Role of apoptosis signal-regulating kinase 1 in complement-mediated glomerular epithelial cell injury

In the rat passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 activates protein kinases in glomerular epithelial cells (GEC), and induces sublethal GEC injury and proteinuria. Complement induces production of reactive oxygen species (ROS) via the NAPDH oxidase, and stimulates phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase in a ROS-dependent manner. In the present study, we demonstrate that apoptosis signal-regulating kinase 1 (ASK1) was activated in glomeruli of rats with PHN, and that incubation of GEC in culture with antibody and sublytic C5b-9 stimulated ASK1 activity. The latter was, in part, mediated via the NADPH oxidase and ROS. Sublytic complement induced JNK and p38 phosphorylation, which was amplified in GEC that stably overexpress ASK1, as compared with Neo (control) GEC. Complement-induced lysis was enhanced in GEC that overexpress ASK1, as compared with Neo, and was attenuated in GEC that overexpress a dominant negative ASK1 mutant. Inhibition of p38, but not JNK, attenuated complement lysis in GEC that overexpress ASK1, but not in Neo GEC. In Neo GEC, generation of ROS restricted complement-mediated GEC injury but the protective effect of ROS was lost when ASK1 was overexpressed. We propose that the level of ASK1 expression determines the functional effect of p38 activation, i.e. when ASK1 is overexpressed, p38 activation is amplified, and C5b-9 assembly leads to GEC injury via ASK1 and p38. The present study thus defines a novel role for ASK1 as a mediator of C5b-9-dependent cell injury.     

Prologation of c-Jun N-terminal kinase is associated with cell death induced by tumor necrosis factor alpha in human chondrocytes

The aim of this study was to elucidate the role of JNK signaling pathway involved in tumor necrosis factor-alpha (TNF-alpha)-induced death of chondrocytes. Primary chondrocyte cultures were obtained from human knee osteoarthritis cartilages. First passage chondrocytes were treated with TNF-alpha and various potentiators, and cell death was measured with MTT assay. C-Jun N terminal kinase (JNK) activation was investigated with the solid phase kinase assay. Expression of apoptosis-related molecule was assayed with Western blot. Chondrocytes were resistant to TNF-alpha-induced cell death. In contrast, pretreatment with actinomycin D, the phosphatase inhibitor vanadate or MAP kinase phosphatase-1 (MKP-1) inhibitor Ro318220 invariably led to chondrocyte death. While TNF-alpha alone stimulated a single, brief JNK activity, a second JNK peak was observed when the cells were pretreated with actinomycin D. When the cells were pretreated with vanadate or Ro318220, TNF-alpha-induced JNK activation was greatly prolonged, which was associated with the induction of cell death. The expression of Bcl-2 and Mcl-1 decreased significantly in conditions of cell death. In conclusions, our data suggest that chondrocyte death induced by TNF-alpha is associated with sustained JNK activation. This effect may be due to downregulation of TNF-alpha induced phosphatase that inactivates JNK and of Bcl-2 family proteins.     

TAK1-binding protein 2 facilitates ubiquitination of TRAF6 and assembly of TRAF6 with IKK in the IL-1 signaling pathway

TAK1 mitogen-activated protein kinase kinase kinase participates in the Interleukin-1 (IL-1) signaling pathway by mediating activation of JNK, p38, and NF-kappaB. TAK1-binding protein 2 (TAB2) was previously identified as an adaptor that links TAK1 to an upstream signaling intermediate, tumor necrosis factor receptor-associated factor 6 (TRAF6). Recently, ubiquitination of TRAF6 was shown to play an essential role in the activation of TAK1. However, the mechanism by which IL-1 induces TRAF6 ubiquitination remains to be elucidated. Here we report that TAB2 functions to facilitate TRAF6 ubiquitination and thereby mediates IL-1-induced cellular events. A conserved ubiquitin binding domain in TAB2, the CUE domain, is important for this function. We also found that TAB2 promotes the assembly of TRAF6 with a downstream kinase, IkappaB kinase (IKK). These results show that TAB2 acts as a multifunctional signaling molecule, facilitating both IL-1-dependent TRAF6 ubiquitination and assembly of the IL-1 signaling complex.     

