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The Journal of Immunology, 2004, 172: 2753-2762.
Copyright © 2004 by The American Association of Immunologists

Phosphatidylinositol 3-Kinase-Dependent Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase 1/2 and NF-{kappa}B Signaling Pathways Are Required for B Cell Antigen Receptor-Mediated Cyclin D2 Induction in Mature B Cells1

Michael J. Piatelli{dagger}, Carrie Wardle{dagger}, Joseph Blois{dagger}, Cheryl Doughty{dagger}, Brian R. Schram*, Thomas L. Rothstein* and Thomas C. Chiles2,{dagger}

* Departments of Microbiology and Medicine, Boston University School of Medicine, Boston Medical Center, Boston, MA 02118; and {dagger} Department of Biology, Boston College, Chestnut Hill, MA 02467


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Phosphatidylinositol 3-kinase (PI-3K) has been linked to promitogenic responses in splenic B cells following B cell Ag receptor (BCR) cross-linking; however identification of the signaling intermediates that link PI-3K activity to the cell cycle remains incomplete. We show that cyclin D2 induction is blocked by the PI-3K inhibitors wortmannin and LY294002, which coincides with impaired BCR-mediated mitogen-activated protein/extracellular signal-related kinase kinase (MEK)1/2 and p42/44ERK phosphorylation on activation residues. Cyclin D2 induction is virtually absent in B lymphocytes from mice deficient in the class IA PI-3K p85{alpha} regulatory subunit. In contrast to studies with PI-3K inhibitors, which inhibit all classes of PI-3Ks, the p85{alpha} regulatory subunit is not required for BCR-induced MEK1/2 and p42/44ERK phosphorylation, suggesting the contribution of another PI-3K family members in MEK1/2 and p42/44ERK activation. However, p85{alpha}-/- splenic B cells are defective in BCR-induced I{kappa}B kinase {beta} and I{kappa}B{alpha} phosphorylation. We demonstrate that NF-{kappa}B signaling is required for cyclin D2 induction via the BCR in normal B cells, implicating a possible link with the defective I{kappa}B kinase {beta} and I{kappa}B{alpha} phosphorylation in p85{alpha}-/- splenic B cells and their ability to induce cyclin D2. These results indicate that MEK1/2-p42/44ERK and NF-{kappa}B pathways link PI-3K activity to Ag receptor-mediated cyclin D2 induction in splenic B cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Signaling by the B cell Ag receptor (BCR)3 leads to distinct cell fates, including survival, proliferation, or apoptosis. The outcome is dependent, in part, on the developmental stage of the B lymphocyte at the time of BCR engagement and on the signals provided by coreceptors (1). BCR ligation is followed by the activation of several src-family protein tyrosine kinases, Bruton’s tyrosine kinase (Btk), Syk, and the lipid kinase, phosphatidylinositol 3-kinase (PI-3K) (1, 2). These kinases coordinate the activation of signal transduction cascades, including phospholipase C-{gamma} (PLC-{gamma}), Vav, Akt/glycogen synthase kinase 3, and Ras/mitogen-activated protein kinase (MAPK) pathways (3). Efforts continue to focus on the contribution of individual signaling pathways in regulating cell fate responses following Ag binding. Notably, PI-3K has been shown to interface BCR signaling with B cell survival (4, 5, 6, 7, 8, 9).

PI-3K members are classified into three primary groups according to their structure and substrate specificity. The most important group for B cell responses are the class IA PI-3Ks, which comprised a catalytic subunit (p110{alpha}, {beta}, {gamma}) and a regulatory subunit (p85{alpha}, p85{beta}, p55{gamma}) (8, 10). The p85 subunit is recruited to the plasma membrane and activates the p110 subunit in response to receptor ligation. Activation of class IA PI-3Ks results in the rapid phosphorylation of membrane phosphatidylinositol lipids on the D3 position of the inositol ring to generate phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-bisphosphate, or phosphatidylinositol 3,4,5-trisphosphate (8, 9, 10, 11, 12). The resulting 3'-phosphorylated phosphoinositides act to recruit to the plasma membrane and facilitate activation of pleckstrin homology domain-containing proteins (9, 13).

Pharmacologic inhibition of PI-3K activity abolishes BCR-dependent proliferation in murine and normal human B cells and human B lymphoma cells (14, 15, 16, 17). Genetic studies of mice deficient for individual PI-3K catalytic and regulatory subunits point to profound defects in B cell function (18, 19). The p85{alpha} subunit is the most abundantly expressed class IA regulatory subunit (20, 21). Mice lacking only the p85{alpha} regulatory subunit exhibit a developmental block at the pro-B cell stage and show a reduction in the peripheral mature B cell compartment (18). p85{alpha}-null splenic B cells cultured ex vivo have pronounced proliferation defects in response to BCR cross-linking (18, 19). Similarly, mice expressing a catalytically inactive form of the p110{delta} catalytic subunit exhibit a block in pro-B cell development and impaired proliferation in response to BCR engagement (22, 23).

D-type cyclins (D1, D2, and D3) function as positive regulatory subunits for cyclin-dependent kinase (cdk)4 and cdk6, and are rate limiting for G1 phase progression (24, 25, 26, 27, 28, 29). Mitogen receptor-coupled signal transduction pathways regulate D-type cyclin accumulation, whereas cyclin E, which associates with cdk2, is regulated autonomously, peaking near the G1/S transition (25). Several findings contribute to the view that cdk4/6 and cdk2 function to drive cells through G1 phase and initiate S phase entry, respectively (30, 31). The principal substrates of cdk4/6 and cdk2 are members of the retinoblastoma gene product (pRb) family (p107, p110, and p130) (25, 32). In quiescent and early G1 phase cells, unphosphorylated and hypophosphorylated pRb members bind transcription factors, such as E2F, thereby negatively regulating gene transcription (32). Sequential phosphorylation of pRb members by cdk4/6 and cdk2 disrupts the association with E2F, leading to the coordinated transcription of genes involved in G1/S transition and DNA metabolism.

Cyclin D2, and to a lesser extent, cyclin D3 are induced in response to BCR cross-linking (33, 34, 35). The principal target of cyclin D2 in mature splenic B lymphocytes is cdk4 (33). Definitive evidence in support of the need for cyclin D2 in BCR-induced proliferation has come from studies of mice deficient for cyclin D2 (36). Although recent studies have identified individual components necessary for cyclin D2 induction, our understanding of the signal transduction pathways that link the BCR to cyclin D2 remains far from complete. Notably, studies point to a requirement for the adaptor protein, B cell linker protein, and the proto-oncogene, Vav, in BCR-mediated cyclin D2 protein induction (37, 38). Interestingly, neither B cell linker- nor Vav-mediated cyclin D2 induction involve Akt or MAPK activation. Despite these findings, we have implicated mitogen-activated protein/extracellular signal-related kinase kinase (MEK)1/2 activity in BCR-mediated cyclin D2 induction, consistent with the recent reported role for MEK1/2 in ex vivo splenic B cell proliferation (39, 40).

To gain insight into the regulation of cyclin D2 by PI-3K, we used both pharmacologic inhibitors of PI-3K and mice deficient in the class IA PI-3K p85{alpha} regulatory subunit to identify signaling pathways that link PI-3K activity to cyclin D2 induction. Results of this study provide the first reported evidence that MEK1/2-p42/44ERK and I{kappa}B kinase (IKK)-inhibitory {kappa}B{alpha} (I{kappa}B{alpha}) pathways function, in part, to couple PI-3K activity to BCR-mediated cyclin D2 induction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

Anti-cyclin D2, anti-mouse cdk4, anti-JunB (N17), anti-c-Myc, anti-I{kappa}B{alpha}, anti-rabbit IgG-HRP, and anti-mouse IgG-HRP Abs (Abs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-MEK1/2 (S217/S221), anti-MEK1/2, anti-IKK{alpha}, anti-phospho-IKK{alpha} (S180)/IKK{beta} (S181), anti-phospho-p44/42 MAPK (T202/Y204), anti-p44/42 MAPK, anti-Akt, anti-phospho-Akt (S473) Abs, and the anti-phospho-I{kappa}B{alpha} (S32/S36) mAb were purchased from Cell Signaling Technology (Beverly, MA). Human pRb mAb (clone G3-245) was obtained from BD PharMingen (San Diego, CA). The anti-PI-3K p85{alpha} Ab was purchased from Upstate Biotechnology (Lake Placid, NY). Anti-cyclin D3 Ab was obtained from NeoMarkers (Fremont, CA). Anti-phospho-pRb (T821) and anti-phospho-pRb (S807/S811) were obtained from BioSource International (Camarillo, CA). F(ab')2 of goat anti-mouse IgM were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Anti-{beta}-actin mAb, PMA, and ionomycin were obtained from Sigma-Aldrich (St. Louis, MO). NF-{kappa}B p50 subunit cell-permeable inhibitory peptide (SN50), corresponding inactive control peptide (SN50M), and wortmannin were obtained from Biomol (Plymouth Meeting, PA). ECL reagents were obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Phosphatidylinositol substrate was supplied by Avanti Polar Lipids (Alabaster, AL) and TLC plates impregnated with 1% potassium oxalate were obtained from Alltech Associates (Deerfield, IL). U0126 was obtained from Calbiochem-Novabiochem International (San Diego, CA).

