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The Journal of Immunology, 2007, 178: 2527-2534.
Copyright © 2007 by The American Association of Immunologists, Inc.

K-ras Is Critical for Modulating Multiple c-kit-Mediated Cellular Functions in Wild-Type and Nf1+/– Mast Cells1

Waleed F. Khalaf*,{ddagger}, Feng-Chun Yang{dagger},{ddagger}, Shi Chen{dagger},{ddagger}, Hilary White{dagger},{ddagger}, Waylan Bessler{dagger},{ddagger}, David A. Ingram{dagger},{ddagger} and D. Wade Clapp2,*,{dagger},{ddagger}

* Department Microbiology & Immunology, {dagger} Department of Pediatrics, and {ddagger} Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
p21ras (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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Ras proteins compose a family of small GTP-binding proteins that transduce extracellular growth factor signals to the nucleus and regulate multiple cellular functions, including proliferation, differentiation, survival, degranulation, and chemotaxis in multiple lineages (1, 2, 3, 4, 5, 6, 7). Following binding of the ligand to its receptor, guanine-nucleotide exchange factors promote allosteric changes that increase the percentage of Ras-GTP in the target cell. Three closely related mammalian Ras isoforms (H-, N-, and K-ras) have been identified and found to share 85% homology at the amino acid level (8, 9, 10). Overexpression of different constitutively active Ras isoforms result in a similar transformed phenotype and have previously led to the suggestion that there is a high functional redundancy within the family of Ras proteins (8, 11). In contrast, each Ras isoform is implicated in distinct forms of cancer (11, 12) and K-ras is required for normal murine development while N-ras and H-ras are dispensable, either separately or in combination (13, 14, 15, 16). Moreover, Ras isoforms undergo differential posttranslational modification (8, 17, 18, 19), intracellular compartmentalization (20, 21, 22), and appear to differentially activate downstream signaling pathways (23, 24, 25). Overexpression models and studies in N-ras-deficient murine embryonic fibroblasts emphasize that K-ras preferentially activates the Raf-MEK-ERK pathway while N-ras and H-ras activate the PI3K pathway (24, 25, 26, 27). A limitation of overexpression studies is that multiple transgene copies of the plasmid may result in nonphysiological binding and/or sequestration of effectors. Furthermore, an emerging theme in recent studies is that there is cell context specificity in intracellular signaling and in the modulation of biological functions among seemingly redundant Ras GTPases. The specific role of each respective Ras isoform in modulating various biological functions in myeloid cells and specifically mast cells has not been examined.

Following activation, Ras proteins in immune cells are inactivated by the conversion of Ras-GTP to Ras-GDP through the action of two highly conserved GTPase-activating proteins (GAPs),3 P120 GAP and neurofibromin (4, 28). Genetic mutations in the tumor suppressor gene NF1, which encodes the GAP protein product neurofibromin, cause neurofibromatosis type 1 (NF1) (29, 30, 31, 32). The NF1 gene is complex in that it has 60 exons, and its protein product, neurofibromin, is 320 kDa (33, 34). Despite this complexity, the GAP-related domain of NF1 (GRD) shares high sequence homology with p120 GAP (30, 35). We previously established that introduction of the NF1 GRD, but not p120 GRD, restores normal Ras activity and rescues the elevated proliferation and survival phenotypes in vitro and in vivo in Nf1 knockout (Nf1–/–) myeloid progenitors and Nf1 heterozygous (Nf1+/–) mast cells (36, 37, 38). These findings demonstrate that alterations in Ras activity in Nf1-deficient myeloid cells is central to disease pathogenesis in NF1 (39), although the specific contribution of individual Ras isoforms to this pathology is not fully understood.

One major cause of morbidity in NF1 patients is the development of the pathognomonic cutaneous and plexiform neurofibromas (40). These complex peripheral nervous system tumors consist of Schwann cells, fibroblasts, endothelial cells, and high concentrations of degranulating mast cells (41, 42). Recent genetic studies in a murine model that has a high penetrance of neurofibromas that closely recapitulate the development of human neurofibromas and indicate that haploinsufficiency of Nf1 in non-neuronal cells in the tumor microenvironment is required for tumor progression (43). Mast cells secrete a wide range of proteases, extracellular matrix remodeling proteins, and preformed mediators of inflammation such as histamine, which is integral to tumor progression (42, 44, 45, 46, 47, 48, 49). These mediators are processed in secretory vesicles in the Golgi and stored in cytoplasmic granules until mast cell activation, during which the contents are released into extracellular space (49). However, the role of Ras isoforms in activating these pathways and modulating these biological processes is incompletely understood.

