Cutting edge: a novel mechanism for rescue of B cells from CD95/Fas-mediated apoptosis.

CD95-induced apoptosis contributes to the maintenance of homeostasis in both B and T lymphocyte-mediated immunity. B cells increase CD95 expression in response to activation signals and become susceptible to CD95-induced apoptosis. Protection from CD95-mediated death signals can be induced in mature B cells by signals delivered through the B cell Ag receptor. In this paper we demonstrate for the first time that rescue from apoptosis can occur independently of de novo protein synthesis. This rescue from apoptosis prevents activation of caspase 8, the apical caspase in the CD95 death pathway, and CD95-FADD (Fas-associated death domain containing protein) association does not occur normally. Thus B cell activation signals can biochemically modify proximal elements of the CD95 death pathway and regulate the sensitivity of cells to apoptosis induction at an early stage in programmed cell death.     

Salarin C inhibits the maintenance of chronic myeloid leukemia progenitor cells

We previously showed that incubation of chronic myeloid leukemia (CML) cells in very low oxygen selects a cell subset where the oncogenetic BCR/Abl protein is suppressed and which is thereby refractory to tyrosine kinase inhibitors used for CML therapy. In this study, salarin C, an anticancer macrolide extracted from the Fascaplysinopsis sponge, was tested as for its activity on CML cells, especially after their incubation in atmosphere at 0.1% oxygen. Salarin C induced mitotic cycle arrest, apoptosis and DNA damage. Salarin C also concentration-dependently inhibited the maintenance of stem cell potential in cultures in low oxygen of either CML cell lines or primary cells. Surprisingly, the drug also concentration-dependently enforced the maintenance of BCR/Abl signaling in low oxygen, an effect which was paralleled by the rescue of sensitivity of stem cell potential to IM. These results suggest a potential use of salarin C for the suppression of CML cells refractory to tyrosine kinase inhibitors     

Implication of calpain in caspase activation during B cell clonal deletion.

In the absence of costimulating signals, B cell receptor (BCR) crosslinking on immature B cells triggers the apoptotic cell death program. In the WEHI-231 B cell lymphoma model, anti-IgM crosslinking triggers activation of caspase-7 independently of caspase-8, followed by apoptosis. Two main mechanisms for caspase-7 activation have been proposed: (i) caspase-8 recruitment to death receptors (Fas or tumour necrosis factor); and (ii) changes in mitochondrial membrane permeability and cytochrome c release, which activate caspase-9. Here we report that caspase-7 activation induced by BCR crosslinking is independent of caspase-8 and cytochrome c translocation from mitochondria to the cytosol, as well as of mitochondrial depolarization. In addition, in a cell-free system, the S-100 fraction of anti-IgM-treated WEHI-231 cells induces a caspase activation pattern different from that activated by cytochrome c and dATP. We demonstrate that calpain specifically triggers activation and processing of caspase-7 both in vitro and in vivo, and that both processes are inhibited by calpain inhibitors. Furthermore, calpain activation is associated with decreased expression levels of calpastatin, which is upregulated by CD40 ligation. These data confirm a role for calpain during BCR crosslinking, which may be critical for cell deletion by apoptosis during B cell development and activation.     

Epstein-Barr virus (EBV) latent membrane protein 2A regulates B-cell receptor-induced apoptosis and EBV reactivation through tyrosine phosphorylation

Epstein-Barr virus (EBV) is a human herpesvirus that establishes a lifelong latent infection of B cells. Within the immune system, apoptosis is a central mechanism in normal lymphocyte homeostasis both during early lymphocyte development and in response to antigenic stimuli. In this study, we found that latent membrane protein 2A (LMP2A) inhibited B-cell receptor (BCR)-induced apoptosis in Burkitt's lymphoma cell lines. Genistein, a specific inhibitor of tyrosine-specific protein kinases, blocked BCR-induced apoptosis and EBV reactivation in the cells. These findings indicate that LMP2A blocks BCR-induced cell apoptosis and EBV reactivation through the inhibition of activation of tyrosine kinases by BCR cross-linking.     

