Antigen-receptor cross-linking and lipopolysaccharide trigger distinct phosphoinositide 3-kinase-dependent pathways to NF-{kappa}B activation in primary B cells

Heather Bone and Neil A. Williams

Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK

Correspondence to: N. A. Williams


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The NF-{kappa}B/Rel transcription factors play an important role in the expression of genes involved in B cell development, differentiation and function. Nuclear NF-{kappa}B is induced in B cells by engagement of either the BCR or CD40 or by stimulation with lipopolysaccharide (LPS). Despite the importance of NF-{kappa}B to B cell function, little is known about the signaling pathways leading to NF-{kappa}B activation. In this report we address the role of phosphoinositide 3'-kinase (PI 3-kinase) in BCR- and LPS-induced NF-{kappa}B activation using populations of primary murine resting B cells. Using the specific pharmacological inhibitors of PI 3-kinase, Wortmannin and LY294002, we demonstrate that PI 3-kinase activity is vital for BCR-induced NF-{kappa}B DNA-binding activity. Furthermore, we show that this is achieved via protein kinase C-dependent degradation of I{kappa}B{alpha}. Similar analyses reveal that PI 3-kinase is also critical in triggering NF-{kappa}B DNA-binding activity and I{kappa}B{alpha} degradation following LPS stimulation. Interestingly, a PKC inhibitor which blocked the BCR-induced I{kappa}B{alpha} degradation had no effect on the degradation of I{kappa}B{alpha} after LPS stimulation. Taken together, our results indicate the involvement of PI 3-kinase in at least two distinct signaling pathways leading to activation of NF-{kappa}B in B cells.

Keywords: BCR, lipopolysaccharide, protein kinase B, protein kinase C


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Engagement of the BCR on mature B cells initiates signaling pathways resulting in multiple cellular responses including activation, proliferation and differentiation (1). Much work has recently gone into defining the signaling events which follow BCR engagement and that ultimately control the fate of mature B cells through activation of various transcription factors. BCR engagement initially results in activation of non-receptor protein tyrosine kinases which include Fyn, Lyn, Syk and Btk (2). One of the critical downstream events following activation of these protein tyrosine kinases is the recruitment of the lipid metabolizing enzymes phospholipase C (PLC)-{gamma}2 and phosphoinositide 3'-kinase (PI 3-kinase) to the plasma membrane. Activated Btk, together with Syk, phosphorylate and activate PLC-{gamma}2 (3,4), resulting in hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], and production of the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (5). In turn, these second messengers stimulate the activity of protein kinase C (PKC) and increase intracellular calcium levels, resulting in activation of downstream transcription factors (6,7).

Activation of PI 3-kinase in response to BCR engagement is not well characterized, but both Lyn and Syk have been shown to be involved (810). Only the class Ia PI 3-kinases have so far been shown to be activated as part of this process (8,9). The class Ia PI 3-kinases are heterodimeric enzymes composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110) (11). Once activated, PI 3-kinase phosphorylates PI(4,5)P2 on the D3 position of the inositol ring to generate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] (11). This process is critical in normal B cell function, as revealed by studies which demonstrate impaired proliferation and differentiation of normal human B lymphocytes and of a human B lymphoma cell line following BCR cross-linking in the presence of PI 3-kinase inhibitors (12,13). Further, mice lacking the regulatory p85 subunit of PI 3-kinase exhibit profound defects in B cell function, including decreased proliferative responses and impaired survival (14,15).

Despite the crucial role PI 3-kinase plays in B cells, the downstream signaling events following PI 3-kinase activation have only recently begun to be addressed. In B cells, the pleckstrin homology (PH) domain-containing kinases Btk and Akt (also termed protein kinase B) are activated in a PI 3-kinase-dependent manner (1618). Btk, which phosphorylates and activates PLC-{gamma}2, is recruited to the plasma membrane via the binding of its PH domain to PI(3,4,5)P3 (4,1921). Mutations in the btk gene result in X-linked immunodeficiency (xid) in mice (22). Peripheral B cells in xid mice are profoundly reduced in numbers and are unable to proliferate in response to BCR stimulation (23). Activation of the serine/threonine kinase Akt similarly involves translocation to the plasma membrane by binding via its PH domain to PI(3,4,5)P3 (24). Akt plays an important role in cell survival in various cell types and recently has been demonstrated to be involved in B cell survival (25). In addition, recruitment of the exchange factors Sos and Vav, which activate Rac1, is facilitated by their association with membrane PI(3,4,5)P3 (26,27), pointing towards a further role for PI 3-kinase in mediating downstream processes following BCR engagement.

Like PI 3-kinase, the transcription factor NF-{kappa}B is activated in response to BCR stimulation. In primary B cells, NF-{kappa}B is activated not only by BCR cross-linking, but also by lipopolysaccharide (LPS), phorbol esters and CD40 ligation (28,29). The NF-{kappa}B/Rel transcription factor family of proteins include p50, p52, RelA, c-Rel and RelB, which form homo- or heterodimers (30). NF-{kappa}B/Rel dimers are sequestered in the cytoplasm in an inactive complex bound to members of I{kappa}B family of inhibitory proteins such as I{kappa}B{alpha} (31). Upon cellular activation, I{kappa}B{alpha} is rapidly phosphorylated by a multi-component I{kappa}B kinase (IKK), which targets the inhibitor for degradation by the ubiquitin–proteasome pathway (31). Degradation of I{kappa}B{alpha} reveals the nuclear localization sequence of the NF-{kappa}B/Rel dimers which subsequently translocate to the nucleus where they regulate transcription of various target genes involved in immune and inflammatory responses (32,33).

