Blood Journal
Leading the way in experimental and clinical research in hematology

Nuclear CD40 interacts with c-Rel and enhances proliferation in aggressive B-cell lymphoma

  1. Hai-Jun Zhou1,
  2. Lan V. Pham1,
  3. Archito T. Tamayo1,
  4. Yen-Chiu Lin-Lee1,
  5. Lingchen Fu1,
  6. Linda C. Yoshimura1, and
  7. Richard J. Ford1
  1. 1Department of Hematopathology, the University of Texas M. D. Anderson Cancer Center, Houston

Abstract

CD40 is an integral plasma membrane–associated member of the TNF receptor family that has recently been shown to also reside in the nucleus of both normal B cells and large B-cell lymphoma (LBCL) cells. However, the physiological function of CD40 in the B-cell nucleus has not been examined. In this study, we demonstrate that nuclear CD40 interacts with the NF-κB protein c-Rel, but not p65, in LBCL cells. Nuclear CD40 forms complexes with c-Rel on the promoters of NF-κB target genes, CD154, BLyS/BAFF, and Bfl-1/A1, in various LBCL cell lines. Wild-type CD40, but not NLS-mutated CD40, further enhances c-Rel–mediated Blys promoter activation as well as proliferation in LBCL cells. Studies in normal B cells and LBCL patient cells further support a nuclear transcriptional function for CD40 and c-Rel. Cooperation between nuclear CD40 and c-Rel appears to be important in regulating cell growth and survival genes involved in lymphoma cell proliferation and survival mechanisms. Modulating the nuclear function of CD40 and c-Rel could reveal new mechanisms in LBCL pathophysiology and provide potential new targets for lymphoma therapy.

Introduction

CD40 is a cell membrane receptor protein of the tumor necrosis factor receptor (TNF-R) superfamily that functions in B lymphocytes for B-cell activation, antigen presentation, immunoglobulin isotype switching, and development of germinal centers and humoral immune memory.1 Besides B lymphocytes, CD40 is expressed in dendritic cells,2 monocytes and macrophages,3 and a variety of nonhematopoietic cells.46 The binding of CD40 to its cognate ligand, CD154 (CD40L), is required for initiating thymus-dependent humoral responses in the immune system.7 Ligation and subsequent triggering of the CD40 plasma membrane receptor lead to a variety of immune and inflammatory responses.8 When CD40L binds to its receptor, CD40 optimally signals as a dimeric or trimeric aggregate from its location in plasma membrane microdomains known as lipid rafts.911 In the lipid rafts, CD40 recruits multiple TNF receptor–associated factors (TRAFs), an important group of intracellular adaptor molecules that bind directly or indirectly to cytoplasmic domains of various members of the TNF receptor superfamily,12,13 and the IKK complex, forming a macromolecular signaling complex called the CD40 signalosome.14 Through CD40 ligand cognate receptor binding, this signalosome transmits signals via the NF-κB pathway to promote cell proliferation and survival and mediate immune and inflammatory responses.14,15

Constitutively activated NF-κB pathway is a molecular signature characteristic of many aggressive B-cell lymphoma cells.16 The CD40 signalosome pathway has also been shown to be important in modulating normal and neoplastic B-cell proliferation and survival, primarily through the activation of NF-κB target genes, such as CD40L,17 BLyS,18 and Bfl-1/A1.19,20 CD40L is endogenously expressed in aggressive B-cell lymphomas, binding to the CD40 receptor, and constitutively activates the canonical NF-κB signaling pathway.17 BLyS signaling activates noncanonical NF-κB signaling pathway21,22 and promotes cell survival, cell-cycle progression and proliferation of B cells,2326 and large B-cell lymphoma (LBCL).18 Bfl-1/A1, an antiapoptotic member of the Bcl-2 family, whose expression is confined primarily to the immune system, also plays an important role in cell survival.27,28 Agents that selectively block CD40-NF-κB pathway have been shown to inhibit cell growth and induce apoptosis in aggressive B-cell lymphoma cells.29

Among the NF-κB family members activated by CD40 pathway, only c-Rel has been shown to be able to transform cells in the lymphoid cell lineage and has been identified as a bona fide oncogene.30 Amplifications of human c-rel are frequently seen in Hodgkin lymphoma, some follicular and mediastinal B-cell lymphomas, and LBCL.31 The molecular mechanism of c-Rel involves its transcriptional function in NF-κB–regulated gene promoters.17,18 Our studies have demonstrated that c-Rel is a pivotal transcription factor that regulates CD40 responsive genes, such as CD40L and BlyS,17,18 through interaction with another important lymphoid transcription factor, NFATc1. Recent studies have also shown that c-Rel exerts its function through recruiting multiple transcription factors, forming a macromolecular enhanceosome in the promoter of Bfl-1/A1.32 However, the precise composition and function of a putative c-Rel enhanceosome in LBCL remain to be characterized.

