Impairment of B cell receptor-mediated Ca2+ influx, activation of mitogen-activated protein kinases and growth inhibition in CD72-deficient BAL-17 cells
Mami Ogimoto1,
Gaku Ichinowatari1,
Noriyuki Watanabe1,4,
Nobuhiko Tada2,
Kazuya Mizuno1 and
Hidetaka Yakura1,3
1 Department of Immunology and Signal Transduction, Tokyo Metropolitan Institute for Neuroscience, Tokyo Metropolitan Organization for Medical Research, Fuchu, Tokyo 183-8526, Japan 2 School of Health Sciences, Tokai University, Isehara, Kanagawa 259-1193, Japan 3 Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan 4 Present address: National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502, Japan
Correspondence to: H. Yakura; E-mail: yakura{at}tmin.ac.jp
Transmitting editor: T. Watanabe
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Abstract
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CD72 is a 45 kDa B cell-specific type II transmembrane protein of the C-type lectin superfamily. It was originally defined as a receptor-like molecule that regulates B cell activation and differentiation; however, its precise function remains unclear since more recent functional analyses, including a gene targeting study, suggest that CD72 may serve as a negative or a positive regulator of B cell signaling. In the present study, we analyzed the cell-autonomous function of CD72 in B cell receptor (BCR) signaling using CD72-deficient cells generated from mature BAL-17 cells. We found that BCR-mediated phosphorylation of CD19, Btk, Vav and phospholipase C
2 and association of CD19 with phosphatidylinositol-3 kinase were impaired in CD72-deficient cells. Inositol trisphosphate synthesis was normally induced initially but ablated at 1 min of stimulation in CD72-deficient cells. In the event, Ca2+ release from intracellular stores remained intact, though influx of extracellular Ca2+ was severely impaired in CD72-deficient cells. Furthermore, BCR-evoked activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase and c-Jun NH2-terminal kinase, and growth inhibition in BAL-17 cells were blocked in the absence of CD72. Significantly, these effects were largely reversed by re-expression of CD72. Thus, CD72 appears to exert a positive effect on BCR signaling pathways leading to Ca2+ influx and MAPK activation, which in turn may determine the fate of BAL-17 cells.
Keywords: B cell antigen receptor, calcium entry, CD19, PI3 kinase
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Introduction
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B cell antigen receptor (BCR) ligation induces activation of three families of protein tyrosine kinases (PTKs): Src-family PTKs (Lyn, Fyn, Lck, Fgr and Blk), Syk and Btk. Activation of these enzymes initiates a number of downstream biochemical events that ultimately determine the final outcomes of B cells, which include activation, proliferation, survival, differentiation into plasma or memory cells, anergy and apoptosis (14). Also participating in this process are various coreceptors, including CD19, CD21, CD22, CD40 and CD72 (48).
Major signaling pathways triggered by BCR ligation involve activation of phospholipase C-
2 (PLC
2), of Ras and Ras-related small G proteins, and of phosphatidylinositol-3 kinase (PI3K) (9). Activated PLC
2 hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which respectively induce Ca2+ mobilization and activation of protein kinase C. For activation of Ras (10,11), tyrosine phosphorylated adaptor protein Shc recruits Grb2, which binds to Sos, a guanine nucleotide exchange factor (GEF) that interacts with Ras, forming a trimolecular complex (12,13). Subsequent translocation of the complexed Sos to the plasma membrane leads to Ras activation, which in turn activates Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) pathways. In addition, phosphorylation of Vav, which serves as a GEF for Rho family GTPases (14), leads to activation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Rho family GTPases also promote sustained PIP2 synthesis by activating PI 4-phosphate 5-kinase, while activation of PI3K leads to accumulation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) in the plasma membrane (15), which in turn induces recruitment of signaling molecules containing pleckstrin homology domainse.g. Btk, PLC
2 and Vavleading to their full activation (2).
CD72 is a 45 kDa type II transmembrane protein belonging to the C-type lectin superfamily (16,17) that is expressed on all B cells except plasma cells (18,19) and has a receptor recently identified as CD100, a class IV semaphorin (20). We originally reported that CD72 is involved in the regulation of antibody responses to T-dependent antigens (21), and that it most likely exerts its effect by modulating an early phase of B cell activation (22,23). Ligation of CD72 with anti-CD72 antibody, alone or together with anti-IgM antibody or IL-1, induces a variety of effects including cell proliferation (23,24), expression of major histocompatibility complex class II (25), IL-4-dependent expression of CD23 (26), rescue of splenic B cells from BCR-mediated apoptosis (27), activation of Lyn, Blk, Btk, PLC
2 and CD19 (28), association of CD72 with CD19 (29), phosphatidylinositol (PI) turnover (30), Ca2+ mobilization (31) and MAPK activation (32). In other words, mAb ligation of CD72 delivers positive regulatory signals in B cells.