NF-{kappa}B is constitutively active in the nucleus of resting, peripheral B lymphocytes (34). However, upon stimulation, additional NF-{kappa}B nuclear translocation occurs. NF-{kappa}B/Rel transcription factors are involved in many processes that are important for B cell development, differentiation and function (30,33). This is highlighted in studies of mice deficient in c-Rel, RelA, RelB or p50, which show a marked reduction in proliferative responses to stimulation with LPS, CD40 ligation and BCR cross-linking (3539). Further, mice expressing a trans-dominant form of I{kappa}B{alpha}, which blocks NF-{kappa}B mobilization to the nucleus, show defects in BCR-induced proliferation and differentiation (40). Interestingly, these defects in B cell function were strikingly similar to those observed in the mice lacking the regulatory subunit of PI 3-kinase (14,15).

Although it is clear that BCR stimulation leads to the activation of NF-{kappa}B, and despite the importance of NF-{kappa}B to B cell proliferation and differentiation, the mechanism leading to NF-{kappa}B activation in B cells is poorly understood. Recent studies in other cells suggest the involvement of PI 3-kinase and its downstream kinase Akt in a pathway leading to activation of NF-{kappa}B (4143). As PI 3-kinase and NF-{kappa}B are both essential for normal B cell function, and in light of these studies, we investigated the significance of PI 3-kinase activation in the NF-{kappa}B signaling pathway in primary murine B cells. Using the specific pharmacological inhibitors of PI 3-kinase, Wortmannin and the structurally distinct LY294002 compound, we provide evidence that the lipid products of PI 3-kinase are an important part of the signaling pathway leading to activation of NF-{kappa}B by the BCR and LPS.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Preparation of primary B cells
Spleens from female NIH mice (obtained from Harlan Olac, Bicester, UK; used between 8 and 12 weeks of age) were excised and cells were dispersed by being pressed through stainless steel gauze. Depletion of erythrocytes was achieved by incubation in ACK lysis buffer (BioWhittaker, Walkersville, MD), followed by washing in HBSS containing 20 mM HEPES. B cells were isolated via magnetic cell sorting by negative selection using anti-CD43 magnetic beads and MACS separation columns (Miltenyi Biotec, Bisley, UK). B cell preparations were 90–95% pure as determined by flow cytometry.

B cell stimulation
B cells were cultured (at 2x107/ml for PI 3-kinase assays and at 1x107/ml for other experiments) and stimulated at 37°C in RPMI 1640 medium supplemented with 20 mM HEPES, 50 µM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine [and 5% FCS for electrophoretic mobility shift assays (EMSA)]. Cells were untreated or pre-treated for 30 min with the PI 3-kinase inhibitors Wortmannin (Calbiochem, Nottingham, UK) or LY294002 (Calbiochem) at the concentrations indicated. Further experiments used the PKC inhibitor, Gö6983 (Calbiochem), which was added to B cells for 30 min prior to stimulation. The optimal concentration of Gö6983 (500 nM) was determined as the lowest concentration required to completely inhibit BCR-induced I{kappa}B{alpha} degradation—a known PKC-dependent process. Cells were then stimulated with 10 µg/ml anti-murine IgM F(ab')2 (Jackson ImmunoResearch, Westgrove, PA) for BCR cross-linking or 50 µg/ml LPS (Sigma, Poole, UK) for the times indicated. Cells that were not stimulated were incubated under the same conditions for the duration of the stimulation.

PI 3-kinase assay
Following stimulation, B cells were pelleted in a microcenrtifuge for 20 s and resuspended in 0.5 ml ice-cold lysis buffer [1% (v/v) Triton X-100, 50 mM Tris-HCl, pH 7.5, 10% (v/v) glycerol, 150 mM NaCl, 5 mM EDTA, 1 mM sodium vanadate, 10 mM sodium fluoride, 1 mM sodium molybdate, 1 mM PMSF, 2 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 5 µg/ml leupeptin and 0.7 µg/ml pepstatin]. The lysates were centrifuged at 14,000 r.p.m. and the supernatants removed. Immunoprecipitation was performed on the supernatants by incubating at 4°C for 30 min with 1 µg 4G10 anti-phosphotyrosine antibody (Upstate Biotechnology, Lake Placid, NY) followed by the addition of Protein A–Sepharose beads [30 µl of a 50% (v/v) slurry] and incubation at 4°C on a rotator for 1 h. The immunoprecipitates were washed 3 times with lysis buffer, once with PBS, twice with 0.5 M LiCl, 100 mM Tris, pH 7.6, once in H2O and once in kinase buffer (5 mM MgCl2, 0.25 mM EDTA and 20 mM HEPES, pH 7.4). The washed immunoprecipitates were resuspended in 40 µl kinase buffer and 50 µl of a lipid mixture [0.5 mg/ml phosphatidylinositol (PtdIns) and 0.5 mg/ml phosphatidylserine (Sigma), dispersed by sonication in 25 mM HEPES, pH 7.4, and 1 mM EDTA]. The reaction was initiated by the addition of 10 µCi of [{gamma}-32P]ATP (ICN Biomedicals, Basingstoke, UK) and 100 µM ATP, and terminated after a 15 min incubation at room temperature by the addition of 100 µl HCl (1 M) and 200 µl chloroform:methanol (1:1). After vigorous mixing and centrifugation to separate the phases, the organic layer was removed. The extracted phospholipids were then spotted into silica gel TLC plates that had been impregnated with 1% potassium oxalate and developed in 1-propanol:acetic acid (2 N) (65:35 (v/v) for 16 h. The TLC plates were stained with iodine to confirm even extraction of substrate lipid between individual samples and 32P-labelled PI 3-P was visualized by autoradiography. The identity of PI 3-P was confirmed by its sensitivity to the PI 3-kinase inhibitors Wortmannin and LY294002.