Preparation of splenic B lymphocytes

BALB/c mice, p85{alpha}-deficient mice and the corresponding wild-type mice (Balb/cAnNTac-Pik3r1 N12) at 8–12 wk were obtained from Taconic Farms (Germantown, NY). Mice were housed, cared for and handled at all times in accordance with National Institutes of Health and Boston College institutional guidelines. Spleens were removed and dissociated in HBSS (Atlanta Biologicals, Norcross, GA) supplemented with 2% heat-inactivated FCS (Atlanta Biologicals). Macrophages (and other adherent cells) were removed by plastic adherence. RBC and nonviable cells were removed by sedimentation on Lympholyte M (Accurate Chemical and Scientific, Westbury, NY). B lymphocytes were then isolated by negative selection using MACS B Cell Isolation kit (Miltenyi Biotec, Auburn, CA). The cells (107) were incubated with 10 µl biotin-Ab mixture at 4°C (10 min) and then with 20 µl anti-biotin microbeads for 4°C (15 min). Cells were washed and resuspended in ice cold PBS containing 0.5% FCS. The cell suspension was then applied onto an LS magnetic separation column (Miltenyi Biotec). The effluent was collected as a pooled fraction containing unlabeled B cells. The B cells were then washed in PBS plus 0.5% FCS and then cultured in RPMI 1640 (Atlanta Biologicals) supplemented with 10 mM HEPES, pH 7.5, 2 mM L-glutamine, 5 x 10-5 M 2-ME, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS. B cells were maintained in a 37°C humidified incubator at 5% CO2.

In vitro PI-3K assay

B cells (106) were resuspended in 1 ml of PI-3K lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, and 1% Triton X-100) supplemented with 1 mM PMSF, 1 µg/ml aprotinin/leupeptin, and 20 mM {beta}-glycerophosphate. Cell lysates were incubated with 5 µg of isotype-matched control rabbit IgG or rabbit anti-p85{alpha} Ab (Upstate Biotechnology) for 5 h at 4°C. The immune complexes were captured by incubation with 50 µl of 1:1 protein A- to protein G-agarose (1 h), washed six times with 1 ml PI-3K lysis buffer, and twice with PI-3K reaction buffer (30 mM HEPES, pH 7.4, 30 mM MgCl2, and 0.45 mM EGTA) (11). Kinase reactions were conducted in 40 µl of PI-3K reaction buffer supplemented with 10 µCi of [{gamma}-32P]ATP (250 mCi/mmol; PerkinElmer Life Sciences, Boston, MA), 10 µM ATP and 0.2 mg/ml phosphatidylinositol substrate (15 min, 30°C). The reactions were terminated with 100 µl of 1 N HCl, followed by sequential extraction in 200 µl 1:1 chloroform to methanol and 200 µl 1:1 1 N HCl to methanol. The organic phase was then removed, lyophilized, dissolved in 10 µl 1:1 chloroform to methanol, and spotted on Silica TLC plates impregnated with 1% potassium oxalate. The plates were developed by ascending chromatography in a 9:7:2 chloroform to methanol to 4 M NH4OH solvent system, dried, and subjected to phosphoimaging using a Molecular Dynamics PhosphoImager cassette (Molecular Dynamics, Sunnyvale, CA).

RNA isolation and RT-PCR

Total cellular RNA was isolated from 2 x 107 splenic B cells using Ultraspec Reagent (Biotecx Laboratories, Houston, TX) denaturing solution following the manufacturer’s protocol. Purified RNA was treated with 1 U DNase I using DNA-free (Ambion, Austin, TX). First-strand cDNA synthesis was conducted with 2 µg of total RNA using RETROscript First-Strand Synthesis kit (Ambion). The concentration of cDNA in each sample was adjusted to produce equal amounts of product following quantitative PCR with primers specific for {beta}2-microglobulin gene ({beta}2-microglobulin sense primer corresponding to: 5'-cggtcgcttcagtcgtcagc-3' and antisense primer to 5'-cccagtagacggtcttggg-3'). The PCR were performed in 25 µl final reaction volumes each consisting of template cDNA using reagents from the GeneAmp PCR kit (Roche Molecular Systems, Branchburg, NJ) supplemented with 10 µCi of [{alpha}-32P]dCTP (6000 Ci/mmol; PerkinElmer Life Sciences). Amplification was conducted in an Eppendorf MasterCycler gradient (Brinkmann Instruments, Westbury, NY) as described (41). PCR products (20 µl) were separated through an 8% polyacrylamide gel and visualized by autoradiography. Under these conditions, the number of PCR cycles and the amount of cDNA used were such that increasing amounts of cDNA template yielded proportionately increasing amounts of {beta}2-microglobulin-specific PCR product (data not shown). The variation of {beta}2-microglobulin PCR product between adjusted samples was ±5%, as judged by densitometric analysis of the resulting autoradiograms using a Molecular Dynamics PhosphoImager. The number of cycles and amount of cDNA used was then adjusted accordingly to yield the same signal output. Cyclin D2 cDNA was amplified from the normalized {beta}2-microglobulin cDNA samples using the identical PCR and temperature cycling parameters. The sequence of the cyclin D2 primer set corresponds to cyclin D2 sense primer 5'-agctgtccctgatccgcaag-3' and antisense primer 5'-gtcaacatcccgcacgtctg-3'.

TAT-coupled I{kappa}B{alpha}-dominant negative (TAT-I{kappa}B{alpha}-DN) purification

The pTAT-HA expression vector contains a six-histidine tag and the TAT transduction domain (provided by Dr. S. Dowdy, University of California at San Diego, La Jolla, CA) (42). The construction of the pTAT-HA expression vector containing I{kappa}B{alpha}-DN has been previously described (43). The pTAT-I{kappa}B{alpha}-DN was used to express the TAT-I{kappa}B{alpha}-DN fusion protein in the BL21 (DE3) strain of Escherichia coli. In brief, the cells were induced for 6 h with 0.1 mM isopropyl {beta}-D-1-thiogalactospyranoside, sonicated in 8 M urea, and then the TAT-I{kappa}B{alpha}-DN protein was purified by sequential chromatography on a nickel-Sepharose column (Qiagen, Valencia, CA) and ion exchange column (Mono Q) in 4 M urea. Shock-misfolding of the protein was conducted by replacing the 4 M urea with aqueous buffer (20 mM HEPES, pH 8.0) during the last ion exchange chromatographic step. TAT-I{kappa}B{alpha}-DN was eluted with a gradient from 50 mM to 1 M NaCl, followed by desalting on a PD-10 column (Amersham Pharmacia Biotech, Sunnyvale, CA) into PBS and storage in 10% glycerol at -80°C. The pTAT-green fluorescence protein (GFP) control plasmid used in these experiments was the gift of Dr. S. Dowdy and was prepared in a similar manner as the TAT-I{kappa}B{alpha}-DN.

EMSA

Nuclei were isolated by hypotonic lysis and extracted in a 450 mM NaCl buffer as previously described (44). Binding reactions were conducted in a final volume of 15 µl and contained 1.5 µg of nuclear protein extract, 0.5 µg of double stranded poly(dI:dC), and 10,000 cpm of a DNA-labeled probe containing an NF-{kappa}B binding element derived from the murine Ig{kappa} L chain enhancer (45). DNA probes were labeled with [{gamma}-32P]ATP by T4 polynucleotide kinase (New England Biolabs, Beverly, MA). After 20 min (23°C), the reaction products were electrophoresed through a 5% polyacrylamide/TBE gel and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PI-3K activity is required for BCR-induced early G1 phase progression in ex vivo splenic B cells

Pretreatment of ex vivo splenic B cells with the PI-3K inhibitor, wortmannin inhibited de novo RNA synthesis induced by 10 µg/ml F(ab')2 of goat anti-mouse anti-IgM (anti-Ig), as measured by [3H]uridine incorporation, in a dose-dependent manner (Fig. 1A) (46); maximal inhibition by wortmannin occurred at a concentration of 50 nM. Similar results were obtained with the selective PI-3K inhibitor, LY294002 (data not shown). The inhibition of RNA synthesis coincided with an early block of endogenous pRb phosphorylation (Fig. 1A, inset, top lane); phosphorylation of pRb on T821 by cyclin E-cdk2 complexes was also impaired in the presence of 50 nM wortmannin, a modification that serves as an indicator of late G1 phase progression (Fig. 1A, inset, bottom lane).



