Gain-of-function mutations of c-kit in human gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors in the human digestive tract, but their molecular etiology and cellular origin are unknown. Sequencing of c-kit complementary DNA, which encodes a proto-oncogenic receptor tyrosine kinase (KIT), from five GISTs revealed mutations in the region between the transmembrane and tyrosine kinase domains. All of the corresponding mutant KIT proteins were constitutively activated without the KIT ligand, stem cell factor (SCF). Stable transfection of the mutant c-kit complementary DNAs induced malignant transformation of Ba/F3 murine lymphoid cells, suggesting that the mutations contribute to tumor development. GISTs may originate from the interstitial cells of Cajal (ICCs) because the development of ICCs is dependent on the SCF-KIT interaction and because, like GISTs, these cells express both KIT and CD34.     

Stem cell factor-induced downregulation of c-kit in human lung mast cells and HMC-1 mast cells

Recent data suggest that local overexpression of the tissue-hormone c-kit ligand (stem cell factor [SCF]) is associated with accumulation of mast cells (MCs) and a decrease in expression of c-kit in the accumulated MCs [28]. In the present study, the effects of recombinant human (rh) SCF on expression of c-kit mRNA and c-kit protein in isolated human MCs and a human mast cell line, HMC-1, were analyzed. Incubation of isolated lung MC with rhSCF (100 ng/mL) for 120 minutes resulted in decreased expression of c-kit mRNA (optical density [OD], control: 100% vs. rhSCF: 37%). Almost identical results were obtained with HMC-1 cells (OD, control: 100% vs. rhSCF: 40 to 45%). As assessed by flow cytometry and monoclonal antibodies (mAbs) to c-kit, the SCF-induced decrease of c-kit mRNA in HMC-1 was associated with a substantial decrease in surface expression of c-kit (MFI, control: 100 +/- 21%, vs. MFI in cells incubated with rhSCF [100 ng/mL at 37 degrees C for 12 hours]: 8 +/- 2%, vs. MFI in cells incubated with rhSCF, 100 ng/mL, at 4 degrees C: 34 +/- 3%). The effects of rhSCF on c-kit expression in HMC-1 cells were dose- and time-dependent with maximum effects observed with 10-100 ng/mL of rhSCF after 4 to 12 hours. The SCF-dependent loss of c-kit was also accompanied by a decreased chemotactic response to rhSCF (control: 100%; rhSCF: 71 +/- 2%). This study shows that exposure of human lung MC and HMC-1 cells to recombinant SCF results in downregulation of c-kit mRNA and surface c-kit expression. These data may explain the partial loss of c-kit on MCs in areas of SCF overexpression.     

Characterization of defective phorbol ester responses in a low secreting rat basophilic leukemia (RBL-2H3) cell variant

The role of protein kinase C (PKC) in the signaling mechanism that stimulates the release of mediators from rat mast cells, for which the RBL-2H3 cell line is a model, is at present unresolved. Current evidence suggests that PKC activation alone is an insufficient stimulus, although it can modulate mast cell exocytosis induced by other agents. In this article we characterize a variant of the RBL-2H3 cell line that has a reduced capacity for mediator secretion in response to an IgE-mediated antigen-induced stimulation. The outcome of our study suggests that at least two PKC isotypes are active in RBL-2H3 cells, and affect the positive and negative modulation of the secretory response.     

Expression of stem cell factor (SCF) and SCF receptor (c-kit) in synovial membrane in arthritis: correlation with synovial mast cell hyperplasia and inflammation