Preparation of nuclear extracts
B cells were stimulated as described above. Cells were washed once in HBSS and resuspended in 400 µl buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 1 mM sodium vanadate, 10 mM sodium fluoride, 0.5 mM PMSF, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 10 mM sodium molybdate, 0.15 mM spermine and 0.75 mM spermidine). After incubation on ice for 15 min, IGEPAL was added to a final concentration of 0.3% and samples vortexed. After centrifugation, cytoplasmic proteins were removed and the pelleted nuclei were resuspended in 50 µl buffer C (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 25% glycerol, 1 mM dithiothreitol, 1 mM sodium vanadate, 10 mM sodium fluoride, 0.5 mM PMSF, 2 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin and 10 mM sodium molybdate). After 15 min agitation at 4°C, the samples were centrifuged and supernatants, containing nuclear proteins, were transfered to a fresh vial and rapidly frozen by immersion in liquid nitrogen and stored at –70°C.

EMSA
Nuclear protein concentrations were determined by BioRad protein assay (BioRad, Hemel Hempstead, UK). Proteins were normalized to a concentration of 2 µg/4 µl buffer C. An oligonucleotide containing the binding sequence for NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega, Southampton, UK) was end-labelled with [{gamma}-32P]ATP using T4 polynucleotide kinase (Boehringer Mannheim, Mannheim, Germany). Binding reactions were performed in a total volume of 20 µl containing 38 mM KCl, 0.6 mM MgCl2, 7.5% (v/v) Ficoll, 1 µg poly(dI–dC) (Promega), 8.5 mM HEPES, 1 mM DTT, labelled probe (4x105 c.p.m./sample) and 2 µg nuclear extract. Samples were separated on non-denaturing 4% polyacrylamide gels at 150 V for 2 h and visualized by autoradiography.

Western blot analysis
For I{kappa}B{alpha} degradation assays, cells were pre-incubated for 15 min in RPMI 1640 containing 50 µg/ml cyclohexamide. Following stimulation, cells were lysed at 5x106 cell equivalents/ 40 µl in ice-cold lysis buffer. Proteins were resolved by SDS–PAGE on 10% acrylamide gels, transferred to nitrocellulose (BioRad), and blocked overnight in 5% BSA and 1% ovalbumin in Tris-buffered saline. Primary antibodies were used at 1 µg/ml for anti I{kappa}B{alpha} (C-21; Santa Cruz), 1:100 dilution of anti-actin (Sigma), 1:1000 dilution of anti-phospho 473 Akt (NEB, Hitchin, UK) and 1:1000 dilution of rabbit polyclonal anti-Akt (a kind gift from Professor J. Tavare, University of Bristol). Goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Dako, High Wycombe, UK) was used at 0.03 µg/ml. Immunoblots were developed using the ECL system and Hyperfilm (Amersham Pharmacia Biotech, Little Chalfont, UK).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
BCR-induced activation of NF- {kappa} B is blocked by PI 3-kinase inhibitors
Preliminary experiments investigated the ability of specific inhibitors to block BCR-induced PI 3-kinase activity in primary B cells. The inhibitors used were the microbial metabolite Wortmannin (44), along with a structurally distinct reversible inhibitor, LY294002 (45), both of which have been shown to specifically block PI 3-kinase in multiple cell types. B cells were stimulated with 10 µg/ml anti-murine IgM F(ab')2 for the times indicated (Fig. 1AGo) and PI 3-kinase activity was determined by assaying the in vitro lipid kinase activity present in anti-phosphotyrosine immunoprecipitates. Enzymatic activity was measured by incubating immunoprecipitates with PtdIns and [{gamma}-32P]ATP and visualizing 32P-labelled PI 3-P by TLC. Treatment of the splenic B cells with anti-murine IgM F(ab')2 resulted in a rapid and transient increase in PI 3-kinase activity, with maximal lipid kinase activity observed following 1–5 min stimulation (Fig. 1AGo). This time course of BCR-induced PI 3-kinase activation is consistent with previously reported results (46). To examine the effects of Wortmannin (Fig. 1BGo) and LY294002 (Fig. 1CGo) on BCR-induced PI 3-kinase activity, the splenic B cells were pretreated with various concentrations of the PI 3-kinase inhibitors for 30 min prior to stimulation with anti-murine IgM F(ab') for 5 min. BCR-induced PI 3-kinase activity was markedly reduced following the addition of 5 nM Wortmannin or 5 µM LY294002. Increasing the concentration of the inhibitors further reduced PI 3-kinase activity with basal levels being reached at 50 nM Wortmannin and 10 µM LY294002.



