Phosphatidylinositol 3-Kinase and NF-κB/Rel Are at the Divergence of CD40-Mediated Proliferation and Survival Pathways

Ligation of CD40 molecule delivers crucial signals that promote survival, proliferation, and Ig class switching of B lymphocytes (1). CD40 ligand is expressed on activated T cells, making this interaction essential for B-T cell cooperation. Because of its multiple functions, the signaling pathways triggered by CD40 are expected to be diverse. Nonetheless, a direct link between specific signaling pathway and particular cellular outcome remains unknown. Upon ligand binding, CD40 receptor trimerization facilitates clustering of the TNF receptor associated factors 2, 3, 5, and 6 molecules. TNF receptor associated factors serve as adapters that connect the cytoplasmic portion of CD40 to downstream effector molecules, such as c-Jun N-terminal kinase and NF-κB/Rel (2, 3, 4). Known signaling events occurring upon CD40 stimulation also include activation of tyrosine kinases (Lyn), activation of phosphatidylinositol 3-kinase (PI-3K),³ phosphorylation of phospholipase Cγ2, and activation of NF-κB/Rel transcription factors (5). Furthermore, CD40 activation has been linked to the up-regulation of several surface proteins (CD23, Fas, ICAM-1, and LFA-1; Ref. 6, 7, 8, 9) as well as anti-apoptotic molecules (Bcl-X, A20; Ref. 10, 11, 12, 13). It is believed that induction of these apoptosis inhibitors causes the augmentation of cell survival induced by CD40, although the actual mechanism accountable for their up-regulation remains elusive. Experiments presented here are aimed at investigating the events involved in CD40-mediated proliferation as well as survival function.

A prominent consequence of CD40 activation is induction of NF-κB/Rel transcription factors. The NF-κB/Rel transcription factor family consists of five members that form homo and heterodimers that translocate to the nucleus, where they regulate transcription of various target genes (14). It is believed that, by activating these genes, NF-κB/Rel participates in both proliferation and survival in B cells. The NF-κB/Rel activity is tightly controlled by IκB proteins that serve to sequester transcription factor complexes in the cytoplasm (14). Data from many laboratories have established the sequence of biochemical events that causes the degradation of IκB proteins and release of NF-κB/Rel (15). Interestingly, through studies performed in the knockout (ko) mice, it became clear that individual members of the NF-κB/Rel family have distinct roles in the immune system. We reported previously that, although the lymphocyte development was normal in the c-Rel ko mouse, profound functional defects were observed in both B and T lymphocyte compartments (16, 17). Namely, c-Rel-deficient B cells were unresponsive to various mitogenic stimuli, including CD40. Surprisingly, whereas c-Rel ko cells showed clear defects in CD40-induced proliferation, CD40-mediated survival was still intact (17). This observation suggested the existence of at least two signaling pathways stemming from the CD40 receptor, distinguishable on the basis of c-Rel involvement.

The recent generation of the PI-3K gene-targeted mouse (p85α ko) also revealed a profoundly impaired B cell phenotype, remarkably similar to that of c-Rel ko mice (18, 19). It has become clear that signaling pathways emanating from the mitogenic receptors in B lymphocytes travel via activation of PI-3K. For example, PI-3K-deficient cells were unable to mount proliferation in response to CD40 ligation, as well as other type of mitogenic stimulation (18). These findings underlie the importance of studying PI-3K in the context of activation pathways in B lymphocytes. PI-3K consists of a 110-kDa catalytic subunit and a tightly associated regulatory subunit encoded by the p85α gene that has splicing isoforms p55α and p50α (20). Inhibition of PI-3K in IL-3-dependent cell lines leads to apoptosis. It has been shown that PI-3K promotes cell survival by activating AKT kinase, which phosphorylates and inactivates pro-apoptotic molecule, Bad (21). In addition, recent reports revealed a connection between AKT and IKKαβ which is responsible for targeting IκBα for degradation and NF-κB/Rel activation (22, 23). Based on these observations, we decided to investigate the role of PI-3K in CD40-mediated pathways that lead to cell proliferation and/or survival and to connect these events to the activation of NF-κB/Rel transcription factors by using c-Rel-deficient mouse.