OBJECTIVE: Stem cell factor (SCF), the ligand for the SCF receptor (c-kit) expressed on precursors and mature mast cells (MC), is a major agonist for human MC (e.g., SCF induces MC development, chemotaxis, activation, proliferation of MC precursors, mediates MC adhesion, and changes MC releasability). We investigated expression of SCF and c-kit in synovial membrane with particular reference to the mechanism of local MC hyperplasia and inflammation in arthritis. METHODS: We conducted single and double labeling immunohistochemistry (ABC, APAAP, indirect immunofluorescence techniques) with antibodies to SCF, c-kit, MC tryptase, Ki-67 antigen (marker for proliferating cells), and CD68 (monocyte/macrophage marker). Synovial specimens analyzed were from 31 patients: traumatic arthritis (TrA, n=9), osteoarthritis (OA, n=12), and rheumatoid arthritis (RA, n=10). Control experiments were performed on human lung, skin, and buccal mucosa tissues, on the HMC-1 mast cell line, and isolated lung MC. Morphometry was performed by computerized image analysis. RESULTS: Synovial c-kit expression was found to be restricted to MC, whereas SCF is detected in synovial lining cells, stromal fibroblasts, monocyte/macrophages, endothelial cells, and in vascular basement membranes. SCF staining was localized to MC as well, but it was not possible to specify whether this represents SCF produced by or bound (via c-kit) to MC. In inflamed synovial membranes/areas, SCF was found to be redistributed into the extracellular matrix. Redistribution of SCF was accompanied by degranulation and/or accumulation of c-kit+ MC, the hyperplasia of which correlated positively with histologic inflammation/inflammatory cell densities, but did not appear to involve MC proliferation in situ. These findings appeared to be common for all the conditions (TrA, OA, RA) studied. CONCLUSION: In addition to the demonstration/characterization of SCF and c-kit protein expression in human synovium, results of this study suggest the hypothesis that, in arthritis, local mobilization of SCF may play a role in the development of synovial MC hyperplasia without inducing in situ proliferation of MC, and that the synovial SCF/MC c-kit system may contribute to the local nonspecific inflammatory response/arthritic flares in TrA, OA, and RA.     

The effect of okadaic acid on histamine release, cell morphology and phosphorylation in rat basophilic leukemia (RBL-2H3) cells, human basophils and rat peritoneal mast cells

To study the involvement of serine/threonine phosphatase in the signal transduction of mast cells, we examined the effects of okadaic acid (OA), an inhibitor of type-1 and -2A phosphatase on histamine release, cell morphology, calcium influx and protein phosphorylation of rat basophilic leukemia (RBL-2H3) cells, human basophils and rat peritoneal mast cells. OA inhibited IgE-mediated histamine release from RBL-2H3 cells and human basophils dose-dependently. There was a remarkable enhancement of IgE-mediated histamine release when rat peritoneal mast cells were suboptimally challenged. OA induced a marked change of cell features, detached RBL-2H3 cells from plastic well and kept the 18- and 68-kD proteins phosphorylated. These findings show that phosphatase may play a role in the modulation of secretion in mast cells.     

Oncogenic mutation in the Kit receptor tyrosine kinase alters substrate specificity and induces degradation of the protein tyrosine phosphatase SHP-1

Activating mutations in the Kit receptor tyrosine kinase have been identified in both rodent and human mast cell leukemia. One activating Kit mutation substitutes a valine for aspartic acid at codon 816 (D816V) and is frequently observed in human mastocytosis. Mutation at the equivalent position in the murine c-kit gene, involving a substitution of tyrosine for aspartic acid (D814Y), has been described in the mouse mastocytoma cell line P815. We have investigated the mechanism of oncogenic activation by this mutation. Expression of this mutant Kit receptor tyrosine kinase in a mast cell line led to the selective tyrosine phosphorylation of a 130-kDa protein and the degradation, through the ubiquitin-dependent proteolytic pathway, of a 65-kDa phosphoprotein. The 65-kDa protein was identified as the src homology domain 2 (SH2)-containing protein tyrosine phosphatase SHP-1, a negative regulator of signaling by Kit and other hematopoietic receptors, and the protein product of the murine motheaten locus. This mutation also altered the sites of receptor autophosphorylation and peptide substrate selectivity. Thus, this mutation activates the oncogenic potential of Kit by a novel mechanism involving an alteration in Kit substrate recognition and the degradation of SHP-1, an attenuator of the Kit signaling pathway.     

