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Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER overload, resulting in ER stress. To cope with ER stress, mammalian cells trigger a specific response known as the unfolded protein response (UPR). Although recent studies have indicated cross-talk between ER stress and oxidative stress, the mechanistic link is not fully understood. By using murine fibrosarcoma L929 cells, in which tumor necrosis factor (TNF) alpha induces accumulation of reactive oxygen species (ROS) and cell death, we show that TNFalpha induces the UPR in a ROS-dependent fashion. In contrast to TNFalpha, oxidative stresses by H2O2 or arsenite only induce eukaroytic initiation factor 2alpha phosphorylation, but not activation of PERK- or IRE1-dependent pathways, indicating the specificity of downstream signaling induced by various oxidative stresses. Conversely, the UPR induced by tunicamycin substantially suppresses TNFalpha-induced ROS accumulation and cell death by inhibiting reduction of cellular glutathione levels. Collectively, some, but not all, oxidative stresses induce the UPR, and pre-emptive UPR counteracts TNFalpha-induced ROS accumulation.

Although it is well established that RhoA signaling pathways play key roles in regulating neuronal morphology, their involvement in other aspects of neuronal function has received little attention. Recent studies have elucidated a novel intracellular signaling pathway used by RhoA to elicit activation of serum response factor (SRF)-mediated transcription. In this pathway, activation of RhoA triggers nuclear translocation of the SRF co-activator, megakaryocytic acute leukemia (MAL). In assessing whether RhoA regulates transcription in neurons via this pathway, we have found that a constitutively active form of Tech (transcript-enriched in cortex and hippocampus), a RhoA guanine nucleotide exchange factor (GEF) that is expressed in forebrain neurons, stimulates SRF reporter activity in extracts of primary cortical cultures and induces nuclear translocation of MAL in cortical neurons. Both of these responses appear to be mediated by Tech's activation of RhoA as they are not mimicked by a mutant Tech construct lacking RhoA GEF activity and are blocked by C3 transferase, a selective inhibitor of RhoA. Furthermore, Tech-induced increases in SRF activity are suppressed by a dominant negative MAL construct. These findings demonstrate that RhoA signaling pathways are able to regulate transcription in neurons by triggering translocation of the SRF co-activator MAL.

Activation of c-Jun N-terminal kinase (JNK) promotes apoptosis in a context-specific fashion. The anti-apoptotic function of NF-κB is mediated in part through its ability to downregulate JNK activation. Candidate molecules, including GADD45β (growth arrest and DNA damage-inducing protein β), XIAP (X-chromosome-linked inhibitor of apoptosis), and reactive oxygen species have recently been reported to be involved in crosstalk between NF-κB and JNK. Here, I discuss recent work from Papa et al. , which proposes that GADD45β modulates JNK activation by binding to, and inhibiting, the JNK kinase, mitogen-activated protein kinase kinase 7 (MKK7).

Reactive oxygen species connect nuclear factor-κB and c-Jun amino-terminal kinase

Full transcriptional activity of the nuclear, DNA-bound form of NF-κB requires additional posttranslational modifications. In this study, we systematically mapped the T cell costimulation-induced phosphorylation sites within the C-terminal half of the strongly trans-activating NF-κB p65 subunit and identified serine 536 as the main phosphorylation site. The transient kinetics of serine 536 phosphorylation paralleled the kinetics of IκBα and IκB kinase (IKK) phosphorylation and also mirrored the principle of T cell costimulation. The TCR-induced pathway leading to serine 536 phosphorylation is regulated by the kinases Cot (Tpl2), receptor interacting protein, protein kinase Cθ, and NF-κB-inducing kinase, but is independent from the phosphatidylinositol 3-kinase/Akt signaling pathway. Loss-of-function and gain-of-function experiments showed phosphorylation of p65 serine 536 by IKKβ, but not by IKKα. Phosphorylation occurs within the cytoplasmic and intact NF-κB/IκBα complex and requires prior phosphorylation of IκBα at serines 32 and 36. Reconstitution of p65−/− cells either with wild-type p65 or a p65 mutant containing a serine to alanine mutation revealed the importance of this phosphorylation site for cytosolic IκBα localization and the kinetics of p65 nuclear import.

