Tyrosine kinase-deficient Wv c-kit induces mast cell adhesion and chemotaxis

Jaroslaw Dastych1, Dennis Taub2, Mary C. Hardison3, and Dean D. Metcalfe3

1 Department of Biogenic Amines, Polish Academy of Sciences, 90-950 Lodz, Poland; 2 Clinical Services Program, Sciences Applications International Corporation, Frederick Cancer Research and Development Center, National Cancer Institute, Frederick, 21702; and 3 Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

W/Wv mice are deficient in tissue mast cells, and mast cells cultured from these mice do not proliferate in response to the c-kit ligand, stem cell factor (SCF). In this paper, we report that mouse bone marrow cultured mast cells derived from W/Wv mice do adhere to fibronectin in the presence of SCF and exhibit chemotaxis to SCF, and we explore this model for the understanding of c-kit-mediated signaling pathways. Both in vitro and in vivo (in intact cells) phosphorylation experiments demonstrated a low residual level of W/Wv c-kit protein phosphorylation. SCF-induced responses in W/Wv mast cells were abolished by the tyrosine kinase inhibitor herbimycin A and by the phospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin but were not affected by protein kinase C inhibitors. These observations are consistent with the conclusions that Wv c-kit initiates a signaling process that is PI 3-kinase dependent and that mutated Wv c-kit retains the ability to initiate mast cell adhesion and migration.

stem cell factor; W/Wv mice; phosphatidylinositol 3-kinase-dependent adhesion; fibronectin

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE PROTOONCOGENE c-kit has been shown to be involved in the induction of a number of critical biological processes related to mast cell function. C-kit signals mast cells to proliferate and differentiate and prevents mast cells from undergoing apoptosis (6, 20, 36, 35). It has also been shown that the c-kit ligand, also known as stem cell factor (SCF), is able to upregulate mast cell adhesion to extracellular matrix and is a potent mast cell chemoattractant (2, 12, 19). The critical role of c-kit in mast cell development in vivo is demonstrated by the phenotypic abnormalities in W mutant mice. Thus several spontaneous mutations in the locus W on chromosome 5, where c-kit is mapped, result in dramatic decreases in tissue mast cell numbers (37). The mutant mice most frequently employed in such in vivo studies are heterozygotes W/Wv (5, 31, 32) in which the W protein product is not present on the cell surface (8). The W mutation in the homozygous state results in death at the neonatal stage of life, and the W mutation has a null phenotypic characteristic in a heterozygous state (3, 16). In contrast, the Wv mutated c-kit gene product consists of a cell surface receptor that differs from the wild-type c-kit by a single-amino acid threonine-to-methionine substitution at position 660 in the kinase domain. This change abolishes or severely diminishes c-kit tyrosine kinase activity (22, 26). Cells bearing only Wv mutated c-kit on their surface do not respond to c-kit ligand in proliferation or apoptosis experiments (22, 35, 36), possibly because Wv c-kit is unable to initiate several signal transduction processes occurring on the wild-type receptor protein (27).

We have reported that the change in mast cell adhesiveness following addition of SCF is partially blocked by the tyrosine kinase inhibitor genistein (2). We employed bone marrow cultured mast cells (BMCMC) derived from W/Wv mice to study the role of tyrosine kinase in the c-kit-induced mast cell adhesion to fibronectin. In initial experiments, we observed an unexpectedly high response of these c-kit-defective mast cells to SCF. After this observation, we elected to investigate this phenomenon in a series of adhesion, chemotaxis, and phosphorylation experiments to gain insight into the pathways involved in promoting adhesion, chemotaxis, and proliferation. As will be shown in this paper, Wv c-kit is able to induce mast cell adhesion and migration through a tyrosine kinase-dependent signal transduction process, even though these cells do not proliferate in response to c-kit ligand. Furthermore, as will be shown, the subsequent signaling events do not require protein kinase C (PKC) and appear to be phospatidylinositol 3-kinase (PI 3-kinase) dependent. Thus the tyrosine kinase-dependent and PI 3-kinase-dependent signals are still transduced by the defective Wv c-kit receptor, and this allows SCF to influence the adhesion and chemotaxis of mast cells derived from W/Wv mice.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. Anti-c-kit monoclonal antibody (MAb) clone ACK2, herbimycin A, murine fibronectin (GIBCO Life Technologies, Gaithersburg, MD); [gamma -32P]ATP, 32P-labeled PBS, myo-[3H]inositol, and [methyl-3H]thymidine (6.7 Ci/mmol) (DuPont NEN, Boston, MA); [35S]methionine (trans-label; ICN Biomedicals, Costa Mesa, CA); G protein-agarose, chelerythrine, and bisindolylmaleimide I (Calbiochem, La Jolla, CA); wortmannin, BSA, and phorbol 12-myristate 13-acetate (PMA) (Sigma, St. Louis, MO); recombinant mouse SCF (rSCF; R&D, Minneapolis, MN); and 96-well Immulon plates (Dynatech Laboratories, Chantilly, VA) were purchased from manufacturers and distributors.

