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• Rapamycin attenuation of BAFF-elevated [Ca2+] i links to its inhibition of B-cell proliferation/survival. • Rapamyicn blunts mTORC1/2-mediated [Ca2+] i elevations induced by BAFF. • Rapamyicn inhibits BAFF-stimulated B-cell proliferation/survival by blocking Ca2+-CaMKII-dependent PTEN/Akt-Erk1/2 signaling. • Rapamycin may be an effective agent for prevention of BAFF-induced aggressive or neoplastic B-cell disorders. B-cell activating factor (BAFF) is a crucial survival factor for B cells, and excess BAFF contributes to development of autoimmune diseases. Recent studies have shown that rapamycin can prevent BAFF-induced B-cell proliferation and survival, but the underlying mechanism remains to be elucidated. Here we found that rapamycin inhibited human soluble BAFF (hsBAFF)-stimulated cell proliferation by inducing G 1 -cell cycle arrest, which was through downregulating the protein levels of CDK2, CDK4, CDK6, cyclin A, cyclin D1, and cyclin E. Rapamycin reduced hsBAFF-stimulated cell survival by downregulating the levels of anti-apoptotic proteins (Mcl-1, Bcl-2, Bcl-xL and survivin) and meanwhile upregulating the levels of pro-apoptotic proteins (BAK and BAX). The cytostatic and cytotoxic effects of rapamycin linked to its attenuation of hsBAFF-elevated intracellular free Ca2+ ([Ca2+] i). In addition, rapamycin blocked hsBAFF-stimulated B-cell proliferation and survival by preventing hsBAFF from inactivating PTEN and activating the Akt-Erk1/2 pathway. Overexpression of wild type PTEN or ectopic expression of dominant negative Akt potentiated rapamycin's suppression of hsBAFF-induced Erk1/2 activation and proliferation/viability in Raji cells. Interestingly, PP242 (mTORC1/2 inhibitor) or Akt inhibitor X, like rapamycin (mTORC1 inhibitor), reduced the basal or hsBAFF-induced [Ca2+] i elevations. Chelating [Ca2+] i with BAPTA/AM, preventing [Ca2+] i elevation using EGTA, 2-APB or verapamil, inhibiting CaMKII with KN93, or silencing CaMKII strengthened rapamycin's inhibitory effects. The results indicate that rapamycin inhibits BAFF-stimulated B-cell proliferation and survival by blunting mTORC1/2-mediated [Ca2+] i elevations and suppressing Ca2+-CaMKII-dependent PTEN/Akt-Erk1/2 signaling pathway. Our finding underscores that rapamycin may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases. [ABSTRACT FROM AUTHOR]

• Cat (Felis catus) BAFF and its receptor BAFF-R were cloned. • csBAFF, EGFP/csBAFF, and cBAFF-R were expressed in E. coli and purified using a Ni2+ column. • csBAFF cellular localization was assessed. • csBAFF promoted B-cell survival in vitro. • Ligand–receptor interaction was assessed. B-cell survival depends on signals induced by binding of B-cell activating factor (BAFF) to its receptor (BAFF-R). In this study, the full-length cDNAs of cat BAFF (cBAFF) and BAFF-R (cBAFF-R) were amplified from the spleen by reverse transcription PCR. The open reading frame of cBAFF cDNA encodes a protein of 285 amino acids containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian, avian, and reptile BAFFs. The cBAFF-R gene encodes a 189 amino acid protein. Real-time quantitative PCR analyses revealed that the two genes are predominantly expressed in the spleen. csBAFF, EGFP/csBAFF, and cBAFF-R were efficiently expressed in Escherichia coli BL21 (DE3), as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses. After purification, the EGFP/csBAFF fusion protein showed a fluorescence spectrum similar to that of EGFP. Confocal laser scanning microscopy showed that EGFP/csBAFF bound to its receptor. In vitro , csBAFF promoted the survival of cat and mouse splenic B cells with/without a priming agent (Staphylococcus aureus Cowan 1, SAC) or anti-mouse IgM. Furthermore, it stimulated the survival of mouse B cells, similar to msBAFF. Recombinant cBAFF-R blocked the function of sBAFF in vitro. These findings indicate that csBAFF plays an important role in the survival of cat B cells and has functional cross reactivity between cats and other mammals, and suggest a role for the BAFF–BAFF-R system in regulating B-cell survival. Therefore, BAFF and BAFF-R show promise for enhancing the immune systems of animals. [ABSTRACT FROM AUTHOR]