The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-κB via the IκB kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-κB activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-κB by the dsRNA-dependent protein kinase., This investigation was supported by research grants BMC2002- 03246 and BIO2000-0340-P4 from Spain and QLK2-CT2002-00954 from the European Union to M.E., by grants SAF2000-0028 and FIPSE to J.A., and by the Fundación Ramón Areces (P.G.-P.). H.N. is supported by a grant from the Human Frontier Science Program. M.A.G. and J.G. were supported by fellowships from the Ministerio de Ciencia y Tecnología, Madrid, Spain.

The TNF receptor-associated factor (TRAF) family of molecules acts as adapter proteins for signaling pathways initiated by several members of the TNF receptor (TNFR) superfamily. TRAF5−/− animals are viable and have normal development of the immune system despite interacting with several TNFR family members. A clear role for TRAF5 has yet to emerge. OX40 (CD134) interacts with TRAF5, suggesting that this pathway could be involved in regulating T cell differentiation into Th1 or Th2 cells. In tissue culture, OX40 stimulation of TRAF5−/− T cells resulted in a pronounced Th2 phenotype with elevated levels of IL-4 and IL-5. Similarly, in vivo immunization with protein in adjuvant in the presence of an agonist anti-OX40 Ab resulted in enhanced Th2 development in TRAF5−/− mice. Additionally, lung inflammation induced by T cells, which is critically controlled by OX40, was more pronounced in TRAF5−/− mice, characterized by higher levels of Th2 cytokines. These results suggest that TRAF5 can limit the induction of Th2 responses, and that TRAF5 can play a role in modulating responses driven by OX40 costimulation.

Epstein-Barr virus latent membrane protein 1 (LMP1) activation of NF-kappaB is critical for Epstein-Barr virus-infected B lymphocyte survival. LMP1 activates the IkappaB kinase complex and NF-kappaB through two cytoplasmic signaling domains that engage tumor necrosis factor receptor-associated factor (TRAF)1/2/3/5 or TRADD and RIP. We now use cells lacking expression of TRAF2, TRAF5, TRAF6, IKKalpha, IKKbeta, IKKgamma, TAB2, IL-1 receptor-associated kinase (IRAK)1, or IRAK4 to assess their roles in LMP1-mediated NF-kappaB activation. LMP1-induced RelA nuclear translocation was similar in IKKalpha knockout (KO) and WT murine embryo fibroblasts (MEFs) but substantially deficient in IKKbeta KO MEFs. NF-kappaB-dependent promoter responses were also substantially deficient in IKKbeta KO MEFs but were hyperactive in IKKalpha KO MEFs. More surprisingly, NF-kappaB responses were near normal in TRAF2 and TRAF5 double-KO MEFs, IKKgamma KO MEFs, TAB2 KO MEFs, and IRAK4 KO MEFs but were highly deficient in TRAF6 KO MEFs and IRAK1 KO HEK293 cells. Consistent with the importance of TRAF6, LMP1-induced NF-kappaB activation in HEK293 cells was inhibited by expression of dominant-negative TAB2 and Ubc13 alleles. These data extend a role for IKKalpha in IKKbeta regulation, identify an unusual IKKbeta-dependent and IKKgamma-independent NF-kappaB activation, and indicate that IRAK1 and TRAF6 are essential for LMP1-induced NF-kappaB activation.

NF-kappaB downregulates tumor necrosis factor (TNF)-induced c-Jun N-terminal kinase (JNK) activation that promotes cell death, but the mechanism is not yet fully understood. By using murine embryonic fibroblasts (MEFs) that are deficient in TNF receptor-associated factor (TRAF) 2 and TRAF5 (DKO) or p65 NF-kappaB subunit (p65KO), we demonstrate here that TNF stimulation leads to accumulation of reactive oxygen species (ROS), which is essential for prolonged mitogen-activated protein kinase (MAPK) activation and cell death. Interestingly, dying cells show necrotic as well as apoptotic morphological changes as assessed by electron microscopy and flow cytometry, and necrotic, but not apoptotic, cell death is substantially inhibited by antioxidant. Importantly, TNF does not induce ROS accumulation or prolonged MAPK activation in wild-type MEFs, indicating that TRAF-mediated NF-kappaB activation normally suppresses the TNF-induced ROS accumulation that subsequently induces prolonged MAPK activation and necrotic cell death