Cell culture. To initiate primary bone marrow cultures, female W/Wv, W/+, or +/+ WBB6F1J mice (6-9 wk) were killed by spinal cord dislocation. Bone marrow cells were obtained from femurs and suspended in RPMI 1640 medium supplemented with 10% WEHI-3 supernatant as a source of interleukin-3 (IL-3), 10% heat-inactivated FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 4 mM L-glutamine, 25 mM HEPES, 50 µM beta -mercaptoethanol, and 100 µg/ml penicillin and streptomycin (complete RPMI 1640). Cells were washed three times by centrifugation, followed by suspension in fresh complete RPMI 1640. Cell number was adjusted to 200,000 cells/ml, and cells were cultured at 37°C in a CO2 incubator. Cultures were fed by replacing the medium on a weekly basis. After 4-5 wk, cultures consisted of >96% BMCMC, as assessed by toluidine blue staining. Cell viability of W/Wv and +/+ mast cells was determined using the trypan blue assay after incubation for 3 h with 2 µM herbimycin A, 2 µM wortmannin, 10 µM bisindolylmaleimide I, and 10 µM chelerythrine. There was no decrease in viability of mast cells exposed to these inhibitors.

[Methyl-3H]thymidine incorporation assay. To measure the proliferative response of mast cells to SCF, the [methyl-3H]thymidine incorporation assay was performed as described (36). Briefly, +/+-derived or W/Wv-derived BMCMC were cultured in RPMI 1640 supplemented with 10% FCS without IL-3 for 12 h and then cultured for 2 h in the presence of [methyl-3H]thymidine with or without SCF.

Cell adhesion assay. The adhesion assay was performed as described (1). Briefly, 96-well plates were coated with fibronectin for 12 h at 4°C, followed by addition of 2% BSA in PBS and incubation for 2 h at 37°C. Forty thousand BMCMC radiolabeled with [methyl-3H]thymidine were then suspended in RPMI 1640 medium supplemented with 1% BSA, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 4 mM L-glutamine, 25 mM HEPES, 50 µM beta -mercaptoethanol, and 100 µg/ml penicillin and streptomycin (BSA-RPMI 1640) and added to each assay well. Increasing concentrations of rSCF or PMA were added to selected wells for a final volume of 150 µl. After culture at 37°C in a CO2 incubator for the indicated time, nonadherent cells were removed. The adherent and nonadherent cells were subsequently harvested (PHD cell harvester, Cambridge Technology, Cambridge, MA). The percentage of adhesion was calculated from the radioactivity associated with adherent cells divided by total radioactivity associated with both nonadherent and adherent cells.

Chemotaxis assay. The modified Boyden chamber technique was used to measure SCF-mediated mast cell migration (4). Briefly, BMCMC were suspended at 4 × 106 cells/ml in RPMI 1640 supplemented with 0.5% BSA and placed in the upper wells of 48-well microchemotaxis chambers using polycarbonate filters (5-µm pore size) coated with vitronectin to separate the upper and lower wells (33). Chambers were incubated for 4-6 h at 37°C in a CO2 incubator. Nonmigrating cells were gently removed from the filter surface, stained, and mounted (4). Mast cell movement was quantitated by counting the number of cells migrating completely through the filter in 10 high-power fields (HPF) in triplicate samples.

Radiolabeling of cell protein. To label mast cell protein with 35S, 107 BMCMC were suspended in 10 ml of methionine-free RPMI 1640 supplemented with 10% heat-inactivated dialyzed FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 4 mM L-glutamine, 25 mM HEPES, 50 µM beta -mercaptoethanol, and 100 µg/ml penicillin and streptomycin. Cells were then incubated in the presence of 50 µCi/ml L-[35S]methionine at 37°C for 3 h.

Phosphorylation of c-kit. To assess the phosphorylation of c-kit in intact cells, 107 BMCMC were suspended in 10 ml of phosphate-free RPMI 1640 supplemented with 10% heat-inactivated dialyzed FCS, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 4 mM L-glutamine, 25 mM HEPES, 50 µM beta -mercaptoethanol, and 100 µg/ml penicillin and streptomycin in the presence of 500 µCi/ml 32P-labeled PBS at 37°C for 3 h. rSCF (100 ng/ml) was then added to selected tubes, followed by a 20-min incubation.

