Kit activates interleukin‐4 receptor and effector signal transducer and activator of transcription 6 independent of its cognate ligand in mouse mast cells

Summary

Signaling by Kit has been extensively studied in hematopoietic cells and is essential for the survival, proliferation and maintenance of hematopoietic stem and progenitor cells. In addition to the activation of intrinsic signaling pathways, Kit has been shown to interact with lineage‐restricted type I cytokine receptors and produce cross signals, e.g. erythropoietin receptor, interleukin‐7 receptor (IL‐7R), IL‐3R. Based on the earlier studies, we hypothesize that Kit activate other type I cytokine receptors in a cell‐specific manner and execute cell‐specific function. To investigate other Kit‐activated receptors, we tested Kit and IL‐4R cross‐receptor activation in murine bone‐marrow‐derived mast cells, which express both Kit and IL‐4R at the surface level. Kit upon activation by Kit ligand (KL), activated IL‐4Rα, γ _C, and signal transducer and activator of transcription 6 independent of its cognate ligand IL‐4. Though KL and IL‐4 are individually mitogenic, combinations of KL and IL‐4 synergistically promoted mast cell proliferation. Furthermore, inhibition of lipid raft formation by methyl‐β‐cyclodextrin resulted in loss of synergistic proliferation. Together the data suggest IL‐4R as a novel Kit‐activated receptor. Such cross‐receptor activations are likely to be a universal mechanism of Kit signaling in hematopoiesis.

Kit has been shown to interact with lineage‐restricted type I cytokine receptors like Epo‐R, IL‐7R, and IL‐3R, and produce cross signals. In the present study, we reported IL‐4R as a novel Kit‐activated receptor in mouse mast cells.

Abbreviations

BMMCs

bone‐marrow‐derived mast cells

Epo‐R

erythropoietin receptor

GMCSF‐R

granulocyte–macrophage colony‐stimulating factor receptor

IL‐3R

interleukin‐3 receptor

IL‐4R

interleukin‐4 receptor

IL‐7R

interleukin‐7 receptor

KL

Kit ligand

MβCD

methyl‐β‐cyclodextrin

STAT6

signal transducers and activators of transcription 6

γ_C

Common γ chain

Introduction

Kit, also known as stem cell factor receptor, belongs to type III subclass of receptor tyrosine kinases and the cognate ligand for Kit is Kit ligand (KL). Some of the other type III receptor tyrosine kinases are platelet‐derived growth factor receptors α and β, Flt3, and CSF1R (c‐fms, macrophage colony‐stimulating factor receptor).1, 2 Signaling by Kit has been extensively studied in hematopoietic cells and has been shown to regulate the survival, proliferation and maintenance of hematopoietic stem and progenitor cells.3 Mutational loss of Kit or KL results in developmental abnormalities of the hematopoietic, nervous, and reproductive systems.

One of the well‐studied mechanisms of Kit is its intrinsic signaling pathway. Kit upon engagement with its cognate ligand is activated and initiates several intrinsic signaling pathways, which includes phosphoinositide 3‐kinase/Akt, mitogen‐activated protein kinase, Janus kinase/signal transducer and activator of transcription (STAT), and Src family kinases. In addition to the activation of intrinsic signaling pathways, Kit has been shown to interact with lineage‐restricted type I cytokine receptors, e.g. erythropoietin receptor (Epo‐R) in erythroid and interleukin‐7 receptor (IL‐7R) in thymic progenitors. The cross‐receptor interactions between Kit and type I cytokine receptors leads to tyrosine phosphorylation of Epo‐R, granulocyte–macrophage colony‐stimulating factor receptor (GM‐CSFR), IL‐7R, IL‐3R and IL‐33R in the absence of their cognate ligand.4, 5, 6, 7, 8 These cross‐receptor interactions are necessary for transduction of Kit‐mediated survival and proliferation signals in these progenitors. A general hypothesis to explain the pleiotropic lineage‐restricted functions of Kit in stem and progenitor populations is that Kit activates different cell‐surface receptors in each cell type, providing a mechanistic basis for the distinct but overlapping, cell type‐specific responses to Kit signaling that have been observed. In an attempt to identify novel interactions between Kit and other receptors in hematopoietic cells, we observed IL‐4R as a potential target of Kit signaling.