In this study, we use a genetic model to examine the role of K-ras in modulating intracellular signaling and biological processes in Nf1+/– mast cells and mast cells that have normal GAP activity. We provide evidence that K-ras modulates both the Raf-MEK-ERK pathway as well as the PI3K pathway in wild-type (WT) and Nf1+/– mast cells. These changes in activity are associated with biological reductions in proliferation, migration, degranulation, and survival. We also provide genetic evidence that Nf1 functions as a critical GAP for K-ras in mast cells and that genetic deletion of K-ras reduced the gain of function phenotypes in Nf1+/– mast cells, in a K-ras gene dose-dependent manner, both in vitro and in vivo. Collectively, these data indicate that K-ras has a central role in mast cell activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice, fetal hemopoietic cell isolation, and mast cell culture

K-ras+/– mice were obtained from The Jackson Laboratory in a 129/Sv background. Nf1+/– mice were obtained from Dr. T. Jacks (Massachusetts Institute of Technology, Cambridge, MA) in a C57BL/6.129 background and were backcrossed for 10 generations into a 129/Sv strain. The Nf1 and K-ras alleles were genotyped by PCR as previously described (15, 50). Studies were conducted with a protocol approved by the Indiana University Animal Care and Use Committee. Nf1+/–; K-ras+/– mice were mated with Nf1+/+;K-ras+/– mice to produce embryos of the six experimental genotypes as outlined: Nf1+/+; K-ras+/+, Nf1+/–; K-ras+/+, Nf1+/+; K-ras+/–, Nf+/–; K-ras+/–, Nf1+/+; K-ras–/–, and Nf+/–; K-ras–/–. Embryonic day 13.5 fetal livers were isolated and single-cell suspensions were prepared by passing the hepatic tissues through a 23-gauge needle as previously described (50). After early attempts to generate mast cell lines, we found that IL-3 (PeproTech) alone does not support the survival of the K-ras–/– cells. Thus, all mast cell lines were generated in the presence of both IL-3 and Kit-ligand (Kit-L; PeproTech). Giemsa staining and FACS analysis (BD Immunocytometry Systems) reveal a homogeneous population of mast cells with similar forward and side scatter and similar surface expression of c-kit (data not shown). All experiments were conducted using at least three lines from each genotype.

Proliferation and survival assays

Proliferation of mast cells was evaluated using the thymidine incorporation assay as previously described (50). Mast cells from each genotype were deprived of growth factors overnight and plated in a 96-well plate at 2 x 105 cells in 200 µl of RPMI 1640 (Invitrogen Life Technologies) containing 10% FBS (Biomedia) and 0–50 ng/ml Kit-L as indicated, in a 37°C/5% CO2 humidified incubator. After 48 h of culture, cells were pulsed with tritiated thymidine (PerkinElmer Life and Analytical Sciences) for 16 h, harvested on glass fiber filters (PerkinElmer), and beta emission was measured using a Beckman Coulter LS 6500 Scintillation Counter. For survival assays, cells were deprived of growth factors overnight and plated in a 96-well plate at 2 x 105 cells in 200 µl of RMPI 1640 with 50 ng/ml Kit-L in a 37°C/5% CO2 humidified incubator. After 24 and 48 h of culture, cells were stained with annexin V-FITC per the manufacturer’s instructions (BD Pharmingen) and analyzed by FACS analysis (BD Biosciences).

Migration assay

Mast cells were assessed for migration in response to Kit-L using the Transwell haptotaxis assay as previously described (37). Cells (1 x 105) in serum-free medium of the different genotypes were loaded onto the upper chamber of a fibronectin-precoated Transwell filter (8-µm pore filter Transwell, 24-well cluster; Costar and fibronectin fragment CH-296; Takara Bio), while the lower chamber contained 500 µl of RPMI 1640 with 10 ng/ml Kit-L or no growth factors as negative control. After a 4-h incubation in a 37°C/5% CO2 humidified incubator, the mast cells in the upper chamber of the Transwell were wiped off with a cotton swab, and the migrated cells attached to the bottom surface of the Transwell were stained with 0.1% crystal violet in 0.1 M borate (pH 9.0) and 2% ethanol for 20 min at room temperature. The average number of migrated mast cells per high-power field was counted fewer than 20 times using an inverted microscope.