Effects of TNF-alpha on expression of ICAM-1 in human airway epithelial cells in vitro. Signaling pathways controlling surface and gene expression

Signaling pathways associated with tumor necrosis factor (TNF)-alpha-induced intercellular adhesion molecule 1 (ICAM-1) surface and gene expression were investigated in well differentiated normal human bronchial epithelial (NHBE) cells in air-liquid interface primary culture. Cells were exposed to human recombinant TNF-alpha (hrTNF-alpha; 0.015 to 150 ng/ml [specific activity, 2.86 x 10(7) U/mg]). TNF-alpha enhanced ICAM-1 surface expression (measured by flow cytometry) and steady-state messenger RNA (mRNA) levels (assessed by Northern hybridization) in concentration- and time-dependent manners. TNF-alpha-induced ICAM-1 surface and gene expression were both blocked by the RNA polymerase II inhibitor actinomycin D (0.1 microg/ml), and surface expression was attenuated by a neutralizing monoclonal antibody directed against the TNF-alpha receptor p55 (TNF-RI). The intracellular signaling pathway leading to enhanced expression appeared to involve activation of a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC) because D609, a specific PC-PLC inhibitor, attenuated TNF-alpha-induced increases in production of diacyl-glycerol (DAG), a hydrolysis product of PC-PLC, and also attenuated TNF-alpha enhancement of ICAM-1 surface and gene expression. Because DAG formed by action of PC-PLC can activate protein kinase C (PKC), involvement of PKC was investigated. The specific PKC inhibitor calphostin C blocked both surface and gene expression of ICAM-1 in response to TNF-alpha in a concentration-dependent manner. Finally, TNF-alpha stimulated binding of p65 and/or c-rel complexes to the nuclear factor (NF)-kappaB consensus binding site found on the ICAM-1 promoter, and binding of these complexes was inhibited by D609. The results support the following pathway, whereby TNF-alpha enhances expression of ICAM-1 in NHBE cells: TNF-alpha --> TNF-RI --> PC-PLC --> DAG --> PKC --> (NF-kappaB?) --> ICAM-1 mRNA --> ICAM-1 surface expression.     

Lipid rafts and oxidative stress-induced cell death

Reactive oxygen species (ROS) are generated in response to a number of physiologic or pathologic conditions. In addition to ROS produced extrinsically, a cell may produce ROS as a result of normal metabolism and signaling processes. When sufficient quantities of ROS are present within the cell, this oxidative stress may have profound effects on the cell, including the induction of cell death. Various signaling pathways are initiated in response to oxidative stress, through which the cell's demise is assured. Many of these signaling pathways involve cholesterol-enriched domains of the cell membrane known as lipid rafts. These lipid rafts are platforms for initiation or transduction of the signal and may modulate protein activity through a direct change in local membrane structure or by allowing protein-protein interactions to occur with higher affinity/specificity or both. Among the examples discussed in this review are death-receptor signaling, induction of membrane-associated tyrosine kinase activation, and activation of transient receptor protein (TRP) channels. Special attention also is given to the RIP1/TRAF2 pathway, which involves the downstream activation of the stress-activated protein kinase JNK. The activation of the JNK pathway plays a key role in the induction of cellular death in response to ROS.     