On the other hand, recent biochemical studies have provided evidence suggesting CD72 serves as a negative regulator of BCR signaling. CD72 contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic domain and, in WEHI-231 B cells, BCR ligation induces tyrosine phosphorylation of CD72 and recruitment of the protein tyrosine phosphatase SHP-1, for which CD72 serves as a substrate (33,34). Because SHP-1 is a negative regulator of BCR signaling (35), this suggests that CD72 may negatively regulate BCR signaling through the action of SHP-1. In fact, expression of CD72 in K46 mouse B lymphoma cells, which expresses hapten-specific µ heavy and
light chains, reduces BCR-induced ERK activation and Ca2+ mobilization, as does preligation of CD72 in splenic B cells (36). CD72 expression in BCR-expressing myeloma J558L µ3 cells also reduces BCR-induced phosphorylation of Ig-
/Ig-ß, Syk and B cell linker protein (BLNK, also named SLP-65) (37). This profile of responses suggests CD72 serves as a negative regulator of B cell activation. A recent gene-targeting study showed that there is a reduction of mature recirculating B cells and an accumulation of pre-B cells in the bone marrow of CD72-deficient (CD72/) mice; that there is a reduction in mature B-2 cells and an increase in B-1 cells in the periphery of these mice; and that responses to anti-IgM antibody and a mitogen are augmented in CD72/ B cells, as is BCR-mediated Ca2+ mobilization (38).
To resolve this discrepancy, we re-evaluated the cell-autonomous function of CD72 in BCR signaling using CD72-deficient cells generated from BAL-17 mature B cells. Our results show that CD72 positively regulates signaling pathways leading to Ca2+ influx and MAPK activation, which may ultimately dictate the fate of BAL-17 cells.
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Methods
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Antibodies and reagents
Goat anti-mouse IgM antibody F(ab')2 fragments were purchased from ICN Pharmaceuticals, Inc. (Aurora, OH). Anti-mouse CD72a mAb (96.1) (21), CD72b/Ly-19.2 mAb (K10.6) and CD72b/Ly-32.2 mAb (B9.320) (39) were used. Anti-BLNK antibody was described previously (40). Anti-phosphotyrosine mAb (PY20) and rabbit anti-mouse antibodies against Lyn, Syk, ERK-2, JNK, p38, PLC
2 and Btk were all from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti- phospho-Lyn (Tyr507), anti-phospho-Src (Tyr416) and anti-CD19 antibodies were purchased from Cell Signaling Technology (Beverly, MA). Rat anti-mouse CD19 mAb (BD Pharmingen, San Diego, CA) was used for immunoprecipitation. Rabbit anti-mouse phospho-specific p38 antibody and phospho-specific JNK antibody were from New England BioLabs (Beverly, MA), and rabbit anti-mouse phospho-specific ERK antibody was from Promega (Madison, WI). Rabbit anti-PI3K regulatory subunit p85 (p85-PI3K) and anti-Vav antibodies were from Upstate Biotechnology (Lake Placid, NY).
Generation of CD72-deficient clones
BAL-17 cells, a murine mature B cell line (IgM-positive, IgD-positive, CD72b), were cultured as described elsewhere (41,42). Initially, the cells were treated with N-methyl-N'-nitro-N-nitrosoguanidine (2 µg/ml) (Sigma, St Louis, MO) for 45 min at 37°C under an atmosphere of 5% CO2/95% air. After washing, the cells were cultured in RPMI 1640 supplemented with 10% FBS, 50 µM 2-ME, 100 µg/ml streptomycin and 100 U/ml penicillin (complete medium) for 12 weeks. To eliminate CD72-positive cells, the cells were incubated with anti-mouse CD72b/Ly-19.2 mAb for 30 min on ice and then with complement for 45 min at 37°C. Viable cells were then cloned by limiting dilution. The process of elimination and limiting dilution was repeated three times.