In the present study, we show evidence that Kit activates both subunits of IL‐4R independent of its cognate ligand. The interaction between Kit and IL‐4R resulted in STAT6 phosphorylation in bone‐marrow‐derived mast cells (BMMCs). This interaction is necessary for the synergy in mast cell proliferation and enhanced STAT6 phosphorylation.

Materials and methods

Reagents and antibodies

Recombinant murine KL, murine IL‐3, and murine IL‐4 were purchased from Peprotech Asia (Rehovot, Israel). The anti‐phosphotyrosine antibodies, clones 4G10 and PY20, were purchased from Merck Biosciences (Darmstadt, Germany). Phospho‐specific antibody against STAT6 (clone J71‐773.58.11) was obtained from BD Biosciences (San Jose, CA). Anti‐STAT6 and anti‐IL‐4Rα antibodies were from Santa Cruz Biotechnology (Dallas, TX). Phospho IL‐4R (Tyr497) polyclonal antibody was obtained from Invitrogen (Waltham, MA).

Mice, cell culture and cytokine stimulation

All procedures involving animals were performed according to protocols approved by the Institutional Animal Ethical Committee at Pondicherry University. Cultures of BMMCs were established, as previously described.9 BM cells obtained from femur and tibia of 4‐ to 8‐week‐old C57BL/6 mice were used to generate BMMCs. Cultures were maintained in BMMC medium in the presence of murine KL and IL‐3 (20 ng/ml each) for 4–6 weeks. For KL and IL‐4 stimulation, BMMCs were growth factor deprived for 12 hr and then incubated in BMMC medium containing no or 2% fetal calf serum for an additional 4 hr. Stimulation of BMMCs was performed with the concentrations of KL and/or IL‐4 as indicated in the figures.

Flow cytometric analysis

To analyze surface expression of Kit, Fcε receptor I (FcεRI), IL‐4Rα and γ _C, growth factor‐deprived BMMCs were labeled with phycoerythrin (PE) ‐conjugated rat anti‐Kit antibody (clone 2B8; BioLegend, San Diego, CA) and with murine anti‐dinitrophenyl immunoglobulin E (clone SPE‐7; Sigma‐Aldrich, St Louis, MO), followed by fluorescein isothiocyanate‐conjugated rat anti‐mouse IgE antibody (clone RME‐1; BioLegend), PE‐conjugated rat anti‐IL‐4Rα (clone IO15F8; BioLegend) or PE‐conjugated rat anti‐γ _C (clone TUGm2; BioLegend), respectively.

To analyze IL‐4Rα phosphorylation, growth‐factor‐deprived BMMCs were stimulated with 250 ng/ml of KL or IL‐4 for the time indicated and cells were fixed using 3·7% paraformaldehyde for 10 min. Permeabilization of cells was performed with ice‐cold methanol for 15 min in ice and cells were incubated with rabbit anti‐mouse/human phospho‐IL‐4R (Tyr497) polyclonal antibody for 2 hr. Cells were then stained with PE‐conjugated donkey anti‐rabbit IgG antibody (Clone Poly4064; BioLegend) for 30 min in room temperature.

Detection of intracellular phospho‐STAT6 was performed after fixation of cells with 3·7% paraformaldehyde for 10 min. Permeabilization of cells was performed with ice‐cold methanol for 15 min in ice and cells were stained with AF488‐conjugated mouse anti‐phospho‐STAT6 antibody (clone J71‐773.58.11; BD Biosciences) for 30 min at room temperature. Antibody‐labeled cells were analyzed using FACS Aria III (BD Biosciences).