Degranulation assay

Degranulation of mast cells was assessed by measuring the release of beta-hexosaminidase. Briefly, mast cells, plated at 2 x 106 cells/ml in a 24-well plate, were sensitized using 1.5 µg/ml IgE-DNP (Sigma-Aldrich) for 2 h in a 37°C/5% CO2 humidified incubator. Cells were then washed with Tyrode’s buffer (10 mM HEPES, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, and 0.05% BSA), followed by stimulation with either 30 ng/ml DNP-human serum albumin (Sigma-Aldrich), 30 ng/ml DNP-human serum albumin and/or 10 ng/ml Kit-L, or no stimulation for 15 min in a 37°C/5% CO2 humidified incubator. After stimulation, the supernatants were collected and the cell pellets were lysed in 0.5% Triton X-100 in Tyrode’s buffer. Aliquots of both the supernatants and the lysates were then taken into a 96-well plate. Samples were then incubated with 1 mM N-acetyl-beta-D-glucosaminidase (Sigma-Aldrich) in citrate buffer (0.1 M citric acid and 0.1 M sodium citrate (pH 4.5)) for 1 h in a 37°C/5% CO2 humidified incubator, followed by the addition of 0.1 M sodium carbonate/sodium bicarbonate buffer for the beta-hexosaminidase colorimetric reaction. Plates were then read for absorbance at 405 nm using an Lmax microplate luminometer (Molecular Devices), and percent release was calculated using the equation: (OD of supernatant)/(OD of supernatant + OD of pellet) x 100.

Ras and Rac activation assays, Ras isoform expression, and ERK1/2 and Akt activity

The activity of Ras was measured by detecting the levels of Ras-GTP using a Ras activation kit (Upstate Biotechnology). Briefly, cells were serum-starved for 16–20 h and stimulated with 10 ng/ml Kit-L. Samples were harvested at 0, 5, 15, and 30 min following Kit-L stimulation. One to1.5 x 106 cells were used for each data point. At each harvest point, cells were lysed, and total Ras-GTP was immunoprecipitated using the Ras-GTP-binding domain of Raf-1 as previously described (50). Levels of Rac-GTP were measured in a similar manner using a Rac activation assay (Upstate Biotechnology). Ras isoform expression was evaluated in freshly isolated fetal liver cells harvested at day 13.5; c-kit+ fetal liver cells and fetal liver-derived mast cells were generated as described above. c-kit enrichment was achieved by sorting through a MACS column (Miltenyi Biotec) to a purity of 80–85% (data not shown). Recombinant Ras proteins were used as controls (Oncogene). For activity of ERK1/2 and Akt, mast cells were deprived of growth factors overnight, stimulated with 10 ng/ml Kit-L, and cell lysates were isolated at 0, 2, and 5 min after Kit-L stimulation. In brief, 1.5 x 106 cells/ml were used for each time point. Levels of different proteins were determined by fractionating cell lysates or an immunoprecipitation product using SDS-PAGE and transferred onto nitrocellulose paper. Membranes were then blocked with TBS-Tween 20 containing 5% milk for 1 h at room temperature and incubated with the following Abs overnight at 4°C: Ras isoform-specific Abs (Santa Cruz Biotechnology and Oncogene); phospho-ERK1/2 and total ERK1/2 Abs (Cell Signaling Technology); and phospho-Akt and total Akt Abs (Cell Signaling Technology). Secondary Abs used were either anti-rabbit or anti-mouse Ig HRP linked (Amersham Biosciences). Proteins were visualized using ECL (Amersham Biosciences) and densitometry of individual bands was determined using NIH Image software.

Adoptive transfer of fetal liver into W/Wv mice

W/Wv mice were purchased from The Jackson Laboratory. To stably reconstitute the hemopoietic system, these mice were irradiated with 400 cGy of ionizing radiation from a Gammacell-40 exactor (Nordion) containing a 137Cs source and injected with 2 x 106 day 13.5 fetal liver cells of the different genotypes isolated as described above. Following stable reconstitution of the donor cells (4 mo), microosmotic pumps (ALZET) filled with either 2 µg/kg per day Kit-L or PBS were surgically placed under the dorsal skin while the mice were under mild Avertin anesthesia. Seven days after placement, the pumps were removed and cutaneous skin sections were harvested around the area of infusion, fixed in buffered formalin, and processed in paraffin-embedded sections. For basal cutaneous mast cell evaluation, 1-cm sections of ears were removed, fixed in buffered formalin, and processed in paraffin-embedded sections. For peritoneal mast cell measurement, peritoneal cells were collected as previously described (51). Ten-milliliter peritoneal lavages were concentrated by centrifugation, and cytospin slides were prepared to quantify total numbers of mast cells per 10 ml of lavage. Specimens were subsequently stained with Giemsa to identify mast cells, which were enumerated in a blinded fashion. Cutaneous mast cells were quantitated from five mice per experimental group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
K-ras and N-ras, but not H-ras, are highly expressed and activated in fetal liver-derived mast cells

Since Ras isoforms are variably expressed in different lineages and during development (52, 53, 54, 55, 56), we examined Ras isoform expression in phenotypically enriched populations of hemopoietic progenitors and mast cells. We detected high expression of both K- and N-ras in freshly isolated day 13.5 fetal liver cells, c-kit+ fetal liver cells, and mast cells (Fig. 1A). In contrast, low levels of H-ras were detected in unfractionated but not in purified populations of fetal liver cells or in fetal liver cells cultured under conditions to progressively enrich for mast cells (Fig. 1A). These findings suggest a role of both K- and N-ras, but not H-ras, in primary mast cells.