The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha-but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that has been shown to induce angiogenesis, apoptosis in tumor cells, and NF-kappaB activation through binding to its receptor, fibroblast growth factor-inducible 14. We have identified TWEAK as an inducer of constitutive NF-kappaB activation by expression cloning, and we report here sequential regulation by TWEAK of two separate signaling cascades for NF-kappaB activation, the NF-kappaB essential modulator-dependent and -independent signaling pathways. Upon TWEAK stimulation, IkappaBalpha is rapidly phosphorylated, generating NF-kappaB DNA-binding complexes containing p50 and RelA in a manner dependent on the canonical IkappaB kinase complex. Unlike TNF-alpha, TWEAK stimulation results in prolonged NF-kappaB activation with a transition of the DNA-binding NF-kappaB components from RelA- to RelB-containing complexes by 8 h, and the latter remained active in binding at least until 24 h post-stimulation. This long lasting activation is accompanied by the proteasome-mediated processing of NF-kappaB2/p100, which does not depend on the NF-kappaB essential modulator but requires IkappaB kinase 1 and functional NF-kappaB-inducing kinase activity. Finally, we show that fibroblast growth factor-inducible 14 with a mutation at its TNF receptor-associated factor (TRAF)-binding site cannot activate NF-kappaB and that TWEAK fails to induce the p100 processing and IkappaBalpha phosphorylation in cells deficient for TRAF2 and TRAF5. Our results thus identify TWEAK as a novel physiological regulator of the non-canonical pathway for NF-kappaB activation.

The transcription factor NF-kappaB is activated when cells are exposed to genotoxic stress. It has been suggested that DNA damage will trigger a cytoplasmic signaling that leads to the activation of IKK and NF-kappaB, but the signaling components upstream of IKK have not yet been identified. Here we report that the receptor interacting protein, RIP, is the IKK upstream component, essential for the activation of NF-kappaB by DNA damage. Also, our findings suggest that this NF-kappaB activation by DNA damage is not mediated by autocrine or TNF-R1 signaling pathway. In wild-type fibroblasts, DNA damage induced by agents such as adriamycin, campthothecin, and ionizing radiation induces NF-kappaB activation. We found, however, that DNA damage failed to activate NF-kappaB in RIP-/- fibroblasts. The induction of IkappaBalpha degradation by DNA damage was normal in TNF-R1-/-, TRAF2-/-, TRAF5-/- and FADD-/- fibroblasts or when de novo protein synthesis was blocked. More importantly, the reconstitution of RIP expression in RIP-/- cells restores DNA damage-induced NF-kappaB activation. We also found that RIP forms a complex with IKK in response to DNA damage. Therefore, our study provides a possible mechanism for the initiation of the cytoplasmic signaling to activate NF-kappaB in response to DNA damage.

Although TRAF6 is essential for both RANKL- and TNFalpha-induced osteoclastogenesis, it has remained unclear whether other members of the TRAF family are involved in osteoclastogenesis. We examined TRAF5 function in both RANKL- and TNFalpha-induced osteoclastogenesis by using osteoclast progenitor cells from TRAF5-deficient mice. The results demonstrated that RANKL or TNFalpha did not effectively induce osteoclast differentiation from osteoclast progenitor cells derived from these mice into mature multinucleated osteoclasts, although c-jun N-terminal kinase (JNK) and NF-kappaB activation was apparently observed in osteoclast progenitor cells. In the parathyroid hormone (PTH)-induced hypercalcemia model, calcium concentration peaked at day 3 after administration. However, in TRAF5-deficient mice, this peak was delayed and found at day 5, showing less effective osteoclast differentiation. Thus, we have provided the first evidence showing that TRAF5 is involved in osteoclastogenesis.

Lymphotoxin-beta receptor (LTbetaR) is a member of the tumor necrosis factor receptor (TNFR) superfamily that activates nuclear factor-kappaB (NF-kappaB) through the IkappaB kinase (IKK) complex, the core of which is comprised of IKK1, IKK2 and NF-kappaB essential modulator (NEMO). We demonstrate here that the LTbetaR signaling to NF-kappaB activation does not necessarily require NEMO, which is essential for TNFR signaling. In the absence of NEMO, the p50 and RelB, but not RelA subunits of NF-kappaB are found in the nuclear DNA binding complexes induced by the LTbetaR signaling. Our results thus disclose NEMO-independent NF-kappaB activation by LTbetaR.