Gene-targeted c-Rel mice were generated previously (16) and together with normal counterparts were bred and maintained at the Weill Graduate School of Medical Sciences of Cornell University Animal Facility under pathogen-free conditions. Six- to 8-wk-old mice were used for experiments. p85α-deficient mice (PI-3K ko) were generated as described previously (18, 19) and maintained at Taconic Farms (Germantown, NY).

PI-3K inhibitor- LY 294002 (Sigma, St. Louis, MO; Ref. 24) was dissolved in DMSO and further diluted in cell culture medium before addition to the cell culture at 10 μM final concentration (final concentration of DMSO did not exceed 0.1%). Anti-CD40 agonistic Ab (clone 1C10, rat IgG2a) was kindly provided by Dr. Maureen Howard (Stanford University, Stanford, CA), affinity purified from hybridoma supernatant, and used at 10μg/ml concentration. For depleting splenic T cells, anti-Thy1.2 mAb (clone J1J) in the form of ascites was kindly provided by Dr. Janko Nikolic-Zugic (Sloan Kettering Institute, New York, NY), and low-tox rabbit complement was obtained from Cedarlane Laboratory (Ontario, Canada). The following flow cytometry staining reagents were purchased from PharMingen (San Diego, CA): PE-conjugated anti-B220, FITC-conjugated anti-CD3, and FITC-conjugated anti-CD54 (ICAM-1). Propidium iodide for DNA fragmentation analysis was purchased from Sigma. Abs for immunoblotting were obtained from the following sources: anti-p27^kip, anti-IκBα, anti-Bcl-2, and anti-c-Rel were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Bcl-X was purchased from Transduction Laboratories (Lexington, KY); anti-AKT and anti-pAKT (detects phosphorylation on S-473 residue) were obtained from New England Biolabs (Beverly, MA); and anti-γ actin was obtained from Sigma. For EMSA, anti-p50 and anti-p65 were affinity purified from rabbit serum.

B lymphocytes were isolated from total splenocytes by depleting T cells with anti-Thy1.2 mAb and complement and by allowing macrophages to adhere on the plastic surface. This protocol typically yields 95–98% pure population of B cells that are B220⁺CD3⁻ as determined by flow cytometry. For in vitro cell culture, B cells were plated at 0.5–1 × 10⁶ cells/ml (for some experiments in triplicates) for indicated time periods in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FBS, essential amino acids, and antibiotics. Cells were pretreated with LY 294002 inhibitor for 1 h before adding anti-CD40 Ab. At the end of culture, cells were collected from individual wells, washed twice with PBS, and further analyzed.

For proliferation assay, cells were plated in triplicate at 2 × 10⁵ cells/well in 96-well round-bottom plates (Fisher Scientific, Pittsburgh, PA). After 48 h of stimulation, cells were incubated with 0.5 uCi [³H]thymidine (Am- ersham, Arlington Heights, IL) for 5–8 h. Cells were then harvested and thymidine incorporation into DNA was quantified by scintillation.

For two- and single-color flow cytometry, cells were stained with directly conjugated mAb on ice for 30 min and washed in PBS (1% FBS, 0.1% NaN₃). Labeled samples were analyzed on a FACScalibur instrument (Becton Dickinson, Mountain View, CA) equipped with CellQuest software. A semiquantitative determination of the DNA content by propidium iodide (PI) staining was used to assess the percentage of cells undergoing DNA fragmentation (25). For PI staining, cultured cells were washed in PBS and resuspended in a hypotonic buffer (0.1% sodium citrate, 0.1% Triton X-100, 20 μg/ml RNaseA) containing 50 μg/ml PI at 1 × 10⁶. Initially, samples were incubated for 30 min at room temperature (to activate RNase); thereafter, samples were kept on ice. For each sample, at least 5 × 10³ events were collected and analyzed using a FACScalibur instrument. The percentage of cells undergoing apoptosis is determined as the relative number of cells with DNA content in subdiploid (subG_0/1) area, characteristic of apoptotic cells (25). Apoptosis data are presented as: percent of specific DNA fragmentation = (test − spontaneous)/(100 − spontaneous) × 100.