Immunoprecipitation. To immunoprecipitate c-kit protein, 107 35S- or 32P-radiolabeled or unlabeled BMCMC were collected by centrifugation and washed with cold PBS. Cell pellets were lysed in 1 ml of cold lysing buffer, which consisted of 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml each aprotinin, leupeptin, pepstatin, E-64, and betastatin, and 20 mM EDTA, in PBS (pH 7.4), without Ca2+ or Mg2+. Mast cell lysates were precleared by incubation with 30 µl of G protein-agarose on a rotary mixer at 4°C overnight, followed by centrifugation. Twenty micrograms of anti-c-kit or control MAb immobilized on 10 µl of G protein-agarose beads were added to the precleared lysate, and the tubes were incubated for 12 h. Agarose beads were collected by centrifugation, suspended in 1 ml of lysing buffer, and centrifuged. This step was repeated five times. The agarose beads were next overlaid with 40 µl of 0.02 M Tris (pH 6.8). Tubes were boiled for 7 min to solubilize immunoprecipitated proteins and centrifuged in a microcentrifuge for 10 min. Supernatants containing immunoprecipitated proteins were loaded on 6% acrylamide gel, and SDS-PAGE was performed according to Laemmli (13). Gels were dried and autoradiographed. The optical densities of bands seen on autoradiography films were measured in the Image Master video documentation system (Pharmacia Biotech, San Francisco, CA).

In vitro kinase assay. To assay activity of c-kit-associated tyrosine kinase, proteins immunoprecipitated with anti-c-kit or control MAb were collected by centrifugation and suspended in an assay buffer containing 0.02 M Tris (pH 7.4), 1 mM PMSF, and 20 mM EDTA. Twenty microliters of beads suspended in forty microliters of assay buffer were placed into separate Eppendorf tubes and incubated in the presence of 10 µCi of [gamma -32P]ATP and increasing concentrations of unlabeled ATP. After a 20-min incubation at room temperature, tubes were boiled for 7 min, chilled on ice, and centrifuged. Supernatants were examined in SDS-PAGE under reducing conditions using 6% acrylamide gels. Gels were dried, rehydrated, incubated in 0.5 M KOH to wash out noncovalently bound radioactivity, again dried, and autoradiographed.

Assay of [3H]inositol phosphates. The assay of total [3H]inositol phosphates was performed as described (17). BMCMC (106 cells/ml) were labeled in inositol-free complete RPMI 1640 medium in the presence of 12 µCi/ml myo-[3H]inositol for 19 h. Radiolabeled cells were washed twice with PBS and suspended in BSA-RPMI 1640. Aliquots of cell suspensions were placed in polypropylene tubes, and rSCF (final concentration 20 ng/ml) or medium alone was added for a final volume of 200 µl, followed by incubation at 37°C. At indicated time points, cell aliquots were mixed with 750 µl of chloroform-methanol (1:2). Cell extracts were vortex mixed, and, after addition of 250 µl of water and 250 µl of chloroform, the phases were separated by centrifugation. The aqueous phase was then loaded on a Dowex 1-X8 (formate form) column. Columns were washed with 5 mM inositol to remove myo-[3H]inositol. Inositol phosphates were eluted with 1.5 ml of 1 M sodium formate in 0.1 M formic acid. The radioactivity associated with inositol phosphates was assayed by liquid scintillation counting. Inositol phosphate release was calculated by dividing the radioactivity of the inositol phosphates by the sum of the total radioactivity incorporated into aqueous and organic phases.

Calculations and statistical analysis. Inhibition of adhesion or chemotaxis was calculated using the formula %inhibition = (aSCF - aI )/(aSCF - aspont) × 100%, where aI is adhesion or chemotaxis in the presence of SCF and inhibitor, aSCF is adhesion or chemotaxis in the presence of SCF, and aspont is spontaneous adhesion or migration. The statistical significance of observed differences was tested using ANOVA followed by the Tukey test.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

To demonstrate that the Wv defect in c-kit tyrosine kinase activity would not prevent mast cells from adhering to fibronectin following addition of SCF, BMCMC derived from W/Wv mice and from +/+ mice were placed in fibronectin-coated plastic wells and stimulated with increasing concentrations of SCF or with PMA. As can be seen in Fig. 1A, both +/+ and W/Wv mast cells adhered to fibronectin in increasing numbers after addition of SCF, although adhesion of W/Wv mast cells was significantly less than that of +/+ mast cells (P = 0.01). The effect of SCF was dose dependent, with a maximal SCF-induced adhesion of 52.9% for W/Wv-derived mast cells at 10 ng/ml SCF and 67.1% for +/+-derived mast cells at 25 ng/ml SCF. The effect of SCF was saturable, and higher concentrations of SCF did not result in additional mast cell adhesion (Fig. 1A, inset).