Figure 1
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  		FIGURE 1. Ras isoform expression in fetal liver cells and fetal liver-derived mast cells (A) and Ras activation in mast cells (B). A, Fetal livers were harvested at day 13.5, sorted using a MACS column to enrich for c-kit+ cells, or used to grow mast cells as described in Materials and Methods. Recombinant Ras proteins were used as controls. Western blot of the results is shown and is a representative of three independent experiments. B, Mast cells were serum-starved for 16–20 h, stimulated with 10 ng/ml Kit-L, and harvested at the indicated time points following Kit-L stimulation. Ras-GTP was immunoprecipitated using a Ras pull-down assay containing the Ras-GTP binding domain of Raf-1. Levels of Ras-GTP were assessed by Western blotting and detected with isoform-specific Ras Abs (Santa Cruz Biotechnology). Western blot of the result is shown and is a representative of three independent experiments. Also shown are the densitometry measurements for activation of each of the Ras isoforms.

 
Previous studies established that Kit-L-mediated stimulation leads to hyperactivation of Ras proteins in Nf1+/– mast cells (50, 57), although the relative role of kit activation of Ras in WT cells or the activation of specific Ras isoforms in cells with normal or hyperactivated Ras has not been examined. As shown in Fig. 1B, both K-ras and N-ras were activated in WT cells in response to Kit-L stimulation. Furthermore, both K-ras and N-ras were hyperactivated in Nf1+/– mast cells as compared to WT mast cells (Fig. 1B), indicating that Nf1is functioning as a GAP for both Ras isoforms in this context.

K-ras modulates c-kit-mediated proliferation of WT and Nf1+/– mast cells

Kit-L is central to normal mast cell development and is critical for multiple mast cell functions including proliferation (58, 59, 60, 61, 62, 63). In addition, previous studies in human neurofibromas indicate that Kit-L is expressed at high concentrations (42) and murine Nf1–/– Schwann cells secrete 7-fold higher concentrations of Kit-L as compared with nonmalignant Nf1+/– Schwann cells (37). To examine the K-ras-dependent effects of Kit-L-mediated proliferation, we intercrossed Nf1 and K-ras heterozygous mice to generate F2 progenies that were mutant at one or both loci. Homozygous genetic disruption of K-ras resulted in a profound reduction in proliferation of mast cells that were WT or heterozygous at the Nf1 locus in response to a range of concentrations of Kit-L (Fig. 2). Consistent with previous work (50), Nf1+/– mast cells had elevated proliferation as compared to WT mast cells (Fig. 2). Genetic disruption of a single allele of K-ras in Nf1+/– mast cells (Nf1+/–; K-ras+/–) resulted in an intermediate proliferative response as compared with Nf1+/– mast cells that are WT (Nf1+/–; K-ras+/+) or nullizygous (Nf1+/–; K-ras–/–) at the K-ras locus (Fig. 2). These data point to the sensitive interaction between neurofibromin and K-ras in c-kit receptor tyrosine kinase-mediated proliferation in both WT and Nf1+/– mast cells.


Figure 2
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  		FIGURE 2. Proliferation of mast cells in response to Kit-L. Mast cells were starved overnight in RPMI 1640, plated in quadruplicate samples, and stimulated with different doses of Kit-L for 48 h. Proliferation was assessed by thymidine incorporation. Graphs are representative of four independent experiments and each value represents the mean and the error bars represent the SEM. *, p < 0.05 for comparison with WT; **, p < 0.05 for comparison with Nf1+/– using Student’s paired t test.

 
Genetic deletion of K-ras reduces the survival of both WT and Nf1+/– mast cells in response to Kit-L

Given previous studies showing that Kit-L is a survival factor for mast cells (63) and observations that Nf1+/– mast cells have increased Ras-dependent survival in response to Kit-L (36), we examined the role of K-ras in modulating WT and Nf1+/– mast cell survival. Homozygous disruption of K-ras (Nf1+/+; K-ras–/–) resulted in a 50% reduction in survival as compared with WT cells, demonstrating a role of K-ras in endogenous c-kit-mediated survival (Fig. 3). In addition, in the setting of hyperactivated Ras in Nf1+/– mast cells, genetic disruption of K-ras reduces survival of Nf1+/– mast cells in a K-ras gene dose-dependent manner to levels below that of WT controls (Fig. 3). Collectively, these findings demonstrate that K-ras is required for c-kit receptor tyrosine kinase-mediated survival and that hyperactivation of K-ras is required for the increased survival of Nf1+/– mast cells in response to Kit-L.