After culture, cells were washed with PBS and lysed in RIPA buffer (25 mM Tris (pH 7.4), 75 mM NaCl, 0.5% Triton X-100, 0.5% sodium deoxycholate, 0.01% SDS, 2.5 mM EDTA) supplemented with the mixture of protease and phosphatase inhibitors (100 μM Na₃VO₄, 50 mM NaF, 10 mM sodium pyrophosphate, and 1 mM aprotinin, leupeptin, and PMSF). For AKT immunoblotting, cell lysis buffer was made according to the manufacturer’s instruction. Equal amounts of whole cell lysates, quantified using the commercial Bio-Rad protein assay (equivalent to 10⁶ cells per sample; Richmond, CA), were loaded onto each lane of a 12.5% SDS-PAGE gel. Polyvinylidene difluoride membrane (Amersham) was used to transfer proteins and was blocked with 2% milk in TBST buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature. Membranes were further incubated with primary Ab (conditions and concentration of the Ab vary) and, after washing, incubated with appropriate secondary HRP-conjugated secondary Ab and visualized using commercial chemiluminescence detection kit (ECL Plus; Amersham).

The nuclear and cytosolic fractionation procedure was performed as previously described (26). Nuclear proteins were quantified by the commercial Bio-Rad protein assay, and EMSA was performed as described (26). Briefly, 10 μg nuclear extract proteins was incubated with 20 fmol of ³²P-labeled κB probe dissolved in an appropriate buffer. The DNA-protein complexes were resolved on a native 6% polyacrylamide gel, dried, and exposed on a film for autoradiography. For Ab-inhibition experiments, optimal amount of Ab was determined by using purified proteins. Appropriate Ab was incubated with a mixture of nuclear extract and poly(dI-dC) in 1× DNA binding buffer on ice for 30 min before the addition of ³²P-labeled κB probe. The reaction was continued for another 15 min before it was loaded on a native 6% polyacrylamide gel.

Cross-linking of CD40 on the cell surface by agonistic Ab or ligand binding generates a very strong proliferation signal for normal B cells. In dissecting the signaling pathway emanating from CD40, we observed that NF-κB/Rel plays a crucial role in CD40-mediated proliferation. Specifically, c-Rel-deficient cells exhibit very low levels of proliferation when stimulated with agonistic anti-CD40 Ab (Ref. 17 and Fig. 1). While searching for upstream signaling molecules that are responsible for CD40-mediated NF-κB/Rel activation, we learned that PI-3K-deficient B lymphocytes were also hyporesponsive to anti-CD40-induced proliferation (18, 27). These observations thus prompted us to further investigate the relationship between PI-3K and NF-κB/Rel. We took advantage of the well-characterized PI-3K inhibitor LY 294002 that specifically blocks the ATP-binding sites of the p110 catalytic subunit (24). We found that LY 294002 completely abrogates proliferation induced via CD40 in both wild-type (wt) and c-Rel ko B cells (Fig. 1). This is not due to the increased rate of cell death, because CD40 stimulation maintains cell viability following the treatment with the PI-3K inhibitor, as shown below in Fig. 5.