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Fig. 1.   Comparison of stem cell factor (SCF)-mediated mast cell adhesion and migration of bone marrow cultured mast cells (BMCMC) cultured from +/+ and W/Wv mice. A: +/+-derived (hatched bars) or W/Wv-derived (solid bars) BMCMC were placed in fibronectin-coated wells and incubated with increasing concentrations of SCF for 30 min. Values are means ± SE from 3 independent experiments performed in duplicate. W/Wv mast cells showed significantly less adhesion than +/+ mast cells (2-way ANOVA, P = 0.01). PMA, phorbol 12-myristate 13-acetate. Inset: mast cell adhesion in presence of high concentrations (ng/ml) of SCF. B: +/+-derived (hatched bars) or W/Wv-derived (solid bars) BMCMC (4 × 106 cells/ml) were placed in upper wells of 48-well microchemotaxis chambers. A polycarbonate filter (5-µm pore size) coated with vitronectin separated upper and lower wells. SCF or medium alone was added to lower well. Mast cell movement at 4 h was quantitated by counting number of cells migrating completely through filter in 10 high-power fields (HPF). Values are means ± SD from 1 experiment performed in triplicate, except at 10 ng/ml SCF, which caused maximal chemotaxis and was repeated 5 times in triplicate. Average migrations of W/Wv mast cells and +/+ mast cells at 10 ng/ml SCF in 5 independent experiments were 90 ± 9 and 99 ± 10 mast cells/HPF, respectively (no significant difference; Student's t-test at P < 0.05). Inset: effect of SCF on [methyl-3H]thymidine incorporation into BMCMC (see MATERIALS AND METHODS). CPM, counts/min.

To further investigate the ability of W/Wv-derived BMCMC to respond to SCF, we similarly examined chemotaxis. As can be seen in Fig. 1B, W/Wv-derived BMCMC migrated toward SCF. Maximal chemotaxis was observed with 10 ng/ml SCF. The average migration (n = 5) of W/Wv-derived BMCMC at 10 ng/ml SCF was 90 ± 10 compared with 99 ± 9 mast cells/HPF observed with normal mast cells (no significant difference; Student's t-test at P < 0.05).

Employing thymidine incorporation as a measure of a proliferative response, we next verified that +/+-derived mast cells but not W/Wv-derived mast cells would respond to SCF as has been described (35). As expected, +/+-derived mast cells, but not W/Wv-derived mast cells, responded to the addition of rSCF at both 10 and 50 ng/ml by incorporation of thymidine (Fig. 1B, inset). W/Wv-derived BMCMC were also stained with anti-c-kit MAb and examined using flow cytometry. Mast cells cultured from W/Wv mice exhibited 50% of the fluorescence intensity shown by +/+-derived BMCMC. This is expected, as the W allele does not produce a membrane-associated protein. Thus BMCMC from W/Wv mice express Wv gene product on their surface and are severely defective in a proliferative response to c-kit ligand, and yet they demonstrate diminished but demonstrable adhesion and also apparently normal migration to c-kit ligand at an optimal concentration of SCF (10 ng/ml).

It has been reported that BMCMC from W/Wv mice do not adhere to fibronectin when stimulated by c-kit ligand (12). To verify the consistency of our observation, we repeatedly examined the ability of BMCMC from independent bone marrow cultures to adhere to fibronectin following addition of SCF. Figure 2 presents data generated in multiple adhesion experiments in which cells harvested from 10 different cultures (each representing the bone marrow from 5 +/+ or W/Wv mice) were incubated with 10 ng/ml SCF for 30 min on fibronectin-coated plastic. As can be seen, in some instances, the percentage of adhesion observed with W/Wv-derived BMCMC was similar to the percentage of adhesion observed with +/+-derived BMCMC. However, when all experimental data (n = 19) are averaged, SCF-induced adhesion of W/Wv-derived BMCMC (mean 43.5%) is significantly lower (P < 0.01) than the adhesion of control BMCMC (mean 62.6%). A review of the techniques involved in measuring adherence in this study compared with those in a previous study (12) reveals methodological differences that may account for the disparate results. In our study, mast cells were labeled with radioactivity, and adherent and nonadherent mast cells were quantitated. However, in the previous study (12), cells were fluorescently labeled, and differences in the fluorescence of adherent cells were determined.


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Fig. 2.   SCF-mediated adhesion of mast cells derived from separate bone marrow cultures. Mast cell adhesion was examined 30 min after addition of 10 ng/ml SCF. Data obtained in multiple independent experiments were grouped according to bone marrow culture used as source of BMCMC. Values are means of data obtained from 1 experiment performed in duplicate. Spontaneous adhesion was subtracted from each value and was never >8%.

We next examined receptor phosphorylation by immunoprecipitating c-kit from normal and W/Wv-derived mast cells. The ACK2 MAb recognizes the extracellular domain of c-kit and precipitated two forms of c-kit protein with molecular masses of 125 and 146 kDa. These two forms represent two c-kit proteins differing in glycosylation, as has been reported (22) and as we verified with pulse-and-chase experiments and digestion with endoglycosidase F (data not shown). C-kit proteins immunoprecipitated from W/+- and W/Wv-derived BMCMC were then used in an in vitro kinase assay. As shown in Fig. 3A, the wild-type c-kit was heavily phosphorylated following incubation with ATP. The level of radioactive label incorporated into receptor protein decreased when increasing concentrations of unlabeled ATP were added to the incubation mixture. The c-kit immunoprecipitated from W/Wv BMCMC incorporated a very small but detectable amount of radioactive phosphate. There is thus a severe loss of kinase activity compared with wild-type receptor. However, radioactivity was still incorporated into c-kit protein, as detected by autoradiography. The molecular mass of the W/Wv-derived protein phosphorylated in the assay was the same as for W/+-derived protein, and the radioactive label was similarly competed against by unlabeled ATP. Thus Wv mutated c-kit exhibits a residual tyrosine kinase activity that was detectable with an in vitro assay.