Recent findings from our lab indicate that the CD40 receptor is also present in the nucleus of both normal and neoplastic human B cells, adding CD40 into a list of membrane receptors whose nuclear localization has revealed a novel mode of action for these receptors, including epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR).3336 We also identified the BLyS/BAFF promoter as a molecular target for nuclear CD40, suggesting an important transcriptional role of CD40 as well as its relationship to the BLyS/BAFF system, however, the molecular mechanism and physiological function of nuclear CD40 are still elusive. We hypothesized that nuclear CD40 may interact with certain transcription factors on the promoters of B-lineage cell growth– and survival-associated genes.

In this study, we show that nuclear CD40 interacts with c-Rel but not with p65, another NF-κB member. This specific interaction forms transcriptional complexes on the promoters of c-Rel target genes, activating gene expression, and enhancing normal and neoplastic B-cell proliferation.

Materials and methods

Cells

Human LBCL cell lines (MS, McA, and FN) were established from diagnostic biopsy tissue or effusions from patients as previously described.37 This study was conducted in accordance with the Helsinki protocol and approved by the M. D. Anderson Cancer Center institutional review board. Informed consent was obtained from all patients in accordance with the declaration of Helsinki. The cells were cultured in RPMI (Gibco, Rockville, MD) containing 10% fetal calf serum (Hyclone, Logan, UT). Normal human B lymphocytes were purified from healthy donors by using the human B-cell enrichment cocktail from StemCell Technologies (Vancouver, BC) as described previously.14 The resulting cell populations were stained 95% to 98% positive for CD20 and analyzed by flow cytometry. Fresh biopsy-derived lymphoma tissues (patients 1-5) were minced in cold RPMI and single-cell suspensions of lymphoma cells were purified by Ficoll-Paque PLUS (GE Healthcare, Piscataway, NJ), and stained positive for CD19, CD20, and CD10, but negative for CD3, by immunohistochemistry.

Plasmids and reagents

The 6xNF-κB CD40L/TKm reporter was obtained by cloning 6 copies of the NF-κB binding site (5′-AGGGATTTCCA-3′) of the CD40L promoter upstream of the minimal thymidine kinase promoter in the pGL3 luciferase reporter plasmid38 (a gift from Dr Peter Vandenberghe, Laboratory for Experimental Hematology, University of Leuven, Belgium). The pCMV-c-Rel expression vector was a gift from Dr Celine Gelinas32 (Center for Advanced Biotechnology and Medicine, Piscataway, NJ). The pCMVp65 expression vector was a gift from Dr Nancy Rice39 (National Cancer Institute-Frederick, Frederick, MD). The pcDNA3.1/myc/his-CD40 and pcDNA3.1/myc/his-CD40NLS mutant plasmids were constructed by cloning the polymerase chain reaction (PCR) product generated using the template pEGFP-CD40 and pEGFP-CD40NLS mutant,33 respectively. The pGL4-basic BLyS-Luc reporter plasmid containing BLyS promoter region was constructed as described previously.18 All expression vectors were verified by DNA sequencing. The following primary antibodies were used: polyclonal Bfl-1/A1, CD154, Oct-1, and monoclonal c-Rel (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal c-Rel (Chemicon, Temecula, CA); polyclonal p65, BLyS (Upstate Biotechnology, Charlottesville, VA); monoclonal β-actin (Sigma-Aldrich, St Louis, MO); monoclonal Myc-tag antibody (Invitrogen, Carlsbad, CA); anti-CD40 monoclonal antibody G28.5 from hybridoma cell line (courtesy of Dr E. Clark, University of Washington, Seattle, WA); secondary antibodies conjugated with horseradish hydrogen peroxidase (HRP) or Cy3 or FITC (Jackson ImmunoResearch Laboratories, West Grove, PA).