B cell antigen receptor-induced activation of Akt promotes B cell survival and is dependent on Syk kinase

Signaling through the B cell Ag receptor (BCR) is a key determinant in the regulation of B cell physiology. Depending on additional factors, such as microenvironment and developmental stage, ligation of the BCR can trigger B lymphocyte activation, proliferation, or apoptosis. The regulatory mechanisms determining B cell apoptosis and survival are not known. Using the chicken B lymphoma cell line DT40 as a model system, we investigated the role of the serine/threonine kinase Akt in B cell activation. While parental DT40 cells undergo apoptosis in response to BCR cross-linking, cells overexpressing Akt show a greatly diminished apoptotic response. By contrast, limiting the activation of Akt, either by inhibiting phosphatidylinositol 3-kinase or by ectopic expression of the phospholipid phosphatase MMAC1, results in a significant increase in the percentage of apoptotic cells after BCR cross-linking. Using various DT40 knockout cell lines, we further demonstrate that the tyrosine kinase Syk is required for Akt activation and that Lyn tyrosine kinase inhibits Akt activation. Taken together, the data demonstrate that Akt plays an important role in B cell survival and that Akt is activated in a Syk-dependent pathway.     

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.     

The adaptor protein HSH2 attenuates apoptosis in response to ligation of the B cell antigen receptor complex on the B lymphoma cell line, WEHI-231

Signals transduced by the B cell antigen receptor (BCR) play a central role in regulating the functional response of the cell to antigen. Depending on the nature of the antigenic signal and the developmental or differentiation state of the B cell, antigen receptor signaling can promote either apoptosis or survival and activation. Understanding the molecular mechanisms underlying BCR-mediated apoptosis constitutes an important area of research because aberrations in programmed cell death can result in the development of autoimmunity or cancer. Expression of the adaptor protein hematopoietic Src homology 2 (HSH2) was found to significantly decrease BCR-mediated apoptosis in the murine WEHI-231 cell line. Analysis of signal transduction pathways activated in response to BCR ligation revealed that HSH2 does not significantly alter total protein tyrosine phosphorylation or Ca2+ mobilization. HSH2 does not potentiate the activation-dependent phosphorylation of AKT either. With respect to MAPK activation, HSH2 was not observed to alter the activation of ERK or p38 in response to BCR ligation, but it does significantly potentiate JNK activation. Analysis of processes directly associated with apoptosis revealed that HSH2 inhibits mitochondrial depolarization to a significant degree, whereas it has only a slight effect on caspase activation and poly ADP-ribose polymerase cleavage. BCR-induced apoptosis of WEHI-231 cells is associated with the loss of endogenous HSH2 expression within 12 h, whereas inhibition of apoptosis in response to CD40-mediated signaling leads to stabilization of HSH2 expression. Thus, endogenous HSH2 expression correlates directly with survival of WEHI-231 cells, which supports the hypothesis that HSH2 modulates the apoptotic response through its ability to directly or indirectly promote mitochondrial stability.     

Bruton's tyrosine kinase regulates B cell antigen receptor-mediated JNK1 response through Rac1 and phospholipase C-gamma2 activation

Bruton's tyrosine kinase (Btk) is essential for B cell development and B cell antigen receptor (BCR) function. Recent studies have shown that Btk plays an important role in BCR-mediated c-Jun NH(2)-terminal kinase (JNK) 1 activation; however, the mechanism by which Btk participates in the JNK1 response remains elusive. Here we show that the BCR-mediated Rac1 activation is significantly inhibited by loss of Btk, while this Rac1 activation is not affected by loss of phospholipase C-gamma2 (PLC-gamma2). Since PLC-gamma2 is also required for BCR-mediated JNK1 response, our results suggest that Btk regulates Rac1 pathway as well as PLC-gamma2 pathway, both of which contribute to the BCR-mediated JNK1 response.     

Synergistic B cell activation by CD40 and the B cell antigen receptor: role of B lymphocyte antigen receptor-mediated kinase activation and tumor necrosis factor receptor-associated factor regulation

Optimal activation of B-lymphocytes depends both upon expression of various cell surface receptors and adequate integration of signaling pathways. This requires signals generated upon recognition of antigen by the B lymphocyte antigen receptor (BCR) as well as additional signals provided by cognate interaction with T helper cells, including the CD40-CD154 interaction. Engagement of both the BCR and CD40 results in synergistic activation of B cells. Previous studies identified tumor necrosis factor receptor-associated factor (TRAF)-2 and TRAF3 in the CD40-signaling pathway together with BCR-activated protein kinase D (PKD) as important cooperative factors in this synergy. To better understand the role of these factors in bridging the BCR and CD40 signaling pathways, BCR signal regulation of TRAF function was examined. Results show that phosphorylation of TRAF2 is increased upon BCR but not CD40 engagement and that of the potentially phosphorylated residues of TRAF2, tyrosine 484 is crucial for BCR-CD40 synergy. Additionally, wild type or constitutively active Bruton's tyrosine kinase (Btk) enhanced, whereas the xid mutant form of Btk prevented, BCR-CD40 synergy. These effects were dependent upon TRAF2 and PKD activity. These findings suggest a model in which Btk contributes to the enhancement of the CD40 response by TRAF2 in a PKD-dependent manner.     

Characterization of the human B cell RAG-associated gene, hBRAG, as a B cell receptor signal-enhancing glycoprotein dimer that associates with phosphorylated proteins in resting B cells

Affinity-purified polyclonal antibodies against the hBRAG (human B cell RAG-associated gene) protein were generated to characterize hBRAG at the biochemical level. Immunoblotting and immunoprecipitation experiments with these antibody reagents demonstrate that this protein can be expressed in B cells as a membrane-integrated glycoprotein disulfide-linked dimer. However, both glycosylated and unglycosylated isoforms of hBRAG are detectable with these reagents. Additionally, their use in cell surface biotinylation and flow cytometry reveals subcellular hBRAG pools both at cell surface and intracellular locations. Co-immunoprecipitation experiments with hBRAG antisera detected the association of hBRAG with phosphorylated proteins in resting B cells, including the protein tyrosine kinase Hck, which is subsequently dephosphorylated upon B cell receptor (BCR) ligation. Consistent with its cell surface expression and possible link to BCR signaling, experiments in which alpha-hBRAG antibodies were used to generate early activation signals suggest a modest but specific element of tyrosine phosphorylation occurring through a putative hBRAG receptor. Additional experiments also suggest that hBRAG may be involved in positively enhancing BCR ligation-mediated early activation events. Collectively, these results are consistent with a function for hBRAG as a B cell surface signaling receptor molecule. Coupled with the earlier observation that hBRAG expression correlates with early and late B cell-specific RAG expression, we submit that hBRAG may mediate regulatory signals key to B cell development and/or regulation of B cell-specific RAG expression.     

Positive signaling through CD72 induces mitogen-activated protein kinase activation and synergizes with B cell receptor signals to induce X-linked immunodeficiency B cell proliferation

CD72 is a 45-kDa B cell transmembrane glycoprotein that has been shown to be important for B cell activation. However, whether CD72 ligation induces B cell activation by delivering positive signals or sequestering negative signals away from B cell receptor (BCR) signals remains unclear. Here, by comparing the late signaling events associated with the mitogen-activated protein kinase pathway, we identified many similarities and some differences between CD72 and BCR signaling. Thus, CD72 and BCR activated the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) but not p38 mitogen-activated protein kinase. Both CD72- and BCR-mediated ERK and JNK activation required protein kinase C activity, which was equally important for CD72- and BCR-induced B cell proliferation. However, CD72 induced stronger JNK activation compared with BCR. Surprisingly, the JNK activation induced by both BCR and CD72 is Btk independent. Although both CD72 and BCR induced Btk-dependent ERK activation, CD72-mediated proliferation is more resistant to blocking of ERK activity than that of BCR, as shown by the proliferation response of B cells treated with PD98059 and dibutyryl cAMP, agents that inhibit ERK activity. Most importantly, CD72 signaling compensated for defective BCR signaling in X-linked immunodeficiency B cells and partially restored the proliferation response of X-linked immunodeficiency B cells to anti-IgM ligation. These results suggest that CD72 signals B cells by inducing BCR-independent positive signaling pathways.     

Protein tyrosine kinase inhibitors modulate radiosensitivity and radiation-induced apoptosis in K562 cells.

We studied the modulating effect of protein tyrosine kinase inhibitors on the response of cells of the human chronic myelogenous leukemia cell line K562 to radiation. The radiosensitivity of the cells was increased by treatment with herbimycin A and decreased by treatment with genistein. This modulating effect of protein tyrosine kinase inhibitors on radiation sensitivity was associated with the alteration of the mode of radiation-induced cell death. After X irradiation, the cells arrested in the G(2) phase of the cell cycle, but these TP53(-/-) cells were unable to sustain cell cycle arrest. This G(2)-phase checkpoint deficit caused cell death. The morphological pattern of cell death was characterized by swelling of the cytoplasmic compartments, cytosolic vacuolation, disruption of the plasma membrane, less evident nuclear condensation, and faint DNA fragmentation, all of which were consistent with oncosis or cytoplasmic apoptosis. The nonreceptor protein tyrosine kinase inhibitor herbimycin A accelerated the induction of typical apoptosis by X irradiation, which was demonstrated by morphological assessments using nuclear staining and electron microscopy as well as oligonucleosomal fragmentation and caspase 3 activity. Herbimycin A is known to be a selective antagonist of the BCR/ABL kinase of Philadelphia chromosome-positive K562 cells; this kinase blocks the induction of apoptosis after X irradiation. Our results showed that the inhibition of protein tyrosine kinase by herbimycin A enhanced radiation-induced apoptosis in K562 cells. This effect was associated with the activation of caspase 3 and rapid abrogation of the G(2)-phase checkpoint with progression out of G(2) into G(1) phase. In contrast, the receptor-type protein tyrosine kinase inhibitor genistein protected K562 cells from all types of radiation-induced cell death through the inhibition of caspase 3 activity and prolonged maintenance of G(2)-phase arrest. Further investigations using this model may give valuable information about the mechanisms of radiation-induced apoptosis and about the radiosensitivity and radioresistance of chronic myelogenous leukemia cells having the Philadelphia chromosome.     

Differential roles for extracellularly regulated kinase-mitogen-activated protein kinase in B cell antigen receptor-induced apoptosis and CD40-mediated rescue of WEHI-231 immature B cells

One of the major unresolved questions in B cell biology is how the B cell Ag receptor (BCR) differentially signals to transduce anergy, apoptosis, proliferation, or differentiation during B cell maturation. We now report that extracellularly regulated kinase-mitogen-activated protein kinase (Erk-MAP kinase) can play dual roles in the regulation of the cell fate of the immature B cell lymphoma, WEHI-231, depending on the kinetics and context of Erk-MAP kinase activation. First, we show that the BCR couples to an early (< or =2 h) Erk-MAP kinase signal which activates a phospholipase A(2) pathway that we have previously shown to mediate collapse of mitochondrial membrane potential, resulting in depletion of cellular ATP and cathepsin B execution of apoptosis. Rescue of BCR-driven apoptosis by CD40 signaling desensitizes such early extracellularly regulated kinase (Erk) signaling and hence uncouples the BCR from the apoptotic mitochondrial phospholipase A(2) pathway. A second role for Erk-MAP kinase in promoting the growth and proliferation of WEHI-231 immature B cells is evidenced by data showing that proliferating and CD40-stimulated WEHI-231 B cells exhibit a sustained cycling pattern (8-48 h) of Erk activation that correlates with cell growth and proliferation. This growth-promoting role for Erk signaling is supported by three key pieces of evidence: 1) signaling via the BCR, under conditions that induce growth arrest, completely abrogates sustained Erk activation; 2) CD40-mediated rescue from growth arrest correlates with restoration of cycling Erk activation; and 3) sustained inhibition of Erk prevents CD40-mediated rescue of BCR-driven growth arrest of WEHI-231 immature B cells. Erk-MAP kinase can therefore induce diverse biological responses in WEHI-231 cells depending on the context and kinetics of activation.     

Activation—induced cell death in B lymphocytes

Upon encountering the antigen(Ag),the immune system can either develop a specific immune response of enter a specific state of unresponsiveness,tolerance.The response of B cells to their specific Ag can be activation and proliferation,leading to the immune response,or anergy and activation-induced cell death(AICD),leading to tolerance.AICD in B lymphocytes is a highly regulated event initiated by crosslinking of the B cell receptor (BCR).BCR engagement initiates several signaling events such as activation of PLCγ,Ras,and PI3K,which generally speaking,lead to survival.However,in the absence of survival signals(CD40 or IL-4R engagement),BCR crosslinking can also promote apoptotic signal transduction pathways such as activation of effector caspases,expression of pro-apoptotic genes,and inhibition of pro-survival genes.The complex interplay between survival and death signals determines the B cell fate and, consequently,the immune response.     

TWEAK induces apoptosis through a death-signaling complex comprising receptor-interacting protein 1 (RIP1), Fas-associated death domain (FADD), and caspase-8

The tumor necrosis factor (TNF) superfamily member TNF-like weak inducer of apoptosis (TNFSF12, CD255) (TWEAK) can stimulate apoptosis in certain cancer cells. Previous studies suggest that TWEAK activates cell death indirectly, by inducing TNFα-mediated autocrine signals. However, the underlying death-signaling mechanism has not been directly defined. Consistent with earlier work, TWEAK assembled a proximal signaling complex containing its cognate receptor FN14, the adaptor TRAF2, and cellular inhibitor of apoptosis protein 1 (cIAP1). Neither the death domain adaptor Fas-associated death domain nor the apoptosis-initiating protease caspase-8 associated with this primary complex. Rather, TWEAK induced TNFα secretion and TNF receptor 1-dependent assembly of a death-signaling complex containing receptor-interacting protein 1 (RIP1), FADD, and caspase-8. Knockdown of RIP1 by siRNA prevented TWEAK-induced association of FADD with caspase-8 but not formation of the FN14-TRAF2-cIAP1 complex and inhibited apoptosis activation. Depletion of the RIP1 E3 ubiquitin ligase cIAP1 enhanced assembly of the RIP1-FADD-caspase-8 complex and augmented cell death. Conversely, knockdown of the RIP1 deubiquitinase CYLD inhibited these functions. Depletion of FADD, caspase-8, BID, or BAX and BAK but not RIP3 attenuated TWEAK-induced cell death. Pharmacologic inhibition of the NF-κB pathway or siRNA knockdown of RelA attenuated TWEAK induction of TNFα and association of RIP1 with FADD and caspase-8. These results suggest that TWEAK triggers apoptosis by promoting assembly of a RIP1-FADD-caspse-8 complex via autocrine TNFα-TNFR1 signaling. The proapoptotic activity of TWEAK is modulated by cIAP1 and CYLD and engages both the extrinsic and intrinsic signaling pathways.     

B cell receptor-stimulated mitochondrial phospholipase A2 activation and resultant disruption of mitochondrial membrane potential correlate with the induction of apoptosis in WEHI-231 B cells

Cross-linking of the Ag receptors on the immature B cell lymphoma, WEHI-231, leads to growth arrest and apoptosis. We now show that although commitment to such B cell receptor (BCR)-mediated apoptosis correlates with mitochondrial phospholipase A(2) activation, disruption of mitochondrial function, and ATP depletion, it is executed independently of caspase activation. First, we demonstrate a pivotal role for mitochondrial function in determining B cell fate by showing up-regulation of cytosolic phospholipase A(2) expression, induction of mitochondrial phospholipase A(2) activity, arachidonic acid-mediated collapse of mitochondrial transmembrane inner potential (Delta psi(m)), and depletion of cellular ATP under conditions of apoptotic, but not proliferative, signaling via the BCR. Importantly, disruption of Delta psi(m), ATP depletion, and apoptosis can be prevented by rescue signals via CD40 or by Delta psi(m) stabilizers such as antimycin or oligomycin. Second, we show that commitment and postmitochondrial execution of BCR-mediated apoptosis are not dependent on caspase activation by demonstrating that such apoptotic signaling does not induce release of cytochrome c from the mitochondria or activation of effector caspases, as evidenced by poly(ADP-ribose) polymerase or Bcl-x(L) cleavage. Indeed, apoptotic signaling via the BCR in WEHI-231 B cells does not stimulate the activation of caspase-3 and, consistent with this, BCR-mediated disruption of Delta psi(m) and commitment to apoptosis take place in the presence of caspase inhibitors. In contrast, BCR signaling induces the postmitochondrial activation of cathepsin B, and resultant apoptosis is blocked by the cathepsin B inhibitor, (23,35)trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester (EST) suggesting a key role for this executioner protease in Ag receptor-driven apoptosis of WEHI-231 immature B cells.     

A balance of Bruton's tyrosine kinase and SHIP activation regulates B cell receptor cluster formation by controlling actin remodeling

The activation of the BCR, which initiates B cell activation, is triggered by Ag-induced self-aggregation and clustering of receptors at the cell surface. Although Ag-induced actin reorganization is known to be involved in BCR clustering in response to membrane-associated Ag, the underlying mechanism that links actin reorganization to BCR activation remains unknown. In this study, we show that both the stimulatory Bruton's tyrosine kinase (Btk) and the inhibitory SHIP-1 are required for efficient BCR self-aggregation. In Btk-deficient B cells, the magnitude of BCR aggregation into clusters and B cell spreading in response to an Ag-tethered lipid bilayer is drastically reduced, compared with BCR aggregation observed in wild-type B cells. In SHIP-1(-/-) B cells, although surface BCRs aggregate into microclusters, the centripetal movement and growth of BCR clusters are inhibited, and B cell spreading is increased. The persistent BCR microclusters in SHIP-1(-/-) B cells exhibit higher levels of signaling than merged BCR clusters. In contrast to the inhibition of actin remodeling in Btk-deficient B cells, actin polymerization, F-actin accumulation, and Wiskott-Aldrich symptom protein phosphorylation are enhanced in SHIP-1(-/-) B cells in a Btk-dependent manner. Thus, a balance between positive and negative signaling regulates the spatiotemporal organization of the BCR at the cell surface by controlling actin remodeling, which potentially regulates the signal transduction of the BCR. This study suggests a novel feedback loop between BCR signaling and the actin cytoskeleton.     

BCR/ABL activates Rap1 and B-Raf to stimulate the MEK/Erk signaling pathway in hematopoietic cells

The BCR/ABL fusion tyrosine kinase activates various intracellular signaling pathways, thus causing chronic myeloid leukemia (CML). Here we demonstrate that the inducible expression of BCR/ABL in a murine hematopoietic cell line, TonB210, leads to the activation of the Ras family small GTPase Rap1, which is inhibited by the ABL kinase inhibitor imatinib. The Rap1 activity in a CML cell line, K562, was also inhibited by imatinib. Inhibition of Rap1 activation by a dominant negative mutant of Rap1, Rap1-N17, or SPA-1 inhibited the BCR/ABL-induced activation of Elk-1. BCR/ABL also activated in a kinase activity-dependent manner the B-Raf kinase, which is an effector molecule of Rap1 and a potent activator of the MEK/Erk/Elk-1 signaling pathway. Together, these data suggest that, in addition to the well-established Ras/Raf-1 pathway, BCR/ABL activates the alternative signaling pathway involving Rap1 and B-Raf to activate Erk, which may play important roles in leukemogenesis.