Zebrafish pdx1 morphant displays defects in pancreas development and digestive organ chirality, and potentially identifies a multipotent pancreas progenitor cell.
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Animals↗
Biomedical subjects
N S Yee
Publications and source records attributed to N S Yee.
Ischemic colitis associated with decongestant use.
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Adult↗
Ischemic colitis associated with factor V Leiden mutation.
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Adult↗
c-kit receptor signaling through its phosphatidylinositide-3'-kinase-binding site and protein kinase C: role in mast cell enhancement of degranulation, adhesion, and membrane ruffling.
In bone marrow-derived mast cells (BMMCs), the Kit receptor tyrosine kinase mediates diverse responses including proliferation, survival, chemotaxis, migration, differentiation, and adhesion to extracellular matrix. In connective tissue mast cells, a role for Kit in the secretion of inflammatory mediators has been demonstrated as well. We recently demonstrated a role for phosphatidylinositide-3' (PI 3)-kinase in Kit-ligand (KL)-induced adhesion of BMMCs to fibronectin. Herein, we investigated the mechanism by which Kit mediates enhancement of Fc epsilon RI-mediated degranulation, cytoskeletal rearrangements, and adhesion in BMMCs. Wsh/Wsh BMMCs lacking endogenous Kit expression, were transduced to express normal and mutant Kit receptors containing Tyr-->Phe substitution at residues 719 and 821. Although the normal Kit receptor fully restored KL-induced responses in Wsh/Wsh BMMCs, Kit gamma 719F, which fails to bind and activate PI 3-kinase, failed to potentiate degranulation and is impaired in mediating membrane ruffling and actin assembly. Inhibition of PI 3-kinase with wortmannin or LY294002 also inhibited secretory enhancement and cytoskeletal rearrangements mediated by Kit. In contrast, secretory enhancement and adhesion stimulated directly through protein kinase C (PKC) do not require PI 3-kinase. Calphostin C, an inhibitor of PKC, blocked Kit-mediated adhesion to fibronectin, secretory enhancement, membrane ruffling, and filamentous actin assembly. Although cytochalasin D inhibited Kit-mediated filamentous actin assembly and membrane ruffling, secretory enhancement and adhesion to fibronectin were not affected by this drug. Therefore, Kit-mediated cytoskeletal rearrangements that are dependent on actin polymerization can be uncoupled from the Kit-mediated secretory and adhesive responses. Our results implicate receptor-proximal PI 3-kinase activation and activation of a PKC isoform in Kit-mediated secretory enhancement, adhesion, and cytoskeletal reorganization.
Actins↗
Differential roles of PI3-kinase and Kit tyrosine 821 in Kit receptor-mediated proliferation, survival and cell adhesion in mast cells.
The pleiotropic effects of the Kit receptor system are mediated by Kit-Ligand (KL) induced receptor autophosphorylation and its association with and activation of distinct second messengers, including phosphatidylinositol 3'-kinase (PI3-kinase), p21ras and mitogen-activated protein kinase (MAPK). To define the role of PI3-kinase, p21ras and MAPK in Kit-mediated cell proliferation, survival and adhesion in bone marrow-derived mast cells (BMMC), mutant Kit receptors were expressed in Wsh/Wsh BMMC lacking endogenous c-kit expression. The introduction of both murine Kit(S) and KitL (isoform containing a four amino acid insert) into Wsh/Wsh BMMC restored KL-induced proliferation, survival and adhesion to fibronectin, as well as activation of PI3-kinase, p21ras and MAPK, and induced expression of c-fos, junB, c-myc and c-myb mRNA. Substitution of tyrosine 719 in the kinase insert with phenylalanine (Y719F) abolished PI3-kinase activation, diminished c-fos and junB induction, and impaired KL-induced adhesion of BMMC to fibronectin. In addition, the Y719F mutation had partial effects on p21ras activation, cell proliferation and survival, while MAP kinase activation was not affected. On the other hand, Y821F substitution impaired proliferation and survival without affecting PI3-kinase, p21ras and MAPK activation, and induction of c-myc, c-myb, c-fos and c-jun mRNA, while KL-induced cell adhesion to fibronectin remained intact. In agreement with a role for PI3-kinase in Kit-mediated cell adhesion, wortmannin blocked Kit-mediated cell adhesion at concentrations known to specifically inhibit PI3-kinase. We conclude, that association of Kit with p85PI3-K, and thus with PI3-kinase activity, is necessary for a full mitogenic as well as adhesive response in mast cells. In contrast, tyrosine 821 is essential for Kit-mediated mitogenesis and survival, but not cell adhesion.
Animals↗
Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C.