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  		FIGURE 1. PI-3K activity is required for anti-Ig-induced cell cycle entry in ex vivo splenic B lymphocytes. A, Splenic B cells were cultured in medium alone (M) or stimulated with 10 µg/ml anti-Ig ({alpha}Ig), following a 30 min preincubation with wortmannin (Wtn) at the indicated concentrations. [3H]Uridine incorporation was measured at 24 h to monitor G1 phase entry. The inset shows the level of total endogenous pRb phosphorylation at 24 h in parallel B cell cultures and pRb phosphorylation by cyclin E-cdk2 in B cells treated in the absence (-) and presence (+) of 50 nM wortmannin, as determined by Western blot with anti-pRb mAb that recognizes both hypo- and hyperphosphorylated pRb and anti-phospho-pRbThr821 Ab, respectively. B, B cells were incubated in the absence (-) and presence (+) of 50 nM wortmannin (30 min) and then cultured for 5 min in medium (M) or 10 µg/ml anti-Ig ({alpha}Ig). Cells were detergent solubilized and incubated with 4 µg nonimmune Ig or anti-p85{alpha} Ab. PI-3K activity was measured in the immune complexes as described in Materials and Methods. The position of the spots corresponds to the origin and phosphatidylinositol 3,4-bisphosphate. C, B cells were cultured in medium (M) or stimulated with anti-Ig ({alpha}Ig) for 15 min, following a 30 min preincubation in the absence (-) and presence (+) of 50 nM wortmannin. Whole cell extracts were prepared for Western blotting with anti-phospho-S473-Akt (pAkt(S473)) and anti-Akt Abs. These data are representative of three independent experiments. Note, the non-wortmannin treated B cell cultures contained an equal percentage of DMSO vehicle. DMSO had no measurable effect on [3H]uridine incorporation, phosphatidylinositol 3,4-bisphosphate production, or total and phospho-S473-Akt levels (data not shown).

 
To confirm that wortmannin inhibits PI-3K activity under the conditions used, we measured PI-3K activity in vitro by immune complex kinase assay. Preincubation of ex vivo splenic B cells with 50 nM wortmannin inhibited BCR-induced PI-3K activity when compared with parallel B cells treated with the solvent control (Fig. 1B). We also investigated anti-Ig-induced Akt activation, a downstream target of PI-3K, by Western blotting with an Ab that detects phosphorylation on S473, a modification known to correlate with its kinase activity (47). We found that anti-Ig-induced Akt phosphorylation was inhibited following preincubation with 50 nM wortmannin before stimulation with anti-Ig (Fig. 1C, top lane).

Requirement for PI-3K activity in BCR-mediated cyclin D2 induction and phosphorylation of endogenous pRb by D-type cyclin-cdk4/6 complexes

We sought to determine whether PI-3K activity is required for BCR-mediated cyclin D2 induction in ex vivo splenic B cells. RNA was isolated from splenic B cells stimulated with 10 µg/ml anti-Ig for 4 and 18 h, and cyclin D2 gene expression was then measured by RT-PCR as described (39). Cyclin D2 mRNA was induced ~3- and 7-fold in response to BCR cross-linking at 4 and 18 h, respectively (Fig. 2A). Anti-Ig-stimulated cyclin D2 mRNA accumulation was markedly reduced by wortmannin when measured at the 4 and 18 h time points (Fig. 2A).



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  		FIGURE 2. Inhibition of PI-3K activity blocks BCR-induced cyclin D2 mRNA and protein accumulation and endogenous pRb phosphorylation in ex vivo splenic B lymphocytes. A, Quiescent splenic B cells (time 0) were incubated for 4 and 18 h with 10 µg/ml anti-Ig ({alpha}Ig) in the absence (-) and presence (+) of a 30 min pretreatment with 50 nM wortmannin (Wtn). RT-PCR was conducted as described in Materials and Methods. Cyclin D2 expression is reported as a proportion (fold induction) of expression present in unstimulated (time 0) B cell samples. The inset shows the corresponding autoradiogram of constitutively expressed {beta}2-microglobulin ({beta}2-MG) and cyclin D2 RT-PCR analysis. These data are representative of three independent experiments. B, Splenic B cells were cultured in medium alone (M) or were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for the indicated times, following a 30 min preincubation period in the absence (-) or presence (+) of 50 nM wortmannin. Whole cell lysates were prepared for Western blotting of the indicated cell cycle proteins. Equal protein loading was controlled by stripping the membranes and probing with anti-{beta}-actin Ab. A representative Western blot of {beta}-actin is shown for the anti-cyclin D2 membrane. Note, the non-wortmannin treated B cell cultures contained an equal percentage of DMSO vehicle. C, B cells were cultured in medium alone (M) or were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for the indicated times, following a 30 min preincubation period in the absence (-) or presence (+) of 10 µM LY294002. Whole cell lysates were prepared, and cyclin D2 and pRb phosphorylation on S807/S811 was detected by Western blot. The non-LY294002-treated B cell cultures contained an equal percentage of solvent vehicle. Also shown is a Western blot of {beta}-actin for the anti-cyclin D2 membrane.

 
The requirement for PI-3K activity was mirrored at the level of cyclin D2 protein induction in that wortmannin led to a nearly complete block of anti-Ig-induced cyclin D2 protein accumulation (Fig. 2B). The inhibition of cyclin D2 protein induction by wortmannin did not arise from changes in the total amount of cellular protein being compared across conditions as evidenced by equal levels of {beta}-actin protein (Fig. 2B). Moreover, there was no detectable induction of cyclin D3 in anti-Ig-stimulated B cells pretreated with wortmannin (Fig. 2B). Although cellular cdk4 levels were not measurably affected by inhibiting PI-3K activity (Fig. 2B), the lack of sustained cyclin D2 (and cyclin D3) accumulation is likely sufficient to prevent activation of endogenous cdk4 necessary for pRb phosphorylation. In support of this, wortmannin-treated splenic B cells exhibited a near complete block in BCR-induced pRb phosphorylation on D-type cyclin-cdk4/6-targeted residues (S807/S811) (Fig. 2B) (48). We observed similar results with the PI-3K inhibitor, LY294002. Notably, anti-Ig induced cyclin D2 protein and endogenous pRb phosphorylation on cdk4/6-targeted residues (S808/811) were blocked in comparison to B cells devoid of LY294002 (Fig. 2C).

BCR-mediated activation of the cyclin D2/cdk4-pRb pathway is impaired in ex vivo splenic B cells from p85{alpha}-deficient mice

To corroborate the findings that pharmacologic inhibition of PI-3K activity results in defective BCR-induced cyclin D2 induction and subsequent pRb phosphorylation by D-type cyclin-cdk4/6 complexes, we examined ex vivo splenic B cells isolated from mice deficient in the p85{alpha} subunit of PI-3K (18). Splenic B cells from p85{alpha}-deficient mice do not express detectable levels of the p85{alpha} subunit (Fig. 3A). In addition, p85{alpha}-deficient B cells exhibited markedly reduced BCR-mediated Akt phosphorylation on S473 in comparison to B cells isolated from the corresponding wild-type mice (Fig. 3A). p85{alpha}-null splenic B cells exhibited a near complete loss of BCR-mediated cyclin D2 mRNA and protein induction in comparison to wild-type B cells (Fig. 3, B and C, respectively). As a positive control for these experiments, cyclin D2 mRNA and protein levels were induced in the p85{alpha}-/- splenic B cells following stimulation with phorbol ester (PMA) plus the calcium ionophore, ionomycin (P/I) (Fig. 3, B and C, end lanes). These reagents are mitogenic for ex vivo splenic B cells and act to circumvent the proximal signaling components of the BCR, including PI-3K (49).



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  		FIGURE 3. Lack of cyclin D2 induction and impaired activation of endogenous D-type cyclin-cdk4/6 complexes in anti-Ig stimulated p85{alpha}-/- ex vivo splenic B cells. A, Whole cell lysates were prepared from p85{alpha}-/- and wild-type (WT) splenic B cells and Western blotted for p85{alpha} subunit expression using an anti-p85{alpha} Ab. Splenic B cells from p85{alpha}-/- and wild-type mice were cultured in medium alone (M) or were stimulated for 15 min with 10 µg/ml anti-Ig ({alpha}Ig). The levels of cellular phospho-S473-Akt (pAkt(S473)) and total Akt were determined by Western blotting. B, Wild-type (WT) and p85{alpha}-/- splenic B cells cultured in medium (M) or were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for the indicated times. p85{alpha}-/- splenic B cells were also stimulated with 300 ng/ml PMA and 400 ng/ml ionomycin (P/I). RT-PCR was conducted as described in Materials and Methods. Cyclin D2 expression is reported as a proportion (fold induction) of expression present in unstimulated (M) B cell samples. The inset shows the corresponding autoradiogram of {beta}2-microglobulin ({beta}2-MG) and cyclin D2 RT-PCR analysis. C, Wild-type (+/+) and p85{alpha}-/- (-/-) B cells were cultured in medium alone (M) or were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for the indicated times. Whole cell lysates were prepared for Western blotting of the indicated cell cycle proteins. Equal protein loading was controlled by stripping the membranes and probing with anti-{beta}-actin Ab. A representative Western blot of {beta}-actin is shown for the anti-cyclin D2 membrane. These data are representative of three independent experiments.

