BAFF-R Receptor Also Functions in the Nucleus of Normal and Neoplastic Human B Lymphocytes.

B-lymphocyte stimulator (BLyS) is a relatively newly described tumor necrosis factor (TNF) superfamily cytokine involved in cell survival and proliferation in normal and neoplastic B cells, particularly in the aggressive B cell non-Hodgkin’s lymphomas (NHL-B). BLyS binds to three receptors: BAFF-R (or BR3), BCMA and TACI. However, recent studies have shown that BLyS regulates B cell survival and proliferation predominately through BAFF-R. Mice with mutant BAFF-R show significant decrease in the peripheral blood B-lymphocyte compartment, similar to the phenotype of BLyS knockout mice. BLyS/BAFF-R signaling functions through activation of the critical transcription factor NF-kB pathways, especially the NF-kB2 pathway, which mainly involves the p52/ rel-B complex.

Our previous studies have shown that BLyS is constitutively expressed in aggressive NHL-B cells leading to increased survival and proliferation of the malignant B cells. In this study, we found by western blotting and confocal microscopy that BAFF-R, the major B cell associated cell membrane receptor for BLyS, was also present in the B cell nucleus, in addition to its location in plasma membrane and cytoplasm in both normal peripheral blood B lymphocytes and NHL-B cells. Nuclear presence can be increased by anti-IgM and soluble CD154 treatment in normal peripheral blood B lymphocytes, and is constitutively expressed in aggressive NHL-B. Inhibition of BLyS expression by specific BLyS siRNA decreases nuclear BAFF-R level and survival in LBCL cells. Immunostaining experiments show that in the cytoplasm of normal and neoplastic B cells, BAFF-R interacts with the improtin a and b which are members of the classic karypherin pathway. A candidate nuclear localization sequence (NLS) was also identified in the BAFF-R protein sequence, and mutation of this putative NLS can block BAFF-R entering nucleus, which is consistent with our hypothesis that BAFF-R undergoes nuclear translocation. To further investigate the functional role of BAFF-R in nucleus, we performed confocal immunostaining analysis and co-immunoprecipitation, that shows that BAFF-R co-localizes with some NF-kB family members such as c-rel in the B cell nucleus. We also found that nuclear BAFF-R/c-rel complex can bind to the NF-kB binding site on the promoters of NF-kB target genes such as BLyS, CD154, Bcl-xL and Bfl-1 /A1 by chromatin immnoprecipitation (ChIP) assay. Luciferase reporter assays show that BAFF-R has transactivation activity on these NF-kB target genes. Furthermore, NLS mutant BAFF-R decreases NF-kB target gene promoter activity, compared to wild type BAFF-R, and NHL-B cell proliferation. These findings indicates that in addition to activating NF-kB pathways at the plasma membrane, BAFF-R may also promote survival and proliferation of both normal and NHL-B cell in the nucleus by directly regulating transcriptional activity of key NF-kB target genes and may functions as a transcriptional co- factor with NF-kB.