Cytoplasmic and nuclear fractionation

Cytoplasmic and nuclear proteins were isolated as described34,36 and modified accordingly. Briefly, cells from fresh culture were washed twice in ice-cold PBS, then suspended in cold homogenization buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) and allowed to swell on ice for 15 minutes. Cells were lysed by addition 10% solution of NP-40 to final 0.5% followed by vigorous vortexing. Nuclei were pelleted by centrifugation at 1500g for 5 minutes, and the resulting supernatant formed the nonnuclear fraction. The cytoplasmic fraction was removed and nuclei were washed twice in homogenization buffer with NP-40 and resuspended in the same buffer containing 0.5 M NaCl to extract nuclear proteins. The extracted material was centrifuged at 15 000g for 10 minutes and the resulting supernatant was designated as the nuclear fraction.

Coimmunoprecipitation assay

Antibodies were cross-linked with Dynabeads protein A (Invitrogen) according to the manufacturer's instructions. Cell lysates were precleared with immunoglobulin G (IgG) Dynabeads protein A for 10 minutes at 4°C before incubation with antibody-linked Dynabeads overnight at 4°C. The immunoprecipitated Dynabeads complexes were washed 5 times with immunoprecipitation (IP) buffer (10 mM Tris [Tris(hydroxymethyl)aminomethane]-HCl, pH 7.8, 1 mM EDTA [ethylenediaminetetraacetic acid], 150 mM NaCl, 1 mM NaF, 0.5% NP-40, 0.5% glucopyranoside, 1 μg/mL aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]). Proteins were eluted by boiling in protein loading buffer and then processed for Western blot analysis.

Confocal microscopic analysis

Cells were fixed with 100% cold methanol for 5 minutes on poly-l-lysine–coated glass slides and air-dried. Nonspecific protein binding was blocked with 5% fetal calf serum (FCS) in PBS. Cells were stained with the appropriate primary antibodies (1:200 dilution) overnight at 4°C. After 3 washes with PBS, the slides were stained with the appropriate donkey secondary antibodies (labeled with FITC or Cy3 [1:200 dilution] and Topro-3 [1:10 000 dilution]) for 45 minutes and washed with PBS. Coverslips were applied with Slow Fade reagent (Molecular Probes, Eugene, OR). The cells were visualized using an Olympus FluoView 500 (FV500) laser scanning confocal microscope (Olympus America, Melville, NY). Images were captured with a PlanApo 60 ×/1.4 oil objective using the appropriate filter sets. Digital images were obtained using the FluoView software.

Electrophoretic mobility shift assays

Nuclear protein extraction and gel shift assay procedures were performed as previously described.14,40 Briefly, a double-stranded NF-κB consensus oligonucleotide 5′-AGT TGA GGG GAC TTT CCC AGG-3′ (Promega, Madison, MI; NF-κB binding site is underlined) was labeled with [γ-32P] ATP. Nuclear extract (10 μg) was incubated 1 × binding buffer, 2 μg poly(dI:dC), 0.5% nonidet P-40, and 1 μL labeled probe (4 fmol/μL) at 37°C for 15 minutes. The samples were then subjected to 5% nondenaturing acrylamide gel electrophoresis and visualized with x-ray film. For supershift analysis, 1 μL antibody was added to the mixture 15 minutes before the labeled probe was added. Unlabeled oligonucleotide (400 fmol) was used to compete with the labeled probe.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assays were performed using the ChIP assay kit and protocol provided by the manufacturer (Upstate Biotechnology) and as described previously.17 DNA fragments were purified for polymerase chain reaction (PCR) amplification with PCR beads from Amersham Pharmacia Biotech (Piscataway, NJ). Oligonucleotide primers specific for the CD154 promoter (forward, 5′-CACCCTCTCGGACAGTTATTCA-3′; reverse, 5′-GATAATTACAAGTTCACTCTGG-3′;), the BLyS promoter (forward, 5′-GAGACAGAACTAAAGCTCACTATTCTT-3′; reverse, 5′- GACCTGTGAGGACTGTTGCA-3′); the Bfl1-A1 promoter (forward, 5′-CAGTGTTGTGCAACTTCCAC-3′; reverse, 5′-GGTAAATCCCCGTCTGTACTAA-3′), and the β-actin promoter (forward, 5′-CAACGCCAAAACTCTCCCTC-3′; reverse, 5′-ATCGGCAAAGGCGAGGCTCTGT-3′) were used. The PCR conditions were as follows: the cDNA template was denatured at 95°C for 1 minute, annealed at 50°C for 30 seconds, and extended at 72°C for 1 minute per cycle for 35 cycles. The PCR product was visualized on a 2.0% agarose gel.