Flow cytometric analysis
To examine the surface phenotype, cells were first incubated with anti-CD72a mAb, anti-CD72b/Ly-19.2 mAb, anti-CD72b/Ly-32.2 mAb, anti-H-2d mAb (34-1-2S), anti-Iad (MK-D6), anti-CD16/CD32 mAb (2.4G2) or buffer alone for 20 min at 4°C and then with FITC-conjugated secondary antibodies for 20 min at 4°C. FITC-conjugated anti-mouse Igs (ICN Pharmaceuticals, Aurora, OH), FITCprotein A (PA) (Amersham Pharmacia Biotech, Uppsala, Sweden), FITCmouse anti-rat Ig
chain mAb (Zymed, San Francisco, CA) and FITCavidin (ICN Pharmaceuticals) were used as secondary antibodies. To detect surface expression of IgM, sIgD, CD19 and CD22, FITC-conjugated F(ab')2 fragments of anti-mouse IgM antibody (ICN Pharmaceuticals), FITC-conjugated anti-IgD allotype (Igh-5a) mAb, FITC-conjugated anti-CD19 mAb, and FITC-conjugated anti-CD22 mAb (BD PharMingen) were used. After washing, the FITC-labeled cells were analyzed with a Beckman-Coulter ELITE flow cytometer (Coulter, Miami, FL).
RTPCR analysis
Total RNA from BAL-17 and 9-59 cells was isolated using the single-step acid guanidium thiocyanate method. cDNA synthesis and RTPCR were performed as previously reported with slight modifications (43). Five micrograms of total RNA from each sample was reverse transcribed at 42°C for 1 h in 20 µl of buffer (50 mM TrisHCl, pH 8.3, 75 mM KCl and 3 mM MgCl2) containing 5 µM random primer, 200 U Superscript II reverse transcriptase (Life Technologies, Grand Island, NY), 0.5 mM dNTP and 10 mM dithiothreitol. Primer sequences for amplification of full-length CD72 and internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows. CD72: sense, 5'-CGGAATTCGCTATGGCTG ACGCTA-3'; antisense, 5'-CTTAGCTGGGCATGCCCCGA GAGTG-3'. GAPDH: sense, 5'-CATCACCATCTTCCAGGAG-3'; antisense, 5'-CCTGCTTCACCACCTTCTTG-3'. The PCR conditions were 25 cycles of denaturation (94°C, 0.5 min), annealing (58°C, 1 min) and extension (72°C, 1 min), followed by a final 5 min extension at 72°C. PCR products were subsequently electrophoresed on 1% agarose gels containing ethidium bromide.
Cell stimulation
Cells were initially harvested from log phase cultures, resuspended in fresh prewarmed complete medium, and incubated for 3 h at 37°C. They were then stimulated with anti-IgM antibody F(ab')2 fragments for 130 min, after which the reactions were terminated with ice-cold PBS containing 2 mM Na3VO4 and 2 mM EDTA (PBS-VE). After centrifugation, the cells were solubilized in TNE lysis buffer (1% Nonidet P-40, 10 mM TrisHCl, pH 7.5, 150 mM NaCl, 2 mM Na3VO4, 2 mM EDTA, 1 mM NaF, 10 mM sodium pyrophosphate) supplemented with protease inhibitors (1 mg/ml E64, 1 mg/ml pepstatin A, 10 mM benzamidine, 2 mg/ml aprotinin and 100 mg/ml TPCK). The resultant lysates were centrifuged at 10 000 g at 4°C for 30 min, and the supernatants were subjected to further analysis.
Measurement of [Ca2+]i
Assays were performed as previously described (41). Briefly, cells were loaded with the membrane-permeant acetoxymethyl ester of the fluorescent Ca2+ indicator Fluo-3 (Fluo-3AM) (Molecular Probes, Eugene, OR) in 1 ml of KrebsRingerHepes (KRH) buffer (6 mM HepesNaOH, 125 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO47H2O, 2 mM CaCl2, 6 mM glucose, pH 7.4) for 45 min at 37°C. After loading, the cells were washed three times and resuspended in KRH buffer at a density of 106 cells/ml. The cells were then stimulated with anti-mouse IgM antibody F(ab')2 fragments (10 µg/ml), and fluctuations in [Ca2+]i were measured over a 510 min period using an ELITE flow cytometer; mean fluorescence intensity was determined using the Multitime program (Phenix Flow Systems, San Diego, CA). In some experiments, EGTA (2 mM) was added to chelate the extracellular Ca2+ prior to making the measurements. To deplete intracellular calcium stores, 0.2 µM thapsigargin (Tg) was added to cells suspended in Ca2+-free KRH buffer, after which capacitative Ca2+ influx was measured following addition of 0.2 mM CaCl2 to the cell suspension.
Western blot analysis
The NP-40-soluble supernatants from unstimulated and anti-IgM-stimulated cells were separated on 10% SDSPAGE gels and transferred to nitrocellulose membranes for 2 h at 80 V. The membranes were blocked with 5% non-fat dried milk; incubated overnight at 4°C with respective antibodies in buffer containing 20 mM TrisHCl (pH 7.5), 150 mM NaCl, 0.1% Tween-20 and 5% BSA, and then incubated for an additional 1 h at room temperature with alkaline phosphatase- or horseradish peroxidase-conjugated secondary antibodies. The blots were visualized using an Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad Laboratories, Hercules, CA) or ECL Western Blotting Detection Reagent (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturers protocols.
