B cell receptor cross-talk: exposure to lipopolysaccharide induces an alternate pathway for B cell receptor-induced ERK phosphorylation and NF-kappa B activation

BCR signaling in naive B cells depends on the function of signalosome mediators; however, prior engagement of CD40 or of IL-4R produces an alternate signaling pathway in which Bruton's tyrosine kinase, PI3K, phospholipase Cgamma2, and protein kinase Cbeta are no longer required for BCR-induced downstream events. To explore the range of mediators capable of producing such an alternate pathway for BCR signaling, we examined the TLR4 agonist, LPS. B cell treatment with LPS at relatively low doses altered subsequent BCR signaling such that ERK phosphorylation and NF-kappaB activation occurred in a PI3K-independent manner. This effect of LPS extended to MEK phosphorylation and IkappaBalpha degradation, and it developed slowly over a period of 16-24 h. The involvement of TLRs is suggested by similar effects observed with a structurally distinct TLR agonist, PAM3CSK4 and by the need for MyD88 for induction of alternate BCR signaling by LPS. Thus, LPS-mediated TLR engagement produces an alternate pathway for BCR-triggered signal propagation that differs from the classical, signalosome-dependent pathway.     

B cell receptor (BCR) cross-talk: CD40 engagement enhances BCR-induced ERK activation

Bystander B cells may be initially stimulated through CD40, which enhances susceptibility to Fas-mediated apoptosis, before encountering Ag, which produces Fas resistance. A key issue in this process is to what extent CD40 cross-talk might affect subsequent BCR signaling. It has previously been shown that CD40 engagement bypasses or mitigates the need for Bruton's tyrosine kinase in subsequent BCR signaling for NF-kappaB activation. However, the full extent of the effects of CD40 on BCR signaling has not been delineated. In the present study we evaluated the possibility that CD40-mediated cross-talk also affects another principal outcome of BCR signaling: MAPK activation. We found that prior stimulation of primary murine B cells with CD40L markedly enhanced the level of ERK and JNK (but not p38 MAPK) phosphorylation produced by subsequently added anti-Ig Ab, and much, but not all, of this enhancement was independent of PI3K and phospholipase C. CD40L treatment similarly enhanced BCR-induced MAPK kinase (MEK) phosphorylation, and MEK was required for enhancement of ERK. Although BCR-induced c-Raf phosphorylation was also enhanced by prior CD40L treatment, c-Raf was not required for MEK/ERK phosphorylation. These results identify a novel system of receptor cross-talk between CD40 and BCR and indicate that the effects of CD40 engagement on subsequent BCR stimulation spread beyond NF-kappaB to involve the MAPK pathway.     

B cell receptor (BCR) cross-talk: IL-4 creates an alternate pathway for BCR-induced ERK activation that is phosphatidylinositol 3-kinase independent

IL-4 has pleiotropic effects on B cells. These effects include alteration of subsequent BCR-triggered responses. To identify a molecular basis for this receptor cross-talk, we examined ERK activation and NF-kappaB induction. We found that treatment with IL-4, but not other cytokines, affected subsequent BCR signaling by creating a new pathway in which the need for PI3K in ERK activation was eliminated. In contrast, the need for PI3K in NF-kappaB induction was not altered. The new pathway for ERK required time to develop, depended on STAT6, and was blocked by inhibition of macromolecular synthesis. As in the classical pathway, BCR-induced ERK activation in the new, PI3K-independent pathway required MEK and was reflected in c-Raf. Thus, IL-4 promotes an alternate pathway through which BCR is coupled to Raf/MEK/ERK that may function to heighten the responsiveness of B cells during times of immunological stress.     

IkappaB kinase beta phosphorylates the K63 deubiquitinase A20 to cause feedback inhibition of the NF-kappaB pathway

Misregulation of NF-kappaB signaling leads to infectious, inflammatory, or autoimmune disorders. IkappaB kinase beta (IKKbeta) is an essential activator of NF-kappaB and is known to phosphorylate the NF-kappaB inhibitor, IkappaBalpha, allowing it to undergo ubiquitin-mediated proteasomal degradation. However, beyond IkappaBalpha, few additional IKKbeta substrates have been identified. Here we utilize a peptide library and bioinformatic approach to predict likely substrates of IKKbeta. This approach predicted Ser381 of the K63 deubiquitinase A20 as a likely site of IKKbeta phosphorylation. While A20 is a known negative regulator of innate immune signaling pathways, the mechanisms regulating the activity of A20 are poorly understood. We show that IKKbeta phosphorylates A20 in vitro and in vivo at serine 381, and we further show that this phosphorylation event increases the ability of A20 to inhibit the NF-kappaB signaling pathway. Phosphorylation of A20 by IKKbeta thus represents part of a novel feedback loop that regulates the duration of NF-kappaB signaling following activation of innate immune signaling pathways.     