Transfection, luciferase, and β-galactosidase assays

Transient transfections of cultured lymphoma cells and normal B lymphocytes were conducted using the nucleofector protocol from Amaxa Biosystems (Cologne, Germany). Luciferase and β-galactosidase (β-gal) assays were performed according to the manufacturer's directions (Promega). Each sample in an experiment was repeated at least 3 times. Luciferase activity values were normalized to transfection efficiency monitored by the cotransfected β-gal expression vector.

In vitro [3H]thymidine incorporation assay

In vitro thymidine incorporation proliferation assays were performed as described previously.41 Cells were plated as triplicate wells in 200 μL RPMI, 10% FCS, with indicated antibody reagents in a 96-well culture plate and incubated in 5% CO2/air at 37°C. After 24 hours, each well was pulsed with 0.5 μCi (0.0185 MBq)/10 μL [3H]thymidine (Amersham Pharmacia Biotech) and incubated for 16 hours; then, cells were harvested and counted in a Beckman LS3800 liquid scintillation counter (Hialeah, FL).

Results

CD40 interacts with c-Rel in the nucleus of LBCL cells

Recently, we have reported that CD40 can enter the nucleus in normal and neoplastic B cells. Nuclear CD40 binds to the promoter of BLyS gene and up-regulates BLyS expression. Because BLyS is also a target gene of c-Rel, we speculated that CD40 might interact with c-Rel in the nucleus. We tested this hypothesis using a coimmunoprecipitation assay. We chose 2 large B-cell lymphoma (LBCL) cell lines (MS, McA) previously established in our lab. Nuclear fractions from these cells were incubated with CD40 antibody and coimmunoprecipitation analyses were performed (Figure 1A). When CD40 was precipitated, c-Rel was also coimmunoprecipitated in both cell lines. These data indicated a physical association between CD40 and c-Rel. However, we did not detect p65 in these experiments, suggesting the interaction between CD40 and c-Rel is specific. The presence of Oct-1 and the lack of β-actin in the nuclear fraction indicate that the nuclear fraction is free of cytosolic contamination. To further confirm the interaction between CD40 and c-Rel, c-Rel and myc-tagged CD40 were cotransfected into MS cells. The nuclear extraction from transfected MS cells was incubated with myc tag antibody, and coimmunoprecipitation analyses were performed. Again, nuclear-expressed myc-tagged CD40 was coprecipitated with c-Rel (Figure 1B). Confocal microscopy also showed that CD40 (green) and c-Rel (red) both colocalized (yellow) in the nucleus of LBCL cells (Figure 1C), indicating that CD40 interacts with c-Rel in the nucleus of large B-cell lymphoma cells.

Figure 1

CD40 interacts with c-Rel in the nucleus of LBCL cells. (A) Coimmunoprecipitation assays were performed with the nuclear extracts of LBCL-MS and LBCL-McA cells. Nuclear extracts (1 mg) from LBCL cells (MS and McA) were immunoprecipitated with a polyclonal CD40 antibody or IgG antiserum (negative control) respectively. Immunoprecipitated complexes were resolved on SDS–polyacrylamide gel electrophoresis (PAGE), and subjected to Western blotting with anti–c-Rel, anti-p65, anti–Oct-1, and anti–β-actin antibodies. Input indicates 25 μg nuclear extract; NE, nuclear extract. (B) MS cells were cotransfected with expression vectors for c-Rel (pCMV-c-Rel) and CD40wt (pcDNA3.1/myc/his-CD40). Forty-eight hours after transfection, the nuclear fraction was extracted and immunoprecipitated with monoclonal anti-Myc tag antibody. Immunoprecipitates were then Western blotted with anti-Myc (detecting recombinant CD40), anti–c-Rel, anti–Oct-1, and anti–β-actin antibodies. Input indicates 25 μg nuclear extract. (C) LBCL cells (MS) were fixed with methanol and stained for CD40 (green), c-rel (red), and nuclear marker Topro-3 (blue) and analyzed by confocal immunofluorescence analysis. Colocalization of CD40 and c-Rel appears yellow. See “Materials and methods, Confocal microscopic analysis” for image acquisition information.

