BAFF activates Erk1/2 promoting cell proliferation and survival by Ca2+-CaMKII-dependent inhibition of PP2A in normal and neoplastic B-lymphoid cells.

B-cell activating factor (BAFF) is involved in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. However, how excessive BAFF promotes aggressive B-cell proliferation and survival is not well understood. Here we show that excessive human soluble BAFF (hsBAFF) enhanced cell proliferation and survival in normal and B-lymphoid (Raji) cells, which was associated with suppression of PP2A, resulting in activation of Erk1/2. This is supported by the findings that pretreatment with U0126 or PD98059, expression of dominant negative MKK1, or overexpression of PP2A prevented hsBAFF-induced activation of Erk1/2 and cell proliferation/viability in the cells. It appears that hsBAFF-mediated PP2A-Erk1/2 pathway and B-cell proliferation/viability was Ca(2+)-dependent, as pretreatment with BAPTA/AM, EGTA or 2-APB significantly attenuated these events. Furthermore, we found that inhibiting CaMKII with KN93 or silencing CaMKII also attenuated hsBAFF-mediated PP2A-Erk1/2 signaling and B-cell proliferation/viability. The results indicate that BAFF activates Erk1/2, in part through Ca(2+)-CaMKII-dependent inhibition of PP2A, increasing cell proliferation/viability in normal and neoplastic B-lymphoid cells. Our data suggest that inhibitors of CaMKII and Erk1/2, activator of PP2A or manipulation of intracellular Ca(2+) may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases.

Molecular cloning, expression, bioinformatics analysis, and bioactivity of TNFSF13 (APRIL) in the South African clawed frog (Xenopus laevi): a new model to study immunological diseases.

TNFSF13 is one of the tumor necrosis factor (TNF) superfamily members that plays important roles in immune homeostasis and proliferation or apoptosis of certain tumor cell lines. This report describes the development of Xenopus laevis TNFSF13 as a model to study its important role in relation to immunological diseases. In brief, TNFSF13 from Xenopus laevis (designated XlTNFSF13) was first amplified by RT-PCR and rapid amplification of cDNA end (RACE) techniques. Bioinformatics analyses revealed the gene structure, three-dimensional structure, and evolutionary relationships. Real-time quantitative PCR (QPCR) analysis identified the tissue distribution of XlTNFSF13 in the major visceral organs. The recombinant plasmid SUMO-XsTNFSF13 was expressed in E. coli Rosseta (DE3). Subsequently, the recombinant protein purified through Ni-NTA affinity chromatography was analyzed by SDS-PAGE and confirmed by Western blot analysis. Laser scanning confocal microscopy analysis revealed the binding activity of pSUMO-XsTNFSF13 to the surface of B cells. WST-8 assays further indicated that purified XsTNFSF13 could cause the survival/proliferation of B cells. In conclusion, we underscore that as a model organism for human disease, Xenopus laevis has been widely used in molecular biology research. Yet while TNFSF13 research in mammalian, fish (e.g., zebrafish), mouse, and human is widely available, studies in the amphibian species are limited. The latter area of OMICS and integrative biology scholarship is directly informed with the present study, with a view to implications for the future study of human immunological diseases.

hsBAFF promotes proliferation and survival in cultured B lymphocytes via calcium signaling activation of mTOR pathway.

B-cell activating factor of the TNF family (BAFF, also called BLyS, TALL-1, THANK, or zTNF4) has revealed its critical function in B lymphocyte proliferation and survival, as well as the pathogenesis of autoimmune disease. However, the molecular mechanisms of excess BAFF-extended aggressive B lymphocytes have not been completely defined. Here we show that excessive hsBAFF-elevated [Ca(2+)]i activated mammalian target of rapamycin (mTOR) signaling pathway, leading to proliferation and survival in B lymphocytes. This is supported by the findings that intracellular Ca(2+) chelator (BAPTA/AM) or mTOR inhibitor (rapamycin) abolished the events. Sequentially, we observed that preventing [Ca(2+)]i elevation using EGTA or 2-APB dramatically inhibited hsBAFF activation of mTOR signaling, as well as cell growth and survival, suggesting that hsBAFF-induced extracellular Ca(2+) influx and ER Ca(2+) release elevates [Ca(2+)]i contributing to B lymphocyte proliferation and survival via activation of mTOR signaling. Further, we noticed that pretreatment with BAPTA/AM, EGTA or 2-APB blocked hsBAFF-increased phosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII), and inhibiting CaMKII with KN93 attenuated hsBAFF-activated mTOR signaling, as well as cell growth and survival, revealing that the effects of hsBAFF-elevated [Ca(2+)]i on mTOR signaling as well as proliferation and survival in B lymphocytes is through stimulating phosphorylation of CaMKII. The results indicate that hsBAFF activates mTOR pathway triggering B lymphocyte proliferation and survival by calcium signaling. Our findings suggest that manipulation of intracellular Ca(2+) level or CaMKII and mTOR activity may be exploited for the prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases.

Cloning, expression, and characterization of TNFSF14 (LIGHT) gene in mefugu, Takifugu obscurus.

LIGHT/TNFSF14 is a member of the tumor necrosis factor ligand superfamily, which plays important roles in inflammatory and immune responses. In this study, the cDNA of mefugu (Takifugu obscures) LIGHT (designated as fLIGHT) was amplified from spleen by reverse transcription polymerase chain reaction. The open reading frame of fLIGHT covers 765 bp and translates into a 254-aa protein. The predicted three-dimensional (3D) structure of the soluble LIGHT of mefugu (designated as fsLIGHT) monomer analyzed by comparative protein modeling revealed that it was very similar to its human counterpart. Real-time quantitative PCR analysis indicated that LIGHT is constitutively expressed in various tissues in mefugu. The soluble LIGHT binding of green fluorescent protein (GFP) (designated as GFP/fsLIGHT) had been cloned into the pET28a vector; SDS-PAGE and western blotting analysis confirmed that the recombinant protein pET28a-GFP/fsLIGHT was efficiently expressed in Escherichia coli BL21 (DE3). After purification, the GFP/fsLIGHT fusion protein obtained similar fluorescence spectrum to those of GFP. Laser scanning confocal microscopy analysis showed GFP/fsLIGHT could bind to its receptors. In view of our basic research, LIGHT may be a potential immunologic factor for enhancing immunological efficacy in fish, and our results might provide a platform for further study into the effects of LIGHT.