Cutting Edge: B cell receptor (BCR) cross-talk: the IL-4-induced alternate pathway for BCR signaling operates in parallel with the classical pathway, is sensitive to Rottlerin, and depends on Lyn

B cell exposure to IL-4 alters subsequent BCR signaling such that ERK phosphorylation becomes signalosome-independent; however, the nature of this new, alternate signaling pathway and its relationship to the classical, signalosome-dependent signaling pathway are not known. In this study, we report that the alternate and classical pathways for BCR signaling are differentially affected by rottlerin, and by Go6976 or LY294002, respectively. Furthermore, in B cells lacking protein kinase C (PKC)beta, the classical pathway for BCR signaling is blocked, whereas the alternate pathway is little affected. Conversely, in B cells lacking Lyn, the alternate pathway for BCR signaling is blocked, whereas the classical pathway is little affected. The rottlerin-sensitive element is not PKCdelta, inasmuch as the alternate pathway is not blocked in PKCdelta-deficient B cells. These results indicate that the rottlerin-sensitive, Lyn-dependent alternate pathway, and the classical pathway, for BCR signaling operate in parallel when BCR engagement follows IL-4 exposure.     

NF-kappaB-inducing kinase restores defective IkappaB kinase activity and NF-kappaB signaling in intestinal epithelial cells

Cytokine-stimulated IkappaBalpha degradation is impaired in HT-29 and primary intestinal epithelial cells. To gain more insight into the mechanism of this defect, we dissected cytokine-induced NF-kappaB signaling pathway in HT-29 cells. IL-1beta and TNF, alone or in combination with IFNgamma, failed to induce IkappaBalpha or IkappaBbeta degradation in HT-29 cells. Despite similar 125I-IL-1beta binding, HT-29 cells displayed no IRAK degradation, a 75% reduction of IKK activity, and decreased IkappaBalpha phosphorylation, NF-kappaB DNA binding activity and IL-8 mRNA accumulation in response to IL-1beta compared to Caco-2 cells. Selective activation of NF-kappaB pathway by adenoviral delivery of NF-kappaB-inducing kinase (Ad5NIK) or IKKbeta (Ad5IKKbeta) strongly activated IKK activity (>20 fold) in HT-29 cells with concomitant endogenous IkappaBalpha serine 32 phosphorylation and total IkappaBalpha degradation. In addition, NF-kappaB DNA binding activity and IL-8 secretion is higher in Ad5NIK-infected than in IL-1beta-stimulated HT-29 cells. These data show that altered NF-kappaB signaling is associated with impaired stimulation of an upstream IKK activator.     

Bruton's tyrosine kinase mediates NF-kappa B activation and B cell survival by B cell-activating factor receptor of the TNF-R family

Loss of Bruton's tyrosine kinase (Btk) function results in mouse Xid disease characterized by a reduction in mature B cells and impaired humoral immune responses. These defects have been mainly attributed to impaired BCR signaling including reduced activation of the classical NF-kappaB pathway. In this study we show that Btk also couples the receptor for B cell-activating factor (BAFF) of the TNF family (BAFF-R) to the NF-kappaB pathway. Loss of Btk results in defective BAFF-mediated activation of both classical and alternative NF-kappaB pathways. Btk appears to regulate directly the classical pathway in response to BAFF such that Btk-deficient B cells exhibit reduced kinase activity of IkappaB kinase gamma-containing complexes and defective IkappaBalpha degradation. In addition, Btk-deficient B cells produce reduced levels of NF-kappaB2 (p100) basally and in response to stimulation via the BCR or BAFF-R, resulting in impaired activation of the alternative NF-kappaB pathway by BAFF. These results suggest that Btk regulates B cell survival by directly regulating the classical NF-kappaB pathway under both BCR and BAFF-R, as well as by inducing the expression of the components of alternative pathway for sustained NF-kappaB activation in response BAFF. Thus, impaired BCR- and BAFF-induced signaling to NF-kappaB may contribute to the observed defects in B cell survival and humoral immune responses in Btk-deficient mice.     