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Fig. 3.   Kinase activity associated with wild-type and Wv c-kit protein immunoprecipitated from BMCMC. A: in vitro kinase activity. W/+-derived (lanes 1-5) or W/Wv-derived (lanes 6-10) BMCMC were lysed and immunoprecipitated as described in MATERIALS AND METHODS. Agarose beads with immunoprecipitated proteins were suspended in assay buffer and incubated in presence of [gamma -32P]ATP and "cold" ATP. After a 20-min incubation, reaction was stopped by boiling in Laemmli loading buffer and proteins were analyzed by SDS-PAGE under reducing conditions. Gel was fixed and dried and then rehydrated, washed in KOH, and dried. Autoradiography was performed for 36 h. Std, mol mass standards (kDa). B: in vivo (in intact cells) phosphorylation of c-kit protein. Top: +/+-derived (lanes 1 and 2) or W/Wv-derived (lanes 3 and 4) BMCMC were labeled with 32P for 3 h. Cells were washed, suspended in BSA-RPMI 1640 medium at 106 cells/ml, and incubated without (lanes 1 and 3) or with (lanes 2 and 4) 50 ng/ml SCF for 10 min. Cells were washed with cold PBS and lysed, and c-kit was immunoprecipitated as described in MATERIALS AND METHODS. Immunoprecipitated proteins were analyzed by SDS-PAGE under reducing conditions. Bottom: in a parallel experiment, mast cells were labeled with [35S]methionine to verify that comparable amounts of radiolabeled proteins were loaded. In vitro kinase assays (A) were performed twice with similar results. In the second in vitro kinase assay, +/+-derived mast cells rather than W/+-derived mast cells were used as a control. Phosphorylation in vivo (in intact cells) experiments (B) were performed 3 times with similar results. In 1 of these assays, mast cells were stimulated with SCF in complete RPMI 1640 medium containing interleukin-3.

To compare phosphorylation of normal and Wv c-kit receptors following SCF activation in intact mast cells, +/+- and W/Wv-derived BMCMC were labeled with 32P and incubated with SCF. As seen in Fig. 3B, when +/+-derived BMCMC were incubated with SCF, immunoprecipitated c-kit protein showed a marked increase in the level of phosphorylation as quantitated by measurement of the optical density of electrophoretic bands using densitometry (437% increase with 146-kDa band and 243% increase with 125-kDa band; n = 2) compared with controls. C-kit proteins precipitated from W/Wv-derived mast cells revealed far less incorporation of 32P but continued to exhibit a small but consistent difference in the level of phosphorylation before and after stimulation: 40 and 72% increases, respectively. Thus SCF stimulation resulted in a change in the level of phosphorylation of normal and Wv c-kit receptors in vivo (in intact cells).

We next used a specific tyrosine kinase inhibitor to determine whether the upregulation of adhesion of W/Wv-derived mast cells to fibronectin depends on the enzymatic activity of the Wv receptor. Preincubation of W/Wv-derived mast cells with increasing concentrations of the tyrosine kinase inhibitor herbimycin A prevented the increase in mast cell adhesion to fibronectin observed in the presence of SCF (Fig. 4A). This inhibition of W/Wv mast cell adhesion was dose dependent, with an IC50 of 12.8 nM. In contrast, concentrations of herbimycin A up to 1.8 µM were less effective in decreasing +/+ mast cell adhesion (28%). Herbimycin A also decreased the chemotaxis of +/+- and W/Wv-derived BMCMC to SCF (Fig. 4B). Preincubation of W/Wv-derived mast cells with 100 nM herbimycin A for 30 min inhibited migration toward SCF by 79%. Normal mast cells also showed a significant decrease in their SCF-mediated migratory response following preincubation with herbimycin A, but the concentration of inhibitor needed to prevent their chemotaxis was higher (58% inhibition at 1.5 µM) than for W/Wv-derived mast cells.