 
Anti-Ig-stimulated cyclin D3 induction was also impaired in p85{alpha}-/- splenic B cells relative to their wild-type counterparts (Fig. 3C). Despite the expression of cdk4 in p85{alpha}-deficient B cells (albeit at a level somewhat lower in comparison to wild-type B cells), endogenous pRb phosphorylation and D-type cyclin-cdk4/6-targeted phosphorylation on S807/S811 following BCR cross-linking was not detected under the conditions used (Fig. 3C). However, these cell cycle events were induced in parallel cultures of p85{alpha}-/- splenic B cells stimulated with PMA and ionomycin (Fig. 3C). These results indicate that BCR-mediated cyclin D2 induction and subsequent phosphorylation of endogenous pRb by D-type cyclin holoenzymes requires expression of p85{alpha} gene products of PI-3K.

BCR-induced cyclin D2 accumulation requires PI-3K-coupled signaling through MEK1/2-ERK in normal splenic B lymphocytes

A previous report from our laboratory demonstrated that MEK1/2-ERK activity is required for cyclin D2 mRNA induction in response to BCR ligation (39). Therefore, we investigated whether impaired induction of cyclin D2 caused by PI-3K inhibition may be due to inhibition of MEK1/2-ERK activation. As shown in Fig. 4A, BCR-induced phosphorylation of MEK1/2 on activation residues S217/S221 was inhibited in splenic B cells pretreated with wortmannin. Moreover, anti-Ig-induced phosphorylation of p42/44ERK on activation residues T202/Y204 was equally sensitive to wortmannin (Fig. 4A). Importantly, PMA and ionomycin-induced MEK1/2 and p42/44ERK phosphorylation was not sensitive to wortmannin (P/I, Fig. 4A, right side lanes). Phosphorylation of p42/44ERK was also inhibited by pretreatment of anti-Ig-stimulated B cells with LY294002 (Fig. 4A). Accordingly, preincubation of splenic B cells with the highly specific inhibitor, U0126, led to a near complete block of anti-Ig-stimulated cyclin D2 mRNA and protein induction (Fig. 4B). Taken together, these data suggest that MEK1/2-p42/44ERK activity is dependent upon a PI-3K activity. Moreover, the data suggest that MEK1/2 functions, at least in part, to link a PI-3K activity to BCR-induced cyclin D2 expression.



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  		FIGURE 4. BCR-mediated cyclin D2 induction requires PI-3K-coupled MEK1/2 and ERK signaling in normal splenic B cells. A, Splenic B lymphocytes (M) were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for the indicated times, with (+Wtn) or without (-Wtn) a 30 min pretreatment with 50 nM wortmannin (Wtn). Whole cell extracts were prepared and Western blotted for phospho-MEK1/2. Cells were pretreated (30 min) with 50 nM wortmannin or 10 µM LY294002 (LY) for 5 and 10 min and whole cell extracts Western blotted for phospho-p42/44ERK. B cells were also stimulated (10 min) with 300 ng/ml PMA and 400 ng/ml ionomycin (P/I), following a pretreatment in the absence (-) and presence (+) of 50 nM wortmannin and Western blotted for phospho-p42/44ERK. The membranes were stripped and reprobed with Abs to the nonphosphorylated forms of MEK1/2 and p42/44ERK. B, B cells were cultured in medium alone (M) or stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for 4 h in the absence (-) or presence (+) of 10 µM U0126. Cyclin D2 and {beta}2-microglobulin ({beta}2-MG) mRNA expression were measured by RT-PCR as described in Materials and Methods. The levels of cyclin D2 and {beta}-actin protein expression were also measured in parallel splenic B cell cultures. These data are representative of four independent experiments.

 
In contrast to the experiments using pharmacologic inhibitors of PI-3K, which inhibit all three classes of PI-3Ks, anti-Ig-induced phosphorylation of MEK1/2 on activation residues was induced in ex vivo p85{alpha}-/- splenic B cells in a manner similar to wild-type B cells (Fig. 5A). There was also no significant difference between the ability of anti-Ig-stimulated p85{alpha}-/- and wild-type splenic B cells to induce p42/44ERK phosphorylation (Fig. 5A). These results, taken together with the pharmacologic inhibitor data (Fig. 4A) suggest that BCR-induced MEK1/2-p42/44ERK phosphorylation in splenic B cells is dependent on a PI-3K family member that does not require the p85{alpha} subunit.



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  		FIGURE 5. Impaired BCR-induced IKK{beta} and I{kappa}B{alpha}-, but not MEK1/2-p42/44ERK phosphorylation in p85{alpha}-/- splenic B cells. A, Wild-type and p85{alpha}-/- splenic B cells were cultured in medium alone (M) or were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for the indicated times. Whole cell extracts were prepared and Western blotted for phospho-MEK1/2 and phospho-p42/44ERK. The corresponding membranes were stripped and reprobed with Abs to the nonphosphorylated forms of MEK1/2 and p42/44ERK. B, Whole cell extracts were also Western blotted with Abs specific for phospho-IKK{alpha}(S180)/IKK{beta}(S181) and phospho-I{kappa}B{alpha} (S32/S36). The corresponding membranes were stripped and reprobed with Abs that recognize the nonphosphorylated forms of IKK{alpha} and I{kappa}B{alpha}. Comparisons of protein loading between B cell types were monitored by Western blotting for constitutively expressed hsp90. C, B lymphocytes (M) were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for the indicated times, with (+Wtn) or without (-Wtn) a 30 min pretreatment with 50 nM wortmannin (Wtn). Whole cell extracts were prepared and Western blotted for phospho-IKK{alpha}(S180)/IKK{beta}(S181) and phospho-I{kappa}B{alpha}(S32/S36). The corresponding membranes were stripped and reprobed with Abs that recognize the nonphosphorylated forms of IKK{alpha} and I{kappa}B{alpha}. Equal protein loading of I{kappa}B{alpha} was confirmed by Western blotting with anti-{beta}-actin. These data are representative of three independent experiments.

 
BCR-induced phosphorylation of IKK{beta} and I{kappa}B{alpha} is impaired in p85{alpha}-deficient splenic B cells

We interpret the observations in p85{alpha}-null splenic B cells to implicate additional signaling components, downstream of p85{alpha}, in cyclin D2 induction. We investigated the activation of several known BCR-coupled signaling pathways and, in data not shown, anti-Ig-stimulated phosphorylation of the two remaining classes of mammalian MAPKs, c-Jun NH2-terminal kinase and p38 MAPK in p85{alpha}-/- splenic B cells was similar to that of wild-type B cells. Moreover, there was no detectable difference between anti-Ig-stimulated calcium mobilization in p85{alpha}-/- splenic B cells and wild-type B cells. We also examined NF-{kappa}B signaling in p85{alpha}-/- splenic B cells. BCR ligation results in the phosphorylation of cytosolic I{kappa}B by the IKK complex, which consists of two catalytic subunits, IKK{alpha} and IKK{beta} and a regulatory subunit, IKK{gamma} (50). Phosphorylation of I{kappa}B targets it for degradation via the ubiquitin-dependent proteasome pathway, which releases NF-{kappa}B/c-Rel subunits for subsequent translocation into the nucleus (51, 52). Stimulation of the wild-type B cells with 10 µg/ml anti-Ig resulted in increased phosphorylation of IKK{alpha}/IKK{beta}, as measured by Western blotting of cellular extracts with a highly specific Ab that recognizes IKK{alpha} and IKK{beta} phosphorylated on S180 and S181, respectively (Fig. 5B); these modifications are known to correlate with catalytic activation of IKK{alpha} and IKK{beta} (53). In contrast, phosphorylation of IKK{beta} was virtually absent and IKK{alpha} phosphorylation was diminished in anti-Ig-treated p85{alpha}-/- splenic B cells (Fig. 5B). As a control, we found that the total cellular levels of IKK{beta} (and IKK{alpha}) were similar in p85{alpha}-deficient and wild-type B cells (Fig. 5B). BCR cross-linking induced phosphorylation of I{kappa}B{alpha} on activation residues, S32/S36 and its degradation was detected at the 10 min time point in the wild-type B cell population, whereas anti-Ig-stimulated phosphorylation of I{kappa}B{alpha} was impaired in p85{alpha}-/- splenic B cells (Fig. 5B).