Phosphatidylinositol 3-kinase-dependent mitogen-activated protein/extracellular signal-regulated kinase kinase 1/2 and NF-kappa B signaling pathways are required for B cell antigen receptor-mediated cyclin D2 induction in mature B cells

Phosphatidylinositol 3-kinase (PI-3K) has been linked to promitogenic responses in splenic B cells following B cell Ag receptor (BCR) cross-linking; however identification of the signaling intermediates that link PI-3K activity to the cell cycle remains incomplete. We show that cyclin D2 induction is blocked by the PI-3K inhibitors wortmannin and LY294002, which coincides with impaired BCR-mediated mitogen-activated protein/extracellular signal-related kinase kinase (MEK)1/2 and p42/44ERK phosphorylation on activation residues. Cyclin D2 induction is virtually absent in B lymphocytes from mice deficient in the class I(A) PI-3K p85alpha regulatory subunit. In contrast to studies with PI-3K inhibitors, which inhibit all classes of PI-3Ks, the p85alpha regulatory subunit is not required for BCR-induced MEK1/2 and p42/44ERK phosphorylation, suggesting the contribution of another PI-3K family members in MEK1/2 and p42/44ERK activation. However, p85alpha(-/-) splenic B cells are defective in BCR-induced IkappaB kinase beta and IkappaBalpha phosphorylation. We demonstrate that NF-kappaB signaling is required for cyclin D2 induction via the BCR in normal B cells, implicating a possible link with the defective IkappaB kinase beta and IkappaBalpha phosphorylation in p85alpha(-/-) splenic B cells and their ability to induce cyclin D2. These results indicate that MEK1/2-p42/44ERK and NF-kappaB pathways link PI-3K activity to Ag receptor-mediated cyclin D2 induction in splenic B cells.     

Nuclear factor kappa B-inducing kinase and Ikappa B kinase-alpha signal skeletal muscle cell differentiation

Nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK), IkappaB kinase (IKK)-alpha and -beta, and IkappaBalpha are common elements that signal NF-kappaB activation in response to diverse stimuli. In this study, we analyzed the role of this pathway during insulin-like growth factor II (IGF-II)-induced myoblast differentiation. L6E9 myoblasts differentiated with IGF-II showed an induction of NF-kappaB DNA-binding activity that correlated in time with the activation of IKKalpha, IKKbeta, and NIK. Moreover, the activation of IKKalpha, IKKbeta, and NIK by IGF-II was dependent on phosphatidylinositol 3-kinase, a key regulator of myogenesis. Adenoviral transduction with the IkappaBalpha(S32A/S36A) mutant severely impaired both IGF-II-dependent NF-kappaB activation and myoblast differentiation, indicating that phosphorylation of IkappaBalpha at Ser-32 and Ser-36 is an essential myogenic step. Adenoviral transfer of wild-type or kinase-deficient forms of IKKalpha or IKKbeta revealed that IKKalpha is required for IGF-II-dependent myoblast differentiation, whereas IKKbeta is not essential for this process. Finally, overexpression of kinase-proficient wild-type NIK showed that the activation of NIK is sufficient to generate signals that trigger myogenin expression and multinucleated myotube formation in the absence of IGF-II.     

B cell receptor-induced cAMP-response element-binding protein activation in B lymphocytes requires novel protein kinase Cdelta

The cAMP-response element-binding protein (CREB) is activated by phosphorylation on Ser-133 and plays a key role in the proliferative and survival responses of mature B cells to B cell receptor (BCR) signaling. The signal link between the BCR and CREB activation depends on a phorbol ester (phorbol 12-myristate 13-acetate)-sensitive protein kinase C (PKC) activity and not protein kinase A or calmodulin kinase; however, the identity and role of the PKC(s) activity has not been elucidated. We found the novel PKCdelta (nPKCdelta) activator bistratene A is sufficient to induce CREB phosphorylation in murine splenic B cells. The pharmacological inhibitor G?6976, which targets conventional PKCs and PKCmu, has no effect on CREB phosphorylation, whereas the nPKCdelta inhibitor rottlerin blocks CREB phosphorylation following BCR cross-linking. Bryostatin 1 selectively prevents nPKCdelta depletion by phorbol 12-myristate 13-acetate when coapplied, coincident with protection of BCR-induced CREB phosphorylation. Ectopic expression of a kinase-inactive nPKCdelta blocks BCR-induced CREB phosphorylation in A20 B cells. In addition, BCR-induced CREB phosphorylation is significantly diminished in nPKCdelta-deficient splenic B cells in comparison with wild type mice. Consistent with the essential role for Bruton's tyrosine kinase and phospholipase Cgamma2 in mediating PKC activation, Bruton's tyrosine kinase- and phospholipase Cgamma2-deficient B cells display defective CREB phosphorylation by the BCR. We also found that p90 RSK directly phosphorylates CREB on Ser-133 following BCR cross-linking and is positioned downstream of nPKCdelta. Taken together, these results suggest a model in which BCR engagement leads to the phosphorylation of CREB via a signaling pathway that requires nPKCdelta and p90 RSK in mature B cells.     

