Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The GH binding protein (GHBP), which exists in many vertebrates, is a circulating high affinity binding protein corresponding to the extracellular domain of the GH receptor (GHR). In humans, rabbits, and several other species, the GHBP is generated by proteolysis of the GHR and shedding of its extracellular domain. We previously showed that GHBP shedding is inducible by the phorbol ester phorbol 12-myristate,13-acetate (PMA) and inhibited by the metalloprotease inhibitor, Immunex Corp. Compound 3 (IC3). The metzincin metalloprotease, tumor necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE), catalyzes the shedding of TNF-alpha from its transmembrane precursor, a process that is also inhibitable by IC3. TACE may hence be a candidate for GHBP sheddase. In this study, we reconstitute fibroblasts derived from a TACE knockout mouse (Null cells) with either the rabbit (rb) GHR alone (Null/R) or rbGHR plus murine TACE (Null/R+T). Although GHR in both cells was expressed at similar abundance, dimerized normally and caused JAK2 activation in response to GH independent of TACE expression, PMA was unable to generate GHBP from Null/R cells. In contrast, PMA caused ample GHBP generation from TACE reconstituted (Null/R + T) cells, and this GHBP shedding was substantially inhibited by IC3 pretreatment. Corresponding to the induced shedding of GHBP from Null/R + T cells, PMA treatment caused a significant loss of immunoblottable GHR in Null/R+T, but not in Null/R cells. We conclude that TACE is an enzyme required for PMA-induced GHBP shedding and that PMA-induced down-regulation of GHR abundance may in significant measure be attributable to TACE-mediated GHR proteolysis.
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PMID:Tumor necrosis factor-alpha converting enzyme (TACE) is a growth hormone binding protein (GHBP) sheddase: the metalloprotease TACE/ADAM-17 is critical for (PMA-induced) GH receptor proteolysis and GHBP generation. 1110 41

Stem cell factor (SCF) is a growth factor that promotes the survival, proliferation, and differentiation of hematopoietic cells. SCF and its receptor, Kit, are normally present in both cell surface and soluble forms. Both forms of Kit can bind SCF. However, the function of soluble Kit is unknown. In order to determine if soluble Kit can modulate SCF activity, we produced a fusion protein, Kit-Fc, comprised of the extracellular domain of murine Kit and the Fc portion of human IgG(1) and investigated its ability to bind 125I-SCF and to inhibit SCF-stimulated hematopoietic colony growth in vitro. Stable cell lines expressing Kit-Fc were generated and Kit-Fc was purified to greater than 95% purity. Scatchard analysis demonstrated that Kit-Fc binds iodinated SCF with high affinity (Kd 570 pM). Kit-Fc also bound to transmembrane SCF displayed on the surface of fibroblasts. The murine mast cell line IC2 was engineered to express murine Kit on the cell surface and was demonstrated to proliferate in the presence of SCF. Kit-Fc completely blocked SCF-stimulated proliferation of IC2-Kit cells, but not IL-3-stimulated growth of IC2-Kit cells, demonstrating the specificity of Kit-Fc. We investigated the ability of Kit-Fc to block SCF-stimulated murine hematopoietic colony growth. Kit-Fc blocked SCF-stimulated erythroid colony growth as effectively as a neutralizing anti-Kit monoclonal antibody, ACK2, but did not block erythropoietin-stimulated erythroid colony growth. Likewise, Kit-Fc blocked SCF-stimulated myeloid colony growth as effectively as ACK2 antibody, but did not block IL-3- or GM-CSF-stimulated myeloid colony growth. These results indicate that a form of soluble Kit binds SCF with high affinity, and can specifically block the ability of SCF to stimulate hematopoietic colony growth, suggesting that one function of soluble Kit may be to modulate SCF bioactivity.
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PMID:Soluble Kit receptor blocks stem cell factor bioactivity in vitro. 1130 Nov 10

Standard reference diesel exhaust particles (DEP) SRM 1650 are often used to evaluate the toxicity of DEP. However, these particles did not necessarily reflect the effects of DEP representative of present diesel automobiles. This study was designed to compare the effects of SRM 1650 to DEP from representative cars (RC-DEP) on airway epithelial cells. Therefore we established a method to recover RC-DEP impacted on filters after emission from diesel automobiles on test beds. Electron microscopy and flow cytometry showed that these two types of particles were phagocytosed to the same extent by epithelial cells. This phagocytosis is not dependent on the adsorbed organic compounds in contrast to the cytotoxic effect evaluated by measurements of LDH release. This is emphasized by the fact that RC-DEP equipped with an oxidation catalyst are less cytotoxic than particles from a non-equipped vehicle or SRM 1650. This type of catalyst also reduces significantly the release of GM-CSF by bronchial epithelial cells. We have shown in the present paper that SRM 1650 may be used as a surrogate of DEP. However, exhaust gas post-treatment devices of current diesel automobiles reduce the cytotoxicity as well as the inflammatory response of these particles.
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PMID:Similar cellular effects induced by diesel exhaust particles from a representative diesel vehicle recovered from filters and Standard Reference Material 1650. 1156 67

