The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 3872-3880
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shinners, N. P.
Right arrow Articles by Khan, W. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shinners, N. P.
Right arrow Articles by Khan, W. N.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene

Bruton’s Tyrosine Kinase Mediates NF-{kappa}B Activation and B Cell Survival by B Cell-Activating Factor Receptor of the TNF-R Family1

Nicholas P. Shinners2,*, Gianluca Carlesso2,*, Iris Castro*, Kristen L. Hoek*, Radiah A. Corn*, Robert L. Woodland{dagger}, Martin L. Scott{ddagger}, Demin Wang§ and Wasif N. Khan3,*

* Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146; {dagger} Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655-0002; {ddagger} Biogen Idec, Cambridge, MA 02142; and § The Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI 53226


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Loss of Bruton’s tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-{kappa}B pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-{kappa}B pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-{kappa}B pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of I{kappa}B kinase {gamma}-containing complexes and defective I{kappa}B{alpha} degradation. In addition, Btk-deficient B cells produce reduced levels of NF-{kappa}B2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-{kappa}B pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-{kappa}B pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-{kappa}B activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-{kappa}B may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Bruton’s tyrosine kinase (Btk)4 is required for B lymphocyte survival, proliferation, and differentiation in response to BCR activation. Accordingly, mutations in the btk gene result in the B cell deficiency disorder xid in mice and X-linked aggammaglobulinemia in humans (1, 2, 3, 4, 5). The xid phenotype, caused by either targeted gene-deletion or a naturally occurring mutation in the pleckstrin homology domain (R28C), which is required for Btk membrane localization, is characterized by a severe reduction in mature follicular B cell numbers relative to wild-type (wt) animals (3). The residual mutant B cells are mostly immature and are severely compromised in their survival potential likely due to, at least in part, an inability to effectively induce the antiapoptotic gene bcl-xL and defective activation of transcription factor NF-{kappa}B following BCR cross-linking (6, 7, 8, 9, 10). We and others have previously shown that Btk and its direct substrate, phospholipase C (PLC)-{gamma}2, are essential for the activation of NF-{kappa}B in response to BCR engagement (9, 10, 11, 12, 13). A recent report has shown that loss of PLC-{gamma}2 results in reduced B cell-activating factor (BAFF)-mediated NF-{kappa}B activation and B cell survival, indicating that Btk likely plays a role in BAFF receptor (BAFF-R) signaling (14). Despite these advances, the entire scope of Btk-dependent signaling in B cell survival and function is not clear.

The NF-{kappa}B/Rel family contains five members: RelA (p65), c-Rel, RelB, NF-{kappa}B1 (p50/105), and NF-{kappa}B2 (p52/100) (15). All members except RelB form homodimers and heterodimers, and RelA and c-Rel heterodimerize with p50 and p52 (15). There are two known pathways to activate NF-{kappa}B (15, 16). The classical pathway involves mainly RelA and c-Rel sequestered in the cytoplasm in an inactive form due to their physical association with I{kappa}B{alpha} that masks the Rel nuclear translocation sequences. Upon cell activation, I{kappa}B{alpha} is phosphorylated by the beta subunit of the I{kappa}B kinase (IKK) complex IKK{alpha}beta{gamma} and subsequently polyubiquitinated (15, 17). The regulatory subunit IKK{gamma} is essential for the assembly of IKK{alpha}beta{gamma} and thus, for the activation of the classical NF-{kappa}B pathway. These inducible signaling events target I{kappa}B{alpha} for degradation by the 26 S proteasome, thereby liberating primarily p50-, RelA-, and c-Rel-containing dimers to translocate to the nucleus to activate target genes (18). The alternative pathway, which targets p52/RelB heterodimers, is known to function independently of IKKbeta and IKK{gamma} and selectively uses IKK{alpha} (15, 16). Once activated by NF-{kappa}B-inducing kinase, IKK{alpha} directs the phosphorylation and subsequent proteolytic processing of NF-{kappa}B2 precursor protein p100 to p52, thereby forming p52/RelB heterodimers that translocate to the nucleus to activate distinct target genes (15, 16).

BAFF (also known as BLyS, TALL-1, THANK, zTNF4 or TNFSF13b) is a member of the TNF family that functions as a prosurvival factor for peripheral immature and mature B cells, in part, by activating both classical and alternative NF-{kappa}B pathways (19, 20, 21, 22, 23, 24, 25). BAFF binds three distinct receptors of the TNFR family: BAFF receptor 3 (BAFF-R, or BR3), transmembrane activator and cyclophilin ligand interactor (TACI), and B cell maturation Ag (26). BAFF-R and TACI are expressed on all B cell subsets; their expression increases with maturation, whereas B cell maturation Ag is expressed primarily on plasma cells (27, 28). Targeted gene deletion studies indicate that loss of either TACI or B cell maturation Ag does not result in defective peripheral B cell development, whereas loss of BAFF or BAFF-R or mice that contain a dysfunctional BAFF-R (A/WySnJ) display a severe deficiency of mature and late immature or transitional type 2 B cells (29, 30, 31). These studies also revealed an important function for BAFF-R in the regulation of Ab production during immune responses, reminiscent of xid phenotype (3, 31). Gene-targeted deletions of either NF-{kappa}B1 (p50) or NF-{kappa}B2 (p100/52) result in defective survival of B cells in response to BAFF (20, 25), indicating that BAFF/BAFF-R survival signals may require both classical (p50) and alternative (p52) NF-{kappa}B activation. Additionally, loss of BAFF-R causes a more severe B cell deficiency than loss of IKKbeta, IKK{alpha}, or NF-{kappa}B2 alone (31, 32, 33, 34, 35, 36). Thus, both the classical and the alternative NF-{kappa}B pathways are likely to contribute to the survival function of BAFF-R (37, 38).

It is well established that Btk is critical for B cell survival and NF-{kappa}B activation in response to BCR engagement, but it is not known whether Btk contributes to B cell survival by other cell surface receptors. We show that Btk plays an important role in both B cell survival and NF-{kappa}B activation in response to BAFF. Btk not only regulates the classical NF-{kappa}B pathway, but also likely facilitates the sustained activation of the alternative NF-{kappa}B pathway by means of regulating the expression of NF-{kappa}B2. These results reveal a novel function for Btk and suggest that Btk may provide a unifying mechanism for the activation of both NF-{kappa}B pathways downstream of BCR and BAFF-R.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

The generation of Btk-, PLC-{gamma}2-, p50-, RelB-, and TACI-deficient mice, as well as xid mice has been previously described (3, 24, 39, 40, 41, 42). For wt controls, 129/SvxC57BL/6 or C57BL/6 mice from The Jackson Laboratory were used. All mice were treated humanely and in accordance with the federal and state government guidelines, and their use was approved by the Vanderbilt Institutional Animal Care Committee.