Virus-activated CD8 T cells and lymphokine-activated NK cells express the mast cell function-associated antigen, an inhibitory C-type lectin

The mast cell function-associated Ag (MAFA) is an inhibitory C-type lectin that was originally identified on the cell surface of a rat mucosal mast cell line, RBL-2H3. We have cloned the mouse homologue of the rat MAFA gene, and Northern blot analysis revealed that mouse MAFA (mMAFA) gene expression was strongly induced in effector CD8 T cells and lymphokine-activated NK cells but not in effector CD4 T cells and in mouse mast cells. Moreover, mMAFA gene expression was only found in effector CD8 T cells that had been primed in vivo with live virus because in vitro activated CD8 T cells did not express mMAFA. Primary sequence comparison revealed a high degree of conservation (89% similarity) between rat MAFA and mMAFA. Thus, the MAFA molecule in the mouse is a putative inhibitory receptor on anti-viral CD8 T cells induced in vivo and on NK cells.     

Dietary intake and functional ability between 70 and 76 years of age

In order to explore the potential existence of human mast cell growth factors other than stem cell factor (SCF), we have compared SCF to L-cell fibroblast supernatants (LCS) during in vitro mast cell differentiation, using human leukaemic mast cells (HMC-1 cells) which contain a gain-of-function mutated SCF receptor (c-Kit) as model. At baseline, cells exhibited an immature phenotype, with <25% being metachromatic or chloroacetate esterase, tryptase and FcepsilonRIalpha positive. Intracellular levels of histamine, tryptase, TNF-alpha and chymase were low, whereas 83% of cells were c-Kit positive. During a 10 day culture with 30% LCS, a significant, time-dependent increase of all mast cell markers, except for chymase and c-Kit, was observed at the protein and for tryptase and FcepsilonRIalpha also at the mRNA level. Cytoplasmatic granulation and stimulated histamine and leukotriene C4 release were increased as well. In contrast to LCS, rhSCF induced none of these changes in HMC-1 cells. On Sephadex G100 fractionation of LCS, HMC-1 cells increased tryptase activity with fractions between 40 and 60, and below 10 kDa, away from the SCF peak. These data show that HMC-1 cells fail to differentiate in response to SCF and that in addition to SCF, LCS contains other human mast cell growth factors.     

Activation of protein-tyrosine kinase Pyk2 is downstream of Syk in FcepsilonRI signaling

Aggregation of the FcepsilonRI, a member of the immune receptor family, induces the activation of proteintyrosine kinases and results in tyrosine phosphorylation of proteins that are involved in downstream signaling pathways. Here we report that Pyk2, another member of the focal adhesion kinase family, was present in the RBL-2H3 mast cell line and was rapidly tyrosine-phosphorylated and activated after FcepsilonRI aggregation. Tyrosine phosphorylation of Pyk2 was also induced by the calcium ionophore A23187, by phorbol myristate acetate, or by stimulation of G-protein-coupled receptors. Adherence of cells to fibronectin dramatically enhanced the induced tyrosine phosphorylation of Pyk2. Although Src family kinases are activated by FcepsilonRI stimulation and tyrosine-phosphorylate the receptor subunits, the activation and tyrosine phosphorylation of Pyk2 were downstream of Syk. In contrast, tyrosine phosphorylation of Pyk2 by stimulation of G-protein-coupled receptors was independent of Syk. Therefore, the FcepsilonRI-induced tyrosine phosphorylation of Pyk2 is downstream of Syk and may play a role in cell secretion.     

Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis

Human mastocytosis is characterized by increased mast cells. It usually occurs as a sporadic disease that is often transient and limited in children and persistent or progressive in adults. The c-KIT protooncogene encodes KIT, a tyrosine kinase that is the receptor for mast cell growth factor. Because mutated KIT can transform cells, we examined c-KIT in skin lesions of 22 patients with sporadic mastocytosis and 3 patients with familial mastocytosis. All patients with adult sporadic mastocytosis had somatic c-KIT mutations in codon 816 causing substitution of valine for aspartate and spontaneous activation of mast cell growth factor receptor (P = 0.0001). A subset of four pediatric onset cases with clinically unusual disease also had codon 816 activating mutations substituting valine, tyrosine, or phenylalanine for aspartate. Typical pediatric patients lacked 816 mutations, but limited sequencing showed three of six had a novel dominant inactivating mutation substituting lysine for glutamic acid in position 839, the site of a potential salt bridge that is highly conserved in receptor tyrosine kinases. No c-KIT mutations were found in the entire coding region of three patients with familial mastocytosis. We conclude that c-KIT somatic mutations substituting valine in position 816 of KIT are characteristic of sporadic adult mastocytosis and may cause this disease. Similar mutations causing activation of the mast cell growth factor receptor are found in children apparently at risk for extensive or persistent disease. In contrast, typical pediatric mastocytosis patients lack these mutations and may express inactivating c-KIT mutations. Familial mastocytosis, however, may occur in the absence of c-KIT coding mutations.     