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Fig. 1. In primary murine splenic B cells, BCR cross-linking induces PI 3-kinase activity which can be inhibited by Wortmannin and LY294002. Primary B cells (2x107) were stimulated with 10 µg/ml anti-IgM F(ab')2 for the times indicated (A) or pretreated for 30 min with various concentrations of Wortmannin (B) or LY294002 (C) prior to stimulation with 10 µg/ml anti-IgM F(ab')2 for 5 min. Lysates from these cells were immunoprecipitated with anti-phosphotyrosine antibodies and the precipitates were assayed for PI 3-kinase activity by in vitro lipid kinase assay using PtdIns as a substrate. Extraction and TLC separation of the lipid products were performed as described in Methods.

 
The effects of the PI 3-kinase inhibitors on BCR-induced NF-{kappa}B DNA-binding activity was next determined. Following pre-treatment with 50 nM Wortmannin, 10 µM LY294002 or media alone, B cells were stimulated with 10 µg/ml anti-murine IgM F(ab')2 for the times indicated, and nuclear extracts prepared and analysed for NF-{kappa}B DNA-binding activity by EMSA (Fig. 2Go). Since inhibition of PI 3-kinase activity will affect cell viability over time, unstimulated samples were incubated with the PI 3-kinase inhibitors for the duration of the experiment. The specificity of the EMSA for the detection of NF-{kappa}B was established by the inclusion of samples containing no nuclear extract, nuclear extracts pre-incubated with an excess of cold NF-{kappa}B oligonucleotides or a cold irrelevant oligonucleotides against AP-1 (Fig. 2AGo; Con i, ii and iii respectively). Nuclear extracts from unstimulated B cells contained some NF-{kappa}B-binding activity as NF-{kappa}B is constitutively active in the nucleus of B lymphocytes (34). Upon BCR cross-linking, enhanced NF-{kappa}B DNA-binding activity was clearly evident after 30 min stimulation (Fig. 2BGo), with DNA-binding activity maximal after 2 h (Fig. 2AGo). Pre-treatment of B cells with Wortmannin (Fig. 2AGo) or LY294002 (Fig. 2BGo) completely inhibited BCR-induced NF-{kappa}B DNA-binding activity. The PI 3-kinase inhibitors did not inhibit the basal level of NF-{kappa}B activity in control cells, indicating that cell viability was not affected by these inhibitors over the relatively short duration of the experiment, and only inhibited the BCR-induced NF-{kappa}B activity to this basal level.



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Fig. 2. PI 3-kinase activity is necessary for BCR-induced NF-{kappa}B DNA-binding activity. Primary B cells (1x107) were pre-treated for 30 min with either 50 nM Wortmannin (A) or 10 µM LY294002 (B), or various concentrations of Wortmannin or LY294002 (C) and then stimulated with 10 µg/ml anti-IgM F(ab')2 for the times indicated. Nuclear extracts were prepared and NF-{kappa}B DNA-binding activity was analysed by EMSA using a 32P-labelled oligonucleotide probe containing the NF-{kappa}B-binding sequence as described in Methods. The control lanes (A) represent probe alone (i), nuclear extracts pre-incubated with cold NF-{kappa}B probe (ii) or cold probe against AP-1-binding sequence (iii) prior to incubation with the 32P-labelled probe containing the NF-{kappa}B-binding sequence.

 
To extend these findings, we next examined the dose-response of the inhibitory effect of Wortmannin and LY294002 on BCR-induced NF-{kappa}B DNA-binding activity (Fig. 2CGo). At 10 nM Wortmannin and 5 µM LY294002, BCR-induced NF-{kappa}B DNA-binding activity was reduced to basal levels. These results strongly suggest that PI 3-kinase activity is required for BCR-induced activation of NF-{kappa}B.

PI 3-kinase inhibitors prevent BCR-induced I {kappa} B {alpha} degradation
In unstimulated lymphocytes, the majority of NF-{kappa}B is retained in the cytoplasm by virtue of its interaction with members of the I{kappa}B family of inhibitors (31). In the majority of cases migration of NF-{kappa}B to the nucleus follows the phosphorylation and subsequent degradation of I{kappa}B{alpha} exposing nuclear localization sequences on the released NF-{kappa}B/Rel dimers. In the nuclear compartment, NF-{kappa}B stimulates transcription of many growth-related genes as well as the gene encoding I{kappa}B{alpha}. To determine whether the inhibitory action of Wortmannin and LY294002 was due to its effects on I{kappa}B{alpha} degradation, levels of I{kappa}B{alpha} protein were examined by Western blotting. In these experiments, the primary murine B cells were first exposed to cyclohexamide to prevent de novo synthesis of I{kappa}B{alpha}. Then, cells were either pre-treated with Wortmannin (Fig. 3AGo) or LY294002 (Fig. 3BGo) and the BCR cross-linked for the times indicated. Consistent with the findings that BCR-induced NF-{kappa}B activation is dependent on PI 3-kinase activity, pre-treatment of B cells with either Wortmannin or LY294002 inhibited BCR-induced degradation of I{kappa}B{alpha}. I{kappa}B{alpha} degradation was visible after 30 min of BCR cross-linking and most prominent after 2 h. These data suggest that PI 3-kinase is involved in a signaling pathway leading to I{kappa}B{alpha} degradation which results in NF-{kappa}B nuclear translocation and DNA-binding activity.