Cells and Reagents

Splenic B cells were enriched by AutoMACS depletion with anti-CD43-conjugated microbeads (Miltenyi Biotec). Immature (AA4.1+) B cells were separated with PE-conjugated anti-C1qRp (AA4.1; eBioscience) followed by anti-PE microbeads (Miltenyi Biotec). From 92 to 99% of cells were B220+ and IgM+ B cells (BD Biosciences). Mammalian B cells were maintained in RPMI 1640 (HyClone Laboratories) with 10% FCS, 50 µM 2-ME, 2 mM L-glutamine, and penicillin/streptomycin at 37°C in 5% CO2. DT40 chicken B cells deficient for Btk, as well as those reconstituted with human Btk, were the gifts of Dr. T. Kuroski (Riken Cell Bank, Riken, Japan). Their generation has been described previously (43). Chicken cells were maintained with the same medium as described, with the inclusion of 1% chicken serum (Sigma-Aldrich) and cultured at 39°C. Cells were either left nonstimulated or stimulated with recombinant human BAFF purified from Chinese hamster ovary cells (44), anti-IgM F(ab')2 (Jackson ImmunoResearch Laboratories), anti-CD40 (BD Pharmingen), or PMA and ionomycin (Sigma-Aldrich). DT40 chicken cells were stimulated with BAFF at a 1/2 dilution of hybridoma supernatants containing anti-chicken IgM mAb (M4) or PMA and ionomycin. Cell surface BAFF-R was detected with biotinylated anti-mouse BAFF-R (28), followed by allophycocyanin-streptavidin (BD Pharmingen). Data were collected on FACSCaliber flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star). Anti-p100/p52, anti-RelB, anti-c-Rel, anti-I{kappa}B{alpha}, anti-IKK{alpha}beta, anti-IKK{gamma}, anti-Btk, anti-beta-actin, anti-p38, and normal rabbit preimmune Abs were purchased from Santa Cruz Biotechnology. Monoclonal anti-IKK{alpha} and anti-IKK{gamma} were purchased from Imgenex.

Cell survival assay

B cells were cultured as described at a density of 3 x 106 cells/ml with either BAFF (100 ng/ml) or anti-CD40 (2.5 µg/ml) or left nonstimulated and stained with allophycocyanin-conjugated anti-IgM and PE-conjugated anti-C1qRp (AA4.1), followed by staining with 7-aminoactinomycin D (BD Biosciences). Cells were analyzed by FACS for 7-aminoactinomycin D negative population and reported as the percentage of survival.

Immunoblotting and in vitro kinase assays

For I{kappa}B{alpha} degradation, primary B cells (1.5 x 106 cells per sample) or DT40 chicken B cells (1.5 x 106 cells per sample) were preincubated for 20 min in medium containing 35 µM cycloheximide and 200 IU/ml polymyxin B (Sigma-Aldrich) at 37° or 39°C, respectively, and stimulated in the continued presence of cycloheximide. Stimulated and nonstimulated cells, as indicated, were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1.0% Nonidet P-40, 0.25% sodium deoxycholate, 2 mM EDTA, and 0.1% SDS) supplemented with Roche Complete Protease Inhibitor Cocktail. A total of 30 µg of total cell lysate per sample was separated by SDS-PAGE, transferred to polyvinylidene difluoride, and immunoblotted with the indicated Abs. For in vitro processing of p100 and analyses of Rel proteins, stimulated and nonstimulated cells, as indicated, were lysed with RIPA buffer and immunoblotted as described. For in vivo processing of p100 and accumulation of p52, wt mice and Btk-deficient (btk–/–) mice were injected i.p. with either PBS or recombinant murine BAFF (25 µg) at 0 and 24 h and splenocytes were harvested at 48 h. Total splenic B cells were enriched by AutoMACS depletion with anti-CD43-conjugated microbeads. Whole cell lysates of splenic B cells were prepared with RIPA buffer and analyzed by immunoblotting as described. For in vitro kinase assay of IKK{alpha}beta{gamma}, purified total (see Fig. 1A) or AA4.1+ immature (see Fig. 1B) wt and btk–/– B cells (20 x 106) were lysed with buffer A (10 mM HEPES (pH 7.4), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1.0% SDS, 5 mM NaF, 1 mM Na3VO4), supplemented with protease inhibitors. Cellular debris was cleared by high-speed spin at 14,000 rpm for 20 min at 4°C. Cytoplasmic extracts were immunoprecipitated with anti-IKK{gamma} (Santa Cruz Biotechnology) and protein A-Sepharose (Zymed Laboratories). Resultant immunocomplexes were processed for in vitro kinase activity as previously described (45).


Figure 1
View larger version (47K):
[in this window]
[in a new window]

  		FIGURE 1. Btk regulates BAFF-induced activation of classical NF-{kappa}B. A, MACS-enriched wt and btk–/– total B cells were stimulated for the indicated times with anti-IgM (20 µg/ml), BAFF (500 ng/ml), PMA and ionomycin (250 nM), or left unstimulated. Cytoplasmic extracts were immunoprecipitated with anti-IKK{gamma}, or rabbit IgG preimmune serum as a negative control, and protein A-Sepharose. Resultant immunocomplexes were subjected to in vitro kinase assay (IKK K.A.) as previously described (45 ). Membranes were additionally probed with anti-IKK{alpha} and anti-IKKbeta as loading control for immunoprecipitation efficiency. Data are representative of at least four independent experiments. B, MACS-enriched AA4.1+ wt and btk–/– immature B cells were stimulated and processed as in A for IKK in vitro activity. Additionally, 35 µg of each lysate was removed before immunoprecipitation and immunoblotted to assess equal amounts of IKK{gamma} due to difficulties detecting immunoprecipitated IKK{gamma} that co-migrates with IgH chain by SDS-PAGE. Data are representative of three independent experiments. C, MACS-enriched wt, btk–/–, and xid total B cells were preincubated with cycloheximide (35 µM) and 200 IU/ml polymyxin B and stimulated with anti-IgM (20 µg/ml), BAFF (500 ng/ml), PMA and ionomycin (250 nM), or left nonstimulated for 90 min. Total cellular extracts (30 µg) were subjected to immunoblotting with the indicated Abs. Data are representative of at least four independent experiments. D, MACS-enriched AA4.1+ wt and btk–/– immature B cells were prepared and treated as in C for I{kappa}B{alpha} degradation. Data are representative of three independent experiments. E, MACS-enriched taci–/– total B cells were analyzed as in C for I{kappa}B{alpha} degradation. Data are representative of three independent experiments. F, MACS-enriched wt and btk–/– total B cells were either left nonstimulated or stimulated with BAFF (200 ng/ml) or anti-IgM (10 µg/ml) for 2 h. Nuclear extracts were directly analyzed for NF-{kappa}B DNA binding or after preincubation with Abs specific to c-Rel. Data are representative of three independent experiments. G, Whole cell extracts from MACS-enriched wt and btk–/– total B cells were subjected to immunoblotting with the indicated Abs. Data are representative of three independent experiments. H, DT40 chicken B cells deficient for Btk or reconstituted with human Btk were pretreated with cycloheximide and polymyxin B as in C. Cells were stimulated with anti-IgM (at a dilution of 1/2 (M4) hybridoma supernatants containing anti-chicken IgM mAb), BAFF (500 ng/ml), PMA and ionomycin (250 nM), or left nonstimulated. Cells were processed as in C for I{kappa}B{alpha} degradation. Data are representative of three independent experiments.

 
EMSA analysis

Equal amounts of nuclear extracts (2 µg) prepared by Nuclear Extraction-Protein Extraction Reagent kit (NE-PER; Pierce) were preincubated for 20 min at 25°C in the presence or absence of polyclonal Abs specific for c-Rel, p52, and RelB (Santa Cruz Biotechnology). Subsequently, [{gamma}-32P]ATP-radiolabeled probe derived from {kappa}B enhancer sequences in the IL-2R promoter (5-CAACGGCAGGGGAATTCCCCTCTCCTT-3) (46) was added and incubated on ice for 15 min. DNA-protein complexes were resolved by electrophoresis on 4% native polyacrylamide gels and exposed to x-ray film.