Immunoprecipitations and immunoblotting

After stimulation with KL or IL‐4, cells were collected in ice‐cold phosphate‐buffered saline containing 1 mm sodium orthovanadate (Sigma‐Aldrich) and lyzed in lysis buffer (1 × 10⁷ to 2 × 10⁷ cells/ml) containing 10 mm Tris–HCl (pH 7·4), 150 mm NaCl, 20 mm sodium phosphate (pH 7·4), 10 mm sodium pyrophosphate (pH 7·4), 5 mm ethylenediaminetetraacetic acid, 1 mm sodium orthovanadate, 1 mm glycerophosphate (Sigma‐Aldrich), and 1% Triton‐X‐100. Proteinase inhibitors (Complete; Roche, Basel, Switzerland) were added, according to the manufacturer's recommendations. Post nuclear supernatants were subjected to one round of pre‐clearing with protein A‐Sepharose (Amersham/Pharmacia, Amersham, UK). A total of 3–6 µg of antibody was used per immunoprecipitation, and antibody–protein complexes were collected with 50–75 µl protein A‐Sepharose. Western blotting was performed, as previously described.9, 10

Cell proliferation assay

Cell proliferation assay was performed using WST‐1 (Roche) according to the manufacturer's protocol. Before measurement of cellular proliferation, BMMCs were cultured in the absence of KL and IL‐4 for 12 hr. Cells were plated in 96‐well tissue‐culture plates at a concentration of 20 000 cells per well in 100 µl of BMMC medium and stimulated with 20 ng/ml KL, 5 ng/ml IL‐4 and 20 ng/ml KL + 5 ng/ml IL‐4. After 48 hr of incubation, 10 µlt of WST‐1 reagent was added and absorbance was measured against a background control as blank using a microplate (enzyme‐linked immunosorbent assay) reader at 440 nm.

Results

KL stimulation results in tyrosine phosphorylation of IL‐4Rα

BMMCs were used to study the potential interactions between Kit and IL‐4R because they are known to express both functional Kit and IL‐4R.11 BMMCs were generated from wild‐type C57BL6/J mice using KL (20 ng/ml) and IL‐3 (20 ng/ml) as growth factors. The purity of BMMCs was confirmed after 6 weeks of culture, with uniform expression of Kit and FCεRI. The BMMCs were also analyzed for surface expression of IL‐4R subunits (IL‐4Rα and γ _C) (Fig. 1).

Figure 1.

Mouse bone‐marrow‐derived mast cells (BMMCs) express interleukin‐4 receptor α (IL‐4Rα) and common γ (γ _C) chains. BMMC culture from wild‐type mice was obtained, as described above and confirmed for surface expression of Kit and FCεRI (top panel). Mature BMMCs were analyzed for surface expression levels of IL‐4Rα and γ _C (bottom panel). Black represents antibodies against Kit, FCεRI, IL‐4Rα and γ _C. Gray represents the corresponding isotype control.

One of the first events in IL‐4‐mediated signaling is tyrosine phosphorylation of IL‐4Rα , a subunit of IL‐4R. We first investigated whether IL‐4Rα is tyrosine phosphorylated in the course of Kit stimulation (0, 1, 5 and 10 min) in BMMCs. We observed that KL stimulation results in the tyrosine phosphorylation of IL‐4Rα in BMMCs (Fig. 2a,b). The dose dependency of Kit‐mediated IL‐4Rα activation was tested by analysis of tyrosine phosphorylation of IL‐4Rα after stimulation with 10, 25, 50, 100 or 250 ng/ml KL. Kit‐induced IL‐4Rα phosphorylation was maximal at 50 ng/ml KL (Fig. 2c). The observation of Kit‐induced IL‐4Rα phosphorylation was confirmed using a different clone of antibody against mouse IL‐4Rα (Monoclonal E1 clone; Fig. 2d).