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Fig. 3. PI 3-kinase is necessary for BCR-induced I{kappa}B{alpha} degradation. Primary B cells (5x106) were pre-incubated with 5 µg/ml cyclohexamide for 15 min. Cells were then treated for 30 min with either 50 nM Wortmannin (A) or 10 µM LY294002 (B) prior to stimulation with 10 µg/ml anti-IgM F(ab')2 for the times indicated. Cytoplasmic extracts were prepared and proteins separated by SDS–PAGE on 10% acrylamide gels as described in Methods. The gels were transferred to nitrocellulose and probed with anti-I{kappa}B{alpha} antibodies (upper panels). The blots were then stripped and reprobed with antibodies against actin (lower panels) to demonstrate equal protein loading. The positions of I{kappa}B{alpha} and actin are indicated.

 
LPS-induced NF- {kappa} B DNA-binding activity and I {kappa} B {alpha} degradation is also inhibited by Wortmannin and LY294002
In B cells, NF-{kappa}B can be activated not only by BCR cross-linking, but also by LPS and CD40 ligation (28,29). However, stimulation of NF-{kappa}B activity by both LPS and CD40 ligation is not affected by incubation overnight with phorbol myristate acetate (PMA), suggesting that it occurs via a PKC-independent pathway (28,29). Conversely, NF-{kappa}B activation through the BCR was markedly inhibited by PKC depletion, indicating that BCR-induced NF-{kappa}B activation is mediated through a PKC-dependent signaling pathway (28,29). Therefore, at least two independent signaling pathways exist in B cells which lead to activation of NF-{kappa}B. Since the data presented here indicate that BCR-induced NF-{kappa}B activation is not only dependent on PKC, but also on PI 3-kinase, we were interested in determining if the LPS-induced pathway to NF-{kappa}B activation also involved PI 3-kinase.

While earlier reports indicated that LPS-induced signalling in B cells occurs independently of lipid turnover (47), a recent study using Wortmannin and LY294002 demonstrated that LPS-mediated B cell proliferation (over 48 h) is dependent on PI 3-kinase (48). It was therefore important to directly measure whether LPS triggers the activation of PI 3-kinase before investigating the effects of Wortmannin and LY294002 on NF-{kappa}B activation. Thus, PI 3-kinase activation in response to LPS in our primary splenic B cells was determined by in vitro lipid kinase assay. An increase in PI 3-kinase activity was observed following 10 min stimulation with LPS, which was sustained for 60 min (Fig. 4Go). This LPS-induced PI 3-kinase activation was less intense and had a delayed onset when compared with BCR-induced PI 3-kinase activity (which was maximal following 1–5 min stimulation; Fig. 1AGo).



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Fig. 4. LPS induces an increase in PI 3-kinase activity, which is sensitive to Wortmannin and LY294002. Primary B cells (2x107) were stimulated with 50 µg/ml LPS for the times indicated (A) or pre-treated for 30 min with 50 nM Wortmannin or 10 µM LY294002 prior to stimulation with LPS for 30 min (B). Anti-phosphotyrosine immunoprecipitates of the resulting cell lysates were then assayed for PI 3-kinase activity by in vitro lipid kinase assay as described in Methods.

 
To confirm the involvement of PKC in the BCR- and LPS-induced pathways leading to NF-{kappa}B activation, the effects on BCR- and LPS-induced I{kappa}B{alpha} degradation by a specific inhibitor of PKC, Gö6983, was determined. Gö6983 specifically inhibits a number of PKC isoforms ({alpha}, ß, {gamma}, {delta} and {zeta}), but the effects of this inhibitor on some novel and atypical PKC have not been tested. LPS has previously been shown to induce I{kappa}B{alpha} degradation in B cells (49). As shown in Fig. 5(A)Go, inhibition of PKC by Gö6983 completely blocked BCR-induced I{kappa}B{alpha} degradation but had no effect on LPS-induced I{kappa}B{alpha} degradation, corresponding with the previously reported results (28,29). The effects of PI 3-kinase inhibition on LPS-induced NF-{kappa}B DNA-binding activity was next assessed. LPS-induced NF-{kappa}B DNA-binding activity was inhibited to basal levels by both Wortmannin and LY294002 (Fig. 5BGo). Again, unstimulated samples were incubated with the PI 3-kinase inhibitors for the duration of the experiment. The inhibitors did not affect the basal level of NF-{kappa}B activity in these unstimulated cells, indicating that viability was not significantly affected by these inhibitors over the short duration of the experiment. To complement these findings, the effects on I{kappa}B{alpha} degradation was assessed. Both Wortmannin and LY294002 inhibited LPS-induced I{kappa}B{alpha} degradation (Fig. 5CGo). To address the sensitivity of these two pathways to PI 3-kinase inhibition, the effects on I{kappa}B{alpha} degradation following treatment with various concentration of LY294002 was determined. As can be seen in Fig. 5(D)Go, both BCR- and LPS-induced degradation of I{kappa}B{alpha} were equally sensitive to the effects of LY294002. Thus, LPS appears to activate NF-{kappa}B through a PI 3-kinase-dependent pathway, suggesting that PI 3-kinase is important in two distinct pathways leading to NF-{kappa}B activation.