Nuclear CD40 enhances c-Rel–mediated BLyS promoter activation in LBCL cells

Since c-Rel is a well-characterized transcription factor, we addressed the question of whether CD40 is involved in the transactivation of c-Rel through their interaction. Here, we used a luciferase pGL3 reporter plasmid (6xNF-κB-CD40L/TKm) containing 6 copies of the CD154-NF-κB binding site oligonucleotide inserted upstream of the thymidine kinase minimal promoter. To emphasize the nuclear function of CD40, we used both wild-type CD40 (CD40wt) and nuclear localization signal (NLS) mutated CD40 (CD40NLSmut) constructs. Because CD40 cannot enter the nucleus after its NLS is mutated, CD40NLSmut loses its nuclear function. The NLS motif of CD40 is a distinct signature that does not overlap with the TRAF binding sites in the CD40 protein33; therefore, the CD40NLSmut should still share the same membrane function with CD40wt. After transfection of the reporter plasmid along with CD40wt, CD40NLSmut, or c-Rel, luciferase activity was measured (Figure 2A). C-Rel activated the promoter through its cognate binding (14.5-fold increase), while CD40wt and CD40NLSmut both up-regulated this reporter through their membrane function-activation of NF-κB (14.3-fold and 11.4-fold increases, respectively). The combination of c-Rel and CD40wt synergized reporter activation (47.9-fold increase); however, this synergy was diminished with the cotransfection of c-Rel and CD40NLSmut (20.2-fold increase). Because CD40NLSmut does not enter the nucleus, this synergy represents a nuclear function of CD40.

Figure 2

Nuclear CD40 synergistically enhance c-Rel–potentiated BLyS promoter activation in LBCL cells. (A) LBCL cells (FN) were cotransfected with 6xNF-κB-CD40L reporter and the expression vectors for c-Rel (pCMV-c-Rel, 5 μg), CD40wt (pcDNA3.1/myc/his-CD40, 5 μg), and CD40NLSmut (pcDNA3.1/myc/his-CD40NLSmut, 5 μg) indicated in the figure. After 24 hours, luciferase activity was determined and normalized for transfection efficiency using β-gal activity. The data are representative of 3 independent experiments. The error bars indicate the standard deviation of triplicate samples. (B) LBCL cells (FN) were cotransfected with BLyS-luciferase reporter and the expression vectors for c-Rel (pCMV-c-Rel, 5 μg), p65 (pCMVp65, 5 μg), CD40wt (pcDNA3.1/myc/his-CD40, 5 μg), and CD40NLSmut (pcDNA3.1/myc/his-CD40NLSmut, 5 μg) indicated in the figure. After 24 hours, luciferase activity was determined as described for panel A.

Since BLyS is a common target for both c-Rel and CD40, we tested whether overexpression of exogenous c-Rel and CD40 proteins would lead to either additive or synergistic BLyS promoter activity in LBCL cells, and whether CD40 would play differential roles between c-Rel and p65 on BLyS promoter activity. Cotransfection of the BLyS-luc reporter plasmid containing BLyS promoter region along with expression vectors containing c-Rel, p65, CD40wt, or CD40NLSmut resulted in differential activation of BLyS promoter activity (Figure 3B). Overexpression of c-Rel or CD40wt increased BLyS promoter activity by 6.4- and 4.1-fold, respectively, and combined c-Rel and CD40wt overexpression resulted in a synergistic 15.6-fold activation of the BLyS promoter. However, combined c-Rel and CD40NLSmut overexpression diminished this synergy (5.1-fold increase). In the case of p65, overexpression of p65 alone increased BLyS promoter activity by 3.2-fold, while both CD40wt and CD40NLSmut enhanced p65 potentiated activity similarly on BLyS promoter (7.2-fold and 9.3-fold increases, respectively). Because p65 did not interact with CD40 in the nucleus and CD40NLSmut does not enter the nucleus, these experiments demonstrated that CD40 interacts with c-Rel in the nucleus of LBCL cells and nuclear CD40 potentiates c-Rel transactivity through the specific interaction of these proteins.

Figure 3

CD40 forms complexes with c-Rel on the promoters of proliferation and survival genes in LBCL cells. (A) Supershift analysis of c-Rel and CD40 proteins binding to the NF-κB site. LBCL cell (MS) nuclear extracts were incubated with the NF-κB cold probe or antibody against c-Rel and CD40 or both. A 32P-labeled NF-κB probe was then added to the reaction mixtures, and samples were analyzed by electrophoretic mobility shift assay (EMSA). comp indicates competition. Arrow indicates the supershift complex. (B) ChIP analysis was performed on 2 LBCL cell lines (MS and McA). The indicated antibodies were used to precipitate chromatin after cross-linking. PCR to detect the BLyS, CD154, Bfl1/A1, and β-actin promoter regions was performed on the precipitated DNA. IgG was used as a nonspecific antibody control.