Figure 3
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  		FIGURE 3. Survival of mast cells in response to Kit-L. Mast cells were starved overnight in RPMI 1640, plated in triplicate samples, then stimulated with 50 ng/ml Kit-L. Cells were assayed for apoptosis at each indicated time point by annexin V staining per the manufacture’s protocol. Each value represents the mean and the error bars represent the SEs of the mean of four independent experiments. *, p < 0.05 for comparison with WT; **, p < 0.05 for comparison with Nf1+/– by Student’s paired t test.

 
K-ras decreases the migration of both WT and Nf1+/– mast cells in response to Kit-L

Kit-L is established as a chemoattractant factor for mast cells in vitro and in vivo (58). Furthermore, previous studies established that Nf1+/– mast cells exhibit increased in vitro migration in response to Kit-L secreted by Nf1–/– Schwann cells, the tumor-initiating cell type in neurofibromas (37). To test whether K-ras modulates the migratory response to Kit-L, we used a Transwell assay as previously described (37). Homozygous deletion of K-ras reduced Kit-L-mediated migration in cells that were either WT or heterozygous at Nf1 (Fig. 4). Furthermore, analogous to findings in Kit-L-mediated proliferation and survival, heterozygous disruption of K-ras reduces the migration of cells that are heterozygous at the Nf1 locus (Fig. 4). This finding suggests that K-ras is critical in transducing signals required for migration of mast cells in response to Kit-L, and that even subtle changes in gene dosage of K-ras alters migration in Nf1+/– mast cells.


Figure 4
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  		FIGURE 4. Migration of mast cell in response to Kit-L. Mast cells were assayed for migration in response to Kit-L using the Transwell migration assay as described in Materials and Methods. At least 40 fields were scored per well in an experimentally blinded fashion. Values represent the means of four independent experiments each done in triplicate and error bars represent the error bars of the mean. *, p < 0.05 for comparison with WT; **, p < 0.05 for comparison with Nf1+/– using Student’s paired t test. HPF, High-power field.

 
K-ras mediates c-kit-dependent degranulation of WT and Nf1+/– mast cells

The cytoskeletal processes regulating the release of stored granules is a key physiological function of mast cells that has important implications in inflammation, innate host defense, and cancer (44, 49). To evaluate the role of K-ras in degranulation, the release of the preformed mediator beta-hexosaminidase was examined in both WT and Nf1+/– mast cells in response to Kit-L stimulation. In four independent experiments, each of which was conducted in three replicates, homozygous inactivation of K-ras alleles reduced degranulation to ~65% in WT controls (Fig. 5). Consistent with clinical observations (42), Nf1+/– mast cells displayed elevated degranulation in response to Kit-L as compared to WT controls (Fig. 5). This gain of function was reduced in a K-ras gene dose-dependent fashion to levels ultimately below those of WT mast cells in Nf1+/–; K-ras–/– mast cells (Fig. 5). Similar results were observed in response to cross-linking the high-affinity Fc receptor for IgE (Fc{epsilon}RI), either alone or in synergy with Kit-L (data not shown). These findings support a role for K-ras in transducing signals that control the degranulation of inflammatory mast cells that have either normal GAP activity or are haploinsufficient at the Nf1 locus.


Figure 5
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  		FIGURE 5. Measurements of in vitro degranulation of mast cells in response to Kit-L. Mast cells were assayed for degranulation by measuring the release of beta-hexosaminidase in response to stimulation with Kit-L as described in Materials and Methods. Values represent the mean of four different experiments, each done in triplicate, and the error bars represent the SEM. *, p < 0.05 for comparison with WT; **, p < 0.05 for comparison with Nf1+/– using Student’s paired t test.

 
Reduced activation of the effector molecules K-ras-deficient mast cells have reduced Akt, Rac, and ERK with genetic activity

Previous studies in immortalized cells overexpressing different Ras isoforms emphasized the role of K-ras in modulating the canonical Raf-MEK-ERK pathway (24, 25). However, mast cell migration and survival in WT and Nf1+/– mast cells is dependent on PI3K activity (37, 51, 63). Therefore, we hypothesized that the reduction in survival of Nf1+/–; K-ras–/– mast cells would be associated with a reduction in PI3K activity. To test this hypothesis, cells from four F2 intercrossed lines were stimulated with Kit-L for 0–5 min, lysed, and examined by Western blot for Akt phosphorylation, a sensitive measure of PI3K activity implicated in survival (64). Because of the large number of samples, we focused the biochemical studies on genotypes that are WT or nullizygous at the K-ras locus. Consistent with observations in cell survival, K-ras–/– mast cells have reduced Akt phosphorylation as compared with WT mast cells (Fig. 6). As expected, Akt phosphorylation is elevated in Nf1+/– mast cells (Fig. 6). However, Akt phosphorylation was reduced in cells mutant at both the Nf1 and K-ras loci (Nf1+/–; K-ras–/–) to levels below that of WT mast cells (Fig. 6). Collectively, the data indicate that K-ras activates the PI3K pathway in mast cells.