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Fig. 5. LPS-induced NF-{kappa}B DNA-binding activity and I{kappa}B{alpha} degradation is dependent on PI 3-kinase activity. Primary B cells (5x106) were pre-incubated with 5 µg/ml cyclohexamide for 15 min (A, C and D). Cells were then treated for 30 min with either 500 nM of the PKC inhibitor Gö6983 (A), 50 nM Wortmannin or 10 µM LY294002 (C), or various concentrations of LY294002 as indicated (D). After stimulation with 10 µg/ml anti-IgM F(ab')2 or 50 µg/ml LPS for 2 h, cytoplasmic extracts were prepared and proteins separated by SDS–PAGE on 10% acrylamide gels. The gels were transferred to nitrocellulose and probed with anti-I{kappa}B{alpha} antibodies (A, C and D: upper panels). The blots were then stripped and re-probed with antibodies against actin (lower panels) to demonstrate equal protein loading. The positions of I{kappa}B{alpha} and actin are indicated. (B) Nuclear extracts were prepared from primary B cells (1x107) pre-treated for 30 min with either 50 nM Wortmannin or 10 µM LY294002 and then stimulated with 10 µg/ml anti-IgM F(ab')2 or 50 µg/ml LPS for 2 h. NF-{kappa}B DNA-binding activity was analysed by EMSA using a 32P-labelled oligonucleotide probe containing the NF-{kappa}B-binding sequence.

 
Lipopolysaccharide induces PI 3-kinase-dependent phosphorylation of Akt on serine 473
A principle downstream mediator of PI 3-kinase-dependent signaling processes is the serine/threonine kinase Akt. The activation of Akt is controlled by phosphorylation of threonine 308 and serine 473 (24). While BCR engagement is known to activate Akt in a PI 3-kinase dependent manner (16,17), the effects of LPS on Akt activity have not been studied. In order to test this, we stimulated primary murine B cells for various times with either LPS or anti-IgM F(ab')2 and performed anti-phospho Akt immunoblots. The results in Fig. 6(A)Go show that Akt is not phosphorylated to a significant extent in unstimulated cells and confirms that BCR ligation causes a substantial increase in phosphorylation of Akt on serine 473. This was evident 2 min after stimulation and was sustained for at least 2 h. Importantly, stimulation with LPS also caused a clear increase in Akt phosphorylation on serine 473. The phosphorylation of Akt after LPS stimulation was slightly delayed (correlating with the delayed onset of LPS-induced PI 3-kinase activation; Fig. 4AGo), being detectable at 30 min and remaining increased at 2 h.



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Fig. 6. BCR cross-linking and LPS stimulation induces Akt phosphorylation on serine 473 in a PI 3-kinase-dependent and PKC-independent manner. Primary B cells (5x106) were either left untreated (A), treated with 50 nM Wortmannin or 10 µM LY294002 (B), or treated with 500 nM Gö6983 (C) for 30 min prior to stimulation with 10 µg/ml anti-IgM F(ab')2 or 50 µg/ml LPS for the times indicated. Cytoplasmic extracts were prepared and proteins separated by SDS–PAGE on 10% acrylamide gels as described in Methods. The gels were transferred to nitrocellulose and probed with anti-phosphoserine 473 Akt antibodies (upper panels). The blots were then stripped and reprobed with antibodies against Akt (lower panels) to demonstrate equal protein loading. The position of Akt is indicated.

 
To test whether LPS-induced phosphorylation of Akt on serine 473 is dependent on PI 3-kinase activity, we tested whether this response could be blocked by Wortmannin and LY294002. Figure 6(B)Go shows that Wortmannin and LY294002 not only blocked BCR-induced phosphorylation of Akt, confirming the previously reported results (16,17), but also blocked stimulation by LPS. Thus, it appears that LPS is capable of activating Akt in a PI 3-kinase-dependent manner.

Finally, we investigated the involvement of PKC in BCR- and LPS-induced phosphorylation of Akt on serine 473. The PKC inhibitor Gö6983 had no effect on BCR- or LPS-induced phosphorylation of Akt (Fig. 6CGo), suggesting that activation of Akt does not occur downstream of activation of PKC in this system.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
NF-{kappa}B was first described as a potent regulator of Ig{kappa} transcription in B cells but is now known to control expression of numerous genes involved in immune and inflammatory responses (32,33). Despite its importance in B cell function, little is known about the downstream events following BCR engagement leading to NF-{kappa}B activation. Using the PI 3-kinase inhibitors Wortmannin and LY294002, the results presented here demonstrate that PI 3-kinase is required for both BCR- and LPS-induced NF-{kappa}B DNA-binding activity and associated degradation of I{kappa}B{alpha}.