Figure 2.

Kit stimulation results in tyrosine phosphorylation of interleukin‐4 receptor α (IL‐4Rα) in mouse bone‐marrow‐derived mast cells (BMMCs). (a) BMMC cultures from wild‐type mice were stimulated with Kit ligand (KL) (250 ng/ml) for 0, 1, 5 and 10 min. BMMCs were analyzed for IL‐4Rα phosphorylation by flow cytometry using rabbit anti‐mouse/human phospho‐IL‐4R (Tyr497) polyclonal antibody and phycoerythrin‐conjugated donkey anti‐rabbit IgG antibody. (b) BMMC cultures from wild‐type mice were stimulated with KL (250 ng/ml) for the time indicated, and IL‐4Rα was immunoprecipitated. Bound fractions of the immunoprecipitations were analyzed for tyrosine phosphorylation and protein amounts by immunoblot using IL‐4Rα (S20) polyclonal antibody. (c) BMMC cultures from wild‐type mice were stimulated with different concentration of KL for 10 min and immunoprecipitations were analyzed for IL‐4Rα tyrosine phosphorylation. (d) BMMC cultures from wild‐type mice were stimulated with 250 ng/ml of KL for 5 min and IL‐4Rα tyrosine phosphorylation were analyzed by immunoprecipitation using IL‐4Rα (E1) monoclonal antibody. The bar graphs represent the densitometry analysis of Western blots. The levels of IL‐4Rα phosphorylation were quantified and normalized with the total protein and presented as mean ± SEM (n = 3). P value is calculated using Student’s t‐test. *P < 0·05, **P < 0·005.

To compare Kit and cognate ligand IL‐4‐mediated IL‐4Rα phosphorylation, we analyzed tyrosine phosphorylation of IL‐4Rα after KL and IL‐4 stimulation. Kit‐induced IL‐4Rα phosphorylation was significantly less compared with cognate ligand IL‐4‐mediated IL‐4Rα phosphorylation (Fig. 3).

Figure 3.

Comparison of Kit ligand (KL) and interleukin‐4 (IL‐4) ‐induced IL‐4 receptor α (IL‐4Rα) phosphorylation in bone‐marrow‐derivedm ast cells (BMMCs). (a) BMMC cultures from wild‐type mice were stimulated with KL (250 ng/ml) or IL‐4 (250 ng/ml) for 10 min. BMMCs were analyzed for IL‐4Rα phosphorylation by flow cytometry using rabbit anti‐mouse/human phospho‐IL‐4R (Tyr497) polyclonal antibody and phycoerythrin‐conjugated donkey anti‐rabbit IgG antibody. Graph shows the MFI of KL‐ and IL‐4‐induced IL‐4Rα phosphorylation. Data represent the mean ± SEM from four independent experiments. *P < 0·05. MFI, Mean fluorescence intensity. (b) BMMC cultures from wild‐type mice were stimulated with (250 ng/ml) of KL or IL‐4 for the time indicated and IL‐4Rα was immunoprecipitated. Bound fractions of the immunoprecipitation were analyzed for tyrosine phosphorylation and protein amounts by Immunoblot. Images were acquired after short and long exposure of the blot to the film (top and middle panel). The bar graphs represent the densitometry analysis of Western blots. The levels of IL‐4Rα phosphorylation was quantified and normalized with the total protein and presented as mean ± SEM (n = 3). P value is calculated using Student’s t‐test. *P < 0·05, **P < 0·005.