We next sought to ascertain whether the impaired IKK{beta} and I{kappa}B{alpha} phosphorylation observed in p85{alpha}-/- B cells could be recapitulated in normal BALB/c splenic B cells pretreated with wortmannin. Stimulation of normal B cells with anti-Ig resulted in increased cellular phospho-IKK{beta}, whereas phospho-IKK{alpha} levels remained relatively unchanged in comparison to unstimulated B cells (Fig. 5C). Interestingly, Saijo et al. (54) recently reported a similar pattern of IKK{alpha}/IKK{beta} phosphorylation in ex vivo splenic B cells from C57BL/6 mice. Anti-Ig induced the phosphorylation of I{kappa}B{alpha}, and at the 20 min time point, appeared to initiate its degradation (Fig. 5C). In contrast, pretreatment of splenic B cells with wortmannin inhibited anti-Ig-induced IKK{beta} and I{kappa}B{alpha} phosphorylation at the time points examined (Fig. 5C).

NF-{kappa}B inhibitor SN50 blocks BCR-induced cyclin D2 induction and endogenous pRb phosphorylation in splenic B cells

To investigate whether disruption of NF-{kappa}B signaling in normal splenic B cells might lead to impaired cyclin D2 induction following BCR cross-linking, we adopted the approach of Lin et al. (55) to block nuclear import of NF-{kappa}B with the in vivo delivery of a synthetic peptide SN50 (55). This peptide contains the nuclear localization sequence (NLS residues 360–369) of NF-{kappa}B p50 subunit coupled to the membrane-permeable signal peptide of Kaposi fibroblast growth factor. The Kaposi fibroblast growth factor domain allows dose-dependent uptake into virtually all cells (55). Results in Fig. 6A demonstrate that preincubation of ex vivo splenic B cells with 75 µM SN50 significantly reduced BCR-mediated cyclin D2 induction. Moreover, BCR-induced phosphorylation of endogenous pRb on cdk4/6-targeted S807/S811 was reduced. Pretreatment of parallel B cells with a control mutant peptide (SN50M), which fails to block NF-{kappa}B nuclear translocation due to an inactive NLS (55), did not decrease BCR-mediated cyclin D2 induction or pRb phosphorylation by cdk4/6 in comparison to nontreated anti-Ig stimulated B cells. In control experiments, we observed that at the concentration used to inhibit BCR-induced cyclin D2 induction, SN50 peptide blocked the induction of c-Myc, an immediate downstream target of NF-{kappa}B activity (Fig. 6B) (56). By contrast, induction of Jun-B in response to BCR ligation was not inhibited by SN50 (44). Note, Jun-B protein migrates as a doublet in SDS-polyacrylamide gels (44). Furthermore, the functional effect of the SN50 peptide on nuclear translocation of NF-{kappa}B/Rel complexes in B cells stimulated by BCR cross-linking was determined by EMSA. As shown in Fig. 6C, BCR-induced NF-{kappa}B binding activity in nuclear extracts was inhibited by SN50 peptide, whereas the mutant SN50M peptide was without measurable effect.



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  		FIGURE 6. Inhibition of NF-{kappa}B with SN50 blocks BCR-mediated cyclin D2 induction in normal splenic B cells. A, Splenic B lymphocytes were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for the indicated times, with or without a 30 min pretreatment with 75 µM SN50 or with the corresponding mutant (SN50M) as described (55 ). Whole cell extracts were prepared and Western blotted for cyclin D2. The membrane was stripped and reprobed sequentially for phospho-pRb(S807/S811) and anti-{beta}-actin. B, B cells (M) were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for 1.5 h, with or without a 30 min pretreatment with 75 µM SN50 or SN50M. Whole cell extracts were prepared and Western blotted for c-Myc and Jun-B. C, B cells were cultured in medium alone (M) or in the presence of 10 µg/ml anti-Ig ({alpha}Ig) for 2 h, with or without a 30 min pretreatment with 75 µM SN50 or SN50M, as indicated. Nuclear extracts were prepared and analyzed by EMSA. These data are representative of three independent experiments. Note, the inhibitors had no effect on constitutive Myc levels or NF-{kappa}B binding activity present in unstimulated B cells (data not shown). P, Migration of the DNA probe in the absence of nuclear extract.

 
TAT-I{kappa}B{alpha}-DN fusion protein blocks anti-Ig-induced cyclin D2 gene expression and pRb phosphorylation in splenic B lymphocytes

We also sought to inhibit NF-{kappa}B activity by selectively targeting a key upstream regulator of NF-{kappa}B. For these studies, a TAT-I{kappa}B{alpha}-DN fusion protein was used that is incapable of phosphorylation on S32/S36 due to alanine substitution at these sites. The TAT-I{kappa}B{alpha}-DN fusion protein contains a short peptide sequence from the HIV TAT protein, which confers the ability to rapidly and efficiently (>90%) transduce proteins up to 115 kDa into primary mammalian cells in a near equivalent concentration (42). The capacity of TAT-I{kappa}B{alpha}-DN to block NF-{kappa}B activation in splenic B cells has been demonstrated in a recent study by Rothstein and coworker (43) inasmuch as induction of nuclear NF-{kappa}B binding activity measured by EMSA was blocked by TAT-I{kappa}B{alpha}-DN, but not TAT-GFP. Moreover, Jun-B/AP-1 DNA-binding activity was not affected by either TAT-I{kappa}B{alpha}-DN or TAT-GFP (43). TAT-I{kappa}B{alpha}-DN was used to determine the effect of blocking NF-{kappa}B activation on BCR-inducible cyclin D2 expression. Results in Fig. 7 indicate that incubation of B cells with TAT-I{kappa}B{alpha}-DN inhibited anti-Ig-induced cyclin D2 induction at the protein and RNA levels (Fig. 7, A and B, respectively). Moreover, endogenous pRb phosphorylation on S807/S811 was effectively blocked at the time points examined. It should be noted that cell viability was not decreased by the TAT-I{kappa}B{alpha}-DN (data not shown). Moreover, the inhibition of cyclin D2 induction and pRb phosphorylation by anti-Ig was not reproduced by incubating B cells with TAT-GFP, which had no effect. Thus, two selective inhibitors of NF-{kappa}B activation (SN50 and TAT-I{kappa}B{alpha}-DN) that target distinct components of the NF-{kappa}B pathway inhibit induction of cyclin D2 produced by BCR engagement.



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  		FIGURE 7. TAT-I{kappa}B{alpha}-DN blocks anti-Ig-mediated cyclin D2 induction and endogenous pRb phosphorylation. A, Splenic B lymphocytes were stimulated with 10 µg/ml anti-Ig ({alpha}Ig) for 12 and 24 h, with or without a 30 min pretreatment with 3 ng/ml TAT-I{kappa}B{alpha}-DN or TAT-GFP as described (43 ). Whole cell extracts were prepared and Western blotted for cyclin D2. The membrane was stripped and reprobed for phospho-pRb(S807/S811). B, B cells (M) were incubated for 6 h with 10 µg/ml anti-Ig ({alpha}Ig) in the absence and presence of 3 ng/ml TAT-I{kappa}B{alpha}-DN or TAT-GFP. RT-PCR was conducted as described in Materials and Methods. Cyclin D2 expression is reported as a proportion (fold induction) of expression present in unstimulated (M) B cell samples. The inset shows the corresponding autoradiogram of constitutively expressed {beta}2-microglobulin ({beta}2MG) and cyclin D2 RT-PCR analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A role for PI-3K activity in BCR-mediated cyclin D2 induction is provided by the findings that PI-3K inhibitors, wortmannin and LY294002 block BCR-mediated cyclin D2 induction in splenic B cells. Moreover, a role for PI-3K is further supported by studies in splenic B cells from mice deficient for the p85{alpha} gene product. In this study, we provide compelling experimental evidence for the lack of detectable BCR-mediated cyclin D2 mRNA and protein induction in p85{alpha}-/- ex vivo splenic B cells. Given the essential role of cyclin D2 in G1-to-S phase progression (36), it is probable that such impairment in BCR-mediated accumulation of cyclin D2 results in the reported defective mitogenic capacity of p85{alpha}-null splenic B cells (18, 19).