TWEAK, a member of the TNF family, induces cell death in some tumor cell lines, but also induces proliferation of endothelial cells and angiogenesis. Recently, fibroblast growth factor-inducible 14 (Fn14) has been identified to be a TWEAK receptor, which may be responsible for the proliferation of endothelial cells and angiogenesis. In this study, we investigated the pro-inflammatory effect of TWEAK on human umbilical vein endothelial cells (HUVEC). We demonstrated that TWEAK could not only induce the proliferation and migration but also upregulate the cell surface expression of adhesion molecules such as ICAM-1 and E-selectin, and induce the secretion of chemokines such as IL-8 and MCP-1 in HUVEC. Moreover, by using an anti-Fn14 mAb that blocks the TWEAK/Fn14 interaction, we demonstrated that Fn14 was constitutively expressed on HUVEC and totally mediated the biological effects of TWEAK on HUVEC. These results indicated that TWEAK could induce pro-inflammatory reactions via Fn14 on HUVEC.

To investigate the role of FADD (Fas-associated protein with death domain) in Fas and tumor necrosis factor receptor 1 (TNFR1)-mediated hepatic injury and inflammatory response in vivo.Fas and TNFR1 are cell surface molecules that trigger apoptosis or inflammation on engagement by a specific ligand or antibody. FADD is recruited to the cytoplasmic domain of these receptors on their activation and works as a common mediator to induce apoptosis. It is known that a blockade of FADD can inhibit apoptosis mediated by Fas or TNFR1 in vitro. However, it is not known whether the blockade can prevent organ injury and whether the inflammatory cascade is affected in vivo.A FADD deletion mutant lacking the death effector domain was introduced into mice by transduction with an adenovirus vector, and the effect of this FADD dominant negative mutant was examined in several liver injury models.Hepatic injury induced by anti-Fas monoclonal antibody or tumor necrosis factor (TNF)-alpha plus D-galactosamine was markedly ameliorated by the FADD dominant negative transduction, which abrogated the death rate. Further, the FADD dominant negative transduction efficiently blocked T cell- mediated concanavalin A-induced hepatitis while not affecting TNF-alpha production or TNF-alpha-induced nuclear factor-kappaB activation in the liver.These results provide the basis for a novel therapeutic modality in which an unfavorable apoptotic process can be inhibited without affecting a favorable response for liver regeneration; this would be relevant to the clinical treatment of acute and chronic liver diseases as well as to some inflammatory disorders with hypercytokinemia, such as sepsis.

CD40 plays a critical role in survival, growth, differentiation, and class switching of B lymphocytes. Although Ku is required for immunoglobulin class switching, how CD40 signal transduction is coupled to Ku is still unknown. Here, we show that CD40 directly interacts with Ku through the membrane-proximal region of cytoplasmic CD40. Ku was confined to the cytoplasm in human primary B cells, and the engagement of CD40 on the B cells cultured in the presence of IL-4 resulted in the dissociation of Ku from CD40, translocation of Ku into the nucleus, and increase in the activity of DNA-dependent protein kinase. These findings indicate that Ku is involved in the CD40 signal transduction pathway and may play an important role in the CD40-mediated events.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28− T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.

CD27, a member of the tumor necrosis factor (TNF) receptor family, has been implicated in T cell activation, T cell development and T-dependent antibody production by B cells. Its ligand CD70 has been identified only in humans, and, thus, physiological and pathological roles of the CD70-CD27 interaction remain to be determined in an experimental animal system. In the present study, we identified murine (m) CD70 by molecular cloning, and characterized its expression and function by generating an anti-mCD70 mAb. The mCD70 cDNA encoded a type II transmembrane glycoprotein of the TNF family, having 56.5% identity to the human CD70 amino acid sequence. The mCd70 gene was assigned in the central region of chromosome 17. To explore the expression and function of mCD70, we generated cDNA transfectants and anti-mCD70 mAb (FR70), which inhibited binding of a murine CD27-Fc fusion protein (mCD27-Ig) to mCD70 transfectants. FR70, as well as mCD27-Ig, immunoprecipitated a 30-33 kDa surface protein from A20 and mCD70-P815 cells but not from P815 cells. The mCD70 transfectants exhibited a potent co-stimulatory activity for anti-CD3-stimulated T cell proliferation, which was blocked by FR70 far more efficiently than mCD27-Ig. FR70 also abrogated the CD28-independent co-stimulatory activity of A20 cells. The expression of mCD70 was detected on splenic T cells after stimulation with anti-CD3 and anti-CD28 mAb, and on splenic B cells after stimulation with anti-CD40 mAb. Cross-linking of surface Ig by anti-IgM mAb did not induce the mCD70 expression but enhanced the anti-CD40-induced mCD70 expression on splenic B cells. These results suggest a contribution of CD70 to murine T-B cognate interaction as proposed in the human system. FR70 will be useful for further investigating the physiological and pathological roles of the CD70-CD27 interaction in T cell development, T-dependent antibody production and various disease models in the murine system.

