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J. Biol. Chem., Vol. 276, Issue 32, 30381-30391, August 10, 2001

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Src Homology 2 Domain-containing Inositol 5-Phosphatase 1 Mediates Cell Cycle Arrest by FcRIIB^*

Odile Malbec, Christian Schmitt^§, Pierre Bruhns, Gerald Krystal^¶, Wolf H. Fridman, and Marc Daëron

From the  Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U.255, Institut Curie, 75005 Paris, France, the ^§ Laboratoire d'Immunologie Cellulaire, CNRS UMR 7627, Hôpital Pitié-Salpétrière, 75013 Paris, France, and ^¶ The Terry Fox Laboratory, Vancouver, British Columbia B5Z 1L3, Canada

Received for publication, December 11, 2001, and in revised form, May 9, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We previously found that low affinity receptors for the Fc portion of IgG, FcRIIB, which are widely expressed by hematopoietic cells, can negatively regulate receptor tyrosine kinase-dependent cell proliferation. We investigated here the mechanisms of this inhibition. We used as experimental models wild-type mast cells, which constitutively express the stem cell factor receptor Kit and FcRIIB, FcRIIB-deficient mast cells reconstituted with wild-type or mutated FcRIIB, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1)-deficient mast cells. We found that, upon coaggregation with Kit, FcRIIB are tyrosyl-phosphorylated, recruit SHIP1, but not SHIP2, SH2 domain-containing protein tyrosine phosphatase-1 or -2, abrogate Akt phosphorylation, shorten the duration of the activation of mitogen-activated protein kinases of the Ras and Rac pathways, abrogate cyclin induction, prevent cells from entering the cell cycle, and block thymidine incorporation. FcRIIB-mediated inhibition of Kit-dependent cell proliferation was reduced in SHIP1-deficient mast cells, whereas inhibition of IgE-induced responses was abrogated. Cell proliferation was, however, inhibited by coaggregating Kit with FcRIIB whose intracytoplasmic domain was replaced with the catalytic domain of SHIP1. These results demonstrate that FcRIIB use SHIP1 to inhibit pathways shared by receptor tyrosine kinases and immunoreceptors to trigger cell proliferation and cell activation, respectively, but that, in the absence of SHIP1, FcRIIB can use other effectors that specifically inhibit cell proliferation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

FcRIIB are widely expressed single-chain low affinity receptors for the Fc portion of IgG antibodies that bind multivalent immune complexes with high avidity. They exist as two (FcRIIB1 and -B2 in humans) or three (FcRIIB1, -B1', and -B2 in mice) alternatively spliced products of the FcgR2b gene (1). FcRIIB isoforms are differentially expressed by lymphoid and myeloid cells; mouse B cells express FcRIIB1 and -B1' (2), T cells express FcRIIB1 (3), and mast cells and macrophages express FcRIIB1, -B1', and -B2 (2, 4). When coaggregated on the same cell with receptors bearing immunoreceptor tyrosine-based activation motifs (ITAMs),¹ murine and human FcRIIB were shown to negatively regulate cell activation. Thus, FcRIIB inhibit BCR-mediated B cell activation (5-7), T cell receptor-mediated T cell activation (3) and high affinity IgE receptor (FcRI)-mediated mast cell activation (3, 8). Long ago, B cell FcR, which were later identified as FcRIIB (9), were also shown to inhibit BCR-mediated B cell proliferation (6), and we found recently that murine FcRIIB can negatively regulate the proliferation of mast cells induced by Kit (10). Kit is a typical receptor tyrosine kinase for stem cell factor (SCF) that belongs to the colony-stimulating factor-1/platelet-derived growth factor receptor subfamily (11) and controls cell proliferation during gametogenesis, melanogenesis, and hematopoiesis (12). The present work aimed at elucidating the mechanism(s) by which FcRIIB could inhibit cell proliferation.

Works published during the last 5 years documented the molecular mechanisms used by murine FcRIIB to negatively regulate cell activation triggered by ITAM-bearing receptors. The inhibitory properties of FcRIIB were found to depend on an immunoreceptor tyrosine-based inhibition motif (ITIM) present in the intracytoplasmic (IC) domain of all murine and human isoforms of FcRIIB (3, 13). The coaggregation of FcRIIB with activating receptors enables the Src family protein tyrosine kinase Lyn to phosphorylate not only ITAMs but also the FcRIIB ITIM (14). The tyrosyl-phosphorylated ITIM recruits the SH2 domain-containing inositol phosphate 5-phosphatase 1 (SHIP1) (15, 16), which inhibits two major signaling pathways triggered by ITAM-bearing receptors, the Ca²⁺ response and the Ras pathway. The preferred substrate of SHIP1 is indeed phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃), generated by phosphatidylinositol 3-kinase (PI3K) (17). PI(3,4,5)P₃ mediates the membrane translocation of a subset of molecules containing a pleckstrin homology (PH) domain. Among these molecules is the Bruton's tyrosine kinase (18), which is mandatory for phospholipase C- (19) to be activated and to generate inositol 1,4,5-trisphosphate, leading to the mobilization of intracellular Ca²⁺. SHIP1 was recently found to inhibit the activation of Erk1/2, the mitogen-activated protein (MAP) kinases of the Ras pathway, independently of its phosphatase activity. When recruited by FcRIIB, SHIP1 is tyrosyl-phosphorylated and serves as an adapter protein. It recruits p62dok, which is in turn tyrosyl-phosphorylated and recruits RasGAP. RasGAP activates the autocatalytic GTPase activity of ras, thereby preventing the activation of the ras pathway. SHIP1 therefore appears as the major effector of FcRIIB-dependent negative regulation of cell activation by acting at different steps of signal transduction via phosphatase activity-dependent and -independent mechanisms. As a consequence, the activation of Ca²⁺-dependent enzymes that promote the nuclear translocation of the nuclear factor of activation NF-AT is prevented, as well as MAP kinase-dependent downstream events. MAP kinase substrates are transcription factors that cooperate with NF-AT to induce the transcription of cytokine genes (20, 21). By recruiting SHIP1, FcRIIB therefore arrest the intracellular propagation of activation signals triggered by ITAM-bearing receptors and subsequent cellular responses. These include exocytosis, in mast cells (3, 8), and cytokine secretion, in mast cells (8), B cells (7) and T cells (3).

Mechanisms used by FcRIIB to inhibit cell proliferation are poorly understood. They are difficult to examine in B cells. Indeed, B cell activation and proliferation can both be triggered by the BCR, which is constitutively associated with several coreceptors whose respective roles in B cell activation and proliferation are not well known. Another reason is that most biochemical studies that unraveled the inhibitory mechanisms used by FcRIIB were conducted in transformed cell lines whose proliferation became independent of the regulatory mechanisms that control the growth of normal cells. By contrast with B cells, the activation and proliferation of mast cells can be triggered independently by FcRI and by Kit, respectively (22). Evidence that FcRIIB can negatively regulate the proliferation of Kit-induced mast cell proliferation originated from our observation that anti-Kit antibodies could induce the proliferation of primary mast cells derived in vitro from mouse bone marrow (BMMCs) provided that their Fc portions could not bind to FcRIIB that are constitutively expressed by these cells. Comparable proliferative responses were induced by F(ab')₂ fragments of anti-Kit antibodies in wt mast cells, by intact anti-Kit antibodies in FcRIIB^/ mast cells, or by intact anti-Kit antibodies in wt mast cells whose FcRIIB were blocked with anti-FcRIIB antibodies. No proliferation was observed if anti-Kit antibodies were allowed to coaggregate Kit with FcRIIB on wt mast cells (10). FcRIIB therefore inhibit mouse mast cell proliferation when coaggregated with Kit, and BMMCs provide an appropriate model to study the effects of FcRIIB on signal transduction pathways leading to cell proliferation.