BCR cross-linking induced the formation of NF-{kappa}B DNA-binding complexes which migrated as two distinct bands as revealed by EMSA. These bands are characteristic of NF-{kappa}B, and correspond to the presence of both homo- and heterodimers of individual NF-{kappa}B/Rel family proteins. Indeed, supershift analysis confirmed the previously reported finding (50) that BCR cross-linking induced DNA-binding activity of complexes containing p65, c-rel, RelB and p50 (data not shown). The levels of all of these components were dramatically reduced after treatment with Wortmannin and LY294002. Taken together with the observation that these inhibitors prevented the BCR-induced degradation of I{kappa}B{alpha}, our findings indicate a critical role for PI 3-kinase in the activation of NF-{kappa}B following antigen recognition by B cells. The mechanisms linking PI 3-kinase to NF-{kappa}B are unknown. While this is the first report which has highlighted a link between PI 3-kinase and NF-{kappa}B following BCR engagement, this link has been established in certain other cell systems. The involvement of PI 3-kinase in NF-{kappa}B activation has been demonstrated following treatment of human transformed epithelial cells and HeLa cells with bradykinin (51,52); exposure of Jurkat T cells to pervanadate (53); and stimulation of a human hepatoma cell line with IL-1 and tumor necrosis factor (TNF)-{alpha} (5456). In addition, a major downstream effector of PI 3-kinase, Akt, has recently been shown to be involved in NF-{kappa}B activation in some systems (4143). In Jurkat T cells, over-expression of Akt was shown to enhance NF-{kappa}B activation, following stimulation with PMA and ionomycin (41). Further, NF-{kappa}B activation stimulated by platelet-derived growth factor in fibroblasts (43) and TNF in embryonic kidney cells (42) was shown to involve the activation of Akt and its interaction with IKK.

While BCR engagement activates Akt via a PI 3-kinase-dependent pathway (16,17 and the results of this study), it is unlikely that PI 3-kinase-dependent BCR-induced activation of NF-{kappa}B occurs via an Akt-dependent route. Our finding that a specific inhibitor of PKC, Gö6983, blocks BCR-induced I{kappa}B{alpha} degradation together with an earlier report which showed that pre-treatment of B cells with PMA prevented NF-{kappa}B activation (28,29) demonstrate a critical role for PKC in this process. The involvement of PKC is not consistent with a putative role for Akt in BCR-induced NF-{kappa}B activation since Akt has been reported to mediate its effects through direct interaction and phosphorylation of IKK (42,43). Furthermore, our results show that Gö6983 had no effect on BCR-induced Akt phosphorylation, indicating that the critical PKC involved in BCR-induced NF-{kappa}B activation is not upstream of Akt in this system. In addition, phosphorylation of Akt at serine 473 occurs normally following BCR cross-linking in Btk-deficient DT40 cells (17) despite the fact that Btk is required for BCR-induced NF-{kappa}B activation (see below). This provides further evidence that BCR-induced NF-{kappa}B activation does not occur through an Akt-dependent pathway.

How might PI 3-kinase act through PKC to trigger NF-{kappa}B activation? The major outcome of PI 3-kinase activity is the generation of PI(3,4,5)P3 which facilitates the recruitment, via PH domains, of a variety of proteins to the cell membrane. One of the key proteins recruited in this way is the Tec family kinase Btk; a critical factor in BCR-induced PLC-{gamma}2 mobilization leading to Ca2+ flux and diacylglycerol-associated PKC activation. Indeed xid mice, which lack functional Btk, exhibit greatly diminished BCR-induced NF-{kappa}B activation (57,58). In addition, PDK1, a serine/threonine kinase, is also recruited to the plasma membrane via its PH domain, and has been shown to phosphorylate and activate PKC {zeta} and {delta} (59). The precise isoform of PKC which is responsible for NF-{kappa}B activation following BCR cross-linking remains unclear. In a variety of systems, PKC {zeta} and {lambda} have been shown to be critical in activation of NF-{kappa}B through the IKK pathway and directly bind the IKK in vitro and in vivo (6062). In B cells PKC {alpha}, ß, {gamma}, {delta}, {zeta}, {eta} and µ have been detected (63,64), and PKC ß (65), µ and {delta} (66) have been shown to be involved in BCR signaling. Thus, following BCR-induced activation of PI 3-kinase, recruitment of Btk and/or PDK1 to the plasma membrane could lead to PKC-induced NF-{kappa}B activation (Fig. 7Go). Elucidation of the relative roles of Btk, PDK1 and the PKC isoforms involved will help determine the precise pathway leading to BCR-induced NF-{kappa}B activation.