The receptor tyrosine kinase Kit and Kit ligand (KL), encoded at the murine white spotting (W) and steel (Sl) loci, respectively, function in hematopoiesis, melanogenesis, and gametogenesis. To understand the mechanism of turnover of Kit in mast cells, mutant receptors generated in vitro were heterologously expressed in Wsb/Wsh mast cells lacking endogenous c-kit expression, and the effects of mutations on KL-induced internalization and ubiquitination/degradation of Kit were studied. Upon binding of KL, KL.Kit receptor complexes were rapidly internalized, and the turnover was accelerated by ubiquitin-mediated degradation. Inactivation of the Kit kinase resulted in a reduced rate of internalization of KL.Kit complexes, degradation of kinase-inactive receptor complexes was relatively slow, and receptor ubiquitination was absent. But abolishment of KL-induced receptor association and activation of phosphatidylinositol 3'-kinase and of tyrosine 821 autophosphorylation did not affect KL-induced internalization and ubiquitination/degradation of Kit. Furthermore, Kit receptors can be down-regulated by proteolytic cleavage induced by either activation of protein kinase C or by isopropyl alcohol. In summary, KL-induced internalization of KL.Kit complexes and ubiquitination/degradation require an active kinase. By contrast, proteolytic cleavage of Kit mediated by protein kinase C activation is independent of kinase activity.
Animals↗
Long-term generation of human mast cells in serum-free cultures of CD34+ cord blood cells stimulated with stem cell factor and interleukin-3.
The generation of murine mast cells is supported by several cytokines, and mast cell lines are frequently established in long-term cultures of normal murine marrow cells. In contrast, growth of human mast cells was initially dependent on coculture with murine fibroblasts. The growth factor produced by murine fibroblasts and required to observe differentiation of human mast cells is attributable in part to stem cell factor (SCF). However, other factors are likely involved. We have previously shown that the combination of SCF and interleukin-3 (IL-3) efficiently sustains proliferation and differentiation of colony-forming cells (CFCs) from pre-CFC enriched from human umbilical cord blood by CD34+ selection. With periodic medium changes and the addition of fresh growth factors, five consecutive cultures of different cord blood samples gave rise to differentiated cells and CFCs for more than 2 months. Although differentiated cells continued to be generated for more than 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue positive, are karyotypically normal, are CD33+, CD34-, CD45+, c-kit-, and c-fms-, and die in the absence of either SCF or IL-3. These cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentration of no less than 10(4) cells/mL. At refeedings, the cultures contain a high number (> 50%) of dead cells and have a doubling time ranging from 5 to 12 days. This suggests that subsets of the cell population die because of a requirement for a growth factor other than SCF or IL-3. These results indicate that the combination of cord blood progenitor and stem cells, plus a cocktail of growth factors including SCF and IL-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor-dependent cell lines. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia.
Antigens, CD↗
Role of kit-ligand in proliferation and suppression of apoptosis in mast cells: basis for radiosensitivity of white spotting and steel mutant mice.
The receptor tyrosine kinase Kit and its cognate ligand KL/steel factor are encoded at the white spotting (W) and Steel (Sl) loci of the mouse, respectively. Mutations at both the W and the Sl loci affect hematopoiesis including the stem cell hierarchy, erythropoiesis, and mast cells, as well as gametogenesis and melanogenesis. In addition, mutant mice display an increased sensitivity to lethal doses of irradiation. The role of KL/c-kit in cell proliferation and survival under conditions of growth factor-deprivation and gamma-irradiation was studied by using bone marrow-derived mast cells (BMMC) as a model. Whereas apoptosis induced by growth factor deprivation in BMMC is a stochastic process and follows zero order kinetics, gamma-irradiation-induced apoptosis is an inductive process and follows higher order kinetics. In agreement with these results, gamma-irradiation-induced apoptosis in BMMC was shown to be dependent on p53 whereas apoptosis induced by deprivation is partly dependent on p53, implying that there are other mechanisms mediating apoptosis in KL-deprived BMMC. In the presence and in the absence of serum, KL stimulated proliferation by promoting cell cycle progression. The presence of KL was required only during the early part of the G1 phase for entry into the S phase. At concentrations lower than those required for proliferation, KL suppressed apoptosis induced by both growth factor-deprivation and gamma-irradiation, and internucleosomal DNA fragmentation characteristic of apoptosis. The ability of KL to suppress apoptosis was independent of the phase of the cell cycle in which the cells were irradiated and suppression of apoptosis was a prerequisite for subsequent cell cycle progression. Moreover, addition of KL to gamma-irradiated and growth factor-deprived cells could be delayed for up to 1 h after irradiation or removal of growth factors when cells became irreversibly committed to apoptosis. KL and IL-3 induce suppression of apoptosis in mast cells by different mechanisms based on the observations of induction of bcl-2 gene expression by IL-3 but not by KL. It is proposed that the increased sensitivity of W and Sl mutant mice to lethal irradiation results from paucity of the apoptosis suppressing and proliferative effects of KL.