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Fig. 4.   Effect of tyrosine kinase inhibitor herbimycin A on SCF-mediated mast cell migration and adhesion. A: +/+-derived (open circle ) or W/Wv-derived (bullet ) BMCMC were placed in fibronectin-coated wells and preincubated with increasing concentrations of herbimycin A or with DMSO (vehicle) for 30 min. Cells were then incubated with SCF (10 ng/ml) for 30 min. Adhesion was assayed, and percent inhibition was calculated, as described in MATERIALS AND METHODS. Values are means ± SE from 5 independent experiments performed in duplicate. B: +/+-derived (open circle ) or W/Wv-derived (bullet ) BMCMC (4 × 106 cells/ml) were preincubated for 30 min with increasing concentrations of herbimycin A or DMSO (vehicle). Cells were placed in upper wells of 48-well microchemotaxis chambers. A polycarbonate filter (5-µm pore size) coated with vitronectin separated upper and lower wells; 10 ng/ml SCF or medium alone was added to lower wells. Mast cell movement at 4 h was quantitated by counting number of cells migrating completely through filter in 10 HPF in triplicate samples. Inhibition was calculated according to formula given in MATERIALS AND METHODS. Values are means ± SE from 3 independent experiments performed in triplicate.

It has been reported that c-kit dimerization results in activation of phospholipase C-gamma (PLC-gamma ) (14, 25, 40), which in turn generates D-myo-inositol 1,4,5-trisphosphate and diacylglycerol (DAG). DAG then activates PKC. To determine whether this pathway was involved in adhesion initiated through Wv c-kit, we compared the kinetics of phosphatidylinositol (PI) hydrolysis mediated by the wild-type c-kit and by the Wv c-kit. As shown in Fig. 5A, addition of SCF initiated a time-dependent accumulation of inositol phosphates in control BMCMC. In contrast, there was no change in the level of the products of PI hydrolysis observed in BMCMC derived from W/Wv mice. We further used two inhibitors (chelerythrine and bisindolylmaleimide I) known to specifically block PKC activity (9, 34) to determine whether c-kit-mediated adhesion of W/Wv-derived mast cells to fibronectin involves this family of kinases. As shown in Fig. 5B, neither chelerythrine nor bisindolylmaleimide I decreased the SCF-mediated mast cell adhesion to fibronectin.


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Fig. 5.   Hydrolysis of inositol phospholipids and effect of protein kinase C (PKC) inhibitors. A: hydrolysis of inositol phospholipids in SCF-stimulated BMCMC. W/Wv-derived (bullet ) or +/+-derived (open circle ) BMCMC were labeled with myo-[3H]inositol for 16 h. Cells were incubated in complete RPMI 1640 medium in presence of SCF (10 ng/ml). At indicated times, aliquots of BMCMC were removed and extracted with chloroform-methanol. Inositol phosphates (IP) were purified by solvent partition followed by ion exchange chromatography on Dowex 1-X8 (formate form) columns. Radioactivity was determined by liquid scintillation counting. Release of [3H]inositol phosphates is expressed as a percent of total [3H]inositol phospholipids present in cell extracts. Values are means ± SE from 3 independent experiments performed in duplicate. B: effect of PKC inhibitors on SCF-mediated mast cell adhesion. W/Wv-derived (solid bars) or +/+-derived (hatched bars) BMCMC were placed in fibronectin-coated wells and preincubated with chelerythrine (10 µg/ml), bisindolylmaleimide (GF 109203X; 10 µg/ml), or DMSO (vehicle) for 30 min. Cells were then incubated with SCF (10 ng/ml) for 30 min, and adhesion was assayed as described in MATERIALS AND METHODS. Values are means ± SE from 2 independent experiments performed in duplicate.

PI 3-kinase is also reported to participate in c-kit-dependent signal transduction (14, 25). To determine whether PI 3-kinase may be involved in Wv c-kit-mediated mast cell adhesion and chemotaxis, we employed a specific inhibitor of PI-3 kinase, wortmannin (30). Preincubation of W/Wv- or +/+-derived BMCMC with wortmannin resulted in a decrease in SCF-mediated adhesion to fibronectin (Fig. 6A). The IC50 was 4.7 nM for W/Wv-derived and 21.2 nM for +/+-derived mast cells. Wortmannin also inhibited chemotaxis of mast cells toward SCF (Fig. 6B), although it was less effective. For example, 250 nM of wortmannin diminished SCF-mediated chemotaxis of W/Wv-derived BMCMC by 75% and of +/+-derived BMCMC by 58%. This evidence is consistent with the conclusion that the PI 3-kinase pathway is involved in signal transduction through c-kit, which in turn is involved in mast cell adhesion and chemotaxis.