IP3 measurement
IP3 production was measured as described by Choi et al. (44), with a minor modification. Briefly, cells (106/ml) were stimulated with anti-mouse IgM antibody F(ab')2 fragments (20 µg/ml), after which the reaction was terminated by adding ice-cold 100% trichloroacetic acid. IP3 was then extracted from the samples and quantified using an IP3 [3H] radioreceptor assay kit (PerkinElmer Life Sciences, Boston, MA).
Stable transfection
cDNA encoding mouse CD72a (a generous gift from Dr J. R. Parnes, Stanford University Medical Center, Stanford, CA) was subcloned into pEGFP-C3 expression vector. This construct was then transfected into CD72b-deficient 9-59 cells by electroporation, and transfectants were selected in the presence of G418 (1 mg/ml). Expression of the transfected cDNA was confirmed by staining cells with anti-CD72a mAb.
Assay for DNA synthesis
Cells (5 x 103) were cultured for 2 days in 0.2 ml of complete medium in triplicate with goat anti-mouse IgM antibody F(ab')2 fragments (520 µg/ml). To assess DNA synthesis, 0.5 µCi of [3H]thymidine was added to each well for the last 4 h. The cells were then harvested on glass fiber filters using a semiautomatic Skatron harvester, and thymidine incorporation was measured in a Beckman liquid scintillation counter. The results were expressed as percent inhibition of DNA synthesis = 100 [1 (anti-IgM antibody added)/(no antibody added)].
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Results
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Generation and characterization of CD72-deficient clones
CD72-deficient clones were isolated from negatively selected BAL-17 cells following mutagenesis (see Methods). Using this approach, we obtained a CD72-deficient clone 9-59 (Fig. 1). This clone was not reactive with two CD72b (anti-Ly-19.2 and anti-Ly-32.2) mAbs directed against different epitopes of CD72 (Fig. 1A). Furthermore, reverse transcription (RT)PCR analysis with primers that cover the whole CD72 sequence demonstrated expression of CD72 at mRNA level was almost undetectable in 9-59 cells (Fig. 1B). Thus, we conclude 9-59 cells express a negligible level of CD72 protein. The cell surface expression of various proteins was also examined in 9-59 cells as compared to BAL-17 parental cells. As shown in Fig. 1(C), molecules such as IgM, IgD, H-2, Ia, CD45, CD16/CD32, CD19 and CD22 were expressed in 9-59 cells, comparable to the level in BAL-17 cells. These results suggest, but do not necessarily prove, that 9-59 cells are selectively defective in CD72 expression. Therefore, to unequivocally demonstrate the function of CD72, we investigated whether the phenotypes observed in 9-59 cells were reversed by re-expression of CD72.



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Fig. 1. Characterization of CD72-deficient cells generated from BAL-17 cells. (A) BAL-17 and CD72-deficient clone 9-59 cells were stained with anti-CD72b/Ly-19.2 and anti-CD72b/Ly-32.2 mAbs (dotted line) followed by FITC-conjugated goat anti-mouse Igs. Cells stained only with secondary antibody served as background controls (solid line). (B) Total cellular RNAs from BAL-17 and clone 9-59 cells were subjected to RTPCR using CD72-specific and GAPDH-specific primers. (C) BAL-17 and 9-59 cells were stained with FITCanti-mouse IgM, FITCanti-mouse-IgD, FITCanti-CD19 (dotted line) and FITCanti-CD22 (thick solid line) antibodies. Cells were also stained with anti-H-2d, anti-Iad, anti-CD45, and anti-CD16/CD32 mAbs followed by FITC secondary antibodies or FITCPA (dotted lines). Cells without staining served as background controls for expression of IgM, IgD, CD19 and CD22 (thin solid line), while cells stained with FITC secondary antibodies or FITCPA alone served as controls for expression of H-2, Ia, CD45 and CD16/CD32 (thin solid line).
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BCR-induced tyrosine phosphorylation is minimally affected in CD72-deficient cells
We first examined early signaling events induced by BCR ligation. As shown in Fig. 2(A), the level of total protein tyrosine phosphorylation was not significantly different in the parental and CD72-deficient 9-59 cells within 5 min. This is also the case for 1530 min (data not shown). The tyrosine phosphorylation of Syk was not affected by the absence of CD72 (Fig. 2B). The phosphorylation of Lyn seemed to be slightly reduced at 25 min in 9-59 cells. We then examined the phosphorylation of the autophosphorylation site, an indicator of activation of Src-PTKs, by immunoblotting with anti-phospho-Src autophosphorylation site antibody that cross-reacts with phosphorylated Y397 of Lyn. As shown in Fig. 2(B), there was little difference between the parental cells and 9-59 cells. The phosphorylation of the negative regulatory site of Lyn was also comparable to the parental cells. Thus, CD72 exerts minimal effects on the phosphorylation and activation of Lyn and Syk.