In dissecting potential mechanisms underlying defective cyclin D2 induction in the absence of p85{alpha} gene products, we find that BCR-induced MEK1/2 and p42/44ERK phosphorylation on activating residues is not significantly diminished in p85{alpha}-/- splenic B cells compared with their wild-type B cell counterparts. This result contrasts with our observations in normal splenic B cells treated with wortmannin in which a requirement for PI-3K is clearly established in BCR-initiated MEK1/2-p42/44ERK phosphorylation. We also observed that LY294004 blocks p42/44ERK phosphorylation following BCR cross-linking, in agreement with a recent report by Jacob et al. (57). The molecular basis underlying this discrepancy is currently unknown; however, because all mammalian class I, II, and III PI-3K members show sensitivity to wortmannin and LY294004, it is plausible that BCR-dependent MEK1/2-p42/44ERK activation requires PI-3K members other than class IA (58). It is also conceivable that MEK1/2-p42/44ERK activation may rely more heavily on p85{beta} subunit expression. Notably, B cells from p85{alpha}-null mice used in this study express p85{beta}, albeit at a relatively low level, in comparison to wild-type B cells (59). Alternatively, it remains possible that a pool of p110 catalytic subunits may be recruited to the membrane and activated by Ras (60). It is interesting to note that we observed a very low level of BCR-induced Akt phosphorylation on S473 in p85{alpha}-null splenic B cells, suggesting residual PI-3K activity. Notwithstanding, the data suggest that BCR signaling selectively uses a p85{alpha}-independent and wortmannin- (and LY294002)-sensitive PI-3K family members to signal MEK1/2-p42/44ERK phosphorylation. These results, taken together with the sensitivity of cyclin D2 induction to wortmannin and the MEK1/2 inhibitor, U0126 (as shown in our study and in Ref. 39), suggest a role for MEK1/2-p42/44ERK in linking PI-3K activity to BCR-mediated cyclin D2 induction.

To identify why cyclin D2 induction is defective in p85{alpha}-/- splenic B cells, we investigated the activation of NF-{kappa}B signaling, which is known to regulate genes involved in cell proliferation and survival (61, 62, 63, 64). We found that BCR-induced IKK{beta} and I{kappa}B{alpha} phosphorylation is impaired in p85{alpha}-/- splenic B cells. It is therefore likely that p85{alpha} expression is required for activation of these key components of the NF-{kappa}B pathway in splenic B cells. In keeping with the defective IKK{beta} and I{kappa}B{alpha} phosphorylation in p85{alpha}-null B cells, analysis of these signaling intermediates in normal BALB/c splenic B cells treated with wortmannin reveals markedly diminished Ag receptor-mediated phosphorylation of IKK{beta} and I{kappa}B{alpha}. Based on these results, we investigated whether impaired NF-{kappa}B signaling might plausibly explain the defect in cyclin D2 induction in p85{alpha}-/- splenic B cells as well as wortmannin-treated normal B cells. In support of this, we find that blocking nuclear translocation of NF-{kappa}B/Rel complexes in normal splenic B cells with a cell permeable SN50 peptide carrying a functional NF-{kappa}B domain NLS (55), markedly reduces BCR-mediated cyclin D2 induction and endogenous pRb phosphorylation by D-type cyclin-cdk4/6. Similar results are obtained following transduction of splenic B cells with a TAT-I{kappa}B{alpha}-DN that blocks NF-{kappa}B activation. Taken together, these results suggest that components of NF-{kappa}B pathway function to link PI-3K family members to cyclin D2 induction in response to BCR ligation.

Our results raise an important question as to how PI-3K activation initiates IKK{beta}-I{kappa}B{alpha} signaling. An important consideration is the downstream effector of PI-3K, Akt, which has been reported to directly interact with and phosphorylate IKK (65, 66). However, the protein kinase C (PKC) inhibitor, Gö6983 blocks BCR-induce I{kappa}B{alpha} degradation without affecting Akt phosphorylation, suggesting that PKC may represent an upstream regulator of IKK in splenic B cells (64, 67). Of note, inhibition of BCR-mediated capacitative calcium entry and PKC activation blocks cyclin D2 induction (37, 64). A direct role for PKC in NF-{kappa}B signaling is supported by a recent report that BCR-induced IKK{alpha}/IKK{beta}-I{kappa}B{alpha} phosphorylation is impaired in PKC-{beta}-/- splenic B cells (54). Accordingly, BCR-induced phosphorylation of I{kappa}B{alpha} is defective in phospholipase C-{gamma}2-null and X-linked immunodeficient B cells, the latter of which expresses a mutant Btk (68, 69, 70). It is widely accepted that PI-3K activity is responsible for the activation of Btk by recruiting it to the plasma membrane through interactions between the pleckstrin homology domain of Btk and phosphatidylinositol 3,4,5-trisphosphate (71). Alternatively, PI-3K might initiate NF-{kappa}B activation via the downstream effector serine/threonine kinase, PDK1, which has been shown to phosphorylate and activate conventional PKCs and PKC-{zeta} and -{delta} (72, 73).

The molecular mechanism underlying NF-{kappa}B-dependent regulation of cyclin D2 induction in response to BCR ligation may involve Myc inasmuch as c-myc transcription is regulated by NF-{kappa}B/Rel factors and inhibition of PI-3K activity blocks nuclear import of NF-{kappa}B1/c-Rel dimers concomitant with a failure to up-regulate c-myc (56, 61, 74, 75). Likewise, expression of a conditional Myc-estrogen receptor fusion protein is sufficient to induce cyclin D2 (76). In contrast, the mechanism by which PI-3K-coupled MEK1/2-ERK signaling regulates cyclin D2 induction in response to BCR ligation has not been defined. It does not appear however, that MEK1/2-ERK is directly involved in induction of NF-{kappa}B/c-Rel, as the MEK1/2 inhibitor, PD98059 does not affect endogenous nor transgenic c-myc expression in murine B cells (61).

In summary, our results indicate that PI-3K-dependent MEK1/2-ERK and NF-{kappa}B signaling contribute to BCR-mediated cyclin D2 induction. The results in this study do not preclude a role for PI-3K controlling the activation of other signaling pathways, which in addition to MEK1/2-ERK and NF-{kappa}B may be essential in BCR-induced cyclin D2 expression.



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  		FIGURE 8. A model representing BCR signaling to cyclin D2 gene in splenic B lymphocytes. The findings in this study support a role for a p85{alpha} subunit-independent, wortmannin-sensitive, MEK1/2 and ERK1/2 signaling module and p85{alpha} subunit-dependent NF-{kappa}B pathway that couples the BCR to cyclin D2 gene expression in B cells. A, BCR signaling to cyclin D2 gene expression in wild-type B cells. B, Corresponding signaling in p85{alpha}-deficient B cells, wherein BCR-coupled MEK1/2 and ERK1/2 signaling is intact and NF-{kappa}B signaling defective (denoted as blur). *, NF-{kappa}B signaling may couple the BCR to cyclin D2 gene induction via a pathway involving c-myc gene expression (56 61 74 ). PM, Plasma membrane; NM, Nuclear membrane.

 

   Acknowledgments
 
We thank Fay Dufort for assistance with the uridine assays.


   Footnotes
 
1 This work was supported by U.S. Public Health Sevice Grant AI49994 (to T.C.C.) and AI40181 (to T.L.R.) awarded by the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Thomas C. Chiles, Department of Biology, Boston College, 414 Higgins Hall, Chestnut Hill, MA 02467. E-mail address: ChilesT{at}bc.edu Back

3 Abbreviations used in this paper: BCR, B cell Ag receptor; Btk, Bruton’s tyrosine kinase; cdk, cyclin-dependent kinase; ERK, extracellular signal-regulated kinase; PI-3K, phosphatidylinositol 3-kinase; GFP, green fluorescence protein; I{kappa}B, inhibitory of NF-{kappa}B; IKK, I{kappa}B kinase; MAPK, mitogen-activated protein kinase; DN, dominant-negative; NLS, nuclear localization sequence; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; PKC, protein kinase C; pRb, retinoblastoma gene product. Back