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Fig. 6.   Effect of the phospatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin on SCF-mediated mast cell migration and adhesion. A: +/+-derived (open circle ) or W/Wv-derived (bullet ) BMCMC were seeded in fibronectin-coated wells and preincubated for 30 min with increasing concentrations of wortmannin or with DMSO (vehicle). Cells were then incubated with SCF (10 ng/ml) for 30 min. Adhesion was assayed, and inhibition was calculated, as described in MATERIALS AND METHODS. Values are means ± SE from 5 independent experiments performed in duplicate. B: +/+-derived (open circle ) or W/Wv-derived (bullet ) BMCMC (4 × 106 cells/ml) were preincubated for 30 min with increasing concentrations of wortmannin or with DMSO (vehicle). Cells were placed in upper wells of 48-well microchemotaxis chambers. A polycarbonate filter (5-µm pore size) coated with vitronectin separated upper and lower wells; 10 ng/ml SCF or medium alone was added to lower well. Mast cell movement at 4 h was quantitated by counting number of cells migrating completely through filter in 10 HPF in triplicate samples. Inhibition was calculated according to formula given in MATERIALS AND METHODS. Values are means ± SE from 3 independent experiments, each performed in triplicate.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

It is known that mast cells cultured from W/Wv mice express deficient c-kit and that these mast cells are not able to proliferate in the presence of SCF and also that SCF is not able to prevent mast cells from undergoing apoptosis (20, 36). For example, W/Wv-derived BMCMC did not incorporate thymidine in response to concentrations of recombinant SCF causing the maximal level of incorporation in normal BMCMC (36) (Fig. 1B, inset). In contrast to the inability of SCF to induce the proliferation of W/Wv-derived BMCMC, we here report that mast cells derived from W/Wv have the ability to respond to SCF by adhesion to fibronectin (Fig. 1A) and by migration (Fig. 1B), although adhesion was significantly less than with +/+ mast cells. For instance, we verified that BMCMC from W/Wv mice consistently adhere to fibronectin in response to SCF, using independent bone marrow cultures (Fig. 2). The average relative difference between W/Wv c-kit-mediated mast cell adhesion and +/+ c-kit-mediated mast cell adhesion to fibronectin in 15 experiments was 30.5%.

We thus hypothesized that the ability of W/Wv mast cells to adhere (~70% of normal level) and engage in chemotaxis (at least 90% of normal level) through a c-kit-mediated signal was in part possible because the Wv c-kit receptor is able to signal through residual tyrosine kinase activity, although this residual activity was only a small fraction of normal. This hypothesis was supported by data from an in vitro kinase assay (Fig. 3A) that demonstrated detectable enzymatic activity associated with Wv c-kit. Similarly, phosphorylation studies performed in intact cells showed that Wv c-kit receptor is phosphorylated in vivo (in intact cells) following addition of SCF, although to a much lesser degree than the wild-type receptor (Fig. 3B). This residual tyrosine kinase activity shown by Wv c-kit may explain the in vivo observation of normalization of mast cell number in Wv/Wv:me/me mice, in which the defect in phosphatase SHP1 appears to compensate for the defect in Wv kinase (17).

The initiation of a signal transduction process by a mutated Wv c-kit was further verified in experiments employing the tyrosine kinase inhibitor herbimycin A. This inhibitor decreased inhibition of SCF-induced adhesion and chemotaxis of W/Wv-derived BMCMC in a dose-dependent manner (Fig. 4), strongly supporting a role for a residual tyrosine kinase activity observed in the in vitro kinase assay in the process of upregulating mast cell adhesion. The IC50 value for herbimycin A in W/Wv-derived BMCMC was lower than that reported for other tyrosine kinase-dependent systems and is consistent with a specific action of this inhibitor on tyrosine kinase (38). A nonselective action of herbimycin A on the phosphatidylinositol cycle was recently reported (28), but at concentrations of inhibitor 16 times higher than that required to completely inhibit Wv c-kit-mediated adhesion. The difference between W/Wv-derived and +/+-derived BMCMC in sensitivity to herbimycin A may well reflect the level of tyrosine kinase activity associated with each type of receptor protein. Thus cell adhesion mediated by Wv c-kit receptor is very sensitive to herbimycin A because both the Wv mutation and herbimycin A act together to decrease tyrosine kinase activity to the point at which no physiological response is detected. In contrast, the same inhibitor failed to significantly decrease +/+ c-kit-mediated cell adhesion because even a small amount of uninhibited kinase activity would lead to this cellular response, as suggested by Wv c-kit receptor-mediated adhesion (Figs. 1, 2, and 3A). Thus the level of tyrosine kinase activity associated with Wv c-kit receptor appears to be sufficient to transduce signals for adhesion and migration but not proliferation.

Addition of SCF to +/+-derived BMCMC resulted in a slow accumulation of phosphoinosites (Fig. 5A). This appears to be similar to phosphatidylinositol hydrolysis in fibroblasts mediated by the platelet-derived growth factor receptor (7), which belongs to the same family of receptors as c-kit (24, 42). PI hydrolysis is mediated by PLC-gamma , and the observed accumulation of inositol phosphates is consistent with the reported activation of this pathway following dimerization of c-kit (14, 25, 40). In contrast to +/+-derived BMCMC, however, there was no increase in PI products when SCF was added to W/Wv-derived BMCMC. Thus stimulation of mast cells through the Wv-mutated c-kit upregulated cell adhesion without initiation of PI hydrolysis (Fig. 5A). The lack of accumulation of phosphoinosites in W/Wv-derived BMCMC is consistent with an absence of activation of PLC-gamma 1.