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Fig. 2. Initial signaling events are minimally affected by the absence of CD72. (A) BCR-mediated total tyrosine phosphorylation of cellular proteins. BAL-17 parental cells and CD72-deficient clone 9-59 were incubated with F(ab')2 fragments of anti-IgM antibody (10 µg/ml) for the indicated times. Total cell lysates were separated by SDSPAGE and then immunoblotted with anti-PY mAb. (B) BCR-induced tyrosine phosphorylation of Lyn and Syk kinases. Cells were cultured as in (A), after which immunoprecipitated Lyn and Syk were subjected to immunoblotting with anti-PY mAb and with anti-Lyn and anti-Syk antibodies. Lyn was also blotted with anti-phospho-Src (Tyr 416) antibody to monitor the phosphorylation of the autophosphorylation site and with anti-phospho-negative regulatory site of Lyn antibody. These results are representative of two or three experiments.
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BCR-induced calcium influx is selectively impaired in CD72-deficient cells
BCR ligation stimulates synthesis of IP3, which leads to the release of Ca2+ from intracellular stores. It has been proposed that this event is coupled to influx of extracellular Ca2+ through store-operated Ca2+ channels (SOCs) or Ca2+ release-activated Ca2+ channels (CRACs) in the plasma membrane. Our analysis of the Ca2+ responses of parental BAL-17 cells and CD72-deficient 9-59 cells revealed that BCR-mediated release of Ca2+ from intracellular stores (initial transient rise in [Ca2+]i) was comparable in the two cell types, but that the later sustained influx of extracellular Ca2+ was markedly diminished in 9-59 cells (Fig. 3A). Chelation of extracellular Ca2+ using 2 mM EGTA blocked the late phase of the Ca2+ response in the parental cells, but had only a minimal effect on the responses in 9-59 cells. Indeed, the profile of untreated 9-59 cells was virtually superimposable on that of EGTA-treated BAL-17 cells. Apparently, Ca2+ influx is selectively impaired in 9-59 cells.
To examine the function of SOCs in 9-59 cells further, cells were initially treated with Tg, an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases that depletes intracellular Ca2+ stores and elicits capacitative Ca2+ influx. In the absence of extracellular Ca2+, Tg (0.2 µM) induced a transient rise in [Ca2+]i due to release from internal stores (Fig. 3B). Once [Ca2+]i had returned to baseline, addition of 0.2 mM CaCl2 elicited a second peak, due to Ca2+ entry through SOCs, that was only slightly smaller in 9-59 cells. This suggests that the function of the SOCs themselves remained intact in 9-59 cells, but that signals leading to Ca2+ influx were in some way altered. Treatment with vehicle (DMSO) had no effect on either Ca2+ release or Ca2+ influx upon CaCl2 addition. Significantly, the impaired Ca2+ influx was partially restored by re-expression of CD72 (Fig. 3C).
BCR-induced phosphorylation of Btk, PI3K and Vav is reduced in CD72-deficient cells
Btk appears to be involved in regulating Tg-sensitive, IP3-gated Ca2+ stores and therefore Ca2+ influx through SOCs (45). Activation of Btk is thought to be regulated by phosphorylation and membrane localization via binding of its pleckstrin homology domain to PIP3, which is a phosphoinositol product of PI3K. When we assessed levels of Btk activation in parental and 9-59 cells by monitoring its phosphorylation state, we found that in the parental cells Btk phosphorylation was sustained for 5 min after BCR ligation whereas the phosphorylation declined at 25 min in 9-59 cells (Fig. 4). Similarly, BCR-induced tyrosine phosphorylation of p85-PI3K was significantly diminished after 5 min in CD72-deficient cells, and these phenotypes were reversed by re-expression of CD72 (Fig. 4). The Vav family of GEFs for Rho family GTPases is also thought to be involved in the regulation of Ca2+ mobilization and to be regulated by tyrosine phosphorylation (14,46,47). We therefore assayed BCR-mediated tyrosine phosphorylation of Vav and found it to be significantly diminished within 1 min of stimulation in 9-59 cells (Fig. 4). Again, introduction of CD72 into 9-59 cells restored the parental phenotype.