Quantitative RT-PCR

RNA was extracted using RNeasy Mini kit (Qiagen) and used to make cDNA. For real-time PCR, we used TaqMan Universal Master mix (Applied Biosystems) and Stratagene Max 3000p Detection System. Primers and FAM-labeled probes were obtained from Applied Biosystems (TaqMan Assay On Demand). The relative mRNA fold induction for each gene was calculated relative to 18 S ribosomal RNA.

Statistical analyses

Data were compared with a Student’s test. All data are represented as mean ± SEM where indicated. Values of p ≤ 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
BAFF-dependent activation of the classical NF-{kappa}B pathway is impaired in btk–/– B cells

NF-{kappa}B appears to play an important role in BAFF-dependent B cell survival (20, 25). To determine whether Btk regulates BAFF-induced activation of the classical NF-{kappa}B pathway, we investigated the activation of IKK{gamma}-containing complexes that target I{kappa}B{alpha} for phosphorylation and subsequent degradation (15). Total cellular extracts from primary wt and btk–/– B cells stimulated with BAFF, anti-IgM, phorbol ester (PMA) and calcium ionophore (ionomycin) or left nonstimulated were immunoprecipitated with Abs specific to IKK{gamma}, and the resulting immunocomplexes were subjected to an in vitro kinase assay. As shown in Fig. 1A, IKK{alpha}beta{gamma} containing complexes from wt cells stimulated with BAFF are catalytically active (Fig. 1A, left panel, compare lane 2 with lane 4), whereas complexes from btk–/– B cells stimulated with BAFF were impaired for this activity (Fig. 1A, left panel, compare lane 6 with lane 8). As expected, BCR-induced IKK activity was also lower in btk–/– than in wt B cells (Fig. 1A, right panel) (10). btk–/– B cells were not inherently defective for this process because stimulation with the pharmacological agents PMA and ionomycin, which bypass membrane proximal signaling events, restored IKK activity (Fig. 1A, compare last to with lanes). Due to the increased ratio of immature to mature B cells in the btk–/– mice compared with wt controls, we further examined IKK catalytic activity in wt and btk–/– immature B cells. As shown in Fig. 1B, BAFF stimulation of wt immature B cells results in similar (activation of) IKK catalytic activity as wt total B cells (Fig. 1B, compare lane 1 with lane 2). Furthermore, BAFF stimulation of btk–/– immature B cells results in reduced IKK catalytic activity as compared with wt controls (Fig. 1B, compare lane 1 with lane 2 and compare lane 3 with lane 4). Because catalytic activation of IKK results in the phosphorylation and degradation of I{kappa}B{alpha}, we next determined whether the reduced IKK activity in btk–/– B cells results in impaired I{kappa}B{alpha} degradation. Given the reduced IKK activity in btk–/– B cells, BAFF treatment of btk–/– B cells also display reduced I{kappa}B{alpha} degradation (Fig. 1C, top middle panel, compare lane 5 with lane 7) relative to wt B cells (Fig. 1C, left panel, compare lane 1 with lane 3). These results suggest that loss of Btk causes a subtle defect in IKK activity, but it is sufficient to cause a profound defect in subsequent I{kappa}B{alpha} degradation. Like btk–/–, xid B cells also failed to induce I{kappa}B{alpha} degradation, indicating that an intact Btk pleckstrin homology domain is required for Btk function in NF-{kappa}B activation by BAFF (Fig. 1C, right panel, compare lane 9 with lane 11). As shown in Fig. 1D, immature B cells respond similarly as do total B cells (immature wt B cells degrade I{kappa}B{alpha} in response to BAFF), whereas immature btk–/– B cells are defective for this process.

Because TACI is expressed in splenic B cells and binds BAFF, we investigated whether TACI contributed to BAFF-dependent I{kappa}B{alpha} degradation. A significant contribution of TACI was ruled out because TACI-deficient (taci–/–) B cells degrade I{kappa}B{alpha} in response to BAFF similarly as do wt control B cells (Fig. 1E). Similarly, LPS contamination of BAFF preparations does not account for the observed degradation of I{kappa}B{alpha} as all samples were pretreated with the endotoxin inhibitor polymyxin B (Fig. 1, C–E). Thus, Btk plays a role in the activation of IKK{gamma}-containing complexes and degradation of I{kappa}B{alpha} and therefore activation of the classical NF-{kappa}B pathway via BAFF-R.

Consistent with the results in Figs. 1, A–E, BAFF stimulates NF-{kappa}B DNA binding activity within 2 h (Fig. 1F, compare lanes 1 and 2), which contains the transactivation subunit c-Rel (Fig 1F, lane 3). In contrast to wt B cells, BAFF stimulation of btk–/– B cells induced significantly less total NF-{kappa}B and c-Rel DNA binding activity (Fig. 1F, left, lanes 4–6). As shown in Fig. 1G, the diminished c-Rel DNA binding in btk–/– B cells is not due to an overall reduction in c-Rel protein. As a control, DNA binding activity by anti-IgM stimulation also occurs in wt B cells, but not in btk–/– B cells (Fig. 1F, right). Taken together, these results indicate that the classical NF-{kappa}B pathway is rapidly induced upon BAFF treatment and that loss of Btk interferes with BAFF-induced DNA binding activity of c-Rel, which is primarily activated by the classical NF-{kappa}B pathway.

To complement these findings, we examined whether BAFF stimulation of Btk-deficient B cells reconstituted with human wt Btk can activate NF-{kappa}B. For this purpose we used chicken DT40 B cells deficient for Btk or DT40.btk–/– B cells reconstituted with human wt Btk as we have previously described (9). As shown in Fig. 1H, expression of human wt Btk restored the ability of Btk-deficient DT40 B cells to induce I{kappa}B{alpha} degradation in response to BAFF. These experiments indicate that BTK is required for NF-{kappa}B activation in response to BAFF.

btk–/– B cells are defective for p100 expression and activation of the alternative NF-{kappa}B pathway by BAFF

Results from Fig. 1 suggest that activation of classical NF-{kappa}B by BAFF involves Btk-dependent mechanisms. Because one known target of classical NF-{kappa}B activation is the gene encoding NF-{kappa}B2/p100 (47), we investigated whether the maintenance and/or production of the precursor protein p100 is regulated by the Btk/classical NF-{kappa}B signaling axis. We first measured steady-state p100 protein levels in mice deficient for Btk, PLC-{gamma}2, and a critical component of the classical NF-{kappa}B pathway, NF-{kappa}B1/p50. As shown in Fig. 2A, top row, btk–/–, plc-{gamma}2–/–, and p50–/– B cells contain less p100 protein than their wt counterparts. Furthermore, the mutant B cells also contain less RelB, the binding partner that preferentially heterodimerizes with p52 (Fig. 2A, upper middle row on left). However, loss of Btk or p50 does not affect c-Rel steady-state protein content (Fig. 2A, lower middle row on left). In addition, p100, p52, and RelB protein levels were reduced in both immature and mature B cells from btk–/– mice (Fig. 2A, right). These results indicate that Btk/PLC-{gamma}2 signaling may be involved in producing or maintaining components of the alternative pathway, NF-{kappa}B2/p100 and RelB.