PKC has been implicated in the regulation of adhesion in lymphocytes and macrophages (11, 19), and it has been reported that SCF-mediated mast cell adhesion may be inhibited by the PKC inhibitor calphostin C (39). In this study, however, two specific PKC inhibitors, bisindolylmaleimide I and chelerythrine, did not inhibit SCF-mediated mast cell adhesion in either +/+-derived or W/Wv-derived mast cells, even when used in high doses (Fig. 5B). This is consistent with the conclusion that the PKC pathway is not necessary for c-kit-mediated mast cell adhesion. This latter conclusion is in disagreement with the reported effect of calphostin C on mast cell adhesion. These differing results may be due to inhibitors exhibiting their action on specific PKCs or some inhibitors also having additional biological activities. Bisindolylmaleimide I itself is a potent and specific PKC inhibitor (Michaelis-Menten inhibition constant 10 nM), easily penetrates into the cell, inhibits a wide variety of PKC kinases, and does not require photoactivation as does calphostin C.

The differences between wild-type and Wv mutated c-kit protein have been mapped to a single-amino acid substitution. It is thus possible that mutated receptor protein retains an ability to interact with some downstream elements of a c-kit signaling pathway. In this regard, the p85 subunit of PI 3-kinase is known to interact with the cytoplasmic part of c-kit, and this interaction is mediated by a hydrophilic insert in c-kit tyrosine kinase (15). Wortmannin is a specific inhibitor of PI 3-kinase that in other systems irreversibly inhibits this kinase with an IC50 of 2-10 nM (30, 41). Wortmannin added to the adhesion assay did inhibit SCF-mediated W/Wv mast cell adhesion with an IC50 of 4.7 nM (Fig. 6A), possibly due to specific inhibition of PI 3-kinase. Adhesion of +/+-derived BMCMC appears to be similarly PI 3-kinase dependent. This suggests activation of the PI 3-kinase signaling pathway in regulating Wv c-kit-dependent adhesion. SCF-mediated migration is less sensitive to wortmannin. This inhibition may thus depend on the action of wortmannin on another enzymatic activity, such as myosin light chain kinase, as has been observed at higher concentrations (41) (Fig. 6B).

The possibility of an interaction between the mutated c-kit molecule and PI 3-kinase was suggested by a study in which a point mutation analogous to Wv was generated in a chimeric receptor consisting of the extracellular domain of human epidermal growth factor (EGF) receptor and the transmembrane and kinase domains of human c-kit. When this chimeric receptor was expressed in human fibroblasts, it exhibited residual tyrosine kinase activity and did coprecipitate and phosphorylate the p85 subunit of PI 3-kinase following the addition of EGF (10). These data appear to be relevant, as there is a high level of homology between human and murine c-kit genes (24, 43). The W/Wv-derived BMCMC employed in our study also are representative of cells that express c-kit in vivo and in which the Wv gene is not overexpressed. Under such conditions, mutated c-kit did signal in a PI 3-kinase-dependent manner for adhesion and migration.

One explanation for differences in responsiveness of W/Wv-derived BMCMC in c-kit-mediated adhesion and proliferation could be the existence of two partially independent pathways signaling for adhesion and proliferation. In an in situ mutagenesis study (29), c-kit-defective murine mast cells were transfected with c-kit substituted at tyrosine-821 with phenylalanine. These transfected BMCMC exhibited a phenotype similar to that of the W/Wv-derived BMCMC, with severely impaired c-kit-dependent cell proliferation but near normal c-kit-mediated cell adhesion to fibronectin. In the same study, c-kit substituted at tyrosine-719 with phenylalanine and thus unable to interact with PI 3-kinase was defective in both c-kit-mediated proliferation and adhesion. These data suggest that phosphorylation of different tyrosines on c-kit may control different cell responses. Our data are compatible with this idea, as they demonstrate that a single point mutation in c-kit can result in differential effects on c-kit-mediated cellular responses. It is thus possible that the tyrosine kinase associated with Wv c-kit that results in quantitatively defective autophosphorylation may preferentially phosphorylate specific tyrosine residues.

The Wv variant of c-kit has very limited, if any, ability to signal for mast cell proliferation; it is, however, capable of inducing some degree of cell adhesion and migration, although less than that induced by normal c-kit. The signal for adhesion involves tyrosine kinase and PI 3-kinase. These observations are consistent with the existence of at least two differentially regulated signal transduction processes initiated by c-kit phosphorylation.

    FOOTNOTES

Address for reprint requests: J. Dastych, Dept. of Biogenic Amines, Polish Academy of Sciences, PO Box 225, 90-950 Lodz, Poland.

Received 20 October 1997; accepted in final form 21 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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