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Fig. 4. Tyrosine phosphorylation of Btk, PI3K and Vav is positively regulated by CD72. BAL-17 parental, CD72-deficient 9-59 and 9-59/CD72a cells were incubated with F(ab')2 fragments of anti-IgM antibody (10 µg/ml) for the indicated times, after which the immunoprecipitated Btk, p85-PI3K and Vav were subjected to immunoblotting with anti-PY mAb and with antibodies against Btk, p85-PI3K or Vav. The blots were subjected to densitometric analysis, and the intensity of each band was normalized to the amount of the protein applied. The numbers below PY blots represent relative intensities, with the intensity of an unstimulated group being arbitrarily set to 1. The results are representative of three separate experiments.
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Tyrosine phosphorylation of CD19 and association of CD19 with PI3K are impaired in CD72-deficient cells
CD19 is implicated in the recruitment and subsequent activation of PI3K, PI hydrolysis and calcium mobilization upon BCR ligation (48,49). We thus examined whether the positive regulatory loop of CD19PI3K association may be disrupted in the absence of CD72. As shown in Fig. 5, phosphorylation of CD19 was strongly induced at 1 min and diminished within 5 min in the parental cells, but this event was severely impaired in 9-59 cells. Accordingly, association of CD19 with PI3K was significantly reduced in CD72-deficient cells, as evidenced by blotting CD19 immunoprecipitates with anti-PI3K antibody (Fig. 5A) and by probing PI3K immunoprecipitates with anti-CD19 antibody (Fig. 5B). This impairment was partially restored by re-expression of CD72.

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Fig. 5. Tyrosine phosphorylation of CD19 and association of CD19 with PI3K are impaired in CD72-deficient cells. (A) BAL-17 parental, 9-59 and 9-59/CD72a cells were incubated with anti-IgM antibody F(ab')2 fragments (10 µg/ml) for the times indicated, and the immunoprecipitated CD19 was subjected to blotting with anti-PY mAb and with anti-PI3K and anti-CD19 antibodies. (B) Cells were stimulated as in (A) and the immunoprecipitated PI3K was subjected to blotting with anti-CD19 and anti-PI3K antibodies. The results representative of three separate experiments are presented as in Fig. 4.
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BCR-induced phosphorylation of PLC
2 and IP3 production is reduced in CD72-deficient cells
Receptor-mediated Ca2+ mobilization is also regulated by BLNK and PLC
2. A current model holds that, upon phosphorylation by Syk and Btk, BLNK recruits PLC
2 and Btk, which leads to phosphorylation and activation of PLC
2 by Btk (50). In addition, CD72 is known to recruit SHP-1 and Grb2 via its ITIMs, enabling Grb2 to associate with BLNK via its SH3 domain (51). We therefore wondered whether CD72 deficiency would affect the phosphorylation states of BLNK and PLC
2. As shown in Fig. 6(A), BCR-mediated phosphorylation of BLNK was not significantly different, but that of PLC
2 was reduced at 5 min in the absence of CD72. Since the phosphorylation state of PLC
2 does not necessarily reflect its activity (45,52,53), we further assayed IP3 production as a downstream indicator of PLC
2 activation and a determining factor for Ca2+ responses. Figure 6(B) shows that IP3 production was not affected at 30 s of stimulation but almost completely ablated at 1 min in 9-59 cells. These phenotypes were reversed by re-expression of CD72. Thus, BCR- mediated PLC
2 activity is positively regulated by CD72, and diminished IP3 production at the peak may contribute to the reduction of Ca2+ influx in CD72-deficient cells.
BCR-induced activation of ERK and JNK, but not p38, is blocked in CD72-deficient cells
We further examined MAPK activation in CD72-deficient cells using phospho-specific MAPK antibodies. In the parental cells, BCR-ligation mediated transient phosphorylation of ERK that peaked within 5 min (Fig. 7). No induction ERK phosphorylation was observed in 9-59 cells; likewise, phosphorylation of JNK was completely blocked in 9-59 cells. Phosphorylation of ERK and JNK was restored by re-expression of CD72. By contrast, phosphorylation of p38 MAPK was minimally affected in 9-59 cells, suggesting that CD72 positively regulates activation of ERK and JNK, but not p38 MAPK, in BAL-17 cells.

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Fig. 7. BCR-mediated activation of ERK and JNK, but not p38 MAPK, is positively regulated by CD72. (A) BAL-17 parental cells, 9-59 cells and 9-59/CD72a cells were incubated with anti-IgM antibody F(ab')2 fragments (10 µg/ml) for the indicated times, after which the total cell lysates were immunoblotted with antibodies against ERK, JNK and p38 and their phosphorylated forms (pERK, pJNK and pp38). The results are representative of four separate experiments. (B) Summarized data are shown as means of fold increase ± SEM in MAPK phosphorylation in the parental cells (solid bars), 9-59 cells (open bars) and 9-59/CD72a cells (gray bars) calculated by densitometric analysis of four separate experiments.