Figure 2
View larger version (52K):
[in this window]
[in a new window]

  		FIGURE 2. BAFF uses Btk/classical NF-{kappa}B for NF-{kappa}B2/p100 expression. A, Whole cell extracts (50 µg) from MACS-enriched wt, btk–/–, plc-{gamma}2–/–, and p50–/– total B cells (left) or AA4.1+ (Immature) and AA4.1– (Mature) B cells (right) were analyzed for p100, RelB, and c-Rel by immunoblotting. Anti-p38 was used as a protein loading control. Data are representative of three independent experiments. B, MACS-enriched wt total B cells were cultured with anti-IgM (10 µg/ml) or BAFF (100 ng/ml). Purified mRNA was used for quantitative PCR analysis expression of NF-{kappa}B2/p100. The relative fold induction of NF-{kappa}B2/p100 was normalized vs 18 S. Data are representative of three independent experiments. C, MACS-enriched btk–/–, plc-{gamma}2–/–, p50–/–, taci–/– total B cells and their respective wt control B cells were stimulated with anti-IgM (10 µg/ml) and BAFF (100 ng/ml) for 16 h, or PMA and ionomycin (100 nM) for 4 h (left) and NF-{kappa}B2/p100 mRNA was analyzed as in B. Data are representative of three independent experiments. *, p ≤ 0.001; **, p = 0.013; and ***, p = 0.068 for wt and btk–/– B cells (first panel). *, p = 0.048 and **, p = 0.02 for wt and plc-{gamma}2–/– B cells (second panel). *, p = 0.068 and **, p = 0.021 for wt and p50–/– (third panel). Differences between wt and taci–/– B cells are not statistically significant (fourth panel). Data are representative of three independent experiments. D, Freshly isolated MACS-enriched wt, btk–/–, plc-{gamma}2–/–, and p50–/– total B cells were analyzed as in C for basal NF-{kappa}B2/p100 mRNA levels. Data are representative of three independent experiments for wt, btk–/–, and p50–/–. *, p = 0.08 and **, p = 0.04. Data for plc-{gamma}2–/– are representative of two independent experiments. E, MACS-enriched AA4.1+ wt and btk–/– immature B cells were treated as in C and purified mRNA was used for quantitative PCR analysis expression of NF-{kappa}B2/p100. *, p = 0.016; **, p = 0.016; and ***, p = 1.0 for wt and btk–/– immature B cells. Data are representative from at least three independent experiments.

 
To understand the mechanism for regulation of p100 protein, we next determined whether stimulation by BAFF or anti-IgM results in NF-{kappa}B2 gene transcription and whether Btk, PLC-{gamma}2, or p50 are involved in this process. Quantitative RT-PCR revealed that BAFF treatment of wt B cells induces p100 mRNA expression (Fig. 2B). In contrast, B cells deficient for Btk, or its downstream target PLC-{gamma}2, are impaired in this process (Fig. 2C, left and left middle panels, respectively). Furthermore, p50–/– cells were also impaired for BAFF-induced p100 expression (Fig. 2C, right middle panel). This defect in the BAFF signal-induced expression of p100 in the mutant cells was not due lower absolute levels of p100 mRNA (Fig. 2D). Additionally, Btk is required for this process even when phenotypically identical cells were compared (Fig. 2E). Thus, the reduced basal p100 protein shown in Fig. 2A is likely due to both transcriptional as well as posttranslational mechanisms involving Btk. The BAFF regulation of p100 likely proceeds via BAFF-R, as taci–/– B cells induce p100 expression at a level comparable to wt control B cells (Fig. 2C, right panel). Prior studies have shown that the alternative NF-{kappa}B pathway plays a critical role in BAFF-dependent B cell survival (19, 25). Our results indicate that Btk deficiency results in diminished BAFF-mediated p100 gene transcription (Fig. 2, C and E). We next investigated whether Btk plays a role in BAFF-induced activation of the alternative NF-{kappa}B pathway. As expected, BAFF stimulation results in p100 processing in wt B cells (Fig. 3A, top, compare lane 1 with lane 3). However, consistent with results from Fig. 2, btk–/– B cells display lower levels of p100 than their wt counterparts (Fig. 3A, top, compare lane 1 with lane 2). The available p100 appears to be processed by BAFF, but results in correspondingly reduced levels of p52 than levels found in wt control cells (Fig. 3A, compare lane 3 with lane 4). Because ex vivo culture of btk–/– B cells may result in increased cell death as compared with wt controls, we next assessed in vivo p100 processing and p52 accumulation after 48 h of BAFF treatment by i.p. injection. The wt B cells process p100 with a corresponding accumulation of p52 upon BAFF treatment, whereas btk–/– B cells process the available p100, but without a significant accumulation of p52 as compared with wt cells (Fig. 3B). Furthermore, injection of BAFF results in an increase of RelB in wt, but not in btk–/– B cells (Fig. 3B, compare lane 3 with lane 4). In addition, The BAFF-induced p100 expression defect in btk–/– B cells was observed in both immature as well as mature B cells (Fig. 3D). As a control, CD40 activation, which signals independently of Btk, efficiently induced p100 processing to p52 as well as p100 protein production equally well in wt and btk–/– B cells (Fig. 3A, compare lane 5 with lane 6). The DNA binding of p52 and RelB is also reduced in btk–/– relative to wt B cells (Fig. 3C, compare lane 4 with lane 9 and lane 5 with lane 10). The processing of p100 likely occurs by BAFF-R as taci–/– B cells process p100 similarly as do wt controls (Fig. 3E). These data suggest that the role for Btk in alternative NF-{kappa}B activation may not be p100 processing, but rather maintaining steady-state RelB and NF-{kappa}B2/p100 as substrate for p52 production.


Figure 3
View larger version (44K):
[in this window]
[in a new window]

  		FIGURE 3. BAFF activation of alternative NF-{kappa}B pathway is reduced in btk–/– B cells. A, MACS-enriched total B cells from wt or btk–/– mice were stimulated for 12 h with BAFF (500 ng/ml), anti-CD40 (2.5 µg/ml), or left nonstimulated (Non). Whole cell extracts (50 µg/lane) were prepared with RIPA buffer and analyzed by immunoblotting with Abs specific for p100/p52. Anti-p38 was used as a protein loading control. Data are representative from at least three independent experiments. B, wt and btk–/– mice were injected i.p. with either PBS or recombinant mouse BAFF (25 µg) at 0 and 24 h or left nonstimulated (N.S.), and splenocytes were harvested at 48 h. Total cellular extracts from MACS-enriched total B cells were prepared in RIPA buffer and immunoblotted as in A. Data are representative from three independent experiments. C, MACS-enriched total B cells from wt and btk–/– mice were stimulated with BAFF for 14 h and analyzed by EMSA as in Fig. 1F, with additional Abs to supershift RelB and p52. Data are representative from three independent experiments. D, MACS-enriched AA4.1+ (Immature) and AA4.1– (Mature) total B cells isolated from wt or btk–/– mice were stimulated and processed as in A. Data are representative from three independent experiments. E, Total cell extracts from MACS-enriched wt and taci–/– total B cells were prepared with RIPA buffer and analyzed by immunoblotting with Abs specific for p100/p52 as in A. Anti-p38 was used as a protein loading control. Data are representative from three independent experiments.