CD40 forms complexes with c-Rel on the promoters of proliferation- and survival-associated genes in lymphoma cells

To investigate whether CD40 and c-Rel form a complex that binds to DNA, we synthesized the consensus NF-κB binding oligonucleotide and performed gel shift assays. When nuclear extracts from LBCL (MS) cells were incubated with this probe, a protein-DNA complex was visualized on a denaturing gel (Figure 3A) and the specificity of the binding was confirmed by competition with the excess (100 ×) unlabeled oligo probe. A supershift was detected with addition of c-Rel antibody (lane 4) and a decreased binding was seen with addition of CD40 antibody (lane 5). C-Rel and CD40 antibodies together further shifted the complex (lane 6). This result suggests that CD40 and c-Rel bind together with the NF-κB consensus binding site.

To further examine whether CD40 is involved in the transcriptional regulation of c-Rel in vivo, we performed chromatin immunoprecipitation (ChIP) assay to test whether CD40 and c-Rel form complexes on the promoters of c-Rel target genes. BLyS, CD154, and Bfl-1/A1 are important c-Rel target genes. The promoters of these genes were precipitated by c-Rel antibody and amplified by PCR as expected as positive controls, while normal IgG was used as negative control (Figure 3B). Interestingly, the same promoters were also precipitated by CD40 antibody. Both LBCL cell lines (MS and McA) confirmed the result and a negative control promoter from β-actin was shown at bottom. These data suggest that CD40 and c-Rel can form complexes on the promoters of c-Rel target genes including CD154, BLyS, and Bfl1/A1.

Nuclear CD40 enhances c-Rel proliferation capacity in LBCL

Next, we asked whether proliferation in lymphoma B cells is functionally enhanced by nuclear interaction between CD40 and c-Rel. To answer this question, thymidine incorporation assays were performed to detect the proliferative characteristics in LBCL cells (FN cells) after different transfections (Figure 4). As shown in Figure 4A, the combination of c-Rel and CD40wt showed almost a 200% increase in proliferation, when compared with c-Rel transfection alone, while the combination of c-Rel and CD40NLSmut resulted in a significantly smaller (90%) increase. Because CD40NLSmut does not enter the nucleus, these data indicate that the nuclear interaction of CD40wt and c-Rel augments proliferation in LBCL cells. To clarify the mechanism of this proliferation difference between CD40wt and CD40NLSmut, Western blotting was performed (Figure 4B). Overexpression of exogenous c-Rel and CD40wt and CD40NLSmut were detected by c-Rel antibody and Myc-tag antibody, respectively. Corresponding to the proliferation assay, cotransfection of c-Rel with CD40wt but not CD40NLSmut markedly up-regulated the expression of BLyS, CD154, and Bfl-1/A1, indicating the nuclear CD40 enhanced the proliferative role of c-Rel through up-regulating its target genes.

Figure 4

Cotransfection of c-Rel and CD40 enhances the proliferation of LBCL cells. (A) [3H] thymidine incorporation assay was performed after transfection of c-Rel (pCMV-c-Rel, 10 μg), CD40wt (pcDNA3.1/myc/his-CD40, 10 μg), and CD40NLSmut (pcDNA3.1/myc/his-CD40NLSmut, 10 μg) into LBCL-FN cells as indicated in the Figure. Twenty-four hours after transfection, live cells from each group were counted and the same number of live cells (20 000) was aliquoted and pulse labeled with [3H] thymidine overnight. Cells were harvested and signals were counted in a Beckman LS3800 liquid scintillation counter. Error bars indicate SD of triplicate samples. (B) Total cell lysis of each transfection group was subjected to Western blotting, and the expression of transfected as well as target gene products was detected by their specific antibodies. β-Actin was used as a loading control.

Nuclear expression of CD40 and c-Rel correlates with the proliferation of normal B lymphocytes

Because neoplastic B cells share many similarities with the activated normal B cells, we addressed the status of nuclear CD40 and c-Rel in the normal B cells. We isolated normal B lymphocytes from healthy donors and extracted cytosolic and nuclear fractions, and Western blots were performed (Figure 5A). Upon B-cell activation, nuclear CD40 was significantly expressed in addition to cytoplasmic CD40, and c-Rel was fully activated, compared with quiescent G0 B cells. Normal B cells were transfected with either the empty vector or vector containing c-Rel and then activated with or without CD40 ligand, and then assayed for cell proliferation (Figure 5B). Stimulation of vector-transfected cells with CD40 ligand enhanced the proliferation of normal B cells (9.6-fold increase). Overexpression of c-Rel further enhanced the ability of CD40 to increase the proliferation of normal B cells (22.4-fold increase). These studies indicate that proliferation of normal activated B cells correlates with nuclear expression of CD40 and c-Rel.