When dimerized by SCF, Kit autophosphorylates and recruits several kinases including PI3K. By generating PI(3,4,5)P₃, PI3K enables the membrane translocation of the protein kinase Akt and the exchange factor Vav via their PH domain. These molecules, altogether, prevent cell death and activate the Rac pathway whose terminal effectors are the MAP kinases p38 and JNK (23). Kit also recruits Shc, which, when phosphorylated, recruits the adapter protein Grb2. Grb2 recruits the exchange factor Sos, which activates the Ras pathway, whose terminal effectors are the MAP kinases Erk1/2 (24). Rac and Ras MAP kinases shuttle into the nucleus, and they cooperate to activate transcription factors that control the expression of cyclin genes (25). Cyclins are the positive regulatory subunits of a class of protein kinases collectively called cyclin-dependent kinases. These phosphorylate proteins of the retinoblastoma family, leading to the release of the transcription factors E2F, which control the coordinated expression of proteins required for the stepwise progression through the cell cycle (26). In the present study, we provide evidence that, when coaggregated with Kit, FcRIIB selectively recruit SHIP1, inhibit the Ras and the Rac pathways, and prevent cells from entering into the cell cycle by blocking the transcription of cyclin genes. The inhibitory effects of FcRIIB were partially suppressed in SHIP1^/ mast cells and could be mimicked by FcRIIB, whose IC domain was replaced with the catalytic domain of SHIP1.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Antibodies-- The mAb anti-mouse FcRIIB K9.361 (27) and the mouse IgE mAb 2682-I (28) were used as culture supernatants. The rat mAbs anti-mouse FcRIIB 2.4G2 (29) and anti-mouse Kit ACK2 (22) were affinity-purified on protein G-Sepharose (Amersham Pharmacia Biotech). ACK2 antibodies were biotinylated as described previously (30). Phycoerythrin (PE)-labeled F(ab')₂ fragments of goat anti-mouse Ig (GAM), F(ab')₂ fragments of anti-biotin mAbs, and F(ab')₂ fragments and intact IgG of polyclonal rabbit anti-mouse Ig (RAM) were purchased from Jackson Immunoresearch Laboratories (West Grove, PA), mouse anti-cyclins D2 and D3 antibodies were from Neomarkers (Union City, CA), rabbit anti-cyclin A antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbit anti-phospho-Erk, Erk, phospho-JNK, JNK, phospho-p38, p38, phospho-Akt, and Akt antibodies were from New England Biolabs (Beverly, MA). Rabbit antibodies anti-FcRIIB IC domain (31) were a gift from Dr. Catherine Sautès-Fridman (Institut Curie, Paris, France). Rabbit anti-SHIP2 antibodies (32) were a gift from Dr. David Wisniewski (Memorial Sloan-Kettering Cancer Center, New York, NY). Horseradish peroxidase (HRP)-conjugated anti-phosphotyrosine (PY) mAb PY-20 was purchased from Chemicon (Temecula, CA), rabbit anti-SHIP1 antibodies were from Upstate Biotechnology (Lake Placid, NY), mouse monoclonal anti-SHP-1 and anti-SHP-2 were from Transduction Laboratories (Lexington, KY), and HRP-conjugated GAM and goat anti-rabbit Ig antibodies were from Santa Cruz Biotechnology.

Cells-- BMMCs were obtained by culturing mouse bone marrow cells in RPMI medium supplemented with 10% fetal calf serum, 100 IU/ml penicillin and 100 µg/ml streptomycin (complete medium), and 2% X63-IL3-conditioned medium. After 4 weeks, cultures contained more than 90% mast cells. Culture reagents were from Life Technologies, Inc.

cDNA Constructs-- cDNA encoding wt FcRIIB1 and IC domain-deleted FcRIIB (FcRIIB(IC1)) were described previously (14). cDNA encoding a chimeric molecule made of the extracellular and transmembrane domains of FcRIIB and, as an IC domain, residues 387-829 of human SHIP1, containing the catalytic domain (residues 428-806) was constructed by polymerase chain reaction using the following primers. Sense, 5'-CTG GTA CCG ATG AAG AAC AAG CAC TCA GAG-3'; antisense, 5'-GGG CAG GAG CTC TTC TTA GGC CTC TAA CCG AAG GGC-3'. KpnI (GGTACC) and SacI (GAGCTC) sites are underlined. The resulting fragment was cloned at KpnI and SacI sites into an NT vector containing sequences encoding the extracellular and transmembrane domains and the six first amino acids of intracytoplamsic domain of FcRIIB1, under the control of the SR promoter and containing a resistance gene to zeocin (NT-zeo) (33). The amplification product was sequenced on the two strains.

Retroviral-mediated Gene Transfer-- A biscistronic retroviral vector (LZRS-IRES.EGFP) was constructed based on the LZRS-LacZ of Nolan and colleagues (34) in which the LacZ gene was replaced by an IRES (35) fused to the EGFP reporter gene. wt and mutant FcRIIB were inserted into LZRS-IRES.EGFP at HindIII and EcoRI sites upstream of the IRES sequence. Viral supernatants were produced from transfected Phoenix packaging cells (ATCC; F-14727) after selection for high green fluorescent protein fluorescence. Titers (0.9-2 × 10⁶ colony-forming units EGFP/ml) were estimated by infection of 3T3 cells with serial dilutions of virus stocks and measurement of EGFP fluorescence. BMMCs were infected by three rounds of 24-h culture with virus supernatant in the presence of 2% X63-IL3-conditioned medium and 8 µg/ml protamine sulfate on fibronectin-coated plates. 2 days after infection, EGFP⁺ cells were selected by cell sorting using a FACStar Plus flow cytometer (Becton Dickinson, Mountain View, CA).

Indirect Immunofluorescence-- Aliquots of 5 × 10⁵ BMMCs were incubated for 1 h at 0 °C with K9.361. Cells were washed and stained for 30 min at 0 °C with 50 µg/ml of PE-labeled F(ab')₂ GAM. Fluorescence was analyzed using a FACScalibur (Becton Dickinson). All BMMCs used in this study expressed comparable levels of Kit as assessed with ACK2.

Cell Stimulation and Thymidine Incorporation-- BMMCs, in RPMI containing 1% fetal calf serum and 0.5% bovine serum albumin (Sigma), were preincubated with or without 10 µg/ml 2.4G2 for 1 h at 37 °C, and aliquots of 3 × 10⁴ BMMCs were incubated with preformed immune complexes for 24 h at 37 °C. 0.5 µCi of [³H]thymidine/well were added (Amersham Pharmacia Biotech), and radioactivity incorporated into cells was measured 4 h later. All BMMCs used incorporated comparable amounts of thymidine in response to SCF.

Serotonin Release-- BMMCs, loaded with [³H]serotonin (Amersham Pharmacia Biotech), were incubated for 1 h at 37 °C with IgE anti-2,4-dinitrophenol and washed, and aliquots of 2 × 10⁵ BMMCs were challenged for 10 min at 37 °C with RAM F(ab'2) or IgG. The percentage of [³H]serotonin released was measured as described (36).