Recent experimental data suggest that one of the major effects of BCR-ABL gene expression in hematopoietic cells is the inhibition of apoptosis. Although the exact mechanisms of this phenomenon are not clear, it is thought to be related to the fact that BCR-ABL induces several signalling pathways also activated by growth factors. In order to determine the anti-apoptotic role of BCR-ABL in a hematopoietic cell line and to by-pass the influence of cytokine-dependence, BCR-ABL gene was expressed in the autonomously growing myelomonocytic U937 cell line using retroviral vectors. There was no resistance to apoptosis induced by either serum deprivation or different doses of etoposide in any U937 clones expressing BCR-ABL protein. In addition to serum deprivation and etoposide, BCR-ABL-expressing clones were not protected from apoptosis induced by TNF, ceramide-C2 and FAS-cross-linking. BCL2 expression was absent in U937 cells and BAX levels were identical between Neo and BCR-ABL clones. To further investigate the mechanisms of this phenomenon, band-shift assays were performed to detect activation of STAT molecules. No constitutive activation of STATs was detected in either NeoR or BCR-ABL-U937 cells, although both IFN-gamma and GM-CSF activated STAT1 and STAT5, respectively, with similar kinetics in both NeoR and BCR-ABL-U937 cells. In addition, the GM-CSF-induced-STAT5 activation was found to be weakened in all clones expressing BCR-ABL. In both control NeoR and BCR-ABL-transfected clones, band-shift assays revealed the presence of an abnormal truncated STAT5 recognized only by an anti-N-terminal but not by an anti-C-Terminal STAT5 antibody. These findings suggest a possible link between the absence of anti-apoptotic potential of BCR-ABL and abnormalities of the STAT5 pathway, including, absence of constitutive activation of STAT5, inhibition of GM-CSF-induced STAT5 activation and expression of a carboxyl-terminal-truncated STAT5.
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PMID:BCR-ABL fails to inhibit apoptosis in U937 myelomonocytic cells expressing a carboxyl-terminal truncated STAT5. 1169 9

Tyrosine phosphorylation is thought to be critical in the regulation of neutrophil functioning, and members of the Src family of tyrosine kinases have recently been shown to be regulated in activated granulocytes. We have used a specific pharmacological inhibitor of Src kinases, pyrazolpyrimidine 1 (PP1), to evaluate the role of Src kinases in cytokine/chemoattractant-induced regulation of neutrophil function. PP1 inhibits PKB phosphorylation but not STAT5 phosphorylation or the activation of MAP kinases by fMLP or GM-CSF. Pretreatment of neutrophils with PP1 and with the PI3K inhibitor LY294002 resulted in a strong inhibition of fMLP-induced superoxide production and cytokine-mediated survival but not fMLP-induced migration. It is interesting that the kinetics of inhibition of actin polymerization and the respiratory burst are very similar. Although initiation of both processes was not affected, sustained activation was inhibited by PP1. Taken together, our results demonstrate a critical role for Src kinases in regulating neutrophil cytotoxic-effector functioning through PI3K-PKB.
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PMID:Src kinases regulate PKB activation and modulate cytokine and chemoattractant-controlled neutrophil functioning. 1178 87

GM-CSF signals through JAK2 and STAT5 and stimulates the expression of STAT5 target genes, such as pim-1 and CIS. Analyzed by EMSA, GM-CSF stimulation led to much stronger STAT5 DNA-binding to pim-1 or CIS GAS elements in primary human monocytes compared with mature macrophages. Similarly, GM-CSF-induced expression of pim-1 and CIS mRNAs was much stronger in monocytes. These differencies were not a result of downregulation of the GM-CSF receptor system or STAT5 expression, because monocytes and macrophages readily expressed GM-CSF receptor, JAK2, STAT5A, and STAT5B mRNAs and proteins. Monocytes expressed significant amounts of truncated STAT5 forms that took part in STAT5-DNA complex formation in GM-CSF-stimulated monocytes. This resulted in faster moving STAT5 complexes compared with macrophages in EMSA. Our results demonstrate that STAT5 isoform expression, GM-CSF-induced STAT5 activation, and STAT5 target-gene expression are altered significantly during monocyte/macrophage differentiation.
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PMID:Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced STAT5 activation and target-gene expression during human monocyte/macrophage differentiation. 1186 89

IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-alpha in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.
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PMID:Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis. 1187 39