KL stimulation results in tyrosine phosphorylation of γ _c

Common γ chain heterodimerizes with ligand‐specific type I cytokine receptor subunits to form the receptors for IL‐2, IL‐4, IL‐7, IL‐9, IL‐15 and IL‐21.12 Cognate signaling by the IL‐4R requires the presence of both subunits at the cell membrane and formation of heterodimers between IL‐4Rα and γ _C. Previous studies by Jahn et al. 6 have shown that γ _C is a direct substrate of Kit via cross‐receptor interactions between activated Kit and IL‐7R in immature T cells (thymocytes). In the present study, we analyzed whether the cross‐receptor interactions between Kit and γ _C occur in mast cells expressing IL‐4R. Consistent with the results in T cells, Kit stimulation resulted in γ _C phosphorylation in wild‐type BMMCs (Fig. 4).

Figure 4.

Kit stimulation results in tyrosine phosphorylation of common γ chain (γ _C). Bone‐marrow‐derived mast cell (BMMC) culture from wild‐type mice were stimulated with Kit ligand (KL) (250 ng/ml) for the time indicated, and γ _C was immunoprecipitated. Bound fractions of the immunoprecipitation were analyzed for tyrosine phosphorylation and protein amounts by Immunoblot. The bar graphs represent the densitometry analysis of Western blots. The levels of γ _C phosphorylation were quantified and normalized with the total protein and presented as mean ± SEM (n = 3). P value is calculated using Student’s t‐test. *P < 0·05.

Kit stimulation results in STAT6 phosphorylation

Previous studies have shown that STAT1α, STAT5A, and STAT5B, but not STAT6 are bound to and directly phosphorylated by activated Kit.13 In contrast, STAT6 is activated after engagement of IL‐4R by its cognate ligand.14, 15, 16. To test whether Kit‐mediated IL‐4Rα phosphorylation activates STAT6, we analyzed STAT6 phosphorylation after KL stimulation in BMMCs. In BMMCs, KL stimulation results in STAT6 phosphorylation (Fig. 5a). We also observed that the STAT6 phosphorylation downstream of cognate ligand activated IL‐4R was fivefold higher than the Kit‐activated IL‐4R (Fig. 5b).

Figure 5.

Kit stimulation results in signal transducer and activator of transcription 6 (STAT6) phosphorylation in bone‐marrow‐derived mast cells (BMMCs). (a) BMMC cultures from wild‐type mice were stimulated with Kit ligand (KL) (10 and 20 ng/ml) for 10 min. BMMC lysates were separated on SDS–PAGE and were analyzed for levels of phosphorylation of STAT6 and total STAT6. (b) BMMC cultures from wild‐type mice were stimulated with (20 ng/ml) of KL or interleukin‐4 (IL‐4) for 10 min. BMMC lysates were separated on SDS–PAGE and analyzed for levels of phosphorylation of STAT6 and total STAT6. The bar graphs represent the densitometry analysis of Western blots. The levels of STAT6 phosphorylation were quantified and normalized with the total protein and presented as mean ± SEM (n = 3). P value is calculated using Student’s t‐test. *P < 0·05.

Co‐stimulation of Kit and IL‐4R induced synergy in BMMC proliferation

Kit and IL‐3R cross‐receptor activation induces synergistic proliferation of erythroid myeloid lymphoid cells upon co‐stimulation with KL and IL‐3.17 Based on earlier studies, we hypothesized that Kit and IL‐4R interaction are required for synergistic proliferation of cells that co‐express Kit and IL‐4R. To determine the biological significance of Kit‐activated IL‐4R, we examined the proliferation of BMMCs after activation of Kit and IL‐4R with their respective cognate ligands. We observed that KL and IL‐4 stimulation results in BMMC proliferation in a dose‐dependent manner. When the BMMCs were co‐stimulated with KL and IL‐4, synergy in proliferation was observed. The synergy index for the BMMC proliferation was calculated as described in the figure legend. We observed that 20 ng/ml of KL and 5 ng/ml IL‐4 are the minimal doses required to obtain the maximum synergy index (Fig. 6a). Lipid rafts are known to play an important role in Kit‐ and IL‐2R‐mediated signaling.18, 19 To understand the importance of lipid rafts for the synergy between KL and IL‐4, the proliferation assay was performed after the disruption of lipid rafts with methyl‐β‐cyclodextrin (MβCD). At the concentration of 1 mm MβCD, Kit‐mediated and IL‐4‐mediated proliferation was not affected. However, synergy in BMMC proliferation was affected. The calculated synergy index for 1 mm MβCD‐treated BMMCs was around 1. This suggests that lipid rafts are necessary for the synergy in BMMC proliferation (Fig. 6b,c).