 
BAFF-dependent B cell survival requires an intact alternative NF-{kappa}B pathway

Recent reports suggest that the alternative NF-{kappa}B pathway is important in BAFF-dependent B cell survival (19, 25). To determine whether diminished alternative NF-{kappa}B activation observed in btk–/– B cells may result in reduced B cell viability, we next assayed for wt and btk–/– B cell survival in the presence of BAFF. BAFF treatment of wt B cells results in greater viability than in btk–/– B cells (Fig. 4A, compare nonstimulated vs treated B cells). To ensure that btk–/– B cells are not inherently defective for survival, anti-CD40 treatment provides equivalent viability for both wt and btk–/– cells (Fig. 4A). Furthermore, the BAFF survival defect of btk–/– B cells is not solely due to their maturation stage as immature btk–/– B cells are less viable than their immature wt counterparts (Fig. 4B).


Figure 4
View larger version (46K):
[in this window]
[in a new window]

  		FIGURE 4. BAFF-mediated B cell survival is compromised by loss of Btk and RelB. A, MACS-enriched total B cells from wt and btk–/– mice were cultured with BAFF (100 ng/ml) or anti-CD40 (2.5 µg/ml) for 24 and 48 h or left nonstimulated (Non), and the ratio of live to dead cells was determined by 7-aminoactinomycin D staining and analyzed by flow cytometry. *, p = 0.002 nonstimulated vs BAFF-treated and **, p = <0.001 nonstimulated vs anti-CD40-treated total B cells for wt 48-h treatments (left). *, p = 0.051 nonstimulated vs BAFF-treated and **, p = <0.001 nonstimulated vs anti-CD40-treated total B cells for btk–/– 48-h treatments (right). Data are representative of at least five independent experiments. B, MACS-enriched AA4.1+ wt and btk–/– immature B cells were cultured with (100 ng/ml) (Figure 4) or without BAFF (Non) ({blacksquare}) for 48 h. The immature phenotype of live cells was confirmed by staining for AA4.1 and IgM after termination of cultures. The cell viability was determined as in A. *, p = 0.037 and **, p = 0.07. Data are representative of three independent experiments. C, MACS-enriched wt, btk–/–, relb+/–, and relb–/– total B cells were analyzed as in B for BAFF-mediated B cell survival. *, p = 0.002; **, p = 0.051; ***, p = <0.001; and ****, p = 0.024. Data are representative of three independent experiments. D, Total cellular extracts from MACS-enriched wt, relb+/–, and relb–/– total B cells were analyzed for p100, RelB, beta-actin, p52, and c-Rel by immunoblotting as in Fig. 2A. Data are representative of three independent experiments. E, Flow cytometric analysis of BAFF-R on freshly isolated MACS-enriched wt, btk–/–, relb+/–, and relb–/– total B cells. Gates were set to identify live B220+ B cells before analysis of control (light gray histograms indicate control) or anti-mouse BAFF-R Ab binding (black line histograms). Data are representative of three independent experiments. F, MACS-enriched wt and taci–/– total B cells were analyzed as in B for BAFF-mediated B cell survival. *, p = 0.009 and **, p = 0.001. Data are representative of three independent experiments.

 
To confirm the contribution of the alternative pathway to BAFF-dependent B cell survival and compare these results with btk–/– B cells, we next analyzed RelB-deficient (relb–/–) B cells for their capacity to survive in the presence of BAFF. In contrast to heterozygous or wt controls, respectively, relb–/– and btk–/– B cells are similarly defective for BAFF-mediated viability (Fig. 4C). Furthermore, similar to btk–/– B cells, relb–/– B cells also contain significantly less p100 and correspondingly less p52 than found in wt control B cells (Fig. 4D, top two panels). Additionally, a failure of btk–/– and relb–/– B cells to survive in response to BAFF was not due to altered cell surface expression of BAFF-R (Fig. 4E). Because B cells also express TACI, which binds BAFF, we performed the same survival assay with taci–/– mice. B cells from taci–/– mice survived as well as B cells from wt control mice in response to BAFF (Fig. 4F). These data indicate that loss of RelB severely reduces the amount of steady-state p100 protein, and correspondingly p52 protein, resulting in the virtual loss of alternative NF-{kappa}B activation. These data further demonstrate that an intact alternative NF-{kappa}B pathway is required for BAFF-mediated B cell survival.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Mutations in the gene encoding Btk result in the B cell deficiency disease xid in mouse (5, 48). Likewise, loss of BAFF/BAFF-R function results in a severe deficiency of peripheral B cells (29, 30, 31). Whereas Btk regulation of B cell survival has been traditionally attributed to BCR signaling, our current studies indicate that Btk also contributes to BAFF/BAFF-R-dependent NF-{kappa}B activation and B cell survival. These data indicate that Btk function is critical for two major mediators of B cell survival, BCR and BAFF-R, and that Btk couples both receptors to NF-{kappa}B. Btk regulates B cell survival by directly regulating the classical NF-{kappa}B pathway in response to stimulation of both BCR and BAFF-R (Fig. 1), and that BAFF-R in turn induces the expression of NF-{kappa}B2 (p100) for sustained alternative NF-{kappa}B activation (Figs. 2 and 3). These findings also suggest that impaired BCR and BAFF/BAFF-R survival signaling may contribute to B cell deficiency and impaired immune responses in btk–/– mice.

Several studies have shown that BAFF activates the alternative NF-{kappa}B pathway (19, 20, 22, 25). Additionally, BAFF interaction with BAFF-R also activates the classical NF-{kappa}B pathway (21, 22), but the evidence is inconsistent and the mechanisms are not well understood. In this study, we provide evidence that Btk mediates BAFF-R-dependent activation of classical NF-{kappa}B: btk–/– B cells are defective for BAFF-mediated activation of IKK{gamma}-containing complexes and degradation of I{kappa}B{alpha}, two hallmarks of classical NF-{kappa}B activation. A role for Btk in BAFF-dependent activation of NF-{kappa}B and B cell survival is consistent with a prior report showing that PLC-{gamma}2, a direct substrate of Btk, is also involved in these processes (14). In addition, because protein kinase C (PKC) is one of the downstream effectors of Btk/PLC-{gamma}2 signaling, Hikida et al. (14) and our results implicate PKC in BAFF-mediated NF-{kappa}B activation and B cell survival. Indeed, a recent study suggests that PKCbeta contributes to the overall metabolic fitness of B cells by directly activating Akt and partially contributing to B cell survival following stimulation by BAFF (49). Thus, Btk, PLC-{gamma}2 and PKCbeta appear to regulate B cell survival in response to BAFF.

Btk deficiency results in a surprising reduction in BAFF-mediated alternative NF-{kappa}B activation. By using mice deficient for regulators of the classical NF-{kappa}B pathway, Btk, PLC-{gamma}2, and p50, we found that these mutants are compromised for BAFF-induced gene transcription and protein production of NF-{kappa}B2/p100 (respectively). Therefore, the reduced activation of BAFF-mediated alternative NF-{kappa}B pathway in btk–/– B cells may be due to these reduced levels of p100, as well as RelB. Thus, it is possible that poor survival of btk–/– B cells in the presence of BAFF is the consequence of defects in both classical and alternative NF-{kappa}B pathways. However, relb–/– B cells, which are intact for the classical pathway, also do not survive in the presence of BAFF, indicating physiologic significance and nonredundant function for the alternative pathway in BAFF/BAFF-R-mediated B cell survival. This function is consistent with the report by Enzler et al. (37). Although our studies suggest a role for Btk in BAFF-dependent B cell survival by both classical NF-{kappa}B and the alternative NF-{kappa}B pathway, it is possible that other mechanisms contribute to BAFF-dependent B cell survival. For example, prior studies have shown that BAFF may promote B cell survival by preventing nuclear translocation of PKC{delta} (38, 50) or by down-regulating a proapoptotic protein of the Bcl-2 family, Bim (51). Thus, multiple pathways under BAFF control can contribute to its survival function. Further studies are required to determine the specific contributions of each pathway to BAFF-dependent B cell viability.