TNF Release-- Aliquots of 7 × 10⁵ BMMCs, previously sensitized by a 1-h incubation at 37 °C with IgE, were incubated for 3 h at 37 °C with 1 µM RAM F(ab')₂ or IgG. Cell-free supernatants were harvested and assayed for TNF. TNF was measured by a cytotoxic assay on L929 cells as described (37).

Cell Cycle Analysis-- BMMCs were incubated with or without 10 µg/ml 2.4G2 for 1 h at 37 °C in culture medium supplemented with 2% WEHI-3B-conditioned medium. Cells at 1 × 10⁶ cells/ml were incubated for 24 h with preformed immune complexes in the same medium. Cells were treated with 75% ethanol for 2 h at 4 °C and then with 50 µg/ml RNase (Roche Molecular Biochemicals), and nuclei were stained for 15 min with 100 µg/ml propidium iodide (Sigma). Fluorescence was analyzed using a FACScalibur. The percentages of cells in G₀ + G₁, S, and G₂ + M were calculated using the Modfit program (Verity Software House, Topchan, ME).

Assessment of Cell Viability-- Aliquots of 5 × 10⁵ BMMCs were incubated for 10 min at 0 °C with propidium iodide and with fluorescein isothiocyanate-conjugated annexin V as recommended by the manufacturer (Immunotech, Marseille-Luminy, France). Fluorescence was analyzed using a FACScalibur.

Western Blot Analysis of Whole Cell Lysates-- BMMCs were incubated for 1 h at 37 °C with or without 10 µg/ml 2.4G2, washed, challenged at 37 °C with immune complexes, and lysed by three cycles of freeze thawing, i.e. 1 min in liquid nitrogen followed by 1 min at 37 °C, in lysis buffer containing 50 mM Tris pH 8, 150 mM NaCl, 1% Nonidet P-40, 1 mM Na₃VO₄, 5 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Lysates were centrifuged at 12,000 × g for 10 min at 4 °C. Proteins were quantitated using a Bio-Rad protein assay. Indicated amounts of proteins were electrophoresed and transferred onto Immobilon-P (Millipore, Bedford, MA). Membranes were saturated with 5% skimmed milk (Régilait, Saint-Martin-Belle-Roche, France) in Western buffer containing 150 mM NaCl, 10 mM Tris, and 0.5% Tween 20 (Merck, Schuchardt, Germany), pH 7.4, and Western blotted with the indicated antibodies followed by HRP goat anti-rabbit Ig antibodies or HRP-GAM. Labeled antibodies were detected using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Immunoprecipitation and Western Blot Analysis-- BMMCs were incubated for 1 h at 37 °C with or without 10 µmg/ml 2.4G2, washed, challenged for 5 min at 37 °C with immune complexes, and lysed in lysis buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM Na₃VO₄, 5 mM NaF, 5 mM sodium pyrophosphate, 0.4 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. Postnuclear lysates were immunoprecipitated with 2.4G2-coated Sepharose beads, electrophoresed, and transferred onto Immobilon-P. Membranes were saturated with either 5% bovine serum albumin or with 5% skimmed milk in Western buffer. FcRIIB immunoprecipitates were Western blotted with HRP-conjugated anti-phosphotyrosine antibodies and with the indicated antibodies followed by HRP goat anti-rabbit Ig antibodies or HRP-GAM. Labeled antibodies were detected by enhanced chemiluminescence.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Anti-Kit Antibodies Can Trigger the Same Signaling Events as SCF-- We found previously that anti-Kit antibodies can exert opposite effects on mast cell proliferation. They can mimic SCF and induce cells to proliferate by aggregating Kit via their Fab portions, and they can inhibit this proliferation by coaggregating Kit with FcRIIB via their Fc portion. To analyze the mechanisms of FcRIIB-dependent inhibition of Kit-induced mast cell proliferation, we first checked that anti-Kit antibodies trigger the same proliferative signals as SCF.

BMMCs were nonstimulated, stimulated with SCF, or stimulated with anti-Kit immune complexes. In the latter case, cells were preincubated with the rat mAb 2.4G2 that blocks the binding site of FcRIIB (29), prior to stimulation with preformed immune complexes made of biotinylated anti-Kit ACK2 (biotin-ACK2) and anti-biotin IgG antibodies as described (10). Intracellular signals and cell responses known to be triggered by SCF were examined when stimulating mast cells under these conditions. Upon dimerization, Kit recruits and activates PI3K (23). Akt activation is a reflection of PI3K-dependent PI(3,4,5)P₃ generation. Kit also recruits adapter proteins that lead to the activation of MAP kinases (24). Akt (Fig. 1A), Erk1/2, JNK, and p38 activation (Fig. 1B) was assessed by examining their phosphorylation by Western blotting 10 min after stimulation. MAP kinases activate transcription factors that promote the expression of cyclin genes (25). The up-regulation of cyclins D2 and D3, and of cyclin A, was assessed by Western blotting 6 and 24 h after stimulation, respectively (Fig. 1C). Cyclins D control the entry of cells into the G₁ phase, and cyclin A controls DNA replication and the entry into the G₂ phase (26). The percentage of cells in the S + G₂M phases of the cell cycle was assessed by flow cytometry following iodide propidium labeling 24 h after stimulation (Fig. 1D). Thymidine incorporation occurs when cells that have entered the cell cycle reach the S phase. Thymidine incorporation was assessed during a 4-h window, 24 h after stimulation (Fig. 1E). All above responses were triggered by SCF and by biotin-ACK2-anti-biotin complexes. Erk1/2 and p38 activation and cyclin D3 induction were of comparable intensities in response to the two stimuli. Akt phosphorylation and cyclin D2 induction were slightly less intense, and JNK activation and cyclin A induction were clearly less intense when induced by antibodies than when induced by SCF. The proportion of cycling cells was 2-fold lower, and thymidine incorporation was 6-fold lower in response to anti-Kit antibodies than in response to SCF.

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Comparison of SCF- and anti-Kit immune complex-induced responses. BMMCs were preincubated with medium or with 2.4G2, washed, and stimulated for various periods of time at 37 °C. BMMCs preincubated with medium were stimulated with 100 ng/ml (or with indicated concentrations) of SCF or with medium (Med); BMMCs preincubated with 2.4G2 were stimulated with immune complexes made of biotin-ACK2 and anti-biotin antibodies (a-Kit). A, Akt phosphorylation; B, Erk, JNK, and p38 phosphorylation. BMMCs were stimulated for 10 min at 37 °C before they were lysed. 20 µg (for Akt and Erk) or 80 µg total proteins (for JNK and p38) were electrophoresed and Western blotted with indicated antibodies. C, cyclin expression. BMMCs were starved in complete medium for 24 h before use. They were stimulated for 6 h (for cyclins D) or 24 h (for cyclin A) before they were lysed. 20 µg of total proteins were electrophoresed and Western blotted with indicated antibodies. D, cell cycle analysis. BMMCs, resuspended at 1 × 106 cells/ml in culture medium supplemented with 2% WEHI-3B-conditioned medium, were stimulated for 24 h at 37 °C. Nuclei were labeled with propidium iodide, and percentages of cells in (G0 + G1), S, and (G2 + M) were determined by flow cytometry. The figure represents the percentage of cells in (S + G2M). E, thymidine incorporation. BMMCs were stimulated for 24 h at 37 °C before thymidine incorporation was measured. BMMCs preincubated with medium were stimulated with indicated concentrations of SCF (left panel); BMMCs preincubated with 2.4G2 were stimulated with complexes made of indicated concentrations of biotin-ACK2 and 0 or 3 µg/ml anti-biotin antibodies (right panel). The figure represents thymidine incorporation as a function of the concentration of SCF (left panel) or of the concentration of biotin-ACK2 (right panel).