Primitive human hematopoietic cells in granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) are more difficult to transduce compared to cells from umbilical cord blood. Based on the hypothesis that MPB cells may require different stimulation for efficient retroviral infection, we compared several culture conditions known to induce cycling of primitive hematopoietic cells. MPB-derived CD34(+) cells were stimulated in the presence or absence of the murine fetal liver cell line AFT024 in trans-wells with G-CSF, stem cell factor (SCF), and thrombopoietin (TPO) (G/S/T; 100 ng/ml) or Flt3-L, SCF, interleukin (IL)-7, and TPO (F/S/7/T; 10-20 ng/ml), and transduced using a GaLV-pseudotyped retroviral vector expressing the enhanced green fluorescence protein (eGFP). Compared to cultures without stroma, the presence of AFT024 increased the number of transduced colony-forming cells (CFC) by 3.5-fold (with G/S/T), long-term culture-initiating cells (LTC-IC) by 4.6-fold (with F/S/7/T), and nonobese diabetic/severe immunodeficiency disease (NOD/SCID)-repopulating cells (SRC) by 6.8-fold (with F/S/7/T). Similar numbers of long-term culture-initiating cells (LTC-IC) and SRC could be transduced using AFT024-conditioned medium (AFT-CM) or a defined medium that had been supplemented with factors identified in AFT-CM. Finally, using our best condition based on transduction with the gibbon ape leukemia virus (GaLV)-pseudotyped vector, we demonstrate a 33-fold higher level of gene transfer (p < 0.001) in SRC using an RD114-pseudotyped vector. In summary, using an optimized protocol with low doses of cytokines, and transduction with an RD114 compared to a GaLV-pseudotyped retroviral vector, the overall number of transduced cells in NOD/SCID mice could be improved 144-fold, with a gene-transfer efficiency in SRC of 16.3% (13.3-19.9; n = 6).
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PMID:Optimization of gene transfer into primitive human hematopoietic cells of granulocyte-colony stimulating factor-mobilized peripheral blood using low-dose cytokines and comparison of a gibbon ape leukemia virus versus an RD114-pseudotyped retroviral vector. 1216 14

Little is known about the distinct roles of the two types of IL-4R on DC. Here we report that IL-4 and IL-13 are able to promote DC maturation, as evaluated by up-regulation of MHC class II and costimulatory molecules, when the concentration of GM-CSF is relatively lower than the dose of IL-4 or IL-13. In addition, under these conditions both cytokines enable DC to respond to maturation stimuli such as bacterial products or proinflammatory cytokines. Both IL-4 and IL-13 act synergistically with weak maturation stimuli such as TNF-alpha or CD40. The IL-4R signaling for DC maturation requires the IL-4R alpha-chain and STAT6, but not Janus kinase 3, indicating that IL-4R type II signaling is preferentially responsible for these effects. In contrast, the production of IL-12 p70, but not IL-10 and TNF, induced by microbial products was enhanced only by IL-4, not by IL-13 or Y119D, a selective type II IL-4R agonist, in vitro and in vivo. This enhancement was dependent on the presence of Janus kinase 3, indicating that this function is exclusively mediated by the type I IL-4R. In short, we discerned the individual roles of the two IL-4R types on DC function, showing that IL-4R type I promotes IL-12 secretion independently of GM-CSF concentration, while IL-4R type II promotes the up-regulation of MHC class II and costimulatory surface markers in a GM-CSF concentration-dependent manner.
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PMID:Differential functions of IL-4 receptor types I and II for dendritic cell maturation and IL-12 production and their dependency on GM-CSF. 1224 47

Human neutrophils were found to express members of the inhibitor of apoptosis (IAP) family, namely cellular IAP1 (cIAP1), cIAP2, and X-linked IAP. Among these members, cIAP2 expression was selectively up-regulated by stimulation with granulocyte colony-stimulating factor (G-CSF), but not with granulocyte-macrophage CSF. The increased expression of cIAP2 mRNA was detected as early as 30 minutes after in vitro stimulation with G-CSF, and the elevated level of cIAP2 protein was detected at 1 hour. The elevated level of cIAP2 protein was also detected in peripheral blood neutrophils obtained from healthy donors receiving G-CSF administration. G-CSF-induced up-regulation of cIAP2 mRNA and protein, phosphorylation of signal transducer and activator of transcription 3 (STAT3), and the antiapoptotic effects were inhibited by pretreatment of cells with AG490, a specific inhibitor of Janus kinase 2 (JAK2). Mature neutrophils from a patient with chronic neutrophilic leukemia exhibited remarkable overexpression of cIAP2 mRNA and prolongation of survival, whereas cIAP2 mRNA expression and survival in mature neutrophils from patients with chronic myelogenous leukemia were essentially similar to those in normal neutrophils. These findings suggest that cIAP2 expression is up-regulated by G-CSF through activation of the JAK2-STAT3 pathway, and increased expression of cIAP2 protein may contribute to G-CSF-mediated antiapoptosis. In addition, overexpression of cIAP2 may be partly responsible for sustained neutrophilia at least in some cases of chronic neutrophilic leukemia.
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PMID:Expression of the inhibitor of apoptosis (IAP) family members in human neutrophils: up-regulation of cIAP2 by granulocyte colony-stimulating factor and overexpression of cIAP2 in chronic neutrophilic leukemia. 1239 23


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