CD72 down-modulates BCR-induced signal transduction and diminishes survival in primary mature B lymphocytes

CD72, a 45-kDa type II transmembrane glycoprotein carrying an ITIM motif, is believed to be an inhibitory coreceptor of the BCR. Mature B cells lacking CD72 show enhanced Ca(2+) mobilization and are hyperproliferative in response to BCR ligation. However, the signal transduction pathways downstream of BCR signaling that transmit the inhibitory effect of CD72 in mature B cells remain unknown. To address this question, we used hen egg lysozyme-specific BCR transgenic mice to elucidate the differential cell signaling between wild-type and CD72-deficient B cells in response to hen egg lysozyme Ag stimulation. Our results demonstrate that CD72 predominantly down-regulates the major signal transduction pathways downstream of the BCR, including NF-AT, NF-kappaB, ERK, JNK, p38-MAPK, and PI3K/Akt in mature B cells. CD72 ligation with anti-CD72 Ab (K10.6), which mimics the binding of CD100 (a natural ligand for CD72) to release the inhibitory function of CD72, augments cell proliferation, Ca(2+) flux, IkappaBalpha activation, and ERK MAPK activity upon Ag stimulation in wild-type B cells. In addition, we show direct evidence that CD72 promotes cell cycle arrest and apoptosis after Ag stimulation in mature B cells. Taken together, our findings conclude that CD72 plays a dominant role as a negative regulator of BCR signaling in primary mature B lymphocytes.     

Differential effects of tumor necrosis factor-alpha and CD40L on NF-kappa B inhibitory proteins I kappa B alpha, beta and epsilon and on the induction of the Jun amino-terminal kinase pathway in Ramos Burkitt lymphoma cells

Interaction between the CD40 ligand and its cognate receptor is known to affect various aspects of B-cell biology. Less is known about the biological consequences of B-cell signaling through tumor necrosis factor alpha (TNF-alpha) and its two receptors. We have used Ramos germinal center (GC)-derived Burkitt's lymphoma (BL) cells as a model system to compare some of the early signaling events of TNF-alpha and CD40L on the NF-kappaB and c-Jun amino-terminal kinase (JNK) pathways. We have previously found that both TNF-alpha and CD40L induced enhanced cell aggregation, adherence and modified cell surface morphology of Ramos cells. In the present report, it was found that treatment with either TNF-alpha or CD40L resulted in a rapid degradation (within 15 min) of IkappaBalpha, followed by a recovery period lasting up to a few hours. The level of IkappaBbeta, another inhibitory molecule of the NF-kappaB pathway, also decreased following treatment with CD40L or TNF-alpha. However, whereas CD40L induced a rapid drop without significant recovery within 2 h, TNF-alpha caused a slow and gradual decline of IkappaBbeta. In addition, treatment with CD40L resulted in a gradual and modest decline of up to 60% of the level of IkappaBepsilon within 2 h, whereas a much smaller decline was seen with TNF-alpha (approx. 20%) Our results thus show that in Ramos cells, TNF-alpha and CD40L have common, as well as differential, signaling effects on the IkappaBalpha, IkappaBbeta and IkappaBepsilon, which form inhibitory complex(es) with the NF-kappaB cytosolic proteins. We also found that CD40L, but not TNF-alpha activates the JNK pathway through transient phosphorylation of its threonine183/tyrosine185 residues. As expected, c-Jun, which is known to be a substrate of JNK, was also phosphorylated at serine residue 73 by treatment with CD40L, but not by TNF-alpha.