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BCR-induced inhibition of DNA synthesis is reduced in CD72-deficient clones
We finally examined the effect of CD72 deficiency on the final outcome. We have previously showed that BCR ligation induces inhibition of DNA synthesis in BAL-17 cells (41,54). Likewise, in the present study, stimulation with anti-IgM antibody induced a dose-dependent 6080% reduction in DNA synthesis in the parental cells (Fig. 8). On the other hand, this inhibition was reduced by 5060% in the CD72-deficient 9-59 clone at all concentrations of anti-IgM antibody tested, and this inhibition was restored by re-introduction of CD72. These results suggest CD72 is positively involved in BCR-induced signaling pathways leading to inhibition of cell proliferation.

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Fig. 8. BCR-mediated inhibition of DNA synthesis is blocked in CD72-deficient clone. BAL-17, clone 9-59 and 9-59/CD72a cells were cultured in the presence of graded concentrations of anti-mouse IgM antibody F(ab')2 fragments, and [3H]thymidine incorporation was determined on day 2. The results are expressed as means of percent inhibition of DNA synthesis ± SEM of two separate experiments.
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Discussion
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The fate of B cells following antigen binding is determined by many factors, including the strength of the binding, the maturational stage of the cells and the contribution of coreceptors such as CD72, CD22 and CD40. The results of some previous studies suggest that CD72 negatively regulates BCR signaling by recruiting and then serving as a substrate for the protein tyrosine phosphatase SHP-1 (33,34). Consistent with those studies, gene-targeting experiments demonstrated that B cells from CD72/ mice are hyperproliferative and exhibit somewhat enhanced Ca2+ responses (38). On the other hand, other reports suggest that CD72 mediates positive regulatory signaling activating a variety of biochemical events, including Ca2+ mobilization and activation of tyrosine kinases, PI3K, PLC
2 and MAPKs (2832).
To resolve this discrepancy, we re-addressed the cell-autonomous function of CD72 using CD72-deficient cells generated from mature BAL-17 cells. Our results show that BCR-mediated initial events, such as total protein tyrosine phosphorylation and activation of Lyn and Syk kinases were not significantly affected by the absence of CD72. BCR ligation induces biphasic Ca2+ mobilization: initial transient increases in [Ca2+]i due to IP3-evoked release from intracellular stores followed by sustained influx of extracellular Ca2+ through SOCs or CRACs in the plasma membrane. CD72 deficiency almost completely inhibits the Ca2+ influx without affecting the initial Ca2+ release from the intracellular stores. Given that Tg-induced capacitative Ca2+ entry was minimally affected in CD72-deficient cells and that re-expression of CD72 reversed the defective BCR-mediated Ca2+ entry, it appears the impairment of Ca2+ influx is not due to a defect in the SOCs per se, but to defective signaling leading to Ca2+ influx.
To explore which signaling pathways are regulated by CD72, we examined signaling events thought to be situated upstream of Ca2+ influx. CD72 contains two ITIMs in its cytoplasmic domain; upon tyrosine phosphorylation, the first (5ITYADL10) recruits SHP-1 (33,34) and the second (37LTYENV42) recruits Grb2, an adaptor protein required for activation of the Ras/MEK/ERK pathway (34). Previous studies, including ours, have shown that SHP-1 does not act on Lyn or Syk but directly dephosphorylates BLNK (40), and that BLNK associates with CD72 via the SH3 domain of Grb2 (51). It was thus possible to speculate that in the absence of CD72, reduced SHP-1 recruited to the membrane may lead to hyperphosphorylation of BLNK. However, phosphorylation of BLNK was not affected by the absence of CD72. Earliest events affected by the absence of CD72 were tyrosine phosphorylation of CD19 and Vav and recruitment of PI3K to CD19. Vav has been implicated in the regulation of PIP 5-kinase, which catalyzes the synthesis of PIP2. This means that a reduction in Vav phosphorylation would be expected to diminish PIP2 availability and therefore IP3 production. Additionally, given that recruitment of PI3K to CD19 may activate its enzymatic activity (48), impaired recruitment of PI3K would be expected to lead to decreased production of PIP3 and perhaps diminished membrane targeting of signaling molecules required for IP3 production, e.g. Btk and Vav. Indeed, IP3 production was ablated at 1 min without being compromised initially in CD72-deficient cells. This train of initial events explains very well the pathways leading to calcium mobilization, and may be targets of CD72 regulation.