A critical consequence of CD40 signaling in B lymphocytes involves high and sustained activation of the NF-κB/Rel transcription factors (Ref. 28 ; S.A. and H.-C.L., unpublished observations). Because PI-3K inhibition resulted in a serious impairment in proliferation that was similar to that of c-Rel-deficient B cells, we investigated whether PI-3K is connected to NF-κB/Rel activation in splenic B lymphocytes. Intriguingly, using the EMSA, we observed that PI-3K blocking completely prevents nuclear translocation and activation of NF-κB/Rel complexes upon CD40 and anti-IgM stimulation (Fig. 2,A). The inhibition was observed in both wt and c-Rel ko B cells. Note that c-Rel-deficient cells mobilize NF-κB/Rel complexes to a lesser degree than the wt cells, due to the lack of the predominant c-Rel member. Translocated complexes in c-Rel ko B cells consist of p65 and p50 proteins as identified by an Ab inhibition assay shown in Fig. 2,B. Furthermore, we found that the inhibition of NF-κB/Rel activity by the PI-3K antagonist is not due to diminishment of NF-κB/Rel proteins, because their expression level in the cytoplasm (in both wt and c-Rel ko cells) remained unchanged regardless of the treatment (Figs. 2 C, 3B, and data not shown). Rather, these results demonstrate that PI-3K specifically affects nuclear translocation of NF-κB/Rel complexes.

In an effort to exclude possible nonspecific effects of the chemical compound serving as pharmacological PI-3K inhibitor, we used PI-3K-deficient mice (p85α ko) (18) to assay for NF-κB/Rel activation. We found that p85α ko B cells stimulated via CD40 ligation completely lack the induction of nuclear NF-κB/Rel complexes (Fig. 3,A), in a manner similar to that observed with the use of PI-3K inhibitor (Fig. 2,A). Therefore, these data strongly support our conclusion that PI-3K action is required for NF-κB/Rel activation. Despite the marked absence of the nuclear NF-κB/Rel proteins, cytoplasmic c-Rel is normally present in p85α ko B cells (Fig. 3,B), suggesting that PI-3K is crucial for permitting nuclear translocation of the NF-κB/Rel complexes. Previous studies have demonstrated that IκBα degradation is required for NF-κB/Rel activation and nuclear translocation (14, 15). Considering recently published data (22, 23), it was reasonable to suggest that the diminishment of nuclear NF-κB/Rel caused by PI-3K inhibition is caused by a block in IκBα degradation. To test this hypothesis, we performed Western blot analysis of the IκBα inhibitor expression in normal and p85α-deficient mice. As shown in Fig. 3 C, degradation of IκBα occurred upon CD40 treatment in wt mice (lane 2), but was prevented in the B cells that lack PI-3K activity (lane 4). This result confirms that PI-3K activation represents the main pathway that conveys activation signals from the CD40 receptor to the complex machinery that directs IκBα degradation via proteosome pathways.

A recent report on the Kit225 cell line stimulated with IL-2 concluded that PI-3K promotes proliferation via down-regulating cell cycle inhibitor p27^kip (29). p27^kip is a cyclin-dependent kinase (CDK) inhibitor whose expression level is reduced by an ubiquitin-proteosome-dependent mechanism during G₁ phase of the cell cycle (30). Degradation of p27^kip CDK inhibitor is necessary for the activation of CDK kinases and subsequent cell cycle progression (30). By examining p27^kip expression levels in B lymphocytes, we discovered that PI-3K inhibitor prevents CD40-induced down-regulation of p27^kip over a 24- to 48-h period (Fig. 4,A). Consistent with the results obtained using the PI-3K inhibitor LY294002, we confirm that p27^kip down-regulation is also prevented in mice deficient for PI-3K activity (Fig. 4 B). Taken together, our data show that PI-3K regulates both NF-κB/Rel activation and p27^kip CDK inhibitor degradation, events that are essential for cellular proliferation.