NF-kappaB is activated by various stimuli including inflammatory cytokines and stresses. A key step in the activation of NF-kappaB is the phosphorylation of its inhibitors, IkappaBs, by an IkappaB kinase (IKK) complex. Recently, two closely related kinases, designated IKKalpha and IKKbeta, have been identified to be the components of the IKK complex that phosphorylate critical serine residues of IkappaBs for degradation. A previously identified NF-kappaB-inducing kinase (NIK), which mediates NF-kappaB activation by TNFalpha and IL-1, has been demonstrated to activate IKKalpha. Previous studies showed that mitogen-activated protein kinase/ERK kinase kinase-1 (MEKK1), which constitutes the c-Jun N-terminal kinase/stress-activated protein kinase pathway, also activates NF-kappaB by an undefined mechanism. Here, we show that overexpression of MEKK1 preferentially stimulates the kinase activity of IKKbeta, which resulted in phosphorylation of IkappaBs. Moreover, a catalytically inactive mutant of IKKbeta blocked the MEKK1-induced NF-kappaB activation. By contrast, overexpression of NIK stimulates kinase activities of both IKKalpha and IKKbeta comparably, suggesting a qualitative difference between NIK- and MEKK1-mediated NF-kappaB activation pathways. Collectively, these results indicate that NIK and MEKK1 independently activate the IKK complex and that the kinase activities of IKKalpha and IKKbeta are differentially regulated by two upstream kinases, NIK and MEKK1, which are responsive to distinct stimuli.

Signals emanated from CD30 can activate the nuclear factor kappaB (NFkappaB). The two conserved subdomains, D1 and D2, in the C-terminal cytoplasmic region of CD30 were tested for interaction with two tumor necrosis factor receptor-associated factor (TRAF) proteins with NFkappaB activating capacity, TRAF2 and TRAF5. TRAF5 is the newest member of the TRAF family that binds to lymphotoxin beta receptor and CD40. TRAF5, as well as TRAF2, interacted with the D2 subdomain of CD30 in vitro and in vivo. Deletion analysis by the yeast two-hybrid system revealed that the C-terminal 22 and 30 amino acid residues are dispensable for interaction of TRAF5 and TRAF2 with CD30, respectively. Substitution of alanine for threonine at 463 abolished the interaction with TRAF2. Overexpression of the TRAF domain of TRAF2 or TRAF5 showed a dominant negative effect on CD30-mediated NFkappaB activation. Simultaneous expression of these TRAF domains further suppressed the NFkappaB activation, suggesting an interplay of these TRAF proteins. Expression of TRAF2 and TRAF5 mRNA was demonstrated in T- and B-cell lines that express CD30. Taken together, our results indicate that TRAF2 and TRAF5 directly interact with CD30 and are involved in NFkappaB activation by CD30 signaling.

Numerous studies have shown that nuclear factor‐κB (NF‐κB) inhibits various types of cell death by upregulating anti‐apoptotic genes, such as cellular FADD‐like interleukin‐1‐converting enzyme (FLICE)‐inhibitory protein ( c‐FLIP ), X‐chromosome‐linked inhibitor of apoptosis ( XIAP ), A1 (also known as Bfl‐1 ) and B‐cell leukaemia/lymphoma‐XL ( Bcl‐XL ). However, the biological role of c‐Jun amino‐terminal kinase (JNK) activation in cell death remains unclear (Karin & Lin, 2002). Generally, transient JNK activation promotes cell survival, whereas sustained JNK activation promotes cell death. Although, until recently, these signalling cascades were usually discussed independently, two reports have described intimate crosstalk between the JNK and NF‐κB signalling pathways (De Smaele et al , 2001; Tang et al , 2001). Tumour necrosis factor‐α (TNF‐α)‐induced activation of JNK is prolonged in cells that are deficient in NF‐κB activation, such as RelA‐ and IκB kinase‐β (IKKβ)‐knockout murine embryonic fibroblasts (MEFs). Consistent with previous findings (Chen et al , 1996), prolonged JNK activation in these cells promotes apoptosis. Together, these studies have shown that one of the anti‐apoptotic functions of NF‐κB is to downregulate JNK activation by upregulating growth arrest and DNA‐damage‐inducing protein‐β (GADD45‐β) or XIAP. A subsequent study reported that reactive oxygen species (ROS) have an essential role in mediating long‐lasting JNK activation and necrotic cell death in NF‐κB activation‐deficient cells (Sakon et al , 2003; Nakano, 2004). Given that enhanced ROS accumulation by TNF‐α is not observed in wild‐type cells (Sakon et al , 2003; Ventura et al , 2004), it seems that …