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Fig. 7. NF-{kappa}B activation via PI 3-kinase. Based on the results presented here and on evidence from previous reports, the following model of PI 3-kinase-dependent NF-{kappa}B activation can be proposed. BCR-induced activation of PI 3-kinase results in accumulation of PI(3,4,5)P3 levels at the membrane to which the PH domains of Btk and PDK1 bind, facilitating their translocation to the plasma membrane. This results in activation of PKC, either via PDK1 or the PLC-{gamma}2 pathway, and consequently triggers the activation of IKK. IKK-mediated phosphorylation of I{kappa}B{alpha} results in its degradation, allowing translocation of NF-{kappa}B to the nucleus. BCR-induced NF-{kappa}B activation is unlikely to occur via Akt (dashed line) since BCR-induced NF-{kappa}B activation is not only dependent on PI 3-kinase but also on PKC and Akt phosphorylation does not occur downstream of PKC. However, LPS-induced NF-{kappa}B activation, which also depends on PI 3-kinase activity, is unlikely to be dependent on PKC. LPS was also shown to be capable of triggering Akt phosphorylation on serine 473, thus it is possible that LPS could activate NF-{kappa}B through this Akt-dependent pathway.

 
However, in B cells, there is a distinct signaling pathway(s), not mediated through the BCR, leading to NF-{kappa}B activation. It has previously been shown, through depletion using PMA, that while PKC is necessary for BCR-induced NF-{kappa}B activation, it is not necessary for CD40- or LPS-induced activation of NF-{kappa}B in B cells (28,29). In addition, we were able to inhibit BCR- but not LPS-induced I{kappa}B{alpha} degradation with the PKC inhibitor Gö6983. Taken together, these findings suggest that LPS-induced NF-{kappa}B activation occurs through a PKC-independent pathway. However, it is important to note that Gö6983 does not block the activation of some novel or atypical PKC and therefore their role in the LPS-induced pathway cannot be ruled out. Despite this, our findings clearly demonstrate the presence of two distinct pathways activated through the BCR and by LPS, leading to I{kappa}B{alpha} degradation (one which is Gö6983 sensitive and the other which is Gö6983 insensitive). Interestingly, the LPS-induced pathway to NF-{kappa}B activation was also dependent on PI 3-kinase activity. We show here for the first time that LPS can directly activate PI 3-kinase activity. Inhibiting this PI 3-kinase activity with either Wortmannin or LY294002 inhibited LPS-induced NF-{kappa}B DNA-binding activity and LPS-induced I{kappa}B{alpha} degradation. As with BCR-induced NF-{kappa}B, supershift analysis revealed the presence of complexes containing p50, p65, c-rel and RelB, all of which were inhibited following pre-incubation with LY294002 (data not shown). Our observations are interesting since a recent report has shown that LPS-induced NF-{kappa}B activation and I{kappa}B{alpha} degradation in Jurkat T cells, human epithelial cells and human glioma cells is dependent on both PI 3-kinase and PKC (67). Therefore, the LPS-induced NF-{kappa}B activation in B cells occurs through a pathway which is distinct from that in other cells types.

The precise processes linking PI 3-kinase to NF-{kappa}B following LPS stimulation is unknown. However, our observation that LPS triggers the phosphorylation of Akt on serine 473 is of interest in this regard. This phosphorylation was PI 3-kinase dependent as revealed by the use of Wortmannin and LY294002. It is noteworthy that this is the first report demonstrating activation of a signaling intermediate downstream of PI 3-kinase following LPS stimulation in B cells. In addition, our observations may provide a clue as to the nature of the PKC-independent pathway leading to NF-{kappa}B activation in B cells. Akt-mediated phosphorylation of IKK is the only currently known pathway by which NF-{kappa}B is activated in a PKC-independent, PI 3-kinase-dependent process (4143). Further, the PKC inhibitor Gö6983 had no effect on LPS-induced Akt phosphorylation. Thus, LPS may mediate NF-{kappa}B activation through an Akt-dependent pathway (Fig. 7Go). Further studies expressing dominant-negative and constitutively active Akt in B cell lines will be needed to confirm the Akt dependency of this process.

In summary, we have demonstrated that BCR- and LPS-induced I{kappa}B{alpha} degradation and NF-{kappa}B DNA-binding activity is dependent on PI 3-kinase activity in primary B cells. When taken together with previous findings, the results presented here suggest that NF-{kappa}B activation stimulated through the BCR is likely mediated through Btk and dependent on PKC. In contrast, NF-{kappa}B activation induced by LPS occurs via a distinct pathway that is independent of the PKC isoforms required for BCR-induced NF-{kappa}B activation. These findings highlight that PI 3-kinase can trigger the activation of NF-{kappa}B through at least two discrete signaling cascades in B cells. Unravelling the precise pathways involved will provide a critical understanding of the mechanisms underlying the activation and proliferation of B cells.


    Acknowledgments
 
This work was supported by a grant from The Wellcome Trust. N. A. W. is in receipt of a Wellcome Trust Research Leave Fellowship.


    Abbreviations
 
EMSA electrophoretic mobility shift assay
IKK I{kappa}B kinase
IP3 inositol 1,4,5-triphosphate
LPS lipopolysaccharide
PH pleckstrin homology
PI 3-kinase phosphoinositide 3'-kinase
PI(3, 4, 5)P3 phosphatidylinositol 3,4,5-trisphosphate
PI(4, 5)P2 phosphatidylinositol 4,5-bisphosphate
PKC protein kinase C
PLC phospholipase C
PtdIns phosphatidylinositol
TNF tumor necrosis factor

    Notes
 
Transmitting editor: M. Neuberger

Received 3 November 2000, accepted 5 March 2001.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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