Apart from causing cellular activation, CD40 is also crucial for maintaining the viability of B lymphocytes both in vitro and in vivo (1, 2). Our studies led us to further test whether PI-3K and NF-κB/Rel contribute to the CD40-mediated survival pathway. In accordance with our previous finding (17), CD40 treatment provides the same level of protection from spontaneous apoptosis to both wt and c-Rel ko B cells (Fig. 5). LY 294002, when applied alone, induces cell death in both wt and c-Rel ko B lymphocytes (Fig. 5), suggesting that PI-3K is important for maintaining the viability of resting splenic B cells. Intriguingly, CD40 stimulation can partially rescue cell death induced via LY 294002, in both wt and c-Rel-deficient B cells (Fig. 5). We confirmed these findings by counting the actual number of live cells, where we observed that in all experiments the number of live cells was comparable in nontreated control samples and samples treated with combination of LY294002 and CD40 stimulation (data not shown). These data suggest that certain aspects of CD40-stimulated apoptosis protection are operational, even in the absence of both c-Rel and PI-3K activity. To find effectors of the CD40-mediated survival, we tested known apoptosis-blocking molecules for their role in this pathway.

Previous studies in several cell systems, including B lymphocytes, demonstrated that AKT kinase is one of the primary targets of PI-3K (31). Because AKT was shown to prevent apoptosis (21, 32), we investigated its role in CD40-mediated survival pathway. As shown in Fig. 6, AKT phosphorylation occurs within 15 min after anti-CD40 treatment, and increases up to 1 h poststimulation. However, pretreatment of cells with LY294002 compound potently blocks phosphorylation, while having no effect on overall levels of nonphosphorylated AKT in the cytoplasm (Fig. 6). This result verifies that, in B lymphocytes, CD40 activates AKT exclusively via PI-3K pathway. Nonetheless, the PI-3K/AKT survival pathway contributes only partially to the CD40-mediated apoptosis protection in splenic B lymphocytes, because we observed that lack of PI-3K activation did not significantly alter CD40-mediated survival function (Fig. 5).

Bcl-X is recognized to be a major anti-apoptotic protein that is induced upon CD40 stimulation (10, 12, 13). To test whether this protein confers CD40-mediated protection from LY 294002-inflicted cell death, we analyzed its expression in wt and c-Rel ko B cells that were stimulated with CD40 in the presence/absence of the PI-3K inhibitor (Fig. 7,A). Surprisingly, we observed a complete absence of Bcl-X expression in c-Rel deficient cells. We further performed experiments to assess Bcl-X expression in p85α ko B cells. As shown in Fig. 7,B, p85α-deficient B cells also lack Bcl-X induction upon CD40 stimulation. This and our other observations (A.O. and H.-C.L., unpublished data) thus indicate that Bcl-X is directly regulated via c-Rel. Bcl-X induction in wt cells was also significantly perturbed by addition of the PI-3K inhibitor and correlates with the compound’s ability to inhibit NF-κB/Rel activity (Fig. 2,A). In contrast to Bcl-X, Bcl-2 is constitutively present in B lymphocytes and remained constant and unaffected by c-Rel deficiency or PI-3K inhibition in all samples tested (Fig. 7 A). Taken together, these data indicate that Bcl-X expression is regulated by both NF-κB/Rel and PI-3K, representing one of the multiple survival pathways triggered via CD40.

It has been unclear as to whether or not CD40-mediated proliferative and survival effects are separable events that are controlled by distinct signaling pathways. Our study argues for the existence of at least two parallel signaling pathways stemming from the CD40 receptor that are distinguishable based on their reliance on the NF-κB/Rel transcription factor. In splenic B cells, we found that PI-3K and NF-κB/Rel pathway is necessary for CD40-induced proliferation. In contrast, this pathway is not the only survival pathways used by CD40, because cells remain viable even though this pathway is blocked. These data thus suggest the existence of other survival pathways that are independent of NF-κB/Rel and Bcl-X.

One of the intriguing findings of this study is a definitive link between PI-3K, NF-κB/Rel activation, and cell proliferation (Fig. 8). Using PI-3K ko mice or pharmacological inhibitor LY294002, we showed that PI-3K controls the nuclear translocation of CD40-induced NF-κB/Rel complexes, through regulation of IκBα degradation. Together with two recent reports showing a direct interaction between AKT and IKKα in cell lines (22, 23), we propose that PI-3K may regulate IκBα degradation in splenic B lymphocytes via AKT. Indeed, we showed that AKT phosphorylation and activation is blocked when PI-3K activity is inhibited. Thus, our study established the necessary signaling pathway responsible for CD40-mediated proliferation that involves PI-3K and NF-κB/Rel activation.

