the differentiation of dendritic cells from CD34(+) progenitors by tumor cells: role of interleukin-6 and macrophage colony-stimulating factor. 984 45

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis by acting as a potent inducer of vascular permeability as well as serving as a specific endothelial cell mitogen. The importance of angiogenic factors such as VEGF, although clearly established in solid tumors, has not been fully elucidated in human hematopoietic neoplasms. We examined the expression of mRNA and protein for VEGF in 12 human hematopoietic tumor cell lines, representing multiple lineages and diseases, including leukemia, lymphoma, and multiple myeloma. Our results revealed that VEGF message was expressed in these cells and that the corresponding protein was secreted into the extracellular environment. Five of the 12 cell lines were also found to express the Flt-1 receptor for VEGF at a moderate to strong level, suggesting an autocrine pathway. When human vascular endothelial cells were exposed to recombinant human VEGF, there was an increase in the mRNA for several hematopoietic growth factors including macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 6. Plasma cells in the bone marrow from patients diagnosed with multiple myeloma were found to express VEGF, whereas both the Flt-1 and KDR high affinity VEGF receptors were observed to be markedly elevated in the normal bone marrow myeloid and monocytic cells surrounding the tumor. These data raise the possibility that VEGF may play a role in the growth of hematopoietic neoplasms such as multiple myeloma through either a paracrine or an autocrine mechanism.
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PMID:Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies. 997 24

Recent studies have shown that long-term repopulating hematopoietic stem cells (HSCs) first appear in the aorta-gonad-mesonephros (AGM) region. Our immunohistochemistry study showed that TEK+ cells existed in the AGM region. Approximately 5% of AGM cells were TEK+, and most of these were CD34(+) and c-Kit+. We then established a coculture system of AGM cells using a stromal cell line, OP9, which is deficient in macrophage colony-stimulating factor (M-CSF). With this system, we showed that AGM cells at 10.5 days postcoitum (dpc) differentiated and proliferated into both hematopoietic and endothelial cells. Proliferating hematopoietic cells contained a significant number of colony-forming cells in culture (CFU-C) and in spleen (CFU-S). Among primary AGM cells at 10.5 dpc, sorted TEK+ AGM cells generated hematopoietic cells and platelet endothelial cell adhesion molecule (PECAM)-1(+) endothelial cells on the OP9 stromal layer, while TEK- cells did not. When a ligand for TEK, angiopoietin-1, was added to the single-cell culture of AGM, endothelial cell growth was detected in the wells where hematopoietic colonies grew. Although the incidence was still low (1/135), we showed that single TEK+ cells generated hematopoietic cells and endothelial cells simultaneously, using a single-cell deposition system. This in vitro coculture system shows that the TEK+ fraction of primary AGM cells is a candidate for hemangioblasts, which can differentiate into both hematopoietic cells and endothelial cells.
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PMID:In vitro hematopoietic and endothelial cell development from cells expressing TEK receptor in murine aorta-gonad-mesonephros region. 1002 83

Dendritic Cell (DC)-based vaccination approaches in man require a reproducible DC generation method that can be performed in conformity with GMP (Good Manufacturing Practice) guidelines and that circumvents the need for multiple blood drawings to generate DC. To this end we modified our previously described method to generate mature DC from CD14 + monocytes by a two step method (priming in GM-SF + IL-4 followed by maturation in monocyte conditioned medium) for use with leukapheresis products as a starting population. Several adaptations were necessary. We established, for example, a modified adherence step to reliably enrich CD14 + DC precursors from apheresis mononuclear cells. The addition of GM-CSF + IL-4 at the onset of culture proved disadvantageous and was, therefore, delayed for 24 h. DC development from apheresis cells occurred faster than from fresh blood or buffy coat, and was complete after 7 days. Monocyte conditioned medium when added on day 6 resulted in fully mature and stable DC (veiled, highly migratory and T cell sensitizing cells with a characteristic phenotype such as 85% CD83 + , p55/fascin + , CD115/M-CSF-R - , CD86 + ) already after 24 h. The mature DC progeny were shown to remain stable and viable if cultured for another 1-2 days in the absence of cytokines, and to be resistant to inhibitory effects of IL-10. Freezing conditions were established to generate DC from frozen aliquots of PBMC or to freeze mature DC themselves for later use. The approach yields large numbers of standardized DC (5-10 x 10(8) mature CD83 + DC/leukapheresis) that are suitable for performing sound DC-based vaccination trials that can be compared with each other.
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PMID:Generation of large numbers of fully mature and stable dendritic cells from leukapheresis products for clinical application. 1003 30


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