Figure 5

C-Rel synergistically enhances the proliferation of normal B lymphocytes upon CD40 ligand stimulation. (A) G0 B cells were purified from healthy donors and the purity was assessed by flow cytometry as described in “Material and methods.” Cytosolic and nuclear fractions of normal B cells were extracted with or without CD40 ligand activation for 24 hours and subjected to Western blotting. C indicates cytosolic fraction; N, nuclear fraction. (B) [3H] thymidine incorporation assay was performed after transfection of c-Rel into normal B cells. Twenty-four hours after transfection, live cells were counted and same number of live cells (20 000) were aliquoted and treated with/without CD40 ligand for 24 hours, and then pulse labeled with [3H] thymidine overnight. Cells were harvested and signals were counted in a Beckman LS3800 liquid scintillation counter. Error bars indicate SD of triplicate samples.

CD40 and c-Rel are expressed in the nucleus of LBCL patient tumor cells

While CD40 and c-Rel are expressed and interact in the nucleus of LBCL cell lines, the presence of these proteins is also shown in the patient lymphoma cell specimens in Figure 6. LBCL patient tumor cells were fractionated and Western blot analysis was performed. As shown in Figure 6A, CD40 and c-Rel are both consistently expressed in the nucleus of these patient lymphoma cells. Confocal microscopy also showed CD40 (green) and c-Rel (red) colocalized (yellow) in the nucleus of patient lymphoma cells (Figure 6B), suggesting the nuclear CD40 and c-Rel also function in LBCL patient cells.

Figure 6

CD40 and c-Rel are expressed in the nucleus of LBCL patient cells. (A) LBCL patient samples (nos. 1-5) were collected as described in “Materials and methods.” Cytosolic and nuclear fractions were extracted and subjected to Western blot analysis. C indicates cytosolic fraction; N, nuclear fraction. (B) Representative patient samples were stained with CD40- and c-Rel–specific antibodies and protein localization was visualized with Cy3 (red) and FITC (green) secondary antibodies under confocal microscope. Nuclear marker Topro-3 (blue) showed the cell nucleus. See “Confocal microscopic analysis” for image acquisition information.

Discussion

In this study, we found that the TNF receptor, CD40, interacts with the NF-κB transcriptional factor c-Rel in the nucleus of large B-cell lymphoma (LBCL) cells. Consistent with our previous findings, CD40 and c-Rel form a transcriptional complex in the promoters of NF-κB target genes including BLyS. Interaction of c-Rel with nuclear CD40 promotes proliferation in LBCL cells.

Nuclear translocation of cell membrane receptors is not limited to CD40. Recent studies have identified a growing list of growth factor receptors, including EGFR and FGFR family members, whose nuclear localization has been described in a variety of normal and neoplastic eukaryotic cell types.3436,42 Functional studies on these nuclear localized plasma membrane–associated growth factors have focused largely on gene transcription processes. ErbB2/HER-2 has been shown to interact with the COX-2 promoter,35 and EGFR interacts with STAT3 and activates the iNOS/NO pathway.43 Our recent demonstration that CD40 is present in the nucleus of normal and neoplastic B cells raised questions regarding its function in the cell nucleus.33 In this study, our demonstration of the nuclear functional interaction between CD40 and c-Rel is of particular interest because CD40, a TNF receptor, not only activates both canonical and noncanonical NF-κB pathways through classical membrane signaling,14,15,44,45 but also enhances the transactivation of NF-κB through nuclear localization and interaction with specific transcription factors in neoplastic B cells. As shown in Figures 2 and 4B, when the NLS of CD40 was mutated, it lost the ability to enter the nucleus,33 and mutated CD40 could not further up-regulate the expression of c-Rel target genes. The difference between plasma membrane and nuclear function of CD40 has also been shown in the proliferation experiments with LBCL cells (Figure 4A), in which fully functional CD40wt has both cell membrane and nuclear capability to mediate cell proliferation, but CD40NLSmut has lost this nuclear role. Many characteristics of activated normal B cells are retained in lymphoma B cells. Upon mitogenic activation, CD40 protein entered the nucleus in normal peripheral B lymphocytes (Figure 5 and Lin-Lee et al33) where c-Rel is also actively expressed in the nucleus. Concurrent nuclear CD40/c-Rel expression in normal B cells mimics the constitutive activation of CD40-NF-κB pathway in many LBCLs.14 Under such conditions, cell proliferation was stimulated and further enhanced by overexpression of c-Rel and CD40 ligand stimulation. This correlation of proliferation and nuclear accumulation of CD40 and c-Rel suggests that these 2 proteins also form a similar nuclear complex that functions in the nucleus of normal B lymphocytes.