The p100 promoter is activated by classical NF-{kappa}B, and p50–/– cells have reduced levels of p100 (47, 52, 53). Our studies reveal that loss of two additional components of the classical NF-{kappa}B pathway, Btk and PLC-{gamma}2, results in reduced p100 mRNA and protein. The key step in activation of the alternative NF-{kappa}B pathway is the processing of p100 to p52 (16). Because proteolysis is irreversible, conversion from p100 to p52 results in the elimination of the precursor protein p100. Thus, sustained activation of the alternative NF-{kappa}B pathway requires continuous supply of p100. Our results suggest that BAFF/BAFF-R use Btk to maintain p100 expression via the classical NF-{kappa}B pathway. As a consequence, the continued expression of p100 provides substrate (p100 protein) for the subsequent activation of the alternative NF-{kappa}B pathway. This intimate link between the two NF-{kappa}B pathways creates a BAFF-R positive autoreinforcing loop in which the alternative pathway draws on the product of classical pathway to sustain its activation (Fig. 5). This model is consistent with previous results that lymphotoxin beta receptor, a potent activator of classical NF-{kappa}B, also regulates p100 expression upon engagement with lymphotoxin beta (54), and indicates this mechanism is likely to occur by other receptors that target both the classical and alternative pathways, including CD40 (Fig. 3). This model may also provide a unifying mechanism for the action of both NF-{kappa}B pathways critical for B cell survival under BAFF-R.


Figure 5
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 5. Model of BCR and BAFF-R activation of both classical and alternative NF-{kappa}B pathways. BCR and BAFF-R activate IKK{gamma}-containing complexes that target the classical NF-{kappa}B pathway via mechanisms involving Btk. In turn, this signaling pathway induces NF-{kappa}B2 (p100) gene transcription, which serves to sustain alternative NF-{kappa}B activation in response to BAFF. Loss of Btk directly interferes with BAFF activation of classical NF-{kappa}B, whereas the alternative NF-{kappa}B pathway is indirectly impaired by diminished NF-{kappa}B2 (p100) protein. Loss of RelB additionally results in diminished p100 protein, likely caused by either a mechanism involving regulation of NF-{kappa}B2 gene transcription (indicated by dashed arrows), reduced p100 protein stability, or both.

 
Like BAFF-R, BCR signaling is also obligatory for B cell survival (55) and induces activation of the classical NF-{kappa}B pathway and expression of p100 (8, 9, 10) (Fig. 2). Thus, it is likely that BCR signaling contributes to B cell survival by maintaining p100 expression and thus, alternative NF-{kappa}B activation by BAFF-R. Consistent with this possibility, we have found that BCR stimulation significantly induces the expression of p100 mRNA and production of p100 protein (Fig. 2 and data not shown). A requirement of Btk in the up-regulation of p100 under both BCR and BAFF-R suggests a central role for Btk in reinforcing the alternative NF-{kappa}B pathway and B cell survival. Taken together, our findings suggest that Btk is involved in NF-{kappa}B activation and B cell survival in response to both BCR and BAFF-R; whereas it directly regulates the classical NF-{kappa}B pathway, it also reinforces the alternative NF-{kappa}B pathway and thus B cell survival. Our findings also indicate that in addition to BCR, Btk function downstream of BAFF-R is associated with pathophysiology of xid disease. Therefore, strategies to specifically interfere with activation of Btk signaling may provide tools for therapeutic intervention in B cell pathologies caused by dysregulated B cell survival, including B cell lymphomas and autoimmune diseases.


   Acknowledgments
 
We thank Drs. Terry Dermody for providing p50–/– mice, Mark Boothby for providing relB–/– mice, and Robert S. Carter and Kevin N. Pennington for expert technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study is supported in part by Grants RO1 AI50213-01 and AI060729-01 (to W.N.K.) and R01 HL073284 (to D.W.) from the National Institutes of Health and by Grants RSG TBE-102299 (to W.N.K.) and RSG CCG-106204 (to D.W.) from the American Cancer Society. N.P.S. and K.H. are supported by Grant T32 HL69715-0 (to J. Hawiger) and I.C. is supported by Grant T32 CA09385-20 (to H. E. Ruley) from the National Institutes of Health. Back

2 N.P.S. and G.C. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Wasif N. Khan, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. E-mail address: Wasif.Khan{at}vanderbilt.edu Back

4 Abbreviations used in this paper: Btk, Bruton’s tyrosine kinase; BAFF, B cell-activating factor; PLC, phospholipase C; wt, wild type; IKK, I{kappa}B kinase; TACI, transmembrane activator and cyclophilin ligand interactor; RIPA, radioimmunoprecipitation assay; PKC, protein kinase C. Back

