Constitutive NF-kappaB and NFAT activation leads to stimulation of the BLyS survival pathway in aggressive B-cell lymphomas

Abstract

B-lymphocyte stimulator (BLyS), a relatively recently recognized member of the tumor necrosis factor ligand family (TNF), is a potent cell-survival factor expressed in many hematopoietic cells. BLyS binds to 3 TNF-R receptors, TACI, BCMA, BAFF-R, to regulate B-cell survival, differentiation, and proliferation. The mechanisms involved in BLYS gene expression and regulation are still incompletely understood. In this study, we examined BLYS gene expression, function, and regulation in B-cell non-Hodgkin lymphoma (NHL-B) cells. Our studies indicate that BLyS is constitutively expressed in aggressive NHL-B cells, including large B-cell lymphoma (LBCL) and mantle cell lymphoma (MCL), playing an important role in the survival and proliferation of malignant B cells. We found that 2 important transcription factors, NF-kappaB and NFAT, are involved in regulating BLyS expression through at least one NF-kappaB and 2 NFAT binding sites in the BLYS promoter. We also provide evidence suggesting that the constitutive activation of NF-kappaB and BLyS in NHL-B cells forms a positive feedback loop associated with lymphoma cell survival and proliferation. Our findings indicate that constitutive NF-kappaB and NFAT activations are crucial transcriptional regulators of the BLyS survival pathway in malignant B cells that could be therapeutic targets in aggressive NHL-B.

Figure 1.

BLyS is constitutively expressed in aggressive NHL-B cells. (A) RT-PCR analysis of BLYS mRNA expression in NHL-B cell lines. (B) Immunoblot analysis of BLyS protein expression in NHL-B cell lines. (C) Immunoblot analysis of BLyS protein expression in cells from representative NHL-B patient biopsies. (D) Confocal immunofluorescence microscopic analysis of BLyS protein localization in LBCL and MCL cell lines and representative biopsy-derived LBCL and MCL patient samples. BLyS protein was stained for Cy2 (green) fluorescence, and the nuclear marker for TOPRO-3 (blue) fluorescence. (E) Flow cytometric analysis of BLyS protein on NHL-B cell surfaces. NHL (LBCL and MCL) cell lines and tumor cells from patient biopsies were stained with FITC-conjugated BLyS antibody (gray histograms) or FITC-conjugated isotype control (open histograms).

Figure 2.

Constitutive BLyS expression contributes to NHL-B cell survival in vitro. (A) XTT proliferation assay of NHL-B (LBCL-MS, MCL-Jeko) cells treated with control IgG or BLyS antibody. The number of live cells was monitored by the absorbance of 490 nm wavelength. (B) TUNEL analysis of NHL-B (LBCL-MS) cells treated with control IgG or BLyS antibody. Free DNA fragments in apoptotic cells were labeled with green fluorescence. (C) Proliferation of NHL-B (LBCL-MS) cells transfected with BLYS siRNA or control nontargeting sequence. DNA synthesis was assessed by [³H]thymidine uptake in vitro. (D) Immunoblot analysis of BLyS protein in NHL-B (LBCL-MS) cells transfected with BLYS siRNA or control siRNA. Actin was used as loading control. The BLyS level was analyzed by BioMax 1D software, normalized with actin levels, and presented as relative fold decrease compared to control samples. (E) TUNEL analysis of NHL-B (LBCL-MS) cells transfected with BLYS siRNA or a control siRNA. Free DNA fragments in apoptotic cells were labeled with green fluorescence. (F) Caspase-3 activity in an NHL-B (LBCL-MS) cell line transfected with BLYS or control siRNA. (G) Immunoblot analysis of Bcl-2 and Bcl-xL in NHL-B (LBCL-MS) cells transfected with BLYS or control siRNA. Bcl-2 and Bcl-xL levels were normalized to those of actin previously described in D. (H) Immunoblot analysis of BLyS, c-myc, and cyclin D1 in NHL-B cell line cells transfected with BLYS or control siRNA. Actin was used as loading control. The BLyS, c-myc, and cyclin D1 levels were normalized to those of actin as previously described in panel D. The data in panels A, C, and F are representative of 2 independent experiments. The error bars indicate standard deviation of triplicate samples.

Figure 3.

Transcription factors NF-κB and NFAT bind to the BLYS promoter. (A) BLYS gene promoter diagram (GenBank accession no. AY129225) showing the putative binding sites for NF-κB (dots) and NFAT (black). (B) EMSA analysis of NF-κB binding to the BLYS promoter. Nuclear extracts from NHL-B (MS) cells were incubated with BLYS–NF-κB binding site oligonucleotides. BLYS-NF-κB cold probe and antibodies to p50, p52, p65, c-rel, or rel-B were added to the binding reaction mixtures. Arrows indicate the DNA-protein and supershifted complex. (C) EMSA analysis of NFAT binding to BLYS promoter. Nuclear extracts from NHL-B (LBCL-MS) cells were incubated with oligonucleotides for the 2 different BLYS-NFAT binding sites. BLYS-NFAT cold probes and antibodies to NFATc1, NFATc2, or NFATc3 were added to the binding reaction mixtures. Arrows indicate the DNA-protein and supershifted complexes.