Figure 6.

Co‐stimulation of Kit and interleukin‐4 (IL‐4) induces synergy in bone‐marrow‐derived mast cell (BMMC) proliferation. (a) BMMC culture from wild‐type mice was stimulated with different concentrations of Kit ligand (KL), IL‐4 or a combination of KL and IL‐4 for 48 hr. BMMC proliferation was measured by WST‐1 assay. (b) BMMC culture from wild‐type mice were stimulated with 20 ng/ml KL (open bar), 5 ng/ml IL‐4 (gray bar) and 20 ng/ml KL + 5 ng/ml IL‐4 (hatched bar) in the presence or absence of methyl‐β‐cyclodextrin (MβCD) and proliferation was measured using WST‐1 assay. The values above the bar in the graph indicate the synergy index. The proliferation synergy index was calculated as ratio of observed optical density at 440 nm (OD₄₄₀) for KL + IL‐4 and the OD₄₄₀ for KL plus the OD₄₄₀ for IL‐4. (c) Dot plot represents synergy index of BMMC proliferation in the absence and presence of MβCD. Each dot in the graph represents an individual experiment performed in triplicates (n = 5). P value was calculated using Student’s t‐test. *P < 0·05, **P < 0·005.

Co‐stimulation of Kit and IL‐4R induces enhanced STAT6 phosphorylation

As co‐stimulation of KL and IL‐4 produce synergy in BMMC proliferation, we analyzed whether the observed effect could be because of enhanced phosphorylation of STAT6. Hence, we studied STAT6 phosphorylation after KL, IL‐4 or KL and IL‐4 stimulation in BMMCs. We observed that IL‐4 stimulation resulted in STAT6 phosphorylation in a dose‐dependent manner with the maximum phosphorylation at concentration of 1 ng/ml (Fig. 7a). Also, a sub‐optimal dose of KL or IL‐4 stimulation induced minimal STAT6 phosphorylation, whereas co‐stimulation with sub‐optimal doses of KL plus IL‐4 resulted in maximum levels of STAT6 phosphorylation (Fig. 7b).

Figure 7.

Co‐stimulation of Kit and interleukin‐4 receptor (IL‐4R) resulted in enhanced phosphorylation of signal transducer and activator of transcription 6 (STAT6). Bone‐marrow‐derived mast cells (BMMCs) were stimulated with (a) different concentrations of IL‐4 for 5 min and (b) different combination of cytokine i.e. 20 ng/ml Kit ligand (KL), 0·5 ng/ml IL‐4, 0·1 ng/ml IL‐4, 20 ng/ml KL + 0·5 ng/ml IL‐4 and 20 ng/ml KL + 0·1 ng/ml IL‐4 for 5 min. Cytokine‐stimulated cells were fixed and stained at intracellular level using AF488‐conjugated mouse anti‐phospho‐STAT6 antibody and STAT6 phosphorylation was analyzed by flow cytometry.