Anti-Kit antibodies can therefore trigger the same proliferative signals as SCF, although less efficiently. We next conducted a backward analysis of signaling events that stand upstream of cell division, comparing the effects of aggregating Kit and of coaggregating Kit with FcRIIB.

When Coaggregated with Kit, FcRIIB Block the Cell Cycle by Inhibiting Cyclin Expression-- We examined the cell cycle and the induction of cyclins, in relation with inhibition of thymidine incorporation, in BMMCs from FcRIIB^+/+ and FcRIIB^/ mice. FcRIIB^+/+ BMMCs, but not FcRIIB^/ BMMCs, expressed FcRIIB as assessed by indirect immunofluorescence with the FcRIIB-specific mAb K9.361 (Fig. 2A). Kit was aggregated by biotin-ACK2-anti-biotin complexes in FcgRIIB^+/+ cells that were preincubated with 2.4G2 and in FcRIIB^/ cells, whether or not they were preincubated with 2.4G2. Kit was coaggregated with FcRIIB by the same complexes in FcRIIB^+/+ cells that were not preincubated with 2.4G2.

View larger version (60K): [in this window] [in a new window]   Fig. 2.   Inhibition of Kit-dependent thymidine incorporation, cell cycle, and cyclin induction by FcRIIB. A, FcRIIB expression. The binding of the anti-FcRIIB mAb K9.361 was assessed by indirect immunofluorescence (gray histograms, cells incubated with K9.361 and PE-GAM F(ab')2; black histograms, cells incubated with PE-GAM F(ab')2 only). B, thymidine incorporation. FcRIIB+/+ BMMCs and FcRIIB/ BMMCs, preincubated with 2.4G2 (Kit aggregation; open symbols) or without 2.4G2 (FcRIIB-Kit coaggregation; closed symbols) were incubated for 24 h with complexes made of the indicated concentrations of biotin-ACK2 and anti-biotin antibodies, and thymidine incorporation was measured. The figure represents thymidine incorporation as a function of the concentration of biotin-ACK2. C, cell cycle analysis. FcRIIB+/+ BMMCs and FcRIIB/ BMMCs, preincubated with 2.4G2 (Kit aggregation; open bars) or without 2.4G2 (FcRIIB-Kit coaggregation; closed bars) were resuspended at 1 × 106 cells/ml in culture medium supplemented with 2% WEHI-3B-conditioned medium and incubated for 24 h with complexes made of 10 µg/ml biotin-ACK2 and the indicated concentrations of anti-biotin antibodies. Nuclei were labeled with propidium iodide, and percentages of cells in (G0 + G1), S, and (G2 + M) were determined by flow cytometry. The figure represents the percentage of cells in (S + G2M). D, cyclin expression. FcRIIB+/+ BMMCs were starved in complete medium for 24 h, preincubated with 2.4G2 (Kit aggregation) or without 2.4G2 (FcRIIB-Kit coaggregation), and incubated for 6 h for cyclins D detection or for 24 h for cyclin A detection with complexes made of 10 µg/ml biotin-ACK2 (Biot-ACK2) and the indicated concentrations of anti-biotin (anti-Biot) antibodies. Cells were lysed, and 20 µg of total proteins were electrophoresed and Western blotted with the indicated antibodies.

Biotin-ACK2-anti-biotin complexes induced thymidine incorporation in FcRIIB^/ BMMCs whether or not they were preincubated with 2.4G2 and in FcRIIB^+/+ BMMCs that were preincubated with 2.4G2. Biotin-ACK2 complex-induced thymidine incorporation varied similarly in the two cell types with the relative concentrations of biotin-ACK2 and anti-biotin antibodies. Optimal responses were induced by complexes formed at equivalence. No thymidine incorporation was induced in FcRIIB^+/+ BMMCs that were not preincubated with 2.4G2 (Fig. 2B).

A dose-dependent increase in the percentage of cells in S + G₂M was observed following stimulation of FcRIIB^+/+ BMMCs with biotin-ACK2-anti-biotin complexes, when preincubated with 2.4G2, but not when not preincubated with 2.4G2. A comparable dose-dependent increase in the proportion of FcRIIB^/ cells in S + G₂M was observed, whether or not cells were preincubated with 2.4G2 (Fig. 2C).

The induction of cyclin D2, cyclin D3, and cyclin A was examined in wt BMMCs following Kit aggregation and following coaggregation of Kit with FcRIIB using the same ligands as above. Kit aggregation increased the intracellular levels of cyclins D2, D3, and A. All three cyclins remained at basal levels following coaggregation of Kit with FcRIIB (Fig. 2D). FcRIIB can therefore prevent BMMCs from entering the cell cycle by inhibiting the induction of cyclins.

When Coaggregated with Kit, FcRIIB Inhibit the Activation of Erk, JNK, p38, and Akt-- Kit was aggregated or coaggregated with FcRIIB for various periods of time in wt BMMCs using the same ligands as in Fig. 2, and the phosphorylation of the MAP kinases Erk1/2, JNKs, and p38 and of the protein kinase Akt were examined. All three MAP kinases were inducibly phosphorylated upon Kit aggregation as early as 3 min after stimulation. Phosphorylation remained at a comparable level at 10 min and decreased at 30 min. The phosphorylation of Erk, JNKs, and p38 occurred normally 3 min after coaggregation of Kit with FcRIIB but was inhibited at 30 min (Fig. 3). Akt was phosphorylated within 3 min following Kit aggregation, and phosphorylation remained constant until 30 min. Akt phosphorylation was inhibited 3 min after coaggregation of Kit with FcgRIIB and abolished at 30 min (Fig. 3). FcRIIB therefore shorten the duration of Kit-dependent Erk, JNK, p38, and Akt phosphorylation.

View larger version (40K): [in this window] [in a new window]   Fig. 3.   Inhibition of Kit-dependent Erk, JNK, p38, and Akt by FcRIIB. BMMCs, preincubated with 2.4G2 (Kit aggregation) or without 2.4G2 (FcRIIB-Kit coaggregation), were incubated for the indicated periods of time with complexes made of 3 µg/ml biotin-ACK2 (Biot-ACK2) and 3 µg/ml anti-biotin (anti-Biot) antibodies. Cells were lysed, and 5 µg of total proteins for Erk and Akt detection, 25 µg for p38 detection, or 50 µg for JNK detection were electrophoresed and Western blotted with the indicated antibodies.

Inhibition of Kit-dependent Cell Proliferation Requires the FcRIIB Intracytoplasmic Domain-- We next analyzed FcRIIB sequences involved in negative regulation of Kit-dependent proliferation. To this aim, FcRIIB^/ BMMCs were reconstituted with FcRIIB1 or with FcRIIB(IC1) (Fig. 4A). Kit was aggregated or coaggregated with FcRIIB under the same conditions as in Fig. 2, and thymidine incorporation was measured. The two types of cells incorporated comparable amounts of thymidine following Kit aggregation. Thymidine incorporation was suppressed when coaggregating Kit with FcRIIB1 but not with FcRIIB(IC1) (Fig. 4B). Inhibition of proliferation, which was abolished in FcRIIB^/ BMMCs, was therefore fully restored following reconstitution with FcRIIB1 but not with FcRIIB(IC1).