Figure 6
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  		FIGURE 6. Measurement of Akt activity in mast cells. Mast cells were serum-starved overnight and stimulated with 10 ng/ml Kit-L, and cell lysates were isolated at 0, 2, and 5 min following Kit-L stimulation. Levels of active Akt were determined by Western blotting using phospho-specific Abs. Western blot of the results is shown and is a representative of three independent experiments. Levels of total Akt are shown as loading controls. Also shown are normalized quantitative densitometry measurements of the different bands.

 
Rac proteins have been previously shown to be key effectors mediating mast cell migration and degranulation (65, 66). Since Rac activation in mast cells has been previously shown to be PI3K dependent (37, 67), we hypothesized that the reduction in mast cell migration would be associated with a reduction in Rac activity. To test this hypothesis, mast cells from the four F2 genotypes were stimulated with Kit-L, lysed at 0–5 min following stimulation, and Rac-GTP was measured using an activity assay. Basal levels of Rac-GTP were lower in mast cells that were nullizygous at the K-ras locus compared with their counterparts that were either WT or heterozygous at Nf1 (Fig. 7). Nf1+/– mast cells that were WT at the K-ras locus had higher Rac activity after stimulation with Kit-L as compared with WT mast cells (Fig. 7). Importantly, genetic disruption of K-ras markedly reduced Kit-L-mediated Rac activation in mast cells that are WT or haploinsufficient at the Nf1 locus, indicating that K-ras is required for c-kit-mediated Rac activation (Fig. 7).


Figure 7
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  		FIGURE 7. Measurement of Rac activation in mast cells. Mast cells were serum-starved for 16–20 h, stimulated with 10 ng/ml Kit-L, and harvested at 0 and 5 min following Kit-L stimulation. Cells (1–1.5 x 106) were analyzed at each data point for expression of active Rac-GTP. Levels of Rac-GTP were assessed by Western blotting and detected with anti-Rac mAb. Western blot of the results is shown and is a representative of three independent experiments. Also shown are normalized densitometry measurements for activation of Rac proteins in the different sample groups.

 
ERK phosphorylation has been previously implicated as an effector critical for mast cell degranulation (68). In addition, we previously provided pharmacological and genetic data indicating that hyperproliferation of Nf1+/– mast cells is a consequence of hyperactivation of ERK (36, 50). Given those data and the current observations that genetic inactivation of K-ras reduces proliferation and degranulation in both WT and Nf1+/– mast cells (Fig. 2), we next examined ERK phosphorylation in response to Kit-L stimulation. As expected, ERK phosphorylation was elevated in Nf1+/–; K-ras+/+ mast cells as compared with WT controls, but Nf1+/–; K-ras–/– mast cells had markedly reduced ERK phosphorylation as compared with WT cells (Fig. 8). Similarly, genetic disruption of K-ras in cells that were WT at the Nf1 locus (Nf1+/+; K-ras–/–) had reduced ERK phosphorylation as compared with WT cells (Fig. 8). Those data indicate that K-ras activates ERK in response to Kit-L stimulation in WT and Nf1+/– mast cells.


Figure 8
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  		FIGURE 8. Measurement of ERK1/2 activity in mast cells. Mast cells were serum-starved overnight and stimulated with 10 ng/ml Kit-L, and cell lysates were isolated at 0, 2, and 5 min following Kit-L stimulation. Levels of active ERK were determined by Western blotting using phospho-specific Abs. Western blot of the results is shown and is a representative of three independent experiments. Levels of total ERK are shown as loading controls. Also shown are normalized quantitative densitometry measurements of the different bands.

 
K-ras modulates basal Nf1+/– and WT mast cell numbers in vivo

To study the role of K-ras in mast cells in vivo, fetal liver cells from the six F2 genotypes were adoptively transferred into irradiated W/Wv mice, which express normal Kit-L but lack endogenous mast cells because of a mutation of the c-kit receptor (69). Thus, following stable reconstitution, the only mast cells in the tissues are donor derived. Four months following reconstitution, basal levels of cutaneous and peritoneal mast cells were examined. The results of those studies are shown in Table I. Mice reconstituted with Nf1+/+; K-ras–/– repopulating cells had decreased numbers of mast cells in the tissues at basal levels as compared with mice reconstituted with WT repopulating cells. Mice reconstituted with Nf1+/–; K-ras+/+ cells had higher numbers of mast cells in the tissues than any other F2 group. Genetic disruption of K-ras reduced basal mast cell numbers in mice reconstituted with cells that were Nf1+/– or WT at the Nf1 locus. These data indicate that K-ras has a role for in vivo mast cell differentiation and maturation. The data also indicate that hyperactivation of K-ras in Nf1+/– mast cells is required for the pathological increase in the number of tissue mast cells.