Complement peptide C3a inhibits IgE-mediated triggering of rat mucosal mast cells

The relationship between mast cells' secretory response to stimulation via their type 1 Fc epsilon receptors (Fc epsilon RI) and that provided by the C3a fragment of the complement system was investigated in the rat mucosal-type mast cell line RBL-2H3. These cells are known to be unresponsive to the so-called 'peptidergic' stimulus provided by cationic agents, such as anaphylatoxins, neuropeptides or polyamines. We now observed that C3a effectively inhibits the Fc epsilon RI clustering induced secretion of RBL-2H3 cells. This inhibition is dose-dependent and takes place at a C3a concentration range of 0.4-12.5 nM, i.e. at least three orders of magnitude lower than those where this anaphylatoxin exerts its secretory stimulus to 'serosal' mast cells. In order to identify where C3a interferes in the Fc epsilon RI coupling cascade, we have studied its effect on the cells' protein phosphorylation pattern, hydrolysis of phosphatidyl inositides, transient rise in free cytosolic Ca2+ ion concentration and Ca2+ uptake. All these processes were found to be inhibited by a similar C3a concentration range.     

The mast cell function-associated antigen exhibits saccharide binding capacity

The mast cell function-associated antigen (MAFA) is a membrane glycoprotein first identified on rat mucosal type mast cells (line RBL-2H3) and known to inhibit the Fc epsilon RI-mediated secretory response. In its extracellular domain, an amino acid stretch homologous to the carbohydrate binding domain of calcium-dependent animal lectins has been found. To investigate its carbohydrate binding capacity, the MAFA has been expressed in the Spodoptera frugiperda insect cell line (Sf9) using the baculovirus expression system. Analysis by flow cytometry and surface labeling with 125I showed that the recombinant MAFA (rMAFA) was expressed as a monomeric and disulfide-linked homodimeric glycoprotein in the membrane of the insect cells, and both forms exhibited the same epitopes as the protein isolated from RBL-2H3 cells. Immunoaffinity-purified rMAFA was then employed for studies of its saccharide binding capacity by using different neoglycans and glycoproteins. The rMAFA was found to bind specifically terminal mannose residues in a Ca(2+)-dependent manner. These results support the notion that the extracellular domain of the MAFA is indeed able to bind ligands, which may be modulatory for the mast cell response.     

Differential effects of the protein kinase C activator phorbol 12-myristate 13-acetate on calcium responses and secretion in adherent and suspended RBL-2H3 mucosal mast cells

Adhesion of RBL-2H3 mucosal mast cells to fibronectin-coated surfaces has been linked to changes in secretion and tyrosine kinase activity. We now show that adhesion affects the sensitivity of RBL cells to the protein kinase C activator phorbol 12-myristate 13-acetate (PMA). In suspended cells, PMA inhibited antigen-induced calcium influx (as measured by manganese influx) and changes in intracellular free calcium and had complex effects on antigen-stimulated secretion. However, in adherent cells PMA had little effect on these responses. Suspended cells only secreted in response to thapsigargin if they were co-treated with PMA, while adherent cells secreted in response to thapsigargin alone. The thapsigargin-induced secretion in adherent cells was inhibited by protein kinase C down-regulation and by the protein kinase C inhibitor GF 109203X, but not by calphostin C. We suggest that protein kinase C is constitutively activated in adherent cells, possibly due to modification of the regulatory domain of the enzyme.