TRAF6 distinctively mediates MyD88- and IRAK-1-induced activation of NF-kappaB

MyD88 and IL-1R-associated kinase 1 (IRAK-1) play crucial roles as adaptor molecules in signal transduction of the TLR/IL-1R superfamily, and it is known that expression of these proteins leads to the activation of NF-kappaB in a TNFR-associated factor 6 (TRAF6)-dependent manner. We found in this study, however, that a dominant-negative mutant of TRAF6, lacking the N-terminal RING and zinc-finger domain, did not inhibit IRAK-1-induced activation of NF-kappaB in human embryonic kidney 293 cells, although the TRAF6 mutant strongly suppressed the MyD88-induced activation. The dominant-negative mutant of TRAF6 did not affect the IRAK-1-induced activation, regardless of the expression level of IRAK-1. In contrast, small interfering RNA silencing of TRAF6 expression inhibited MyD88-induced and IRAK-1-induced activation, and supplementation with the TRAF6 dominant-negative mutant did not restore the IRAK-1-induced activation. Expression of IRAK-1, but not MyD88, induced the oligomerization of TRAF6, and IRAK-1 and the TRAF6 dominant-negative mutant were associated with TRAF6. These results indicate that TRAF6 is involved but with different mechanisms in MyD88-induced and IRAK-induced activation of NF-kappaB and suggest that TRAF6 uses a distinctive mechanism to activate NF-kappaB depending on signals.     

NADPH oxidases in the gastrointestinal tract: a potential role of Nox1 in innate immune response and carcinogenesis

The gastrointestinal epithelium functions as physical and innate immune barriers against commensal or pathogenic microbes. NADPH oxidase 1 (Nox1) and dual oxidase 2 (Duox2), highly expressed in the colon, are suggested to play a potential role in host defense. Guinea-pig gastric pit cells and human colonic epithelial cells (T84 cells) express Nox1. With regard to activation of Nox1, the gastric epithelial cells are primed with Helicobacter pylori lipopolysaccharide, whereas T84 cells preferentially use the Toll-like receptor (TLR) 5, rather than TLR4, against Salmonella enteritidis infection. Thus, gastric and colonic epithelial cells may use different TLR members to discern pathogenicities among bacteria, depending on their environments and to activate Nox1 appropriately for host defense. Nox1-derived reactive oxygen species (ROS) have been implicated in the pathogenesis of inflammation-associated tumor development. The human stomach does not express Nox1. Helicobacter pylori infection alone does not induce it, whereas Nox1 is specifically expressed in gastric adenocarcinomas. In the human colon, Nox1 is differentiation-dependently expressed, and its expression is upregulated in adenomas and well-differentiated adenocarcinomas. Although Nox1 expression may not be directly linked to mitogenic activity, Nox1-derived ROS may exert a cancer-promoting effect by increasing resistance to programmed cell death of tumor cells.     

Rapid CD40-mediated rescue from CD95-induced apoptosis requires TNFR-associated factor-6 and PI3K

The activation molecule CD40 and the death receptor CD95/Fas play important roles in regulating B cells so that effective antimicrobial immunity occurs without autoimmunity. CD40 signaling increases CD95 expression, sensitizing cells to apoptosis, but sustained CD40 signals rescue B cells from CD95 killing. Here we describe a mechanism of early CD40-mediated rescue from CD95-induced apoptosis in B cells. Maximal rescue was achieved when CD40 signals were given within 1-2 h of initiating CD95 apoptosis. CD40 signaling did not block association of Fas-associated death domain-containing protein with CD95, but decreased CD95-induced activation of caspases 3 and 8. Rapid CD40 rescue did not require NF-kappaB activation and was independent of de novo protein synthesis, but was dependent upon active PI3 K. Signaling via a CD40 mutant that does not bind TNFR-associated factor (TRAF)1, TRAF2, and TRAF3 rescued B cells from CD95-induced apoptosis. TRAF1/2/3-independent rescue was confirmed in B cell lines made deficient in these TRAF molecules by gene targeting. In contrast, CD40 rescue was completely abrogated in TRAF6-deficient B cells, which showed reduced activation of Akt in response to CD40 engagement. These results reveal a new rapid mechanism to balance B cell activation and apoptosis.     