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  		Table I. Effects of genetic deficiency of K-ras on mast cell numbers in different tissues in WT and Nf1+/– micea

 
Genetic deletion of K-ras reduces the in vivo response of both WT and Nf1+/– mast cells to local continuous infusion of Kit-L

Tsai et al. (62) previously established that local administration of Kit-L results in a local proliferation and accumulation of mast cells at the site of administration. Using a similar methodology, we subsequently found that Nf1+/– mast cells have increased proliferation in vivo (50). To evaluate the role of K-ras in modulating Kit-L-mediated proliferation in vivo, slow release micro-osmotic pumps containing Kit-L were inserted s.c. in the backs of W/Wv mice reconstituted with fetal liver repopulating cells from of the F2 intercrosses. There was a consistent reduction in the number of local cutaneous mast cells observed in recipients reconstituted with K-ras null hemopoietic cells as compared to WT controls (n = 5/genotypes; Fig. 9A). A marked increase in the local accumulation of Nf1+/– mast cells was observed in areas where Kit-L was administered as compared with all other groups (Fig. 9A). This increase in mast cell numbers was reduced in a K-ras gene dose-dependent manner to levels slightly lower than WT in the Nf1+/–; K-ras–/– mast cells (Fig. 9A). In addition to modulating Kit-L-mediated proliferation, K-ras influences degranulation in a genotypically comparable pattern (Fig. 9B). Thus, K-ras is influencing multiple cellular functions in vivo and the hyperactivation of K-ras in Nf1+/– mast cells results in both an increase in basal and Kit-L-mediated mast cell functions in vitro and in vivo.


Figure 9
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  		FIGURE 9. In vivo response of mast cells to local continuous infusion of Kit-L. Total number of cutaneous mast cells per square millimeter (A) or percentage of degranulating mast cells (B) in response to local infusion of Kit-L. Microosmotic pumps were loaded with 2 µg/kg per day of Kit-L, placed s.c. in the dorsal side of W/Wv mice stably reconstituted with day 13.5 fetal liver cells of the different genotypes, and removed 7 days later. Skin sections were harvested at the site of pump placement and stained with Giemsa. Total and degranulating cutaneous mast cells were quantitated in a blinded fashion. Each value represents the mean of five mice per experimental group and the error bars represent the SEM. *, p < 0.05 for comparison with WT; **, p < 0.05 for comparison with Nf1+/– using Student’s paired t test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Mast cells are increasingly recognized for not only their roles in innate host defense and allergic hypersensitivity (49, 70, 71), but also for their role in altering the tumor microenvironment in a range of malignancies (44, 45, 46, 72). Mast cells mediate these processes by their ability to migrate, proliferate, and release preformed mediators including proteases and the secretion of multiple cytokines. In this study, we evaluate the role of K-ras in modulating Kit-L-mediated mast cell functions in primary cells that have normal GAP activity and in the context of a disease model where the hemopoietic system is haploinsufficient in neurofibromin, a GAP for Ras. Since K-ras knockout mice die in utero, we used K-ras–/– fetal liver cells as a source of reconstituting hemopoietic stem cells to adoptively transfer the hemopoietic system into syngeneic W/Wv mutant mice that lack endogenous mast cells and to generate primary mast cells for detailed examination of cellular functions and biochemical activities.

The physiological importance of K-ras and the interactions between K-ras and Nf1 in the development and maintenance of endogenous mast cell populations in vivo are exemplified by observations in reconstituted W/Wv mice (Table I). Mice reconstituted with K-ras–/– stem cells have reduced basal populations of both cutaneous and peritoneal mast cells as compared with mice reconstituted with WT repopulating stem cells. Similar findings are noted in mice reconstituted with Nf1+/–; K-ras–/– stem cells as compared with mice reconstituted with cells that were mutant at the Nf1 locus only (Nf1+/–; K-ras+/+). In addition, in these in vivo studies, loss of a single allele of K-ras alters basal mast cell levels in mice reconstituted with Nf1+/– repopulating cells while loss of a single K-ras allele in cells that are WT at the Nf1 locus is not sufficient to reduce endogenous mast cell populations. These data indicate the close regulation between Nf1 and K-ras in modulating Ras activity and mast cell homeostasis. Similar results are observed when evaluating mast cell functions in vitro.