The relationship between the level of gamma delta T cells and cellular immunity of T lymphocytes was assessed in the peripheral blood of active sarcoidosis patients and healthy controls. We divided the active sarcoidosis patients into two groups: a group of patients with a normal distribution of circulating gamma delta T cells (group A; n = 11, less than 8.2% lymphocytes) and another group with increased gamma delta T cell levels (group B; n = 11, greater than 8.2% lymphocytes). The proportion and absolute count of CD4+ lymphocytes in group B (28.6 +/- 11.2%, 374.1 +/- 193.8/microliters) were remarkably smaller than control subjects (45.7 +/- 6.8%, 818.3 +/- 290.2/microliters, P < 0.001, P < 0.002, respectively). Group A, however, showed a moderate reduction in CD4+ lymphocytes when compared with controls. Serial measurements of T cell subtypes were performed on five patients in group B. gamma delta T cells and CD4+ lymphocytes were inversely correlated over the observation period which ranged from 2 to 18 months. When peripheral blood T cells were stimulated with PHA or PPD in vitro, the responses were weaker in group B compared with both group A and control subjects. These results suggest that the increase in circulating gamma delta T cells in sarcoidosis closely relates to a defect in cellular immunity which progresses during the disease process.

CD3 zeta and eta chains are components of the T cell antigen receptor (TCR) complex and are transcribed from a common gene by alternative splicing. TCR complexes containing the zeta eta dimer have been thought to mediate different functions than complexes containing the zeta 2 dimer. To analyze the role of eta in the development and function of T cells, we generated eta-deficient mice without affecting zeta by gene targeting in embryonic stem cells. Homozygous mutant embryos developed normally. Unexpectedly, however, these mice exhibited high mortality soon after birth for unknown reason(s). Analysis of surviving homozygous animals revealed that the development and function of T cells were normal in the absence of the eta chain. Recently, the zeta/eta locus was reported to encode a transcription factor, Oct-1, on the opposite DNA strand. Our targeting strategy resulted in modulation of Oct-1 transcription--reduction of the authentic Oct-1 mRNA and induction of aberrant transcripts. Although differences in tissue distribution and DNA binding capacity of Oct-1 between wild-type and eta-deficient mice were not evident from in situ hybridization and gel shift analysis, the high mortality in the eta-deficient strain may well be due to the disturbance of Oct-1 transcription by the mutation in the zeta/eta locus. Such possible complexities have to be taken into account in the interpretation of gene targeting experiments.