Discussion

In the present study, we demonstrated that Kit signaling activates IL‐4R and its downstream signaling pathways in mast cells. Previous studies have indicated that such cross‐receptor interactions between Kit and Epo‐R, GM‐CSFR, IL‐7R, and IL‐3R occur in erythroid, megakaryoblastic, T lymphoid, and multipotent erythroid myeloid lymphoid progenitors, respectively. Similar to earlier reported Kit‐interacting receptors, IL‐4R also belongs to the type I cytokine receptor family. Lacking intrinsic tyrosine kinase activity, the type I cytokine receptors instead depend on signaling through the Janus kinase–STAT cascade after engagement of their respective cognate ligands and receptor dimerization.20 In contrast to signaling through cognate interactions, cross‐receptor activation of each subunit of the heterodimeric Type I cytokine receptors appears to occur independently of the other subunit. In the present study, we observed that activated Kit could phosphorylate at tyrosine residues of both IL‐4Rα and γ _C subunits (Figs 2 and 4). In the cellular contexts examined so far, the independent phosphorylation may not be relevant as both subunits appear to be expressed coordinately in the respective cell types. We also observed that the Kit‐mediated IL‐4Rα activation was significantly less than the cognate‐ligand‐mediated activation (Fig. 3). Hence, cross‐receptor activation of Type I cytokine receptors differs biochemically from that induced by cognate ligand.

We have demonstrated that cross‐receptor activation of IL‐4R by Kit captures some of the downstream signaling pathways specifically mediated by the IL‐4R, e.g. STAT6. A previous analysis of the STAT proteins bound to and activated by Kit showed that STAT6 is not a direct substrate for activated Kit.13 In contrast, Hundley et al.,21 demonstrated that activation of Kit in BMMCs led to STAT6 phosphorylation. Our data suggest that Kit‐mediated STAT6 phosphorylation might be through cross‐receptor activation of IL‐4R. We also observed that Kit‐mediated STAT6 phosphorylation was fivefold less when compared with cognate‐ligand‐induced STAT6 phosphorylation.

Co‐activation of the Kit and IL‐4R pathways causes synergy in BMMC proliferation. Earlier studies have reported that Kit–IL‐3R cross‐receptor interaction results in synergistic proliferation of rrythroid myeloid lymphoid cells.17 Similar to earlier reports, Kit and IL‐4R cross‐receptor activation could be a potential mechanism for the cause of synergy in BMMC proliferation. In addition, Kit and Epo‐R cross‐receptor activation is reported to be necessary for normal erythroid cell expansion.22, 23, 24, 25, 26 Previous in vivo analyses using Kit and γ _C mutant mice revealed synergistic effects of Kit and γ _C signaling in thymopoiesis.27, 28 In natural killer cells, activation of Kit and IL‐2/15 signaling causes synergy in proliferation through enhanced mitogen‐activated protein kinase activity.29 Our results also suggest that Kit and IL‐4R cross‐receptor activation would regulate signaling and functions of IL‐4R in a defined manner in addition to the classical Kit and IL‐4R pathways. This could be a possible mechanism for observed synergy in BMMC proliferation, which needs further study.

We further demonstrated that Kit‐ and IL‐4R‐mediated synergy in BMMC proliferation is lipid raft dependent. Lipid rafts are shown to regulate IL‐2R signaling in T cells and immobilization of components of lipid rafts resulted in inhibition of IL‐2‐induced proliferation of T cells.19 Studies have also reported that lipid rafts are essential for regulation of Kit signaling. Kit upon engagement with KL is recruited to lipid rafts and interacts with its downstream targets like phosphoinositide 3‐kinase and Src family kinases. Recruitment of Kit to lipid rafts is also shown to be necessary for activation of Kit‐mediated proliferative pathways.18 Our observation of lipid‐raft‐dependent Kit‐ and IL‐4R‐mediated synergy in BMMC proliferation suggest the possibility that lipid rafts are essential and would contribute to such cross‐receptor interactions.

Our study of Kit and IL‐4R cross‐receptor activation, along with previous studies of Kit with Epo‐R, GM‐CSFR, IL‐7R, and IL‐3R, suggest that Kit in addition to Kit‐specific pathways also activate other signals mediated by type I cytokine receptors. These cross‐receptor activations are likely to be universal mechanism to execute synergistic and cell type‐specific signaling by Kit in several Kit‐positive cells.

Disclosures

The authors declare no commercial or financial conflict of interests.