Although previous studies in immortalized cell lines emphasize the role of K-ras in modulating the Raf-MEK-ERK pathway (24, 25), genetic data in these experiments identify a role for K-ras in modulating PI3K signaling as well as activation of the Raf-MEK-ERK signaling pathway. Numerous studies in mast cells implicate the importance of the activation of PI3K by c-kit in primary mast cell maturation, survival, migration, and proliferation (37, 50, 67, 73). Recent work by our group (37, 50) and Kalesnikoff et al. in RabGEF1–/– mice (74) indicates a clear role for c-kit-mediated Ras activation of PI3K in primary mast cells. In this study, we show that c-kit stimulation leads to activation of both N-ras and K-ras in primary mast cells. Genetic data indicate that K-ras isoform activation is sufficient to modulate PI3K activity in mast cells that are WT or heterozygous at the Nf1 locus. Furthermore, Rac activation, which has previously been shown to be dependent on PI3K in mast cells (50, 67), is reduced in K-ras–/– cells. Thus, although activation of the classical Raf-MEK-ERK pathway remains a key K-ras-mediated signal transduction cascade in mast cells, parallel activation of the PI3K-Rac pathway also occurs and is important in regulating multiple cellular processes in primary mast cells.

There are apparent differences in the utilization of Ras isoforms in different tissues. In astrocytes, the tumorigenic cell in optic gliomas, K-ras, N-ras, and H-ras are all highly expressed (75). However, stimulation of Nf1–/– astrocytes with epidermal growth factor leads to activation of the K-ras isoform only (75). Furthermore, overexpression of oncogenic K-ras using a heterologous promoter provides a similar phenotype as genetic disruption of Nf1, while overexpression of oncogenic H-ras causes the development of astrocytomas (75). Our data indicate that K-ras has a significant role in Kit-L-mediated mast cell functions and that loss of K-ras reduces mast cell migration, survival, and degranulation by ~50%. Furthermore, K-ras has an even greater role in modulating c-kit-mediated proliferation where genetic disruption of K-ras reduced mast cell proliferation by nearly 10-fold in cells that were WT at the Nf1 locus. Although it is apparent that K-ras has a major role in c-kit-mediated mast cell functions, we do not exclude a role for N-ras in mediating overlapping or specific mast cell activities. For instance, although endogenous levels of mast cells in mice reconstituted with Nf1+/–; K-ras–/– stem cells are significantly lower than those of mice reconstituted with Nf1+/–; K-ras+/+ cells, mast cells in mice reconstituted with Nf1+/–; K-ras–/– repopulating cells are higher than those of mice reconstituted with WT cells. These data, along with increased activation of N-ras-GTP in response to Kit-L, demonstrate a clear role for N-ras in c-kit-mediated mast cell activation. The availability of N-ras–/– mice will allow detailed genetic analysis examining the activation of N-ras in mast cells and its functional role in both cells that are WT or haploinsufficient at the Nf1 locus in future studies.

Our findings that K-ras has a role in modulating Kit-L-mediated functions may have clinical importance as it relates to the role of mast cells contributing to the tumor microenvironment in neurofibroma formation. There currently are no specific inhibitors that modulate K-ras activity. Previous studies have attempted to use farnesyltransferase inhibitors to modulate activated forms of K-ras as well as other Ras isoforms in cancer (76, 77, 78, 79, 80). However, K-ras undergoes both farnesylation and geranylation in its processing and inhibition of farnesylation alone is not sufficient to prevent K-ras activation or transformation (81, 82, 83). Given the role of K-ras in modulating multiple mast cell functions in WT and Nf1+/– mast cells, the development of effective targets that modulate K-ras activity might provide efficacy for the treatment of neurofibromas. Alternatively, it may be possible to target key downstream effectors such as PI3K or Rac to modulate mast cell functions. Genetic studies are currently underway to evaluate the potential of these effectors in neurofibroma formation.


   Acknowledgments
 
We thank Natascha Karlova for assistance in preparing this manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by the National Institutes of Health (NCI R01 CA 74177) and the American Heart Association (0410094Z). Back

2 Address correspondence and reprint requests to Dr. D. Wade Clapp, Indiana University School of Medicine, Cancer Research Institute, 1044 West Walnut Street, R4 402, Indianapolis, IN 46202. E-mail address: dclapp{at}iupui.edu Back

3 Abbreviations used in this paper: GAP, GTPase-activating protein; NF1, neurofibromatosis type 1; GRD, GAP-related domain of NF1; WT, wild type; Kit-l, Kit ligand. Back

Received for publication June 29, 2006. Accepted for publication November 15, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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