Animals↗
Mechanism of kit ligand, phorbol ester, and calcium-induced down-regulation of c-kit receptors in mast cells.
The proto-oncogene c-kit is allelic with the white spotting locus (W) on mouse chromosome 5 and it encodes a transmembrane protein tyrosine kinase which belongs to the platelet-derived growth factor and macrophage-colony stimulating factor (CSF-1) receptor subfamily. In an effort to study the function of the c-kit receptor, specifically the physiological mechanism of controlling the signal induced by the ligand, the effect and mechanism of down-regulation of the c-kit receptor by the kit ligand (KL) was investigated in mast cells. Following preincubation with KL, the capacity of mast cells to bind kit antibody was reduced and binding of radiolabeled KL to mast cells decreased with similar kinetics, suggesting that KL stimulates the loss of c-kit receptor from the cell surface. After binding to the c-kit receptor, KL was rapidly internalized, and degradation of the receptor was accelerated. The c-kit receptor was transmodulated by the protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA) and by the calcium ionophore ionomycin. TPA- and ionomycin-induced down-regulation of the c-kit receptor was accompanied by release of the extracellular domain of the receptor, presumably by proteolytic cleavage near the transmembrane domain. Release of the extracellular domain of the c-kit receptor occurred also in untreated cells but at a slow rate. In addition, ionomycin induced shedding of the intact c-kit receptor. In mast cells depleted of protein kinase C, the c-kit receptor remained sensitive to down-regulation induced by KL and ionomycin, but not by treatment with TPA. Therefore, the down-regulation of the c-kit receptor induced by KL, activated protein kinase C, and an increased level of intracellular calcium is mediated through independent mechanisms.
3T3 Cells↗
Ectopic expression of a c-kitW42 minigene in transgenic mice: recapitulation of W phenotypes and evidence for c-kit function in melanoblast progenitors.
The proto-oncogene c-kit encodes a transmembrane tyrosine kinase receptor that is allelic with the murine white-spotting locus (W). W mutations affect melanogenesis, gametogenesis, and hematopoiesis during development and adult life, and they result from the partial or complete loss of c-kit function. The W42 allele is a W mutation with severe effects in both the homozygous and the heterozygous states. Previous analysis of the W42 allele identified a missense mutation in an essential amino acid of the c-kitW42 kinase domain that abolishes the in vitro kinase activity of the c-kitW42 protein but does not affect its normal expression. These results suggested that the c-kitW42 allele was a dominant negative mutation within the context of c-kit-mediated signal transduction. To further explore the dominant negative characteristics of the W42 mutation, we have generated transgenic mice in which ectopic expression is driven by the human beta-actin promoter (hAP). Two mouse lines carrying the hAP-c-kitW42 transgene show an effect on pigmentation and the number of tissue mast cells. The patchy coat color pattern of the line 695 mice may reflect variable expression of the transgene in melanoblast progenitors and their descendants and, consequently, is indicative of a function for c-kit in early melanoblasts. Germ cell development and erythropoiesis, however, do not appear to be affected by the transgene. Mice expressing the c-kitW42 transgene therefore recapitulate some of the phenotypes of mice with W mutations. These results are therefore in agreement with the molecular basis of the W42 mutation and the dominant-negative characteristics of the c-kitW42 protein product.
Alleles↗
Effect of cimetidine or ranitidine pretreatment on hepatic mixed function oxidase activity in the rat.
This study compared the effect of single equimolar oral doses of cimetidine (100 mg/kg) or ranitidine (139 mg/kg) on rat hepatic mixed function oxidases. Cimetidine significantly (p less than 0.05) increased hexobarbital sleeping time and prolonged aminopyrine and theophylline elimination. In contrast, ranitidine did not significantly affect hexobarbital sleeping time and theophylline elimination but significantly (p less than 0.025) increased aminopyrine elimination. Aminopyrine N-demethylase activity in vitro was significantly (p less than 0.05) inhibited by cimetidine pretreatment but significantly (p less than 0.025) increased by ranitidine pretreatment. The direct addition of cimetidine or SKF 525A to the 10,000g supernatant fraction from controlled liver homogenates decreased aminopyrine N-demethylase activity, whereas the direct addition of ranitidine tended to increase aminopyrine N-demethylase activity. A significant correlation (r = 0.65, p less than or equal to 0.005) was observed between hexobarbital sleeping time in vivo and aminopyrine N-demethylase activity in vitro in the same rat. The results of this study showed that cimetidine inhibited mixed function oxidases, whereas ranitidine had no effect or tended to stimulate mixed function oxidases.
Aminopyrine↗