Received for publication June 30, 2003. Accepted for publication December 15, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Craxton, A., K. L. Otipoby, A. Jiang, E. A. Clark. 1999. Signal transduction pathways that regulate the fate of B lymphocytes. Adv. Immunol. 73:79.[Medline]
  2. Sefton, B. M., J. A. Taddie. 1994. Role of tyrosine kinases in lymphocyte activation. Curr. Opin. Immunol. 6:372.[Medline]
  3. Campbell, K. S.. 1999. Signal transduction from the B cell antigen-receptor. Curr. Opin. Immunol. 11:256.[Medline]
  4. Banerji, L., J. Glassford, N. C. Lea, S. B. Thomas, G. B. G. Klaus, E. W.-F. Lam. 2001. BCR signals target p27Kip1 and cyclin D2 via the PI3-K signalling pathway to mediate cell cycle arrest and apoptosis of WEHI 231 B cells. Oncogene 20:7352.[Medline]
  5. Scheid, M. P., J. R. Woodgett. 2001. PKB/AKT: functional insights from genetic models. Nat. Rev. Mol. Cell Biol. 2:760.[Medline]
  6. Pogue, S. L., T. Kurosaki, J. Bolen, R. Herbst. 2000. B cell antigen receptor-induced activation of Akt promotes B cell survival and is dependent on Syk kinase. J. Immunol. 165:1300.[Abstract/Free Full Text]
  7. Muscarella, D. E., S. E. Bloom. 2003. Cross-linking of surface IgM in the Burkitt’s lymphoma cell line ST486 provides protection against arsenite- and stress-induced apoptosis that is mediated by ERK and phosphoinositide 3-kinase signaling pathways. J. Biol. Chem. 278:4358.[Abstract/Free Full Text]
  8. Fruman, D. A., L. C. Cantley. 2002. Phosphoinositide 3-kinase in immunological systems. Semin. Immunol. 14:7.[Medline]
  9. Gold, M. R., M. P. Scheid, L. Santos, M. Dang-Lawson, R. A. Roth, L. Matsuuchi, V. Duronio, D. L. Krebs. 1999. The B cell antigen receptor activates the Akt (protein kinase B)/glycogen synthase kinase-3 signaling pathway via phosphatidylinositol 3-kinase. J. Immunol. 163:1894.[Abstract/Free Full Text]
  10. Koyasu, S.. 2003. The role of PI3K in immune cells. Nat. Immun. 4:313.
  11. Gold, M. R., R. Aebersold. 1994. Both phosphatidylinositol 3-kinase and phosphatidylinositol 4-kinase products are increased by antigen receptor signaling in B cells. J. Immunol. 152:42.[Abstract]
  12. Gold, M. R., V. W. Chan, C. W. Turck, A. L. DeFranco. 1992. Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes. J. Immunol. 148:2012.[Abstract]
  13. Gold, M. R.. 2000. Intermediary signaling effectors coupling the B-cell receptor to the nucleus. Curr. Top. Microbiol. Immunol. 245:77.[Medline]
  14. Brennan, P., A. M. Mehl, M. Jones, M. Rowe. 2002. Phosphatidylinositol 3-kinase is essential for the proliferation of lymphoblastoid cells. Oncogene 21:1263.[Medline]
  15. Aagaard-Tillery, K. M., D. F. Jelinek. 1996. Phosphatidylinositol 3-kinase activation in normal human B lymphocytes. J. Immunol. 156:4543.[Abstract]
  16. Beckwith, M., R. G. Fenton, I. M. Katona, D. L. Longo. 1996. Phosphatidylinositol-3-kinase activity is required for the anti-Ig-mediated growth inhibition of a human B-lymphoma cell line. Blood 87:202.[Abstract/Free Full Text]
  17. Venkataraman, C., P. J. Lu, A. M. Buhl, C. S. Chen, J. C. Cambier, S. Bondada. 1998. CD72-mediated B cell activation involves recruitment of CD19 and activation of phosphatidylinositol 3-kinase. Eur. J. Immunol. 28:3003.[Medline]
  18. Suzuki, H., Y. Terauchi, M. Fujiwara, S. Aizawa, Y. Yazaki, T. Kadowaki, S. Koyasu. 1999. Xid-like immunodeficiency in mice with disruption of the p85{alpha} subunit of phosphoinositide 3-kinase. Science 283:390.[Abstract/Free Full Text]
  19. Fruman, D. A., S. B. Snapper, C. M. Yballe, L. Davidson, J. Y. Yu, F. W. Alt, L. C. Cantley. 1999. Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85{alpha}. Science 283:393.[Abstract/Free Full Text]
  20. Fruman, D. A., L. C. Cantley, C. L. Carpenter. 1996. Structural organization and alternative splicing of the murine phosphoinositide 3-kinase p85 {alpha} gene. Genomics 37:113.[Medline]
  21. Inukai, K., M. Funaki, T. Ogihara, H. Katagiri, A. Kanda, M. Anai, Y. Fukushima, T. Hosaka, M. Suzuki, B. C. Shin, et al 1997. p85{alpha} gene generates three isoforms of regulatory subunit for phosphatidylinositol 3-kinase (PI 3-kinase), p50{alpha}, p55{alpha}, and p85{alpha}, with different PI 3-kinase activity elevating responses to insulin. J. Biol. Chem. 272:7873.[Abstract/Free Full Text]
  22. Jou, S. T., N. Carpino, Y. Takahashi, R. Piekorz, J. R. Chao, N. Carpino, D. Wang, J. N. Ihle. 2002. Essential, nonredundant role for the phosphoinositide 3-kinase p110{delta} in signaling by the B-cell receptor complex. Mol. Cell. Biol. 22:8580.[Abstract/Free Full Text]
  23. Okkenhaug, K., A. Bilancio, G. Farjot, H. Priddle, S. Sancho, E. Peskett, W. Pearce, S. E. Meek, A. Salpekar, M. D. Waterfield, et al 2002. Impaired B and T cell antigen receptor signaling in p110{delta} PI 3-kinase mutant mice. Science 297:1031.[Abstract/Free Full Text]
  24. Pardee, A. B.. 1989. G1 events and regulation of cell proliferation. Science 246:603.[Abstract/Free Full Text]
  25. Ekholm, S., S. I. Reed. 2000. Regulation of G1 cyclin-dependent kinases in the mammalian cell cycle. Curr. Opin. Cell Biol. 12:676.[Medline]
  26. Sicinski, P., J. L. Donaher, S. B. Parker, T. Li, A. Fazeli, H. Gardner, S. Z. Haslam, R. T. Bronson, S. J. Elledge, R. A. Weinberg. 1995. Cyclin D1 provides a link between development and oncogenesis in the retina and breast. Cell 82:621.[Medline]
  27. Resnitzky, D., M. Gossen, H. Bujard, S. I. Reed. 1994. Acceleration of the G1/S phase transition by expression of cyclins D1 and E with an inducible system. Mol. Cell. Biol. 14:1669.[Abstract/Free Full Text]
  28. Baldin, V., J. Lukas, M. J. Marcote, M. Pagano, G. Draetta. 1993. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 7:812.[Abstract/Free Full Text]
  29. Matsushime, H., D. E. Quelle., S. A. Shurtleff, M. Shibuya, C. J. Sherr, J.-Y. Kato. 1994. D-type cyclin-dependent kinase activity in mammalian cells. Mol. Cell. Biol. 14:2066.[Abstract/Free Full Text]
  30. Lundberg, A. S., R. A. Weinberg. 1998. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes. Mol. Cell. Biol. 18:753.[Abstract/Free Full Text]
  31. Harbour, J. W., R. X. Luo, A. Dei Santi, A. A. Postigo, D. C. Dean. 1999. Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell 98:859.[Medline]
  32. Dyson, N.. 1998. The regulation of E2F by pRB-family proteins. Genes Dev. 12:2245.[Free Full Text]
  33. Tanguay, D. A., T. C. Chiles. 1996. Regulation of the catalytic subunit (p34PSK-J3/cdk4) for the major D-type cyclin in mature B lymphocytes. J. Immunol. 156:539.[Abstract]
  34. Solvason, N., W. W. Wu, N. Kabra, X. Wu, E. Lees, M. Howard. 1996. Induction of cell cycle regulatory proteins in anti-immunoglobulin-stimulated mature B lymphocytes. J. Exp. Med. 184:407.[Abstract/Free Full Text]
  35. Reid, S., E. C. Snow. 1996. The regulated expression of cell cycle-related proteins as B-lymphocytes enter and progress through the G1 cell cycle stage following delivery of complete versus partial activation stimuli. Mol. Immunol. 33:1139.[Medline]
  36. Solvason, N., W. W. Wu, D. Parry, D. Mahony, E. Lam, J. Glassford, G. Klaus, P. Sicinski, R. Weinberg, Y. J. Liu, M. Howard, E. Lees. 2000. Cyclin D2 is essential for BCR-mediated proliferation and CD5 B cell development. Int. Immunol. 12:631.[Abstract/Free Full Text]
  37. Tan, J. E.-L., S.-C. Wong, S. K. Gan, S. Xu, K.-P. Lam. 2001. The adaptor protein BLNK is required for B cell antigen receptor-induced activation of nuclear factor-{kappa}B and cell cycle entry and survival of B lymphocytes. J. Biol. Chem. 276:20055.[Abstract/Free Full Text]
  38. Glassford, J., M. Holman, L. Banerji, E. Clayton, G. G. B. Klaus, M. Turner, E. W.-F. Lam. 2001. Vav is required for cyclin D2 induction and proliferation of mouse B lymphocytes activated via the antigen receptor. J. Biol. Chem. 276:41040.[Abstract/Free Full Text]