Figure 4.

Constitutive BLyS expression through the NF-κB pathway activates a positive feedback loop contributing to NHL-B cell survival. (A) EMSA analysis of NF-κB protein binding to the BLYS promoter. Nuclear extracts from NHL-B cells were transfected with dominant-negative (DN) NF-κB or control plasmid and analyzed with an oligonucleotide probe for the BLYS–NF-κB binding site. (B) Immunoblot analysis of BLyS expression in NHL-B (LBCL-MS) cells transfected with DN NF-κB or control plasmid. Actin was used as loading control. BLyS levels were normalized to those of actin as previously described and presented as relative fold decrease compared to control samples. (C) EMSA analysis of NF-κB protein binding to the BLYS promoter in NHL-B (MCL-SP53) cells treated with various doses of the IκB inhibitor BAY11-7082. Lamin B was used as nuclear protein loading control. (D) Immunoblot analysis of BLyS in NHL-B (MCL-SP53) cells treated with various doses of IκB inhibitor BAY11-7082. (E) Immunoblot analysis for BLyS, p52, and Rel-B expression in NHL-B (LBCL-MS) cells treated with 100 nM p52 or Rel-B siRNA. Actin was used as loading control. (F) EMSA analysis of NF-κB binding to the BLYS promoter. Nuclear extracts from an NHL-B cell line (LBCL-MS) were transfected with BLYS or control siRNA and analyzed with an oligonucleotide probe for the BLYS–NF-κB binding site. Lamin B was used as nuclear protein loading control. (G) Immunoblot analysis of phosphorylated IκBα (pIκBα) and p52 in NHL-B cells transfected with BLYS or control siRNA. pIκBα and p52 protein expression levels were normalized to those of actin as previously described in panel B.

Figure 5.

Inhibition of NFAT activity reduces BLyS protein expression in NHL-B cells. (A) EMSA analysis of NFAT protein binding to BLYS promoter. Nuclear extracts from NHL-B (LBCL-MS) cells were transfected with dominant-negative (DN) NFAT or control plasmid and analyzed with oligonucleotide probes from the 2 BLYS-NFAT binding sites. NFAT binding levels were analyzed by BioMax 1D software, presented as before, as relative fold decrease compared to control samples. Lamin B was used as nuclear protein loading control. (B) Immunoblot analysis of BLyS expression in NHL-B (LBCL-MS) cells transfected with DN NFAT or control plasmid. Actin is used as loading control. BLyS protein levels were analyzed by BioMax 1D software, normalized with actin levels as before, and presented as before. (C) EMSA analysis of NFAT binding to the BLYS promoter in a representative NHL-B cell line (MCL-SP53). Nuclear extracts from NHL-B (MCL-SP53) cells treated with cyclosporine A (CsA) were analyzed with oligonucleotide probes from the 2 BLYS-NFAT sites to detect NFAT binding activity. NFAT binding levels were analyzed as previously described in panel A. Similar results observed in LBCL-MS cells too (data not shown). (D) Immunoblot analysis of BLyS protein in NHL-B (MCL-SP53) cells treated various doses of CsA. Actin was used as loading control. BLyS protein expression levels were analyzed as previously described in panel B. Similar results observed in LBCL-MS cells too.

Figure 6.

Induction of cellular NF-κB and NFAT activation leads to BLyS protein expression in normal B lymphocytes and low-grade follicular lymphoma (FL) cells. (A) EMSA analysis of normal G0 and activated peripheral blood B cells using the NF-κB or NFAT binding site oligonucleotides from the BLYS promoter. (B) RT-PCR analysis of BLyS expression in normal peripheral blood B cells. (C) Western blot analysis of BLyS in normal peripheral blood B cells. (D) EMSA analysis of tumor cells from a patient with follicular lymphoma (> 90% small cleaved cells: grade 1 FL) using oligonucleotide probes from BLYS-NF-κBor BLYS-NFAT binding sites on the BLYS promoter. (E) RT-PCR analysis of BLyS expression in the FL cells (described in panel D). (F) Western blot analysis of BLyS expression in follicular lymphoma (small cleaved cell lymphoma, grade 1) patient biopsy cells. Actin was used as loading control. BLyS protein expression levels were analyzed by BioMax 1D software as before.

Figure 7.

Both NF-κB and NFAT binding sites in the BLyS promoter are important for its activity. (A) Diagram of the BLYS promoter showing locations of site-directed mutagenesis. Luciferase activity in NHL-B (LBCL-MS) cells that had been transfected with wild-type, NF-κB, or NFAT mutant BLYS promoter-luciferase reporter constructs. (B) EMSA analysis of NF-kB or NFAT binding levels in NHL-B (LBCL-MS) cells incubated with wild-type BLYS–NF-κB or BLYS-NFAT binding site oligonucleotide probes or the corresponding mutant oligonucleotide probes from the BLYS promoter. Lamin B is used as nuclear extract loading control. (C) Luciferase activity in NHL-B (LBCL-MS) cells that had been transfected with control vector, c-rel expression vector, or NFATc1 expression vector. The error bars in panels A and C indicate the standard deviation of triplicate samples.