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Requirement of FcRIIB intracytoplasmic sequences for inhibition of Kit-dependent cell proliferation. A, FcRIIB expression. The binding of the anti-FcRIIB mAb K9.361 was assessed by indirect immunofluorescence (gray histograms, cells incubated with K9.361 and PE-GAM F(ab')2; black histograms, cells incubated with PE-GAM F(ab')2 only). B, thymidine incorporation. FcRIIB/ BMMCs reconstituted with FcRIIB1 (FcRIIB/B1) or FcRIIB(IC1) (FcRIIB/IC1), preincubated with 2.4G2 (Kit aggregation; open circles) or without 2.4G2 (FcRIIB-Kit coaggregation; closed circles) were incubated for 24 h with complexes made of indicated concentrations of biotin-ACK2 and 3 µg/ml anti-biotin antibodies, and thymidine incorporation was measured. The figure represents thymidine incorporation as a function of the concentration of biotin-ACK2. C, Erk and Akt phosphorylation. BMMCs, preincubated with 2.4G2 (Kit aggregation) or without 2.4G2 (FcRIIB-Kit coaggregation), were incubated for the indicated periods of time with complexes made of 3 µg/ml biotin-ACK2 (Biot-ACK2) and 3 µg/ml anti-biotin (anti-Biot) antibodies. Cells were lysed, and 5 µg of total proteins for Erk detection or 20 µg for Akt detection were electrophoresed and Western blotted with the indicated antibodies.

Biochemical events associated with Kit-dependent proliferation were also examined in the same two BMMCs under the same conditions as in Fig. 3. Kit aggregation induced Erk and Akt phosphorylation at 3 min, and phosphorylation remained at comparable levels until 30 min in the two cell types. Erk phosphorylation was partially inhibited 10 min and abolished 30 min after coaggregation of Kit with FcRIIB1. It was unaffected by coaggregating Kit with FcRIIB(IC1). Akt phosphorylation was dramatically inhibited 3 min and remained abolished 30 min after coaggregation of Kit with FcRIIB1. It was unaffected by coaggregating Kit with FcRIIB(IC1) (Fig. 4C). Thus, to inhibit Kit-dependent proliferation, FcRIIB require the conservation of their IC domain.

Upon Coaggregation with Kit, FcRIIB Becomes Tyrosyl-phosphorylated and Recruits SHIP1 but Not SHIP2, SHP-1, or SHP-2-- Negative regulation of cell activation by FcRIIB is correlated with the recruitment of SH2 domain-containing phosphatases by tyrosyl-phosphorylated FcRIIB (15, 16, 38). We investigated which phosphatases were recruited by FcRIIB upon coaggregation with Kit. FcRIIB^/ BMMCs reconstituted with FcRIIB1 were stimulated or not with biotin-ACK2-anti-biotin immune complexes, and FcRIIB1 were immunoprecipitated. Their phosphorylation was assessed by Western blotting with anti-phosphotyrosine antibodies, and phosphatases coprecipitated with FcRIIB1 were identified by Western blotting with specific antibodies. As observed previously (30), FcRIIB1 became tyrosyl-phosphorylated when coaggregated with Kit. SHIP1, but not SHIP2, SHP-1, or SHP-2, coprecipitated with tyrosyl-phosphorylated FcRIIB (Fig. 5). FcRIIB-mediated inhibition of Kit-dependent proliferation therefore correlated with the selective recruitment of SHIP1.

View larger version (56K): [in this window] [in a new window]   Fig. 5.   Recruitment of SHIP1, but not SHIP2, SHP-1, or SHP-2, by FcRIIB when tyrosyl-phosphorylated upon coaggregation with Kit. FcRIIB were immunoprecipitated from BMMCs stimulated for 5 min with medium or with complexes made of 3 µg/ml biotin-ACK2 (Biot-ACK2) and 3 µg/ml anti-biotin (anti-Biot) antibodies. Immunoprecipitates were electrophoresed and sequentially Western blotted with anti-phosphotyrosine antibodies (anti-PY), anti-FcRIIB antibodies to check that comparable amounts of materials were immunoprecipitated, and anti-SHIP2, anti-SHIP1, anti-SHP-2, and anti-SHP-1 antibodies to identify coprecipitated phosphatases. Whole cell lysates (WCL) were used as positive controls.

SHIP1 Deletion Abrogates FcRIIB-mediated Inhibition of Akt and Erk Activation and Attenuates Inhibition of Cyclin D3 Induction-- To investigate the role of SHIP1 in the negative regulation of Kit-dependent cell proliferation by FcRIIB, we compared the effects of coaggregating Kit with FcRIIB in BMMCs derived from SHIP1^/ and SHIP1^+/+ mice. We first checked the phenotype of SHIP1^/ BMMCs. SHIP1^/ BMMCs expressed SHP-1, SHP-2, and SHIP2 (not shown) but not SHIP1 (Fig. 6A), and as previously suggested (39), they responded more vigorously to SCF. Akt and Erk phosphorylation were indeed more intense and lasted longer in SHIP1^/ BMMCs than in SHIP1^+/+ BMMCs, in response to SCF stimulation (Fig. 6A).

View larger version (51K): [in this window] [in a new window]   Fig. 6.   Suppression of FcRIIB-mediated inhibition of Akt and Erk activation and reduction of inhibition of cyclin D3 induction by SHIP1 deletion. A, absence of SHIP1 expression and enhanced responses to SCF in SHIP1/ BMMCs. Aliquot of SHIP1+/+ and SHIP1/ BMMCs were lysed with SDS, and proteins were precipitated with cold aceton. Whole cell lysates were electrophoresed (5 × 105 cells/lane) and Western blotted with anti-SHIP1 antibodies. SHIP1+/+ and SHIP1/ BMMCs were starved overnight in complete medium and stimulated with SCF for the indicated times. SDS-solubilized lysates (5 × 105 cells/lane) were subjected to Western blot analysis with anti-phospho-Akt and anti-phospho-Erk antibodies, and the membrane was reprobed with anti-Erk antibodies. B, Erk and Akt phosphorylation. SHIP1+/+ and SHIP1/ BMMCs, preincubated with 2.4G2 (Kit aggregation) or without 2.4G2 (FcRIIB-Kit coaggregation), were incubated for the indicated periods of time with complexes made of 3 µg/ml biotin-ACK2 (Biot-ACK2) and 3 µg/ml anti-biotin (anti-Biot) antibodies. Cells were lysed, and 10 µg of total proteins for Erk detection or 40 µg for Akt detection were electrophoresed and Western blotted with the indicated antibodies. C, cyclin D3 induction. FcRIIB/ BMMCs, FcRIIB/ BMMCs reconstituted with FcRIIB1, and SHIP1/ BMMCs were starved in complete medium for 24 h, preincubated with 2.4G2 (Kit aggregation) or without 2.4G2 (FcRIIB-Kit coaggregation), and incubated for 6 h with complexes made of 3 µg/ml biotin-ACK2 and 3 µg/ml anti-biotin antibodies. Cells were lysed, and 20 µg of total proteins were electrophoresed and Western blotted with anti-cyclin D3 antibodies.