  39. Piatelli, M. J., C. Doughty, T. C. Chiles. 2002. Requirement for a hsp90 chaperone-dependent MEK1/2-ERK pathway for B cell antigen receptor-induced cyclin D2 expression in mature B lymphocytes. J. Biol. Chem. 277:12144.[Abstract/Free Full Text]
  40. Richards, J. D., S. H. Davé, C.-H. G. Chou, A. A. Mamchak, A. L. DeFranco. 2001. Inhibition of the MEK/ERK signaling pathway blocks a subset of B cell responses to antigen. J. Immunol. 166:3855.[Abstract/Free Full Text]
  41. Huo, L., T. L. Rothstein. 1995. Receptor-specific induction of individual AP-1 components in B lymphocytes. J. Immunol. 154:3300.[Abstract]
  42. Nagahara, H., A. M. Vocero-Akbani, E. L. Snyder, A. Ho, D. G. Latham, N. A. Lissy, M. Becker-Hapak, S. A. Ezhevsky, S. F. Dowdy. 1998. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nat. Med. 4:1449.[Medline]
  43. Schram, B. R., T. L. Rothstein. 2003. NF-{kappa}B is required for surface Ig-induced Fas resistance in B cells. J. Immunol. 170:3118.[Abstract/Free Full Text]
  44. Chiles, T. C., T. L. Rothstein. 1992. Surface Ig receptor-induced nuclear AP-1-dependent gene expression in B lymphocytes. J. Immunol. 149:825.[Abstract]
  45. Liu, J. L., T. C. Chiles, R. J. Sen, T. L. Rothstein. 1991. Inducible nuclear expression of NF-{kappa}B in primary B cells stimulated through the surface Ig receptor. J. Immunol. 146:1685.[Abstract]
  46. DeFranco, A. L., E. S. Raveche, R. Asofsky, W. E. Paul. 1985. Separate control of B lymphocyte early activation and proliferation in response to anti-IgM antibodies. J. Immunol. 135:87.[Abstract]
  47. Burgering, B. M., P. J. Coffer. 1995. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376:599.[Medline]
  48. Zarkowska, T., S. Mittnacht. 1997. Differential phosphorylation of the retinoblastoma protein by G1/S cyclin-dependent kinases. J. Biol. Chem. 272:12738.[Abstract/Free Full Text]
  49. Rothstein, T. L., T. R. Baeker, R. A. Miller, D. L. Kolber. 1986. Stimulation of murine B cells by the combination of calcium ionophore plus phorbol ester. Cell. Immunol. 102:364.[Medline]
  50. Senftleben, U., Y. Cao, G. Xiao, F. R. Greten, G. Krahn, G. Bonizzi, Y. Chen, Y. Hu, A. Fong, S. C. Sun, M. Karin. 2001. Activation by IKK{alpha} of a second, evolutionary conserved, NF-{kappa}B signaling pathway. Science 293:1495.[Abstract/Free Full Text]
  51. Karin, M., Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu. Rev. Immunol. 18:621.[Medline]
  52. Silverman, N., T. Maniatis. 2001. NF-{kappa}B signaling pathways in mammalian and insect innate immunity. Genes Dev. 15:2321.[Free Full Text]
  53. Delhase, M., M. Hayakawa, Y. Chen, M. Karin. 1999. Positive and negative regulation of I{kappa}B kinase activity through IKK{beta} subunit phosphorylation. Science 284:309.[Abstract/Free Full Text]
  54. Saijo, K., I. Mecklenbräuker, A. Santana, M. Leitger, C. Schmedt, A. Tarakhovsky. 2002. Protein kinase C {beta} controls nuclear factor {kappa}B activation in B cells through selective regulation of the I{kappa}B kinase {alpha}. J. Exp. Med. 195:1647.[Abstract/Free Full Text]
  55. Lin, Y.-Z., S. Yao, R. A. Veach, T. R. Torgerson, J. Hawiger. 1995. Inhibition of nuclear translocation of transcription factor NF-{kappa}B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J. Biol. Chem. 270:14255.[Abstract/Free Full Text]
  56. Lee, H., M. Wu, F. A. La Rosa, M. P. Duyao, A. J. Buckler, G. E. Sonenshein. 1995. Role of the Rel-family of transcription factors in the regulation of c-myc gene transcription and apoptosis of WEHI 231 murine B-cells. Curr. Top. Microbiol. Immunol. 194:247.[Medline]
  57. Jacob, A., D. Cooney, M. Pradhan, K. M. Coggeshall. 2002. Convergence of signaling pathways on the activation of ERK in B cells. J. Biol. Chem. 277:23420.[Abstract/Free Full Text]
  58. Vanhaesebroeck, B., M. D. Waterfield. 1999. Signaling by distinct classes of phosphoinositide 3-kinases. Exp. Cell Res. 253:239.[Medline]
  59. Suzuki, H., S. Matsuda, Y. Terauch, M. Fujiwara, T. Ohteki, T. Asano, T. W. Behrens, T. Kouro, K. Takatsu, T. Kadowaki, S. Koyasu. 2003. PI3K and Btk differentially regulate B cell antigen receptor-mediated signal transduction. Nat. Immun. 4:280.
  60. Rodriguez-Viciana, P., P. H. Warne, B. Vanhaesebroeck, M. D. Waterfield, J. Downward. 1996. Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation. EMBO J. 15:2442.[Medline]
  61. Grumont, R. J., A. Strasser, S. Gerondakis. 2002. B cell growth is controlled by phosphatidylinositol 3-kinase-dependent induction of Rel/NF-{kappa}B regulated c-myc transcription. Mol. Cell 10:1283.[Medline]
  62. Grumont, R. J., I. J. Rourke, L. A. O’ Reilly, A. Strasser, K. Miyake, W. C. Sha, S. Gerondakis. 1998. B lymphocytes differentially use the Rel and nuclear factor {kappa}B1 (NF-{kappa}B1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells. J. Exp. Med. 187:663.[Abstract/Free Full Text]
  63. Baldwin, A. S., Jr. 1996. The NF-{kappa}B and I{kappa}B proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649.[Medline]
  64. Glassford, J., I. Soeiro, S. M. Skarell, L. Banerji, M. Holman, G. G. B. Klaus, T. Kadowaki, S. Koyasu, E. W.-F. Lam. 2003. BCR targets cyclin D2 via Btk and the p85{alpha} subunit of PI3-K to induce cell cycle progression in primary mouse B cells. Oncogene 22:2248.[Medline]
  65. Romashkova, J. A., S. S. Makarov. 1999. NF-{kappa}B is a target of AKT in anti-apoptotic PDGF signalling. Nature 401:88.
  66. Ozes, O. N., L. D. Mayo, J. A. Gustin, S. R. Pfeffer, L. M. Pfeffer, D. B. Donner. 1999. NF-{kappa}B activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature 401:82.[Medline]
  67. Bone, H., N. A. Williams. 2001. Antigen-receptor cross-linking and lipopolysaccharide trigger distinct phosphoinositide 3-kinase-dependent pathways to NF-{kappa}B activation in primary B cells. Int. Immunol. 13:807.[Abstract/Free Full Text]
  68. Petro, J. B., S. M. Rahman, D. W. Ballard, W. N. Khan. 2000. Bruton’s tyrosine kinase is required for activation of I{kappa}B kinase and nuclear factor {kappa}B in response to B cell receptor engagement. J. Exp. Med. 191:1745.[Abstract/Free Full Text]
  69. Bajpai, U. D., K. Zhang, M. Teutsch, R. Sen, H. H. Wortis. 2000. Bruton’s tyrosine kinase links the B cell receptor to nuclear factor {kappa}B activation. J. Exp. Med. 191:1735.[Abstract/Free Full Text]
  70. Petro, J. B., W. N. Khan. 2001. Phospholipase C-{gamma}2 couples Bruton’s tyrosine kinase to the NF-{kappa}B signaling pathway in B lymphocytes. J. Biol. Chem. 266:1715.
  71. Fukuda, M., T. Kojima, H. Kabayama, K. Mikoshiba. 1996. Mutation of the pleckstrin homology domain of Bruton’s tyrosine kinase in immunodeficiency impaired inositol 1, 3, 4, 5-tetrakisphosphate binding capacity. J. Biol. Chem. 271:30303.[Abstract/Free Full Text]
  72. Le Good, J. A., W. H. Ziegler, D. B. Parekh, D. R. Alessi, P. Cohen, P. J. Parker. 1998. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281:2042.[Abstract/Free Full Text]
  73. Dutil, E. M., A. Toker, A. C. Newton. 1998. Regulation of conventional protein kinase C isozymes by phosphoinositide-dependent kinase 1 (PDK-1). Curr. Biol. 8:1366.[Medline]
  74. Perez-Roger, I., S. H. Kim, B. Griffiths, A. Sewing, H. Land. 1999. Cyclins D1 and D2 mediate myc-induced proliferation via sequestration of p27Kip1 and p21Cip1. EMBO J. 18:5310.[Medline]
  75. Bouchard, C., K. Thieke, A. Mairer, R. Saffrich, J. Hanley-Hyde, W. Ansorge, S. Reed, P. Sicinski, J. Bartek, M. Eilers. 1999. Direct induction of cyclin D2 by Myc contributes to cell cycle progression and sequestration of p27. EMBO J. 18:5321.[Medline]
  76. Coller, H. A., C. Grandori, P. Tamayo, T. Colbert, E. S. Lander, R. N. Eisenman, T. R. Golub. 2000. Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc. Natl. Acad. Sci. USA 97:3260.[Abstract/Free Full Text]



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