The A20 protein interacts with the Epstein-Barr virus latent membrane protein 1 (LMP1) and alters the LMP1/TRAF1/TRADD complex.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) interacts with the tumor necrosis factor receptor (TNFR)-associated factor (TRAF) molecules, which are important for LMP1-mediated signaling. Two domains of LMP1 can independently activate NF-kB, carboxyl-terminal activating region 1 (CTAR1) and CTAR2. The activation of NF-kB by CTAR1 occurs through direct interaction of LMP1 with the TRAF molecules, whereas CTAR2 interacts with the TNFR-associated death domain protein (TRADD) to activate NF-kB and the c-Jun N-terminal kinase (JNK). A20, which is induced by LMP1 through NF-kB, can block NF-kB activation from both domains of LMP1 and inhibit JNK activation from CTAR2. A20 also has been shown to associate with TRAF1 and TRAF2. In this study, an interaction between LMP1 and A20 was detected that was increased by TRAF2 overexpression. A20 did not affect the association of TRAF1 with TRAF2 but did displace TRAF1 from the LMP1 complex. The interaction of LMP1 and TRADD was decreased in the presence of A20, and the LMP1-A20 association was decreased by TRADD, suggesting that A20 and TRADD both interact with LMP1 and may compete for binding. These data indicate that A20 alters the interactions between LMP1 and the TRAF molecules and TRADD, affecting the activation of NF-kB, JNK, and perhaps other TRAF-mediated signaling events.     

Sensitivity of TLR4- and -7-induced NF kappa B1 p105-TPL2-ERK pathway to TNF-receptor-associated-factor-6 revealed by RNAi in mouse macrophages

Tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF6) is an adaptor protein involved in Toll-like receptor (TLR) signaling. Recent studies using macrophages from TRAF6 knockout mice have revealed that TRAF6 is required for TLR7 signaling. However, an essential role of TRAF6 in TLR4 signaling and cytokine production is slightly controversial. Using an RNAi approach to reduce the cellular levels of TRAF6, we tested the role of this adaptor protein on the sensitivity of the various components of the ERK pathway mediated by TLR4 and -7 in Raw264.7, a mouse macrophage cell line. ERK activation in macrophages by TLR4 and -7 is mediated via a MAP3K, called TPL2/COT, which under unstimulated conditions is associated with NF kappa B1 p105, a member of the I kappa B family of proteins. Upon stimulation with TLR ligands, p105 is phosphorylated by I kappa B kinase (IKK) complex and partially degraded, which releases TPL2. The free TPL2 is active and stimulates the ERK pathway via MEK1/2. The free TPL2, however, is also unstable and is targeted for degradation. We demonstrate here that reduced level of TRAF6 ( approximately 80% decrease) in macrophages does not significantly affect any of the components of the TLR4-stimulated ERK pathway, including p105 phosphorylation, TPL2 degradation and ERK1/2 phosphorylation. Surprisingly, however, TLR4-induced JNK1/2 phosphorylation is significantly blocked by TRAF6 knockdown, suggesting that ERK and JNK pathways are differentially sensitive to TRAF6 levels. Furthermore, although TLR4-mediated IKK-induced p105 phosphorylation is not sensitive to TRAF6 knockdown, I kappa B alpha phosphorylation (also, IKK-induced) is significantly blocked, suggesting that TLR4 activation results in a TRAF6-sensitive and -insensitive IKK activation in macrophages. In contrast to TLR4 signaling, TLR7 activation of ERK, JNK pathways and phosphorylation of p105 and I kappa B alpha are completely inhibited in TRAF6 knockdown cells. Compared to the signaling data, while TLR4-induced TNFalpha mRNA expression is not significantly inhibited by TRAF6 knockdown, TLR7-induced TNFalpha mRNA is significantly blocked. In contrast, both TLR4- and TLR7-induced IL6 mRNA are significantly blocked by TRAF6 knockdown. These results suggest that while TRAF6 is absolutely essential for TLR7 activation of ERK, JNK and NF kappa B pathways, TLR4-induced ERK, JNK pathways and IKK-mediated phosphorylation of I kappa B family members as well as cytokine expression are differentially sensitive to the cellular levels of TRAF6. These results have important implications in terms of therapeutic targeting of TRAF6 complexes in diseases where TLR4 and -7 are involved.     

Differential activation and regulation of mitogen-activated protein kinases through the antigen receptor and CD40 in human B cells.