Signaling events occurring further downstream from BCR ligation than Ca2+ mobilizatione.g. activation of ERK and JNK (but not p38 MAPK) and inhibition of DNA synthesisare also affected by CD72 deficiency. Impaired activation of ERK and JNK in CD72-deficient cells may be due respectively to the loss of the Grb2 docking site and the reduced activation of PI3K, which activates Rho and Rac upstream of JNK. Our earlier studies demonstrated that BCR ligation inhibits proliferative responses as measured by DNA synthesis by the concerted actions of JNK and p38 MAPK (41,42,54). The present study showed that BCR-mediated inhibition of DNA synthesis is reduced by 5060% in CD72-deficient clones. The fact that only ERK and JNK are regulated by CD72 might explain why the blockade of BCR-mediated inhibition of DNA synthesis was only partial in CD72-deficient cells. In any case, all of these results indicate CD72 to be a positive regulator of cellular responses induced by BCR ligation.
It is notable that BCR signaling pathways affected by CD72 deficiency are reminiscent of pathways affected by CD72 ligation. Treatment with anti-CD72 antibody stimulates Btk but not Syk PTK, resulting in Syk-independent PLC
2 activation, Ca2+ mobilization and B cell proliferation (24,28). It also induces activation of PI3K recruited to CD19, a transient association of CD72 with CD19 (29), and activation of ERK and JNK but not p38 MAPK (32). Moreover, the observations that CD72-induced B cell proliferation is blocked by PI3K inhibitors and is reduced in B cells from CD19/ mice (29) suggest that activation of CD19-associated PI3K is critical for CD72-triggered B cell responses.
In CD72/ mice, B cell development is blocked at the transitions from pre-B cells to immature B cells and from immature to mature B cells. In the periphery of CD72/ mice, the numbers of mature, long-lived B-2 cells and follicular B cells are reduced, whereas the numbers of B-1 cells and marginal zone B cells are increased. Thus CD72 seems to exert positive and negative effects at distinct maturational stages. In vitro studies of B cells from CD72/ mice show increased proliferation in response to anti-IgM antibody and lipopolysaccharide, and slightly enhanced Ca2+ responses. Given the biased B cell subpopulations in the CD72/ spleeni.e. greater numbers of marginal zone B cells and fewer follicular B cellsthe alterations in Ca2+ mobilization may be masked in part by differences in the responsiveness of the various subpopulations. However, enhanced proliferative responses of resting B cells in CD72/ mice are difficult to reconcile in this study.
There are other indications that CD72 serves as a negative regulator of B cell as revealed by experiments using cell lines. For example, expression of CD72 in K46 mouse B lymphoma cells, into which hapten-specific µ heavy and
light chains were introduced, reduces BCR-induced ERK activation and Ca2+ mobilization (36). Moreover, introduction of CD72 in BCR-expressing myeloma cells (J558L µ3) also inhibits BCR-induced phosphorylation of Ig-
/Ig-ß, Syk and BLNK (37). One caveat is that the cellular milieu of myeloma cells is different from B cells and both of these cell lines lack expression of CD72. This may suggest that CD72 does not have a significant function in these cells. These factors may delicately contribute to the apparently discrepant capacity of CD72 to stimulate or inhibit BCR signaling in different cell types. Additionally, which regulatory molecules, including SHP-1, Grb2 or even as-yet-unidentified proteins, are dominantly recruited to CD72 may dictate the mode of CD72 action in a cell.
In summary, CD72 apparently positively regulates BCR-mediated signaling pathways including phosphorylation of CD19, Vav, Btk and PLC
2, recruitment of PI3K to CD19, IP3 production and Ca2+ influx. Consequently, the signals are relayed to activate ERK and JNK, determining the fate of BAL-17 cells. Given that CD72 is able to recruit both positive and negative regulatory molecules, signals generated by CD72 may be intricately balanced by the stoichiometry of the molecules recruited to it.
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Acknowledgements
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We thank Dr Janes Parnes for plasmid containing CD72a gene, Dr David Saffen for preprints and informative discussion, Dr Tatsuo Katagiri for his initial contribution and Dr Haruo Takemura for technical advice. This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology.
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Abbreviations
|
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BCRB cell antigen receptor
BtkBrutons tyrosine kinase
[Ca2+]icytosolic free Ca2+ concentration
ERKextracellular signal regulated kinase
IP3inositol 1,4,5-trisphosphate
JNKc-Jun NH2-terminal kinase
MAPKmitogen-activated protein kinase
PI3Kphosphatidylinositol 3-kinase
PIP2phosphatidylinositol 4,5-bisphosphate
PIP3phosphatidylinositol 3,4,5-trisphosphate
PLC
2phospholipase C-
2
PTKprotein tyrosine kinase
SOCstore-operated channel
Tgthapsigargin
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