Abstract B cell activating factor from the TNF family (BAFF) is implicated in not only the physiology of normal B cells, but also the pathophysiology of aggressive B cells related to malignant and autoimmune diseases. Autophagy plays a crucial role in balancing the beneficial and detrimental effects of immunity and inflammation. However, little is known about whether and how excessive BAFF mediates autophagy contributing to B-cell proliferation and survival. Here, we show that excessive human soluble BAFF (hsBAFF) inhibited autophagy with a concomitant reduction of LC3-II in normal and B-lymphoid (Raji) cells. Knockdown of LC3 not only potentiated hsBAFF inhibition of autophagy, but also attenuated hsBAFF activation of Akt/mTOR pathway, thereby diminishing hsBAFF-induced B-cell proliferation/viability. Further, we found that hsBAFF inhibition of autophagy was Akt/mTOR-dependent. This is supported by the findings that hsBAFF increased mTORC1-mediated phosphorylation of ULK1 (Ser757); Akt inhibitor X, mTORC1 inhibitor rapamycin, mTORC1/2 inhibitor PP242, expression of dominant negative Akt, or knockdown of mTOR attenuated hsBAFF-induced phosphorylation of ULK1, decrease of LC3-II level, and increase of cell proliferation/viability. Chelating intracellular free Ca2+ ([Ca2+] i) with BAPTA/AM or preventing [Ca2+] i elevation using EGTA or 2-APB profoundly blocked hsBAFF-induced activation of Akt/mTOR, phosphorylation of ULK1 and decrease of LC3-II, as well as increase of cell proliferation/viability. Similar effects were observed in the cells where CaMKII was inhibited by KN93 or knocked down by CaMKII shRNA. Collectively, these results indicate that hsBAFF inhibits autophagy promoting cell proliferation and survival through activating Ca2+-CaMKII-dependent Akt/mTOR signaling pathway in normal and neoplastic B-lymphoid cells. Our findings suggest that manipulation of intracellular Ca2+ level or CaMKII, Akt, or mTOR activity to promote autophagy may be exploited for prevention of excessive BAFF-induced aggressive B lymphocyte disorders and autoimmune diseases. Graphical abstract Unlabelled Image Highlights • hsBAFF promotes B-cell proliferation and survival by inhibiting autophagy. • Downregulation of LC3 protein level is critical for hsBAFF inhibition in B cells. • hsBAFF-activated Akt/mTOR signaling represses autophagy via phosphorylating ULK1 (Ser757) in B cells. • hsBAFF inhibits autophagy by activating Ca2+-CaMKII-dependent Akt/mTOR signaling pathway in B cells. [ABSTRACT FROM AUTHOR]

Abstract: B cell activating factor (BAFF) and its receptors were regarded as elements of the immune system, regulating the fate of B cell. In recent years, these molecules were identified in a number of normal and pathological tissues, expanding their potential functions beyond the immune system. In this study, on the basis of molecular clone and prokaryotic expression of equine BAFF, we reported that equine adipose-derived stem cell (ASC) expressed BAFF and its receptors, which exhibited the increased expression during ASC adipogenic differentiation in vitro. Moreover, with the addition of recombinant protein His6-sBAFF, an increased differentiation of equine ASC towards adipocyte was detected. These results suggested that BAFF and its receptors might be associated with the differentiation process of ASC towards adipocyte in horse. [Copyright &y& Elsevier]

Abstract: 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 Ca2+-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 Ca2+-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 Ca2+ may be exploited for prevention of excessive BAFF-induced aggressive B-cell malignancies and autoimmune diseases. [Copyright &y& Elsevier]

Abstract: B cell activating factor (BAFF), a member of the tumor necrosis factor family, is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. In the present study, the full-length cDNA of BAFF from the South African clawed frog (Xenopus laevis, designated xlBAFF) was cloned using rapid amplification of cDNA ends (RACE) techniques and RT-PCR. The full-length cDNA of xlBAFF consists of 1204 bases including an open reading frame (ORF) of 801 nucleotides that are translated into a predicted 266 amino acid protein. Sequence comparison indicated that the amino acids of xlBAFF possessed the TNF signature, including a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the xlBAFF monomer revealed that it was very similar to its counterparts. Real-time quantitative PCR analysis revealed that xlBAFF could be detected in various tissues and predominantly expressed in the spleen and other lymphoid tissue. The soluble xlBAFF had been cloned into a pET28a vector to express the recombinant protein. The His6-xlBAFF was efficiently expressed in Escherichia coli. BL21 (DE3) and its expressions were confirmed by SDS-PAGE and Western blotting analysis. After purification, laser scanning confocal microscopy analysis showed that xlBAFF could bind to its receptors on B cells. CCK-8 assays revealed that xlBAFF is not only able to promote survival/proliferation of South African clawed frog lymphocytes but also able to stimulate survival/proliferation of mouse B cells. These results will allow for further investigation the use of X. laevis as an in vivo model for related studies. [Copyright &y& Elsevier]

Abstract: Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-BAFF. After four rounds of panning against BAFF, thirty-two out of 92 phage clones displayed BAFF binding activity. One of the positive clones, designated F8, bound to BAFF with relatively high affinity and neutralized BAFF bioactivity in vitro. F8 clone was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC). The purified scFv recognized BAFF with the affinity constant (K aff) of 2.5×107 M−1 without cross-reaction to APRIL. In addition to binding, the purified scFv could does-dependently inhibit BAFF-induced mouse spleen B lymphocyte proliferation. Together with its fully human mature, F8 scFv may have therapeutic implications in therapy of autoimmune disorders mediated by BAFF. [Copyright &y& Elsevier]