In human B cells, antigen receptor ligation and CD40 ligation are known to activate the extracellular-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) pathways, which in turn regulate many important B cell functions. We previously reported that antigen receptor ligation activated the ERK pathway whereas CD40 ligation activated the JNK/stress-activated protein kinase (SAPK) pathway. Here, we demonstrate that another SAPK, p38/Hog1, is activated by both antigen receptor ligation or CD40 ligation in a human B-lymphoblastoid cell line and tonsillar B cells. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, partially inhibited ERK2 and p38 activation triggered through the B cell receptor whereas activation of JNK1 and p38 through CD40 was not affected. PD98059, a specific inhibitor of mitogen-activated extracellular response kinase kinase (MEK), significantly inhibited ERK2 activation and partially inhibited p38 activation triggered by anti-IgM antibody treatment, but did not affect CD40-dependent signaling events. In addition, anti-IgM antibody-induced signaling pathways were shown to be PKC-dependent in contrast to the CD40-induced signaling pathways. Thus, the B cell receptor and CD40 recruit the ERK, JNK and p38 pathways by using different upstream effectors.     

TRAF2 differentially regulates the canonical and noncanonical pathways of NF-kappaB activation in mature B cells

To examine the role of the TNF-R superfamily signaling protein TRAF2 in mature B cell development and NF-kappaB activation, conditionally TRAF2-deficient mice were produced. B cells lacking TRAF2 expression in these mice possessed a selective survival advantage, accumulated in the lymph nodes and splenic marginal zone, were larger in size, and expressed increased levels of CD21/35. These TRAF2-deficient B cells could not proliferate or activate the canonical NF-kappaB pathway in response to CD40 ligation. By contrast, noncanonical NF-kappaB activation was constitutively hyperactive, with TRAF2-deficient B cells exhibiting close to maximal processing of NF-kappaB2 from p100 to p52 and high levels of constitutive p52 and RelB DNA binding activity. These findings establish TRAF2 as a multifunctional regulator of NF-kappaB activation that mediates activation of the canonical pathway but acts as a negative regulator of the noncanonical pathway. This dual functionality explains the contrasting roles of TRAF2 in B cell maturation and activation.     

Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling

The adaptor and signaling proteins TRAF2, TRAF3, cIAP1 and cIAP2 may inhibit alternative nuclear factor-kappaB (NF-kappaB) signaling in resting cells by targeting NF-kappaB-inducing kinase (NIK) for ubiquitin-dependent degradation, thus preventing processing of the NF-kappaB2 precursor protein p100 to release p52. However, the respective functions of TRAF2 and TRAF3 in NIK degradation and activation of alternative NF-kappaB signaling have remained elusive. We now show that CD40 or BAFF receptor activation result in TRAF3 degradation in a cIAP1-cIAP2- and TRAF2-dependent way owing to enhanced cIAP1, cIAP2 TRAF3-directed ubiquitin ligase activity. Receptor-induced activation of cIAP1 and cIAP2 correlated with their K63-linked ubiquitination by TRAF2. Degradation of TRAF3 prevented association of NIK with the cIAP1-cIAP2-TRAF2 ubiquitin ligase complex, which resulted in NIK stabilization and NF-kappaB2-p100 processing. Constitutive activation of this pathway causes perinatal lethality and lymphoid defects.     

Distinct contributions of different CD40 TRAF binding sites to CD154-induced dendritic cell maturation and IL-12 secretion

The mechanisms by which CD40 controls the maturation and antigen presentation functions of dendritic cells (DC) remains largely undefined in this critical cell type. To examine this question, we have employed retroviral transduction of primary bone marrow-derived mouse DC. Mutation of the distinct binding sites for TNF receptor-associated factor 6 (TRAF6) and for TRAF 2, 3, and 5 in the CD40 cytoplasmic domain revealed their independent contributions to DC maturation and activation of NF-kappaB. In contrast, disruption of the TRAF6 but not the TRAF 2,3,5 binding site markedly decreased IL-12 p40 secretion along with p38 and JNK activation in response to CD154 stimulation. These data document a clear bifurcation of the CD40 signaling cascade in primary DC at the level of the receptor's two distinct and autonomous TRAF binding sites, and reveal the predominant role of the TRAF6 binding site in CD40-induced pro-inflammatory cytokine production by these cells.