Received for publication June 26, 2007. Accepted for publication June 29, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Conley, M. E., M. D. Cooper. 1998. Genetic basis of abnormal B cell development. Curr. Opin. Immunol. 10: 399-406. [Medline]
  2. Khan, W. N.. 2001. Regulation of B lymphocyte development and activation by Bruton’s tyrosine kinase. Immunol. Res. 23: 147-156. [Medline]
  3. Khan, W. N., F. W. Alt, R. M. Gerstein, B. A. Malynn, I. Larsson, G. Rathbun, L. Davidson, S. Müller, A. B. Kantor, L. A. Herzenberg, et al 1995. Defective B cell development and function in Btk-deficient mice. Immunity 3: 283-299. [Medline]
  4. Rawlings, D. J.. 1999. Bruton’s tyrosine kinase controls a sustained calcium signal essential for B lineage development and function. Clin. Immunol. 91: 243-253. [Medline]
  5. Rawlings, D. J., D. C. Saffran, S. Tsukada, D. A. Largaespada, J. C. Grimaldi, L. Cohen, R. N. Mohr, J. F. Bazan, M. Howard, N. G. Copeland, et al 1993. Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID mice. Science 261: 358-361. [Abstract/Free Full Text]
  6. Anderson, J. S., M. Teutsch, Z. Dong, H. H. Wortis. 1996. An essential role for Bruton’s [corrected] tyrosine kinase in the regulation of B-cell apoptosis. Proc. Natl. Acad. Sci. USA 93: 10966-10971. [Abstract/Free Full Text]
  7. 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: 1735-1744. [Abstract/Free Full Text]
  8. Petro, J. B., I. Castro, J. Lowe, W. N. Khan. 2002. Bruton’s tyrosine kinase targets NF-{kappa}B to the bcl-x promoter via a mechanism involving phospholipase C-{gamma}2 following B cell antigen receptor engagement. FEBS Lett. 532: 57-60. [Medline]
  9. 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. 276: 1715-1719. [Abstract/Free Full Text]
  10. 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: 1745-1754. [Abstract/Free Full Text]
  11. Antony, P., J. B. Petro, G. Carlesso, N. P. Shinners, J. Lowe, W. N. Khan. 2003. B cell receptor directs the activation of NFAT and NF-{kappa}B via distinct molecular mechanisms. Exp. Cell Res. 291: 11-24. [Medline]
  12. Humphries, L. A., C. Dangelmaier, K. Sommer, K. Kipp, R. M. Kato, N. Griffith, I. Bakman, C. W. Turk, J. L. Daniel, D. J. Rawlings. 2004. Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase C{gamma} Src homology 2-Src homology 3 linker. J. Biol. Chem. 279: 37651-37661. [Abstract/Free Full Text]
  13. Kim, Y. J., F. Sekiya, B. Poulin, Y. S. Bae, S. G. Rhee. 2004. Mechanism of B-cell receptor-induced phosphorylation and activation of phospholipase C-{gamma}2. Mol. Cell Biol. 24: 9986-9999. [Abstract/Free Full Text]
  14. Hikida, M., S. Johmura, A. Hashimoto, M. Takezaki, T. Kurosaki. 2003. Coupling between B cell receptor and phospholipase C-{gamma}2 is essential for mature B cell development. J. Exp. Med. 198: 581-589. [Abstract/Free Full Text]
  15. Ghosh, S., M. Karin. 2002. Missing pieces in the NF-{kappa}B puzzle. Cell 109: (2 Suppl. 1):S81-S96. [Medline]
  16. 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: 1495-1499. [Abstract/Free Full Text]
  17. Siebenlist, U., K. Brown, E. Claudio. 2005. Control of lymphocyte development by nuclear factor-{kappa}B. Nat. Rev. Immunol. 5: 435-445. [Medline]
  18. Birbach, A., P. Gold, B. R. Binder, E. Hofer, R. de Martin, J. A. Schmid. 2002. Signaling molecules of the NF-{kappa}B pathway shuttle constitutively between cytoplasm and nucleus. J. Biol. Chem. 277: 10842-10851. [Abstract/Free Full Text]
  19. Claudio, E., K. Brown, S. Park, H. Wang, U. Siebenlist. 2002. BAFF-induced NEMO-independent processing of NF-{kappa}B2 in maturing B cells. Nat. Immunol. 3: 958-965. [Medline]
  20. Hatada, E. N., R. K. Do, A. Orlofsky, H. C. Liou, M. Prystowsky, I. C. MacLennan, J. Caamano, S. Chen-Kiang. 2003. NF-{kappa}B1 p50 is required for BLyS attenuation of apoptosis but dispensable for processing of NF-{kappa}B2 p100 to p52 in quiescent mature B cells. J. Immunol. 171: 761-768. [Abstract/Free Full Text]
  21. Morrison, M. D., W. Reiley, M. Zhang, S. C. Sun. 2005. An atypical tumor necrosis factor (TNF) receptor-associated factor-binding motif of B cell-activating factor belonging to the TNF family (BAFF) receptor mediates induction of the noncanonical NF-{kappa}B signaling pathway. J. Biol. Chem. 280: 10018-10024. [Abstract/Free Full Text]
  22. Ramakrishnan, P., W. Wang, D. Wallach. 2004. Receptor-specific signaling for both the alternative and the canonical NF-{kappa}B activation pathways by NF-{kappa}B-inducing kinase. Immunity 21: 477-489. [Medline]
  23. Rolink, A. G., J. Tschopp, P. Schneider, F. Melchers. 2002. BAFF is a survival and maturation factor for mouse B cells. Eur. J. Immunol. 32: 2004-2010. [Medline]
  24. Yan, M., H. Wang, B. Chan, M. Roose-Girma, S. Erickson, T. Baker, D. Tumas, I. S. Grewal, V. M. Dixit. 2001. Activation and accumulation of B cells in TACI-deficient mice. Nat. Immunol. 2: 638-643. [Medline]
  25. Zarnegar, B., J. Q. He, G. Oganesyan, A. Hoffmann, D. Baltimore, G. Cheng. 2004. Unique CD40-mediated biological program in B cell activation requires both type 1 and type 2 NF-{kappa}B activation pathways. Proc. Natl. Acad. Sci. USA 101: 8108-8113. [Abstract/Free Full Text]
  26. Kalled, S. L.. 2005. The role of BAFF in immune function and implications for autoimmunity. Immunol. Rev. 204: 43-54. [Medline]
  27. Ng, L. G., A. P. Sutherland, R. Newton, F. Qian, T. G. Cachero, M. L. Scott, J. S. Thompson, J. Wheway, T. Chtanova, J. Groom, et al 2004. B cell-activating factor belonging to the TNF family (BAFF)-R is the principal BAFF receptor facilitating BAFF costimulation of circulating T and B cells. J. Immunol. 173: 807-817. [Abstract/Free Full Text]
  28. Shulga-Morskaya, S., M. Dobles, M. E. Walsh, L. G. Ng, F. MacKay, S. P. Rao, S. L. Kalled, M. L. Scott. 2004. B cell-activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation. J. Immunol. 173: 2331-2341. [Abstract/Free Full Text]
  29. Gorelik, L., K. Gilbride, M. Dobles, S. L. Kalled, D. Zandman, M. L. Scott. 2003. Normal B cell homeostasis requires B cell activation factor production by radiation-resistant cells. J. Exp. Med. 198: 937-945. [Abstract/Free Full Text]
  30. Miller, D. J., C. E. Hayes. 1991. Phenotypic and genetic characterization of a unique B lymphocyte deficiency in strain A/WySnJ mice. Eur. J. Immunol. 21: 1123-1130. [Medline]
  31. Sasaki, Y., S. Casola, J. L. Kutok, K. Rajewsky, M. Schmidt-Supprian. 2004. TNF family member B cell-activating factor (BAFF) receptor-dependent and -independent roles for BAFF in B cell physiology. J. Immunol. 173: 2245-2252. [Abstract/Free Full Text]
  32. Caamaño, J. H., C. A. Rizzo, S. K. Durham, D. S. Barton, C. Raventós-Suárez, C. M. Snapper, R. Bravo. 1998. Nuclear factor (NF)-{kappa}B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses. J. Exp. Med. 187: 185-196. [Abstract/Free Full Text]
  33. Franzoso, G., L. Carlson, L. Poljak, E. W. Shores, S. Epstein, A. Leonardi, A. Grinberg, T. Tran, T. Scharton-Kersten, M. Anver, et al 1998. Mice deficient in nuclear factor (NF)-{kappa}B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture. J. Exp. Med. 187: 147-159. [Abstract/Free Full Text]