Recombinant proteins prepared by genetic engineering are utilized for various purposes in molecular biology such as production of specific antibodies or investigation of the mechanism of protein—DNA or protein-protein interactions (1). There are several methods commonly used to produce recombinant proteins in bacteria or insect cells (2). Expression of a recombinant protein in E. coli as a fusion protein with glutathione S-transferase (GST) is one of the most popular and easiest methods (3). However, we often encounter the serious problems that some fusion proteins are rapidly degraded or cannot be solubilized in bacterial cells. To prepare a polyclonal antibody against the 7 chain of the high affinity IgE receptor (FCCRIY), we constructed GST-FceRI-y, which was composed of the GST gene joined to the cytoplasmic portion of the FceRty cDNA. In initial experiments, we utilized two commonly used bacteria strains, DH5a and TGI, for production of the fusion protein. Although the GST-fusion protein was induced with isopropyl-/3-D-thiogalactopyranoside and solubilized in the supernatant of the bacterial lysate after sonication, the fusion protein was rapidly cleaved at the fusion joint between the GST and FwRI-y during purification (Fig l.A). This cleavage could not be prevented by the addition of several available protease inhibitors such as phenylmethyl sulfonyl fluoride, aprotinin, leupeptin, bestatin and ethylenediaminetetraacetic acid (data not shown). Several trials for alternative methods such as shortening the induction time, changing the incubation temperature, or lysis with freezing and thawing method instead of sonication did not improve the results at all (data not shown). To overcome this problem, we then used several protease negative E.coli strains (Table 1). In one of these strains, AD202, which is defective in ompT encoding an outer membrane-associated protease (4), the fusion protein was not cleaved at all and recovered from bacterial lysate with an expected molecular size (Fig. 1A). The prevention of degradation was observed not only with this construct but also with other GST-fusion proteins including GST-CD3e (Fig. IB). It is noteworthy that a fusion protein, GST-CD3f, which could not be detected in DH5a, could be induced and purified by using AD202 (Fig. 1C). These results suggest that an extremely unstable fusion protein such as GSTCDS f can be stabilized in AD202. We next examined the efficiency of this strain by using a larger fusion protein construct. A 75 kD GST-CTBPl, which contains a coding frame of a singlestranded DNA and RNA binding protein of the yeast, was purified efficiently in AD202, whereas most of the products were degraded in DH5a (Fig. ID). This suggests that AD202 is also effective for fusion proteins as large as 75 kD and for those encoding nucleic acid binding proteins. From these observations, we conclude that AD202 is one of the most appropriate strains for the purpose of producing GST-fusion proteins in E. coli.

Smad3 inhibits activation of the smooth muscle actin promoter and functions as a timer for myogenic programming in the epithelium., Epithelial–myofibroblast (MF) transition (EMyT) is a critical process in organ fibrosis, leading to α–smooth muscle actin (SMA) expression in the epithelium. The mechanism underlying the activation of this myogenic program is unknown. We have shown previously that both injury to intercellular contacts and transforming growth factor β (TGF-β) are indispensable for SMA expression (two-hit model) and that contact disruption induces nuclear translocation of myocardin-related transcription factor (MRTF). Because the SMA promoter harbors both MRTF-responsive CC(A/T)-rich GG element (CArG) boxes and TGF-β–responsive Smad-binding elements, we hypothesized that the myogenic program is mobilized by a synergy between MRTF and Smad3. In this study, we show that the synergy between injury and TGF-β exclusively requires CArG elements. Surprisingly, Smad3 inhibits MRTF-driven activation of the SMA promoter, and Smad3 silencing renders injury sufficient to induce SMA expression. Furthermore, Smad3 is degraded under two-hit conditions, thereby liberating the myogenic program. Thus, Smad3 is a critical timer/delayer of MF commitment in the epithelium, and EMyT can be dissected into Smad3-promoted (mesenchymal) and Smad3-inhibited (myogenic) phases.

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.

Tumor necrosis factor (TNF)-induced activation of the c-jun N-terminal kinase (JNK, also known as SAPK; stress-activated protein kinase) requires TNF receptor-associated factor 2 (TRAF2). The apoptosis signal-regulating kinase 1 (ASK1) is activated by TNF and stimulates JNK activation. Here we show that ASK1 interacts with members of the TRAF family and is activated by TRAF2, TRAF5, and TRAF6 overexpression. A truncated derivative of TRAF2, which inhibits JNK activation by TNF, blocks TNF-induced ASK1 activation. A catalytically inactive mutant of ASK1 is a dominant-negative inhibitor of TNF- and TRAF2-induced JNK activation. In untransfected mammalian cells, ASK1 rapidly associates with TRAF2 in a TNF-dependent manner. Thus, ASK1 is a mediator of TRAF2-induced JNK activation.

The relationship between activation-induced growth inhibition and regulation of the cell cycle progression was investigated in T cell hybridomas by studying the function of the cell cycle-regulating genes such as G1 cyclins and their associated kinases. Activation of T cell hybridomas by anti-T cell receptor antibody induces growth arrest at G1 phase of the cell cycle and subsequently results in activation-driven cell death. Rapid reduction of both messenger RNA and protein level of the cyclin D3 is accompanied by growth arrest upon activation. Although the residual cyclin D3 protein forms a complex with cdk4 protein, cyclin D3-dependent kinase activity is severely impaired. Stable transfectants engineered to express cyclin D3 override the growth arrest upon activation. These results imply that the activation signal through T cell receptor induces the down-regulation of cyclin D3 expression and cyclin D3-dependent kinase activity, leading to growth arrest in G1 phase of the cell cycle in T cells.