c-Rel is particularly important among the NF-κB family members because of its oncogenic transformation capacity in chicken splenocytes and its amplification in some lymphoma patients.31 Our study, which has revealed the nuclear interrelationship between CD40 and c-Rel, suggests involvement of nuclear CD40 in lymphomagenesis. Nuclear localization of c-Rel has been demonstrated in several clinical settings. Correlation of c-Rel with other important molecular markers, such as amplification of REL locus as well as expression of TRAF1 and STAT1, has been shown as a diagnostic tool to identify classical Hodgkin lymphoma and mediastinal large B-cell lymphoma.4648 Our finding that both CD40 and c-Rel are coexpressed constitutively in the nucleus of LBCL patient cells (Figure 6) suggests a clinical relevance for our findings.

Rel family proteins have a particularly strong propensity to function as components in macromolecular enhanceosomes by recruiting other transcription factors and cofactors into specific promoters,49 such as best-studied interferon-β regulatory element.50 Gelinas' group has demonstrated c-Rel–dependent assembly of an enhanceosome-like complex in the promoter region of bfl-1/A1, which is required for induced expression (Edelstein et al32). Our study suggests CD40 as a likely component of such an enhanceosome complex. Further investigation is needed to determine whether additional components are also involved in this transcriptional complex for activating cell growth and survival genes regulated by the NF-κB signaling pathway.

Along with the discovery of the nuclear expression of the plasma membrane–associated CD40 protein, downstream cytoplasmic components of CD40 signaling pathways, such as NF-κB family regulatory components—the IKKs5153 (components of the CD40 signalosome)—have also been recently shown to enter the nucleus. In the nucleus, IKKα binds specific promoter regions in conjunction with the CREB-binding protein, resulting in phosphorylation of histone H3 and the promotion of transcriptional activity.52,53 TRAF6, another component of the CD40 signalosome,14 has also been shown to have a nuclear presence in lymphoma cells,54 indicating that components of the CD40 signalosome may translocate together or individually into the nucleus and possibly reassemble in various combinations to function as enhanceosomes or related types of macromolecular complexes to regulate the transcription of NF-κB target genes.

In addition to CD40 forming a signalosome on the plasma membrane of aggressive B-lymphoma cells, we have described in this study that CD40 can enter the nucleus of B cells and interact with c-Rel on the promoters of NF-κB target genes—CD40L, BLyS, and Bfl-1/A1—thereby further up-regulating the expression of these genes and enhancing cell proliferation. The potential targets of nuclear CD40 appear to correspond to the pathways activated by the CD40 plasma membrane counterpart, suggesting that blocking CD40-NF-κB pathway may involve not only signal transduction for cytoplasmic NF-κB activation, but also CD40 migration into the nucleus for full activation of NF-κB–regulated gene promoters. These findings may influence future therapeutic targeting strategies for aggressive B-cell lymphomas, such as LBCL.

Authorship

Contribution: H.-J.Z. designed and performed experiments and wrote the paper; L.V.P. and Y.-C.L.-L., performed experiments; A.T.T., L.F., and L.C.Y. helped perform experiments; and R.J.F. supervised the project and wrote the paper.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Richard J. Ford, Department of Hematopathology, Box 54, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: rford{at}mdanderson.org.

Acknowledgments

This work was supported by National Cancer Institute grants CA-RO1-100836 and CA-16672-26 (R.J.F.), Farahi Family Foundation and grants from the Lymphoma Research Foundation, New York, NY (R.J.F.). L.V.P. was supported by the Odyssey Program and the Kimberly-Clark Foundation Award for Scientific Achievement at the University of Texas M.D. Anderson Cancer Center.

Footnotes

  • An Inside Blood analysis of this article appears at the front of this issue.

  • The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

  • Submitted February 8, 2006.
  • Accepted June 9, 2007.

References

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