In dissecting the mechanism by which CD40 controls cell proliferation, we also found that PI-3K-mediated cell cycle progression involves p27^kip down-regulation. There is no experimental evidence to connect p27^kip down-regulation and NF-κB/Rel activation. However, it is most likely that these two PI-3K-mediated events are independently regulated. First, it is noticeable that these two events occur at different times upon CD40 ligation. NF-κB/Rel nuclear translocation begins 2 h after stimulation and is sustained for several hours. In contrast, down-regulation of p27^kip-cell cycle inhibitor appears only upon 24 h of CD40 stimulation. Furthermore, our study indicates that the down-regulation of p27^kip isindependent from c-Rel, because in c-Rel ko B cells, p27^kip deg- radation still occurs normally upon B cell receptor engagement.⁴

In contrast to the effects PI-3K exerts on proliferation upon CD40 stimulation, apoptosis protection seems to proceed normally even in the absence of the PI-3K activity. Surprisingly, even though NF-κB/Rel has been implicated in protecting cells from various apoptotic insults, we found that NF-κB/Rel is not the only survival pathway mediated by CD40 (Fig. 8). This is concluded based on our data that establish that CD40-mediated apoptosis protection is normal in c-Rel ko mouse and in cells treated with the PI-3K inhibitor. In dissecting the survival pathway, we examined several known anti-apoptotic molecules. First, AKT kinase, known to be a direct target of the PI-3K phosphorylation and to prevent apoptosis, was not activated when PI-3K action has been blocked. Still, CD40 prevented apoptosis of these cells, suggesting that CD40-activated cells can remain viable in the absence of AKT. Remarkably, we demonstrated that Bcl-X expression is regulated by PI-3K and NF-κB/Rel; and its absence in the c-Rel ko or PI-3K deficient cells (H.S., T.K., and S.K., unpublished observation) does not affect the ability of these cells to survive when stimulated via CD40 receptor. In searching for the remaining CD40-mediated survival pathways or molecules that confer the protection against death mediated by PI-3K inhibition, we have indirectly excluded the c-IAP1 and c-IAP2 members (inhibitor of apoptosis) of caspase inhibitors (33, 34), as well as transcription factor A-20 (Ref. 11 ; S. Andjelic, J. Tumang, and H.-Chi Liou, unpublished data), because these molecules were shown to be dependent on the NF-κB/Rel activity (3, 35), which is blocked in this case. Moreover, XIAP (X-linked member of the IAP family molecule; Ref. 36) is also not involved according to several experiments we performed on XIAP ko mice (kindly provided by Dr. Craig Thompson, University of Pennsylvania Medical Center; data not shown), which revealed that the CD40 survival pathway was not affected by the absence of XIAP (that is NF-κB/Rel-independent).

Thus, the CD40-mediated survival pathways are more complex than we anticipated, and they are not relying completely on the induction of known anti-apoptotic molecules, such as Bcl-X, A20, and IAPs. Previously, we have reported that CD40 stimulation blocks caspase-3-like activity in normal splenic B lymphocytes (37). Therefore, it is likely that CD40-induced events block caspase action through a direct posttranslational modification of certain signaling molecules that are unknown at present, which in turn inactivate caspases. In addition to searching for these molecules, our future study will be focused on investigating the effect of CD40 stimulation on preventing the apoptotic events occurring in mitochondria during the course of programmed cell death (38).

We thank Drs. Maureen Howard, Craig Thompson, and Janko Nikolic-Zugic for gifts of reagents and mice, and Dr. Philip King for helpful discussion. We also thank members of the Liou laboratory for their support and help. This work was funded by National Institutes of Health, American Cancer Society of America Junior Faculty Research Award, and March of Dimes Basil O’Connor Award (to H.-C.L.) and by a grant from the Japan Society for the Promotion of Science (to S.K.).