  34. Kaisho, T., K. Takeda, T. Tsujimura, T. Kawai, F. Nomura, N. Terada, S. Akira. 2001. I{kappa}B kinase {alpha} is essential for mature B cell development and function. J. Exp. Med. 193: 417-426. [Abstract/Free Full Text]
  35. Li, Z. W., S. A. Omori, T. Labuda, M. Karin, R. C. Rickert. 2003. IKKbeta is required for peripheral B cell survival and proliferation. J. Immunol. 170: 4630-4637. [Abstract/Free Full Text]
  36. Pasparakis, M., M. Schmidt-Supprian, K. Rajewsky. 2002. I{kappa}B kinase signaling is essential for maintenance of mature B cells. J. Exp. Med. 196: 743-752. [Abstract/Free Full Text]
  37. Enzler, T., G. Bonizzi, G. J. Silverman, D. C. Otero, G. F. Widhopf, A. Anzelon-Mills, R. C. Rickert, M. Karin. 2006. Alternative and classical NF-{kappa}B signaling retain autoreactive B cells in the splenic marginal zone and result in lupus-like disease. Immunity 25: 403-415. [Medline]
  38. Sasaki, Y., E. Derudder, E. Hobeika, R. Pelanda, M. Reth, K. Rajewsky, M. Schmidt-Supprian. 2006. Canonical NF-{kappa}B activity, dispensable for B cell development, replaces BAFF-receptor signals and promotes B cell proliferation upon activation. Immunity 24: 729-739. [Medline]
  39. Sha, W. C., H. C. Liou, E. I. Tuomanen, D. Baltimore. 1995. Targeted disruption of the p50 subunit of NF-{kappa}B leads to multifocal defects in immune responses. Cell 80: 321-330. [Medline]
  40. Wang, D., J. Feng, R. Wen, J. C. Marine, M. Y. Sangster, E. Parganas, A. Hoffmeyer, C. W. Jackson, J. L. Cleveland, P. J. Murray, J. N. Ihle. 2000. Phospholipase C{gamma}2 is essential in the functions of B cell and several Fc receptors. Immunity 13: 25-35. [Medline]
  41. Weih, F., D. Carrasco, S. K. Durham, D. S. Barton, C. A. Rizzo, R. P. Ryseck, S. A. Lira, R. Bravo. 1995. Multiorgan inflammation and hematopoietic abnormalities in mice with a targeted disruption of RelB, a member of the NF-{kappa}B/Rel family. Cell 80: 331-340. [Medline]
  42. Woodland, R. T., M. R. Schmidt, S. J. Korsmeyer, K. A. Gravel. 1996. Regulation of B cell survival in xid mice by the proto-oncogene bcl-2. J. Immunol. 156: 2143-2154. [Abstract]
  43. Takata, M., T. Kurosaki. 1996. A role for Bruton’s tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-{gamma}2. J. Exp. Med. 184: 31-40. [Abstract/Free Full Text]
  44. Thompson, J. S., S. A. Bixler, F. Qian, K. Vora, M. L. Scott, T. G. Cachero, C. Hession, P. Schneider, I. D. Sizing, C. Mullen, et al 2001. BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science 293: 2108-2111. [Abstract/Free Full Text]
  45. Carter, R. S., B. C. Geyer, M. Xie, C. A. Acevedo-Suárez, D. W. Ballard. 2001. Persistent activation of NF-{kappa}B by the tax transforming protein involves chronic phosphorylation of I{kappa}B kinase subunits IKKbeta and IKK{gamma}. J. Biol. Chem. 276: 24445-24448. [Abstract/Free Full Text]
  46. Boothby, M. R., A. L. Mora, D. C. Scherer, J. A. Brockman, D. W. Ballard. 1997. Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-{kappa}B. J. Exp. Med. 185: 1897-1907. [Abstract/Free Full Text]
  47. Weih, F., S. K. Durham, D. S. Barton, W. C. Sha, D. Baltimore, R. Bravo. 1997. p50-NF-{kappa}B complexes partially compensate for the absence of RelB: severely increased pathology in p50–/– relB–/– double-knockout mice. J. Exp. Med. 185: 1359-1370. [Abstract/Free Full Text]
  48. Thomas, J. D., P. Sideras, C. I. Smith, I. Vorechovsky, V. Chapman, W. E. Paul. 1993. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261: 355-358. [Abstract/Free Full Text]
  49. Patke, A., I. Mecklenbräuker, H. Erdjument-Bromage, P. Tempst, A. Tarakhovsky. 2006. BAFF controls B cell metabolic fitness through a PKCbeta- and Akt-dependent mechanism. J. Exp. Med. 203: 2551-2562. [Abstract/Free Full Text]
  50. Mecklenbräuker, I., S. L. Kalled, M. Leitges, F. Mackay, A. Tarakhovsky. 2004. Regulation of B-cell survival by BAFF-dependent PKC{delta}-mediated nuclear signalling. Nature 431: 456-461. [Medline]
  51. Craxton, A., K. E. Draves, A. Gruppi, E. A. Clark. 2005. BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway. J. Exp. Med. 202: 1363-1374. [Abstract/Free Full Text]
  52. de Wit, H., W. H. Dokter, S. B. Koopmans, C. Lummen, M. van der Leij, J. W. Smit, E. Vellenga. 1998. Regulation of p100 (NFKB2) expression in human monocytes in response to inflammatory mediators and lymphokines. Leukemia 12: 363-370. [Medline]
  53. Liptay, S., R. M. Schmid, E. G. Nabel, G. J. Nabel. 1994. Transcriptional regulation of NF-{kappa}B2: evidence for {kappa}B-mediated positive and negative autoregulation. Mol. Cell Biol. 14: 7695-7703. [Abstract/Free Full Text]
  54. Dejardin, E., N. M. Droin, M. Delhase, E. Haas, Y. Cao, C. Makris, Z. W. Li, M. Karin, C. F. Ware, D. R. Green. 2002. The lymphotoxin-beta receptor induces different patterns of gene expression via two NF-{kappa}B pathways. Immunity 17: 525-535. [Medline]
  55. Lam, K. P., R. Kuhn, K. Rajewsky. 1997. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90: 1073-1083. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
I. Castro, J. A. Wright, B. Damdinsuren, K. L. Hoek, G. Carlesso, N. P. Shinners, R. M. Gerstein, R. T. Woodland, R. Sen, and W. N. Khan
B Cell Receptor-Mediated Sustained c-Rel Activation Facilitates Late Transitional B Cell Survival through Control of B Cell Activating Factor Receptor and NF-{kappa}B2
J. Immunol., June 15, 2009; 182(12): 7729 - 7737.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. F. Andrews and D. J. Rawlings
Transitional B Cells Exhibit a B Cell Receptor-Specific Nuclear Defect in Gene Transcription
J. Immunol., March 1, 2009; 182(5): 2868 - 2878.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. Sasaki, D. P. Calado, E. Derudder, B. Zhang, Y. Shimizu, F. Mackay, S.-i. Nishikawa, K. Rajewsky, and M. Schmidt-Supprian
NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells
PNAS, August 5, 2008; 105(31): 10883 - 10888.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
C. Spatuzza, M. Schiavone, E. Di Salle, E. Janda, M. Sardiello, G. Fiume, O. Fierro, M. Simonetta, N. Argiriou, R. Faraonio, et al.
Physical and functional characterization of the genetic locus of IBtk, an inhibitor of Bruton's tyrosine kinase: evidence for three protein isoforms of IBtk
Nucleic Acids Res., August 1, 2008; 36(13): 4402 - 4416.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
L. Yu, A. J. Mohamed, O. E. Simonson, L. Vargas, K. E. M. Blomberg, B. Bjorkstrand, H. J. Arteaga, B. F. Nore, and C. I. E. Smith
Proteasome-dependent autoregulation of Bruton tyrosine kinase (Btk) promoter via NF-{kappa}B
Blood, May 1, 2008; 111(9): 4617 - 4626.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow A correction has been published
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shinners, N. P.
Right arrow Articles by Khan, W. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shinners, N. P.
Right arrow Articles by Khan, W. N.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS