DNA-binding activity of NF-kappaB and phosphorylation of p65 are induced by N-acetylcysteine through phosphatidylinositol (PI) 3-kinase

N-Acetylcysteine (NAC) has been widely used as an antioxidant in research, however, it has also been found to reduce the binding of TNF to its receptor independent of its antioxidative role. In this study, we investigated the effect of NAC on NF-kappaB activation. In HeLa cells, Hep3B cells, and A549 cells, DNA-binding activity of NF-kappaB was induced by NAC without any other stimulation but not by tetramethylthiourea (TMTU) or vitamin C, suggesting that ROS is not involved in the effect of NAC. The degradation of IkappaBalpha and nuclear translocation of NF-kappaB were not induced by NAC. The phosphorylation of p65 at serine 536 was induced by NAC, which is known to contribute to the enhancement of DNA-binding activity of NF-kappaB, however, NAC did not directly phosphorylate p65. The NAC-induced DNA-binding activity of NF-kappaB and phosphorylation of p65 were sensitive to a phosphatidylinositol (PI) 3-kinase inhibitor, partially sensitive to an IkappaB kinase (IKK) inhibitor, but not sensitive to a Bruton's tyrosine kinase (Btk) inhibitor. Moreover, both the DNA-binding activity and phosphorylation induced by NAC were reduced by the overexpression of a dominant negative Akt in HeLa cells. These results suggest that NAC activates mainly PI3K to phosphorylate p65 and subsequently induces DNA-binding activity of NF-kappaB, independent of its antioxidative function.     

Bacterial lipopolysaccharide induced B cell activation is mediated via a phosphatidylinositol 3-kinase dependent signaling pathway.

Bacterial lipopolysaccharide (LPS) is a potent stimulant of B cells and macrophages. LPS induces B cell proliferation and differentiation into antibody secreting cells. In addition, LPS also stimulates IL-6 secretion in mature B cells and in immature B cell lines such as WEHI-231. Although sufficient literature is available on LPS induced signaling events in monocytes and macrophages, the mechanisms involved in LPS induced B cell activation are not well understood. In this report, it is shown that both LPS mediated B cell proliferation and IL-6 secretion are dependent on phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathways. The B cell specific co-receptor, CD19 is not tyrosine phosphorylated in LPS stimulated B cells. Thus, in contrast to B cell antigen receptor (BCR) signaling, the activation of PI 3-kinase appears not to be related to the recruitment of PI 3-kinase to tyrosine phosphorylated CD19. This is the first demonstration of the importance of PI 3-kinase signaling pathway in LPS mediated B lymphocyte activation.     

Syk mediates BCR- and CD40-signaling integration during B cell activation

CD40 is essential for optimal B cell activation. It has been shown that CD40 stimulation can augment BCR-induced B cell responses, but the molecular mechanism(s) by which CD40 regulates BCR signaling is poorly understood. In this report, we attempted to characterize the signaling synergy between BCR- and CD40-mediated pathways during B cell activation. We found that spleen tyrosine kinase (Syk) is involved in CD40 signaling, and is synergistically activated in B cells in response to BCR/CD40 costimulation. CD40 stimulation alone also activates B cell linker (BLNK), Bruton tyrosine kinase (Btk), and Vav-2 downstream of Syk, and significantly enhances BCR-induced formation of complex consisting of, Vav-2, Btk, BLNK, and phospholipase C-gamma2 (PLC-γ2) leading to activation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase, Akt, and NF-κB required for optimal B cell activation. Therefore, our data suggest that CD40 can strengthen BCR-signaling pathway and quantitatively modify BCR signaling during B cell activation.     

Potential role of CARMA1 in CD40-induced splenic B cell proliferation and marginal zone B cell maturation

NF-kappaB activation through B cell receptor (BCR) ligation is critical for B cell development, survival and antigen-mediated activation of B cells. CARD domain and MAGUK-domain containing protein-1 (CARMA1), recently identified adaptor molecule, has been shown to play an essential role in BCR-induced NF-kappaB activation. CARMA1-deficient B cells fail to proliferate upon BCR stimulation, leading to defective humoral responses. Surprisingly, CARMA1-deficient B cells are also defective in CD40-induced proliferation. The mechanisms responsible for CD40-induced proliferation defect have not yet been characterized. In this study, we show that signaling cascades activated by CD40 stimulation are largely unaffected in CARMA1-deficient B cells. Instead, we have found that the defective proliferation of CARMA1-deficient B cells is due to two events. First, CARMA1-deficient B cells show defective cell-cycle progression. Secondly, the numbers of marginal zone (MZ) B cells, which are the main responders upon CD40 stimulation, are greatly diminished in CARMA1-deficient mice. Since B cell maturation requires basal signaling through BCR and NF-kappaB activation, we propose that impaired BCR signaling in CARMA1-deficient mice leads to defective maturation of MZ B cell population, which in turn, contributes to impaired proliferation upon CD40 stimulation.     

The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.     

The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.     

Effect of cross-tolerance between endotoxin and TNF-alpha or IL-1beta on cellular signaling and mediator production.

Endotoxin [lipopolysaccharide (LPS)] tolerance suppresses macrophage/monocyte proinflammatory-mediator production. This phenomenon also confers cross-tolerance to other stimuli including tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta. Post-receptor convergence of signal transduction pathways might occur after LPS, IL-1beta, and TNF-alpha stimulation. Therefore, it was hypothesized that down-regulation of common signaling molecules induces cross-tolerance among these stimuli. LPS tolerance and cross-tolerance were examined in THP-1 cells. Phosphorylation of MAP kinases and degradation of inhibitor kappaBalpha (IkappaBalpha) DNA binding of nuclear factor-kappaB (NF-kappaB), and mediator production were examined. In naive cells, LPS, TNF-alpha, and IL-1beta induced IkappaBalpha degradation, kinase phosphorylation, and NF-kappaB DNA binding. LPS stimulation induced production of TNF-alpha or TxB2 and degradation of IRAK. However, neither TNF-alpha nor IL-1beta induced IRAK degradation or stimulated TNF-alpha or TxB2 production in naive cells. Pretreatment with each stimulus induced homologous tolerance to restimulation with the same agonist. LPS tolerance also suppressed LPS-induced TxB2 and TNF-alpha production. LPS pretreatment induced cross-tolerance to TNF-alpha or IL-1beta stimulation. Pretreatment with TNF-alpha induced cross-tolerance to LPS-induced signaling events and TxB2 production. Although pretreatment with IL-1beta did not induce cross-tolerance to LPS-induced signaling events, it strongly inhibited LPS TNF-alpha and TxB2 production. These data demonstrate that IL-1beta induces cross-tolerance to LPS-induced mediator production without suppressing LPS-induced signaling to MAP kinases or NF-kappaB activation.     

PI3K and Btk differentially regulate B cell antigen receptor-mediated signal transduction

Phosphoinositide-3 kinase (PI3K) is thought to activate the tyrosine kinase Btk. However, through analysis of PI3K-/- and Btk-/- mice, B cell antigen receptor (BCR)-induced activation of Btk in mouse B cells was found to be unaffected by PI3K inhibitors or by a lack of PI3K. Consistent with this observation, PI3K-/- Btk-/- double-deficient mice had more severe defects than either single-mutant mouse. NF-kappaB activation along with Bcl-xL and cyclin D2 induction were severely blocked in both PI3K-/- and Btk-/- single-deficient B cells. Transgenic expression of Bcl-xL restored the development and BCR-induced proliferation of B cells in PI3K-/- mice. Our results indicate that PI3K and Btk have unique roles in proximal BCR signaling and that they have a common target further downstream in the activation of NF-kappaB.     

Ligation of the T cell co-stimulatory receptor CD28 activates the serine-threonine protein kinase protein kinase B

The intracellular signaling pathways activated upon ligation of the co-stimulatory receptor CD28 remain relatively ill-defined, although CD28 ligation does result in the strong association with, and activation of, phosphatidylinositol (PI) 3-kinase. The downstream effector targets of the CD28-activated PI 3-kinase-dependent signaling pathway remain poorly defined, but recent evidence from other systems has shown that Akt/protein kinase B (PKB) is a major target of PI 3-kinase and have indicated that a major function of PKB is the regulation of cell survival events. Given the strong coupling of CD28 to PI 3-kinase and the known protective effects of both CD28 and PI 3-kinase against apoptosis in different cell models, we investigated the effects of CD28 on PKB activation. We demonstrate that ligation of CD28 by either anti-CD28 monoclonal antibodies or the natural ligand B7.1, results in the marked activation of PKB in both the leukemic T cell line Jurkat and freshly isolated human peripheral blood-derived normal T lymphocytes. Our data suggest therefore, that PKB may be an important intracellular signal involved in CD28 signal transduction and demonstrate CD28 coupling to downstream elements of a signaling cascade known to promote cell survival.     

Differences in CXCR4-mediated signaling in B cells

Among all chemokine receptors CXCR4 possesses a unique response profile and distinguishes itself through a prolonged signaling capacity. Here, we investigated the signaling capacity of CXCR4 to its so far known unique ligand CXCL12 in B cell lines and primary CD19(+) B lymphocytes. During lymphopoiesis, CXCR4 is continuously expressed on the surface of B cells. However, its signaling profile changes inasmuch preB and proB cells migrate towards CXCL12, mobilize intracellular calcium and activate the small GTPases Rac1 and Cdc42, whereas mature B cells do not show these responses, albeit the cells retain the capability to migrate in response to CXCL13 and CCL21. By contrast, stimulation of B cells with CXCL12 at all stages of development results in the activation of the MAP-kinase cascade and in rapid CXCR4 internalization. The pathways leading to ERK1/2 activation are different in preB and mature B cell lines. In either case, ERK1/2 activation is pertussis toxin sensitive, but only in mature B-cells inhibition of PI3-kinase causes an almost complete block of ERK1/2 activation. Taken together, the results show that CXCR4 changes its coupling to downstream signal-transduction pathways in B cells, suggesting that receptor activity may depend on accessory proteins.     

Beta2-adrenergic receptor regulates Toll-like receptor-4-induced nuclear factor-kappaB activation through beta-arrestin 2

Toll-like receptors (TLRs) play an important role in innate immunity while, beta(2)-adrenergic receptors (beta(2)AR) provide the key linkages for the sympathetic nervous system to regulate the immune system. However, their role in macrophages remains uncertain. Here, we demonstrate the cross-talk between beta(2)AR and TLR signalling pathways. Expression of beta(2)AR was down-regulated by TLR4 ligand lipopolysaccharide (LPS) stimulation. To investigate the physiological consequence of this down-regulation RAW264 cells, a macrophage cell line, were transfected with a beta(2)AR expression vector (RAWar). Both LPS-stimulated inducible nitric oxide synthase (NOS II) expression and NO production were markedly suppressed in the RAWar cells. The activation of nuclear factor-kappaB (NF-kappaB) and degradation of the inhibitor of NF-kappaB (IkappaBalpha) in response to LPS were markedly decreased in these cells. The level of beta-arrestin 2, which regulates beta(2)AR signalling, was also reduced in RAW264 cells after stimulation with LPS, but not in RAWar cells. Overexpression of beta-arrestin 2 (RAWarr2) also inhibited NO production and NOS II expression. Furthermore, we demonstrated that beta-arrestin 2 interacted with cytosolic IkappaBalpha and that the level of IkappaBalpha coimmunoprecipitated by anti-beta-arrestin 2 antibodies was decreased in the RAW264 cells but not in RAWar or RAWarr2 cells. These findings suggest that LPS-stimulated signals suppress beta(2)AR expression, leading to down-regulation of beta-arrestin 2 expression, which stabilizes cytosolic IkappaBalpha and inhibits the NF-kappaB activation essential for NOS II expression, probably to ensure rapid and sufficient production of NO in response to microbial attack.     

Inhibition of IKK down-regulates antigen + IgE-induced TNF production by mast cells: a role for the IKK-IkappaB-NF-kappaB pathway in IgE-dependent mast cell activation

Mast cells (MC) are major effector cells for allergic diseases. Cross-linking of immunoglobulin E (IgE) and its high-affinity receptor, FcepsilonRI, by antigen initiates a cascade of signaling events leading to nuclear factor (NF)-kappaB activation and tumor necrosis factor (TNF) production. Here, we demonstrated that inhibition of inhibitor of kappaB (IkappaB) kinase (IKK) by a peptide IKK inhibitor or by four individual chemical IKK inhibitors including 15-deoxy-prostaglandin J(2), BMS-345541, SC-514, or sulindac significantly blocked IgE + trinitrophenyl (TNP)-induced TNF production by mouse bone marrow-derived MC (BMMC). Moreover, IgE + TNP induced a rapid phosphorylation of IKKalpha but not IKKbeta in BMMC. IgE + TNP-induced phosphorylation of IKKalpha was accompanied with phosphorylation and degradation of IkappaBalpha, subsequent NF-kappaB activation, and TNF production. Inhibition of IKK by sulindac decreased IKKalpha phosphorylation, IkappaBalpha phosphorylation and degradation, NF-kappaB activation, and TNF production by BMMC. It is interesting that IgE + TNP stimulation also induced a prominent synthesis of IKKalpha and IkappaBalpha. Inhibition of NF-kappaB activity by pyrrolidine dithiocarbomate (PDTC) blocked IgE + TNP-induced IkappaBalpha synthesis. NF-kappaB activity and TNF production were also inhibited when PDTC was used even after IgE + TNP stimulation, suggesting a potential role for the newly synthesized IkappaBalpha in MC activation. In addition, IgE + TNP-induced IKKalpha and IkappaBalpha phosphorylation was inhibited by a protein kinase C (PKC) inhibitor Ro 31-8220. Taken together, our results support a role for the IKK-IkappaB-NF-kappaB pathway, which likely involves PKC in IgE-dependent TNF production by MC. Thus, IKK may serve as a new target for the regulation of MC function in allergy.