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Query: UNIPROT:P15088 (mast cell)
 14,925 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We recently developed a new progenitor assay using murine fetal liver cells that provides a source of pluripotent progenitors, bipotent progenitors, and committed macrophage, megakaryocyte, erythroid, and mast cell progenitors. This clonal cell culture system was used to examine the direct effects of Harvey sarcoma virus on murine hemopoietic progenitors. Very large erythroid colonies containing 100,000 to 200,000 cells were seen in the infected group. Only small erythroid colonies were seen in the uninfected control cultures. The cells in the large erythroid colonies from infected cultures expressed the ras gene as demonstrated by immunofluorescence with a monoclonal antibody to p21, the ras gene product. The infected cells were not immortal since they did not yield secondary colonies upon replating. Sequential observation of individual colonies showed that maturation was not blocked by infection with the virus. The size of other colony types, including granulocyte/macrophage, mast cell, and mixed, was unaffected even though some of these colonies expressed the ras gene. Thus, infection with Harvey sarcoma virus appears to give a growth advantage primarily to committed erythroid progenitors.
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PMID:Enhancement of the proliferation of murine fetal liver erythroid progenitors by infection with Harvey sarcoma virus. 215 15

Recent studies have shown that retroviruses can serve as efficient vectors of exogenous genes that can be inserted and expressed in a variety of mammalian cell types. Several investigators have exposed total bone marrow populations to retroviruses in vitro and have demonstrated the presence of exogenous genes after inoculation into irradiated mice. Our approach was to identify individual pluripotent hemopoietic progenitors in vitro and to use these single cells as targets for retroviral gene transfer. This approach was made possible by our previous identification of in vitro colonies containing pluripotent, undifferentiated blast cells with very high secondary replating efficiencies. By using a monoclonal antibody to detect the product of the transferred gene, we were able to document infection of single multipotent cells and to quantitate the percentage of the progeny cells that expressed the transferred gene. Specifically, individual blast cells were obtained by micromanipulation, exposed to Harvey sarcoma virus, and ras gene expression was detected by immunofluorescence in individual colonies. A variety of types of p21-positive colonies were seen, including a macrophage (m)-neutrophil (n)-erythroid (E)-mast cell (mast)-megakaryocyte (M) colony, an mEmastM colony, an nmmast colony, mnE colonies, mn colonies, and m colonies. These results demonstrated that multipotent progenitors were recipients of exogenous genes and that these genes were expressed in the differentiated progeny. Initial experiments failed to demonstrate that the cells in the infected colonies were transformed. Retroviral infection of isolated blast cells may provide a unique method for studies of the effects of a variety of genes, including oncogenes, in hemopoietic cells.
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PMID:In vitro retroviral transfer of ras genes to single hemopoietic progenitors. 310 76

The endogenous nucleoside adenosine is thought to play a role in the pathophysiology of asthma by stimulating mast cells. We previously showed that the human mast cell line HMC-1 expresses A2A and A2B receptors, and that both receptors activate adenylate cyclase via Gs-protein but that only A2B receptors are also coupled to phospholipase C via Gq proteins. Stimulation of A2B but not A2A receptors induced production of interleukin-8 (IL-8) from HMC-1 cells. The mechanism by which adenosine promotes IL-8 synthesis has not been defined. In this study, we tested the hypothesis that mitogen-activated protein kinase (MAPK) signaling pathways are involved in this process. Stimulation of HMC-1 with the stable adenosine analog NECA (5'-N-ethylcarboxamidoadenosine) activated p21(ras) and both p42 and p44 isoforms of extracellular signal-regulated kinase (ERK). NECA (10 microM) induced a 1.9 +/- 0. 06-fold increase in ERK activity, whereas 10 microM of the selective A2A agonist CGS 21680 (4-((N-ethyl-5'-carbamoyladenos-2-yl)-aminoethyl)-phenylpropionic acid) had no effect. NECA, in parallel with the activation of ERK, also stimulated the p46 isoform of c-Jun N-terminal kinase (MEK) and p38 MAPK. Furthermore, the selective MAPK/ERK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone), and p38 MAPK inhibitors SB 202190 (4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole) and SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H- imidaz ole) blocked A2B receptor-mediated production of IL-8. These results indicate that extracellular adenosine can regulate ERK, c-Jun N-terminal kinase, and p38 MAPK signaling cascades and that activation of ERK and p38 MAPK pathways are essential steps in adenosine A2B receptor-dependent stimulation of IL-8 production in HMC-1.
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PMID:Role of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinase kinase in adenosine A2B receptor-mediated interleukin-8 production in human mast cells. 1010 Oct 31

M-Ras, a recently identified homologue of p21 Ras, is widely expressed, with levels of the 29-kD protein in spleen, thymus, and NIH 3T3 fibroblasts equaling or exceeding those of p21 Ras. A G22V mutant of M-Ras was constitutively active and its expression in an interleukin-3 (IL-3)-dependent mast cell/megakaryocyte cell line resulted in increased survival in the absence of IL-3, increased growth in IL-4, and, at high expression levels, in factor-independent growth. Expression of M-Ras G22V, however, had a negative effect on growth in the presence of IL-3, suggesting that M-Ras has both positive and negative effects on growth. Expression of M-Ras G22V in NIH-3T3 fibroblasts resulted in morphological transformation and growth to higher cell densities. M-Ras G22V induced activation of the c-fos promoter, and bound weakly to the Ras-binding domains of Raf-1 and RalGDS. Expression of a mutant of M-Ras G22V that was no longer membrane-bound partially inhibited (40%) activation of the c-fos promoter by N-Ras Q61K, suggesting that M-Ras shared some, but not all, of the effectors of N-Ras. An S27N mutant of M-Ras, like the analogous H-Ras S17N mutant, was a dominant inhibitor of activation of the c-fos promoter by constitutively active Src Y527F, suggesting that M-Ras and p21 Ras shared guanine nucleotide exchange factors and are likely to be activated in parallel. Moreover, M-Ras was recognized by the monoclonal anti-Ras antibody Y13-259, commonly used to study the function and activity of p21 Ras. Mammalian M-Ras and a Caenorhabditis elegans orthologue exhibit conserved structural features, and these are likely to mediate activation of distinctive signaling paths that function in parallel to those downstream of p21 Ras.
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PMID:M-Ras, a widely expressed 29-kD homologue of p21 Ras: expression of a constitutively active mutant results in factor-independent growth of an interleukin-3-dependent cell line. 1049 16

Stable transduction of genetic material, in combination with sensitive methodologies for in vivo study of cell physiology, provides an opportunity to efficiently evaluate the functions of regulatory proteins. To dissect the minimal therapeutic function of such proteins, we have stably expressed protein microdomains as fusions, composed of short peptides, and detected specific subfunctions distinct from holoprotein function, using flow cytometry and other techniques. We demonstrate that retroviral delivery of the 24-amino-acid proliferating cell nuclear antigen-binding motif (p21C), derived from the C-terminus of the cell cycle inhibitor protein, p21, is sufficient to induce cell cycle arrest. Cells expressing this peptide motif reversibly execute both G1- and G2-checkpoint controls that are normally activated subsequent to interference with DNA synthesis. The p21C effect is distinct from results obtained with an intact p21 protein that also binds cyclin-CDK complexes and arrested cells exclusively at the G1/S transition. Thus, microdomains can exert unique biological effects compared to the parental molecules from which they were derived. To further evaluate the peptide delivery strategy, we analyzed the role of various kinases in IgE-mediated stimulation of mast cell exocytosis. Primary bone marrow-derived mast cells were transduced with retroviral constructs encoding short-kinase inhibitor motifs and analyzed by flow cytometry for effects on exocytosis. We found that a specific protein kinase A (PKA) inhibitor peptide suppressed IgE-mediated stimulation of mast cell exocytosis. This anti-exocytotic effect was mimicked by a small molecule inhibitor of PKA (KT5720). Thus, the ability to express protein microdomains can be a powerful means to subtly perturb cellular physiology in manners that reveal new paths for therapeutic intervention. We believe that such approaches might allow for new forms of gene therapy to become available.
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PMID:Retroviral delivery of peptide modulators of cellular functions. 1093 65

In this study, we present evidence for the critical role of proteinase-3 (PR3) in the proliferation of myeloid cells via the proteolytic regulation of the cyclin-dependent kinase inhibitor p21(waf1). Expression of recombinant PR3 in rat (RBL) or human (HMC1) mast cell lines increased bromodeoxyuridine incorporation and CDK2 activity compared with RBL and HMC1 cells transfected with an enzymatically inactive PR3 mutant (PR3(S203A)) or with human neutrophil elastase. Western blot analysis of p21(waf1) showed an absence of detectable protein, despite normal levels of p21 mRNA. Ectopic overexpression of p21 restored normal levels of p21 in the RBL/PR3/p21 double transfectants and reverted the proliferative effect of PR3. Inhibition of the 26 S proteasome by lactacystin or of caspases by benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone did not inhibit p21 proteolysis. p21 cleavage correlated with PR3 expression in HMC1 cells infected with recombinant adenoviral vector Ad/PR3. During in vitro studies, purified p21 was cleaved by PR3, resulting in a 10-kDa p21 fragment. Employing double immunofluorescence confocal microscopy, subcellular fractionation, and co-immunoprecipitation, we found that PR3 and p21 colocalized in the cytosol. In human neutrophils treated with tumor necrosis factor-alpha, which induces PR3 re-expression, we observed that p21 disappeared and was reversed by Pefabloc, a serine proteinase inhibitor. The physiopathological implications of the cleavage of p21 by PR3 have to be determined.
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PMID:Cleavage of p21waf1 by proteinase-3, a myeloid-specific serine protease, potentiates cell proliferation. 1235 76

Uterine leiomyoma is a mesenchymal tumor composed of smooth muscle cells with fibrous tissues and many mast cells. Tranilast is known to suppress fibrosis or to work as a mast cell stabilizer and is reported to inhibit proliferation of vascular smooth muscle cells. In this study, we examined the effects of tranilast on cultured human leiomyoma cells in vitro to evaluate whether this agent has the potential to inhibit the growth of uterine leiomyomas. Tranilast inhibited the proliferation of cultured leiomyoma cells in a dose-dependent manner without any cytotoxic effect or induction of apoptosis. In association with the inhibitory effect, tranilast induced the cyclin-dependent kinase (CDK) inhibitor p21(waf1) and tumor suppressor gene p53 and decreased CDK2 activity. These results suggest that tranilast arrests the proliferation of uterine leiomyoma cells at the G0/G1 phase, through the suppression of CDK2 activity via an induction of p21(waf1) and p53. Tranilast was concluded to be a potent agent to inhibit proliferative activity of uterine leiomyoma cells.
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PMID:Tranilast inhibits the proliferation of uterine leiomyoma cells in vitro through G1 arrest associated with the induction of p21(waf1) and p53. 1246 60

Genetic loss of surface Fas antigen expression leads to reduced apoptosis of myeloid and lymphoid progenitor cells, and a propensity to develop autoimmunity and myeloid leukemia in mouse models. Oncogenic p21(ras) decreases surface Fas antigen expression and renders fibroblasts resistant to Fas mediated apoptosis. Neurofibromin, which is encoded by NF1, is a GTPase activating protein that negatively regulates p21(ras) activity. NF1 loss leads to deregulation of p21(ras)-effector pathways, which control myeloid cell survival. Heterozygous inactivation of Nf1 increases mast cell numbers in Nf1 +/- mice, and enhances mast cell survival in response to c-kit ligand (kit-L). Here, we show that Nf1-deficient mast cells have reduced surface Fas antigen expression in response to kit-L and are resistant to Fas ligand-mediated apoptosis. Using genetic intercrosses between Nf1 +/- and class I (A)-PI-3K-deficient mice, we demonstrate that hyperactivation of the p21(ras)-class I(A) PI-3K pathway is the mechanism for this phenotype. Finally, we demonstrate that mast cells from both Fas antigen-deficient mice and Nf1 +/- mice are resistant to apoptosis following kit-L withdrawal in vivo. Thus, therapies designed to decrease p21(ras) activity and up-regulate Fas antigen expression may limit the pathological accumulation of myeloid cells in disease states where p21(ras) is hyperactivated.
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PMID:Loss of the nf1 tumor suppressor gene decreases fas antigen expression in myeloid cells. 1503 34

The objective of this study was to evaluate by immunohistochemical means the nuclear expression of p27 and p21 proteins in cutaneous mast cell tumors and histiocytomas of dogs. In mast cell tumors, nine of the 13 grade I tumors, 13 of the 19 grade II tumors, and 10 of the 15 grade III tumors showed no detectable or mild p27 immunoreactivity. In contrast, one of the 13 grade I tumors, 12 of the 19 grade II tumors, and 11 of the 15 grade III tumors showed moderate or marked p21 immunoreactivity. Nineteen of the 28 histiocytomas showed no detectable or mild p27 immunoreactivity, and 24 cases showed moderate or marked p21 immunoreactivity. These findings indicate that a loss or absence of p27 expression is an early pathogenic event in mast cell and histiocyte tumorigenesis and that p21 expression may be a marker of mast cell tumor progression and histiocytoma cell proliferation.
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PMID:Immunohistochemical expression of p27 and p21 in canine cutaneous mast cell tumors and histiocytomas. 1513 84

p21(ras) (Ras) proteins and GTPase-activating proteins (GAPs) tightly modulate extracellular growth factor signals and control multiple cellular functions. The specific function of each Ras isoform (H, N, and K) in regulating distinct effector pathways, and the role of each GAP in negatively modulating the activity of each Ras isoform in myeloid cells and, particularly, mast cells is incompletely understood. In this study, we use murine models of K-ras- and Nf1-deficient mice to examine the role of K-ras in modulating mast cell functions and to identify the role of neurofibromin as a GAP for K-ras in this lineage. We find that K-ras is required for c-kit-mediated mast cell proliferation, survival, migration, and degranulation in vitro and in vivo. Furthermore, the hyperactivation of these cellular functions in Nf1(+/-) mast cells is decreased in a K-ras gene dose-dependent fashion in cells containing mutations in both loci. These findings identify K-ras as a key effector in multiple mast cell functions and identify neurofibromin as a GAP for K-ras in mast cells.
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PMID:K-ras is critical for modulating multiple c-kit-mediated cellular functions in wild-type and Nf1+/- mast cells. 1727 61


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