Akt phosphorylation was also observed following Kit aggregation with antibodies in SHIP1^/ and SHIP1^+/+ BMMCs, and as observed in response to SCF, it was more intense in SHIP1^/ cells than in SHIP1^+/+ cells. It was inhibited at 3 min and abolished at 10 and 30 min in SHIP1^+/+ BMMCs, but not in SHIP1^/ BMMCs, following the coaggregation of Kit with FcRIIB (Fig. 6B). Erk phosphorylation was also observed following Kit aggregation in both types of cells, and as observed in response to SCF, it was more intense in SHIP1^/ cells than in SHIP1^+/+ cells. It was abolished at 30 min in SHIP1^+/+ BMMCs, but not in SHIP1^/ BMMCs, following the coaggregation of Kit with FcRIIB (Fig. 6B).

We also examined the requirement of SHIP1 for FcRIIB to inhibit the induction of cyclin D3. Cyclin D3 was induced following Kit aggregation in FcRIIB^/ BMMCs, in FcRIIB^/ BMMCs reconstituted with FcRIIB1, and in SHIP1^/ BMMCs that had been preincubated with 2.4G2 and stimulated with biotin-ACK2-anti-biotin complexes. Cyclin D3 induction was suppressed in FcRIIB^/ BMMCs reconstituted with FcRIIB1 but not in FcRIIB^/ BMMCs that had been stimulated with the same complexes without preincubation with 2.4G2. Under the same conditions, cyclin D3 induction was also inhibited in SHIP1^/ BMMCs, but it did not return to basal level (Fig. 6C). Thus, SHIP1 deletion abolished FcRIIB-mediated inhibition of Akt and Erk activation and decreased FcRIIB-mediated inhibition of cyclin D3 induction.

SHIP1 Deletion Partially Suppresses FcRIIB-mediated Inhibition of Kit-dependent Cell Proliferation-- We next compared the effects of coaggregating Kit with FcRIIB on thymidine incorporation in SHIP1^/ and SHIP1^+/+ BMMCs, using the same conditions as in Fig. 2. Thymidine incorporation induced by Kit aggregation was abolished following coaggregation of Kit with FcRIIB in SHIP1^+/+ BMMCs and still inhibited, though only partially, in SHIP1^/ BMMCs (Fig. 7A). This result was unexpected, because SHIP1 deletion was reported to abrogate FcRIIB-mediated inhibition of BCR-induced cell activation in DT40 cells (40). This led us to investigate the consequences of coaggregating FcRIIB with FcRI in SHIP1^/ mast cells. SHIP1^+/+ and SHIP1^/ BMMCs were sensitized with murine IgE and challenged with F(ab')₂ fragments of RAM antibodies to aggregate FcRI or with intact RAM IgG antibodies to coaggregate FcRI with FcRIIB. Both serotonin release (Fig. 7B) and TNF secretion (Fig. 7C) induced by FcRI aggregation were inhibited in SHIP1^+/+ BMMCs following coaggregation of FcRI with FcRIIB. Inhibition of both responses was abolished in SHIP1^/ BMMCs (Fig. 7, B and C).

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Partial suppression of FcRIIB-mediated inhibition of Kit-dependent cell proliferation by SHIP1 deletion. A, thymidine incorporation. SHIP1+/+ and SHIP1/ BMMCs, preincubated with 2.4G2 (Kit aggregation; open circles) or without 2.4G2 (FcRIIB-Kit coaggregation; closed circles) were incubated for 24 h with complexes made of the indicated concentrations of biotin-ACK2 and 3 µg/ml anti-biotin antibodies, and thymidine incorporation was measured. The figure represents thymidine incorporation as a function of the concentration of biotin-ACK2. B, serotonin release. SHIP1+/+ and SHIP1/ BMMCs were sensitized with mouse IgE before they were challenged for serotonin release with the indicated concentrations of RAM F(ab')2 (FcRI aggregation; open circles) or IgG (FcRI-FcRIIB coaggregation; closed circles). The figure represents the percentage of serotonin released as a function of the concentration of RAM. C, TNF secretion. SHIP1+/+ and SHIP1/ BMMCs were sensitized with mouse IgE before they were challenged for 3 h with or without 1 µM RAM F(ab')2 (FcRI aggregation; open symbols) or IgG (FcRI-FcRIIB coaggregation; closed symbols). Cell-free supernatants were harvested, and 2-fold dilutions were tested for cytotoxicity on L929 cells. The figure represents the percentage of cytotoxicity as a function of the dilution of supernatants. D, Cell viability. SHIP1+/+ and SHIP1/ BMMCs, preincubated with 2.4G2 (Kit aggregation) or without 2.4G2 (FcRIIB-Kit coaggregation), were incubated for 24 h with complexes made of 3 µg/ml biotin-ACK2 (Biot-ACK2) and 3 µg/ml anti-biotin (anti-Biot) antibodies. Dead cells and apoptotic cells were visualized with propidium iodide and annexin V. FITC, fluorescein isothiocyanate.

The residual inhibition of thymidine incorporation in SHIP1^/ BMMCs could be explained if BMMCs underwent apoptosis following coligation of Kit with FcRIIB. SHIP1 was indeed reported to act as an anti-apoptotic molecule in B cells (40, 41). SHIP1^+/+ and SHIP1^/ BMMCs were therefore cultured for 24 h with medium alone or with biotin-ACK2-anti-biotin complexes after they had been preincubated with or without 2.4G2. Dead cells were visualized with annexin-V and propidium iodide. Comparable proportions of viable cells (80-90%) were observed in the two cell types in all three conditions (Fig. 7D). Apoptosis therefore does not account for residual inhibition of SHIP1^/ BMMC proliferation.

Thus, whereas FcRIIB-mediated inhibition of FcRI-dependent cell activation was abolished in SHIP1^/ BMMCs, and although SHIP1 only coprecipitated with FcRIIB phosphorylated upon coaggregation with Kit in SHIP1^+/+ BMMCs, FcRIIB-mediated inhibition of Kit-dependent cell proliferation was only partially abrogated in SHIP1^/ BMMCs. This partial effect correlates with the partial inhibition of cyclin D3 induction observed in SHIP1^/ cells.

The Catalytic Domain of SHIP1 Is Sufficient to Negatively Regulate Kit-dependent Mast Cell Proliferation-- The above genetic approach provided negative evidence that SHIP1 is involved in FcRIIB-mediated negative regulation of Kit-dependent cell proliferation. To obtain direct, positive evidence that SHIP1 can be an effector of this regulation, we constructed a cDNA encoding a chimeric molecule made of FcRIIB whose IC domain was replaced with the catalytic domain of SHIP1. This cDNA was expressed in FcRIIB^/ BMMCs, and the chimera was compared with FcRIIB expressed by wt BMMCs for its ability to negatively regulate Kit-dependent proliferation. The expression of the FcRIIB-SHIP1 chimera was confirmed by indirect immunofluorescence with K9.361 (Fig. 8A). Comparable thymidine incorporation was observed following Kit aggregation in both cells. Thymidine incorporation was similarly inhibited following coaggregation of Kit with wt FcRIIB or with the FcRIIB-SHIP1 chimera (Fig. 8B). A SHIP1 chimera could therefore mimic FcRIIB-mediated negative regulation of Kit-dependent mast cell proliferation.

View larger version (24K): [in this window] [in a new window]   Fig. 8.   The catalytic domain of SHIP1 is sufficient to negatively regulate Kit-dependent mast cell proliferation. A, FcRIIB expression. The binding of the anti-FcRIIB mAb K9.361 to wt BMMCs and FcRIIB/ BMMCs reconstituted with a chimera made of FcRIIB whose IC domain was replaced by the catalytic domain of SHIP1 (FcRIIB/ FcR-SHIP1) was assessed by indirect immunofluorescence (gray histograms, cells incubated with K9.361 and PE-GAM F(ab')2; black histograms, cells incubated with PE-GAM F(ab')2 only). B, thymidine incorporation. wt BMMCs and FcRIIB/ FcR-SHIP1 BMMCs, preincubated with 2.4G2 (Kit aggregation; open circles) or without 2.4G2 (FcRIIB-Kit coaggregation; closed circles), were incubated for 24 h with complexes made of the indicated concentrations of biotin-ACK2 and 1 µg/ml anti-biotin antibodies, and thymidine incorporation was measured. The figure represents thymidine incorporation as a function of the concentration of biotin-ACK2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The present work aimed at investigating the mechanism by which FcRIIB negatively regulate cell proliferation. We used, as an experimental model, Kit-dependent mast cell proliferation that we previously found to be negatively regulated by FcRIIB (10). We show here that SHIP1 is selectively recruited by tyrosyl-phosphorylated FcRIIB and plays a critical but not exclusive role in inhibiting transduction pathways that lead to the transcription of cyclin genes and the progression of cells through the cell cycle.

We observed previously a dual effect of IgG anti-Kit antibodies, they can activate cell proliferation by aggregating Kit via their Fab portions, and they can inhibit cell proliferation by coaggregating Kit with FcRIIB via their Fab and Fc portions (10). We therefore used anti-Kit antibodies to analyze the mechanism of FcRIIB-dependent inhibition of Kit-induced proliferation. Beforehand, we checked that anti-Kit antibodies triggered the same intracellular signals as SCF. When dimerized by SCF, Kit is probably under an optimal spatial configuration on the cell membrane but possibly not when aggregated by anti-Kit antibodies. We found no qualitative difference between signals examined upon stimulation of BMMCs with SCF and with anti-Kit antibodies, but we did find quantitative differences. Antibodies were indeed less efficient than SCF, and apparently, late signals were more attenuated than early signals following stimulation with antibodies. Whatever the reason of these differences, these preliminary results validated the use of anti-Kit antibodies for studying FcRIIB-dependent inhibition of signals generated by Kit and leading to mast cell proliferation.

Inhibition of thymidine incorporation correlated with the tyrosyl phosphorylation of FcRIIB induced upon coaggregation with Kit. Inhibition observed in FcRIIB^/ BMMCs reconstituted with wt FcRIIB1 was not seen in FcRIIB^/ BMMCs reconstituted with an IC domain-deleted FcRIIB. Inhibition is therefore not a consequence of steric hindrance between extracellular domains and ligands but requires the IC domain of FcRIIB. Four tyrosines are contained in this domain, one being within the ITIM (3, 13), which when tyrosyl-phosphorylated, has been shown to mediate the recruitment of SHIP1 in mast cells and B cells (3, 15) and SHIP2 in B cells (42, 43), but not SHP-1 or SHP-2 in both cells (16, 44), following coaggregation of FcRIIB with ITAM-bearing receptors. When coaggregated with Kit, tyrosyl-phosphorylated FcRIIB was found to recruit SHIP1, but not SHIP2, SHP-1, or SHP-2, as assessed by coprecipitation. Early events associated with FcRIIB-mediated inhibition of Kit-induced mast cell proliferation therefore resemble early events associated with FcRIIB-mediated inhibition of FcRI-induced mast cell activation (15, 16).

Akt phosphorylation induced by Kit aggregation was inhibited following coaggregation of Kit with FcRIIB. Inhibition of Akt phosphorylation was not observed when Kit was coaggregated with an IC domain-deleted FcRIIB, suggesting that this inhibition is a consequence of the recruitment of SHIP1 by tyrosyl-phosphorylated IC sequences of FcRIIB. Indeed, inhibition of Akt phosphorylation did not occur in SHIP1^/ cells. Interestingly, both SCF-induced and anti-Kit-induced Akt phosphorylation were more intense, particularly at later time points, in SHIP1^/ BMMCs than in SHIP1^+/+ BMMCs, and FcRIIB-dependent inhibition of Akt phosphorylation seen in SHIP1^+/+ cells was more pronounced at the same late time points. When activated, Kit recruits and activates PI3K (23), which generates PI(3,4,5)P₃, enabling the membrane translocation of proteins bearing a PH domain including Akt. Akt phosphorylation, which requires the membrane recruitment of Akt, is a reflection of PI3K activation, and inhibition of Akt phosphorylation is a likely reflection of the catalytic activity of SHIP1, whose preferred substrate is PI(3,4,5)P₃. Similar observations were made in B cells following coaggregation of FcRIIB with BCR (45, 46). We also found that the phosphorylation of p38 and JNK, the terminal effector MAP kinases of the Rac pathway, was inhibited following coaggregation of Kit with FcRIIB. This inhibition is also likely due to the recruitment of SHIP1 and the subsequent degradation of PI(3,4,5)P₃, which mediates the membrane recruitment of Vav.

Erk1/2 phosphorylation observed following Kit aggregation was of a shorter duration following the coaggregation of Kit with FcRIIB. This effect was not observed when Kit was coaggregated with an IC domain-deleted FcRIIB. Inhibition of Erk phosphorylation is also a likely consequence of the recruitment of SHIP1 by tyrosyl-phosphorylated FcRIIB, because it was prevented in SHIP1^/ BMMCs. Like Akt phosphorylation, both SCF-induced and anti-Kit-induced Erk phosphorylation lasted longer in SHIP1^/ BMMCs than in SHIP1^+/+ BMMCs, and FcRIIB-dependent inhibition of Erk phosphorylation seen in SHIP1^+/+ cells was more pronounced at late time points. Several mechanisms were proposed to explain how SHIP1 could inhibit immunoreceptor-induced Erk activation. One leads to a decreased production of molecules that activate Ras. Thus, by preventing the Bruton's tyrosine kinase-dependent full activation of phospholipase C-, SHIP1 may decrease the conversion of phosphatidylinositol 4,5 bisphosphate into inositol 1,4,5-trisphosphate and diacylglycerol. Diacylglycerol activates protein kinase C, which activates Ras. Another mechanism consists in the sequestration of molecules that are necessary for the activation of Ras. SHIP1 was indeed reported to compete with Gbr2 for binding Shc, thereby preventing the constitution of the complex of adapters that connect immunoreceptors to the Ras pathway (47). Finally, SHIP1 has recently been shown to inhibit Erk activation by functioning as an adapter molecule. When recruited by FcRIIB in B cells, SHIP1 is indeed a substrate of Lyn, and when tyrosyl phosphorylated, it recruits Dok via its phosphotyrosine-binding domain. Dok is itself phosphorylated by Lyn and recruits RasGAP via its SH2 domain. RasGAP antagonizes with Sos by accelerating the hydrolysis of GTP into GDP on Ras (48). Kit also recruits adapter proteins that, via the exchange factor Sos, activate the Ras pathway, whose ultimate effectors are the MAP kinases Erk1/2 (24). Whether one or several of these nonexclusive mechanisms account(s) for FcRIIB-mediated inhibition of Kit-dependent activation of Erk1/2 remains to be investigated.

The induction of cyclins D2, D3 (we found no cyclin D1 in BMMCs), and A observed following Kit aggregation was inhibited when Kit was coaggregated with FcRIIB. This effect was not observed in FcRIIB^/ BMMCs. This inhibition likely results from the inhibition of MAP kinase activation. MAP kinases were indeed shown to control the transcription of cyclin genes (25). Inhibition of cyclin D3 induction was, however, reduced but not suppressed in SHIP1^/ BMMCs, although inhibition of MAP kinase activation was abolished. This result suggests that, among the mechanisms that control cyclin D3 expression and that are inhibited by FcRIIB, one can distinguish mechanisms whose inhibition by FcRIIB is SHIP1-dependent and mechanisms whose inhibition by FcRIIB is SHIP1-independent.

As a consequence of the inhibition of cyclin induction, the increased proportion of cells entering the S phase observed following Kit aggregation was not seen following the coaggregation of Kit with FcRIIB. This correlates with inhibition of thymidine incorporation. Expectedly, FcRIIB-induced inhibition of thymidine incorporation was reduced in SHIP1^/ BMMCs, but unexpectedly, it was not abolished. This partial inhibition is consistent with the partial inhibition of cyclin D3 induction in the absence of SHIP1. In contrast, FcRIIB-mediated inhibition of FcRI-dependent serotonin release and TNF secretion was abrogated in SHIP1^/ BMMCs. Incidentally, this is the first demonstration of the mandatory role of SHIP1 in FcRIIB-mediated negative regulation of IgE-dependent mast cell activation. The differential effect of SHIP1 deletion on inhibition of FcRI-induced mast cell activation and of Kit-induced mast cell proliferation suggests that FcRIIB utilize SHIP1 to inhibit pathways shared by FcRI and Kit such as GTP-binding protein-dependent MAP kinase activation but that FcRIIB can also inhibit Kit-specific pathways in the absence of SHIP1. SHIP1 has been reported to prevent cell death by acting as an antiapoptotic factor in B cells (41). The residual inhibition of thymidine incorporation seen in SHIP1^/ BMMCs, however, could not be explained by a decreased cell viability. Residual inhibition could be accounted for by mechanisms that complement SHIP1-dependent mechanisms in wt cells or by mechanisms that compensate SHIP1-dependent mechanisms in the absence of SHIP1 but are not operating in wt cells. Whatever they are, SHIP1-independent mechanisms are not sufficient to abolish cell proliferation, because inhibition of proliferation was only reduced in SHIP1^/ cells. To determine whether SHIP1-dependent mechanisms may suffice to abolish cell proliferation, we constructed a chimeric molecule, the IC domain of which was constituted by the catalytic domain of SHIP1. This chimera inhibited thymidine incorporation as efficiently as FcRIIB1, when coaggregated with Kit. This result indicates that the enzymatic activity of SHIP1 is sufficient for inhibiting Kit-dependent mast cell proliferation.

In conclusion, we used here anti-Kit antibodies as analytical tools to study the mechanism of FcRIIB-dependent negative regulation of cell proliferation. We found that this regulation depends primarily on the recruitment of SHIP1 by tyrosyl-phosphorylated FcRIIB when these receptors are coaggregated with Kit by anti-Kit antibodies. Under these conditions, SHIP1 was found to extinguish Kit-induced signals depending on the recruitment of molecules that have a PH domain to the membrane and downstream signals, as well as the activation of the Ras pathway. Noticeably, signaling pathways triggered by Kit, but not by FcRI and therefore possibly-specific for cell proliferation, could be inhibited by FcRIIB in the absence of SHIP1. However, although FcRIIB could inhibit not only pathways shared by cell activation and cell proliferation, but also pathways specific for cell proliferation, the inhibition of common pathways, by SHIP1, was sufficient to prevent cell proliferation. Our work also provides information on the mechanisms by which SHIP1 negatively regulates signals triggered by Kit. Hematopoietic progenitors from SHIP1^/ mice were indeed reported to be hyper-responsive to several growth factors including SCF (39), and we show here that Akt and Erk phosphorylation were more intense and lasted longer in SHIP1^/ BMMCs than in SHIP1^+/+ BMMCs, in response to both anti-Kit antibodies and SCF stimulation. Whether triggered by SCF or by anti-Kit antibodies, Kit-derived signals are under the control of SHIP1 through the inhibition of PI(3,4,5)P₃-dependent early events. By recruiting more SHIP1, FcRIIB may therefore enhance the constitutive negative regulation of Kit signaling by SHIP1.

Based on these results, anti-Kit antibodies may be envisioned as being more than analytical tools. Abnormal cell proliferation may indeed arise from mutations of Kit that render this receptor constitutively activated. A few well characterized oncogenic Kit mutations were found in mastocytosis, mastocytomas, mast cell leukemias, and intestinal tumors derived from Cajal interstitial cells, and these mutations are thought to be the etiology of these proliferative diseases. Our results provide the molecular grounds for a potential therapeutic use of anti-Kit antibodies in such malignant diseases.

	ACKNOWLEDGEMENTS

We are grateful to Dr. Ulrich Hämmerling (Memorial Sloan-Kettering Cancer Center, New York, NY) for anti-FcRIIB allotypic mAb K9.361, Dr. Catherine Sautès-Fridman (Institut Curie, Paris, France) for rabbit polyclonal antibodies against the IC domain of FcRIIB, Dr. Marco Colonna (Basel Institute for Immunology, Basel, Switzerland) for ACK2 cells, Dr. David Wisniewski (Memorial Sloan-Kettering Cancer Center, New York, NY) for anti-SHIP2 antibodies, and Hélène Hardré-Liénard (Institut Curie, Paris, France) for SHIP1 cDNA.

	FOOTNOTES

^* This work was supported in part by INSERM, the Association pour la Recherche sur le Cancer (ARC), and the Institut Curie. P. B. is the recipient of a fellowship from the ARC. G. K. is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada (NCI-C) supported by funds from the Canadian Cancer Society and the Terry Fox Run.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratoire d'Immunologie Cellulaire et Clinique, INSERM U.255, Institut Biomédicale des Cordeliers, 15 rue de l'Ecole de Médecine, 75006 Paris, France. Tel.: 33-1-5310-0406; Fax: 33-1-4051-0420; E-mail: Marc.Daeron@U255.bhdc.jussieu.fr.

Published, JBC Papers in Press, May 18, 2001, DOI 10.1074/jbc.M011094200

	ABBREVIATIONS

The abbreviations used are: ITAM, immunoreceptor tyrosine-based activation motif; BCR, B cell receptor for antigen; BMMC, bone marrow-derived mast cells; GAM, goat anti-mouse Ig; HRP, horseradish peroxidase; IC, intracytoplasmic; ITIM, immunoreceptor tyrosine-based inhibition motif; MAP, mitogen-activated proteins; PE, phycoerythrin; PH, pleckstrin homology; PI(3, 4,5)P₃, phosphatidylinositol (3,4,5)-trisphosphate; PI3K, phosphatidylinositol-3 kinase; PY, phosphotyrosine; RAM, rabbit anti-mouse Ig; SH2, Src homology 2; SCF, stem cell factor; SHIP, SH2 domain-containing inositol polyphosphate 5-phosphatase; SHP, SH2 domain-containing protein tyrosine phosphatase; wt, wild-type; JNK, c-Jun NH₂-terminal kinase; mAb, monoclonal antibody; FcRIIB(IC1), IC domain-deleted FcRIIB; IRES, internal ribosomal entry sequence; EGFP, enhanced green fluorescence protein; TNF, tumor necrosis factor; biotin-ACK2, biotinylated anti-Kit ACK2.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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