Molecular cloning.

Transcription factor c-Jun is a member of AP-1 transcription complex that can be induced by various pathogens and plays a broad regulatory role in vertebrate immune response. In this study, the complete c-Jun cDNA of large yellow croaker Larimichthys crocea (Lcc-Jun) was cloned, whose open reading frame (ORF) is 984 bp long and encodes a protein of 327 amino acids (aa). The deduced Lcc-Jun protein contains three highly conserved domains, a transactivation domain (TAD, Met1-His118), a DNA binding domain (DBD, Lys218-Arg243), and a Leucine zipper domain (LZD, Leu271-Leu299), as found in other specie c-Jun. Lcc-Jun was constitutively expressed in all examined tissues, with the higher levels in blood, heart, and head kidney. Its transcripts were not only induced in spleen and head kidney by poly (I: C) or LPS, but also up-regulated in primary head kidney leukocytes (PKL), macrophages (PKM), and granulocytes (PKG), suggesting that Lcc-Jun may be involved in immune responses induced by poly (I: C), a viral mimic, and LPS, a Gram-negative bacterial component. Overexpression of Lcc-Jun in PKL increased the expression of cytokines and transcription factors involved in T helper 1 (Th1: TNF-α, IFN-γ, and T-bet) and Th2 (IL-4/13 A/B, IL-6, and GATA3) cell development and differentiation, suggesting that Lcc-Jun may play a role in regulation of Th1/Th2 cell response. These results therefore led us to suggest that the c-Jun-mediated signaling pathways may have an important immune-modulatory function in teleost fish.

Histone deacetylase 2 is essential for LPS-induced inflammatory responses in macrophages.

The role of specific histone deacetylase (HDAC) proteins in regulating the lipopolysaccharide (LPS)-induced inflammatory response and its underlying mechanisms are unclear. Here, HDAC2, a class I HDAC family protein, is essential for the LPS-triggered inflammatory response in macrophages. LPS stimulation increases HDAC2 expression in macrophages. Knockdown of HDAC2 decreases the expression of proinflammatory genes, such as IL-12, TNF-α and iNOS following stimulation with LPS. The adoptive transfer of HDAC2 knockdown macrophages attenuates the LPS-triggered innate inflammatory response in vivo, and these mice are less sensitive to endotoxin shock and Escherichia coli-induced sepsis. Mechanistically, the c-Jun protein is the main target of HDAC2-mediated LPS-induced production of proinflammatory cytokines. Moreover, HDAC2 knockdown increases the expression of c-Jun, which directly binds the promoters of proinflammatory genes and forms nuclear receptor corepressor complexes to inhibit the transcription of proinflammatory genes in macrophages. These effects are rescued by c-Jun expression. According to the chromatin immunoprecipitation analysis, HDAC2 also selectively suppresses c-Jun expression by directly binding to its promoter and modifying histone acetylation after LPS stimulation. Our findings define a new function and mechanism of the HDAC2/c-Jun signaling network that regulates the LPS-induced immune response in macrophages.

Immunostimulatory effect of laminarin on RAW 264.7 mouse macrophages.

This study investigated the immunostimulatory effects of laminarin with respect to inflammatory mediators such as hydrogen peroxide, calcium, nitric oxide, various cytokines, transcription factors, and immune response gene in RAW 264.7 mouse macrophages. Laminarin did not reduce the cell proliferation of RAW 264.7 mouse macrophages at concentrations up to 500 µg/mL. Laminarin significantly increased the release of hydrogen peroxide, calcium, nitric oxide, monocyte chemotactic protein-1, vascular endothelial growth factor, leukemia inhibitory factor, and granulocyte-colony stimulating factor with enhancing expression of Signal Transducer and Activator of Transcription 1 (STAT1), STAT3, c-Jun, c-Fos, and cyclooxygenase-2 mRNA in RAW 264.7 cells. The results suggest that laminarin has immunostimulatory properties, strengthening immune reactions via the transcription factor pathway in macrophages.

Ran overexpression leads to diminished T cell responses and selectively modulates nuclear levels of c-Jun and c-Fos.

Ras-related nuclear protein (Ran) is a Ras family GTPase, and its documented functions are the regulation of DNA replication, cell cycle progression, nuclear structure formation, RNA processing and exportation, and nuclear protein importation. In this study, we performed detailed mapping of Ran expression during mouse ontogeny using in situ hybridization. High Ran expression was found in various organs and tissues including the thymus cortex and spleen white pulp. Ran was induced in T cells 24 h after their activation. The function of Ran in the immune system was investigated using Ran transgenic (Tg) mice. In Ran Tg T cells, there was compromised activation marker expression, lymphokine secretion, and proliferation upon T cell receptor activation in vitro when compared with wild type T cells. Tg mice also manifested defective delayed type hypersensitivity in vivo. Upon PMA and ionomycin stimulation, Tg T cells were defective in nuclear accumulation of AP-1 factors (c-Jun and c-Fos) but not NF-kappaB family members. Our experiments showed that Ran had important regulatory function in T cell activation. One of the possible mechanisms is that intracellular Ran protein levels control the nuclear retention for selective transcription factors such as c-Jun and c-Fos of AP-1, which is known to be critical in T cell activation and proliferation and lymphokine secretion.

JunD/AP-1 and STAT3 are the major enhancer molecules for high Bcl6 expression in germinal center B cells.

The Bcl6 proto-oncogene, which encodes a transcriptional repressor, is ubiquitously expressed and predominantly in germinal center (GC) B cells. Although the promoter region of the human Bcl6 gene has been reported, enhancer molecules for its high expression in GC B cells were largely unknown. Here we show that transcriptional start sites of the murine Bcl6 gene were different from the reported human one. DNA sequence around the new promoter region is highly conserved between mice and humans and has no canonical TATA or CCAAT box. Two AP-1-binding elements in the promoter region were the major enhancer elements in GC-derived B lymphoma cells, and JunD/AP-1 was detected in GC B cells. In addition, we identified the silencer region with three Bcl6-binding elements around the start site. Bcl6 bound to the silencer elements and its over-expression repressed the promoter activity through the elements. Activated STAT factors (STATs), especially activated STAT3, also bound to the silencer elements in GC B cells and competed with Bcl6 for the binding, suggesting that JunD/AP-1 and activated STATs drive high Bcl6 expression in GC B cells. Since stimulation of splenic B cells with IL-4 or IL-21 induced high Bcl6 expression with induction of junD and activation of STATs, these cytokines may be inducers for its high expression in GC B cells. However, IL-21 but not IL-4 stimulation activated STAT3 in splenic B cells. Thus, IL-21 may be a major inducer for high Bcl6 expression in GC B cells.

Transcriptional regulation of the human TLR9 gene.

To clarify the molecular basis of human TLR9 (hTLR9) gene expression, the activity of the hTLR9 gene promoter was characterized using the human myeloma cell line RPMI 8226. Reporter gene analysis and EMSA demonstrated that hTLR9 gene transcription was regulated via four cis-acting elements, cAMP response element, 5'-PU box, 3'-PU box, and a C/EBP site, that interacted with the CREB1, Ets2, Elf1, Elk1, and C/EBPalpha transcription factors. Other members of the C/EBP family, such as C/EBPbeta, C/EBPdelta, and C/EBPepsilon, were also important for TLR9 gene transcription. CpG DNA-mediated suppression of TLR9 gene transcription led to decreased binding of the trans-acting factors to their corresponding cis-acting elements. It appeared that suppression was mediated via c-Jun and NF-kappaB p65 and that cooperation among CREB1, Ets2, Elf1, Elk1, and C/EBPalpha culminated in maximal transcription of the TLR9 gene. These findings will help to elucidate the mechanism of TLR9 gene regulation and to provide insight into the process by which TLR9 evolved in the mammalian immune system.

Molecular basis for functional maturation of thymocytes: increase in c-fos translation with positive selection.

In the process of positive selection, immature CD4+8+ double positive (DP) thymocytes expressing TCR reactive to self-MHC by appropriate avidity develop into mature thymocytes. Positive selection involves not only down-regulation of either CD4 or CD8 but also acquisition of immunocompetent potential such as cell proliferation and cytokine production. To understand the molecular basis for such functional maturation during the positive selection process, we examined whether nonselected DP, selected DP, and CD4+8- single positive thymocytes possess the activation potential for signaling pathways from mitogen-activated protein kinases (extracellular signal-regulated kinase and c-Jun N-terminal kinase) to AP-1. In response to stimulation, a marked induction of c-Fos protein expression as well as cell proliferation is detected only in CD4+8- single positive cells but not in selected and nonselected DP cells, though mitogen-activated protein kinase activities and c-fos transcripts are equally induced. In the presence of proteasome inhibitors, c-Fos protein became detectable in selected DP cells but still not in nonselected DP cells, suggesting that DP cells receiving positive selection signals acquire the capacity to translate the c-fos gene, but it may not be sufficiently high to overcome the degradation of c-Fos protein. These data indicate that the translating ability of the c-fos gene is up-regulated in the thymic positive selection process, from nonselected DP to CD4+8- single positive cells through positively selected DP cells. The distinguished responsiveness to stimulation in thymocytes with and without positive selection may be a result in part of the distinct regulation of the c-fos gene at the translational level.

CD28-mediated regulation of the c-jun promoter involves the MEF2 transcription factor in Jurkat T cells.

Within a few minutes of T-cell activation, transcription of a set of genes including c-fos and c-jun is activated. For maximal induction of c-jun, at least two major signal pathways are required. One can be triggered by T-cell receptor engagement or phorbol esters and the other by anti-CD28 engagement. The c-jun promoter region between -117 and -50 contains binding sites for the transcription factors Spl, CTF, ATF/CREB, and MEF2. In this study, we sought to map the sequences in the c-jun promoter responsible for CD28-mediated induction in activated Jurkat T cell by point mutational analysis. We found that mutation of the c-jun MEF2 site strongly reduces CD28 induction of the promoter in Jurkat T cells and that MEF2D is the major binding molecule to the c-jun MEF2 site in Jurkat T cells. Mutation of the c-jun ATF site also partially reduced CD28 induction of the promoter. In addition, pretreatment with an endolysomotropic agent NH4Cl, an acidic sphingomyelinase inhibitor, completely inhibited the activation of the c-jun promoter by anti-CD28 antibody treatment, whereas pretreatment with wortmannin, a PI3-kinase inhibitor, did not affect the induction of the c-jun promoter. These results suggest that CD28 signaling leading to the c-jun promoter involves acidic sphingomyelinase, but not PI3-kinase, to activate factors binding to the MEF2 and ATF sites.

Involvement of NF-kappaB p50/p65 heterodimer in activation of the human pro-interleukin-1beta gene at two subregions of the upstream enhancer element.

A region between-3134 and -2729 bp upstream from the transcription site of the human pro-interleukin 1beta (proIL-1beta) gene was identified as an LPS-responsive enhancer element. In this study, the influence of the sequences located between -3134 and -2987 on the transcriptional activity of the proIL-1beta gene in LPS-stimulated Raw 264.7 cells was examined in detail. The results obtained by transient transfection of fos -CAT constructs that contained serial 5'-deletion mutations showed that the region between -3134 and -3059 appears to be required for the induction of transcription by LPS. Gel shift assay studies with synthetic oligonucleotides corresponding to partial sequences of the latter region and nuclear extracts from stimulated cells revealed specific protein binding sites between -3110 and -3090 and between -3079 and -3059. These specific bindings were time and LPS dose dependent. The results of supershift analysis using specific antibodies against transcription factors suggested that both binding complexes contained the NF-kappaB components p50 and p65, and did not contain other NF-kappaB proteins (p52, c-Rel, Rel B), AP-1 proteins (c-Fos, C-Jun), CREB or C/EBPbeta (NF-IL6). Mutation of either of the putative NF-kappaB-binding sites in the enhancer element decreased the LPS-stimulated transcriptional activity. These data indicated that two NF-kappaB-binding sites, which are located between -3134 and -3059, are critical for the activation of proIL-1beta gene transcription.

Lipopolysaccharide-induced desensitization of junB gene expression in a mouse macrophage-like cell line, P388D1.

Treatment of a mouse macrophage cell line, P388D1, for 1 h with bacterial LPS caused a transient increase in the level of junB mRNA expression. These cells became refractory in terms of the junB gene response to exposure to a second round of LPS or lipid A, but not to PMA. The LPS-induced desensitized state was not due to the shortening of the half-life of junB mRNA, but was suggested, by nuclear run-on analysis, to be caused by reduction of junB gene transcription. Pretreating cells with herbimycin A, a tyrosine kinase inhibitor, substantially inhibited LPS-induced expression of junB mRNA and decreased tyrosine phosphorylation of 38- to 42-kDa proteins, which comigrated with p38 and p42 mitogen-activated protein (MAP) kinases. Parallel to down-regulation of junB mRNA expression, activation of the p38 MAP kinase was markedly reduced in LPS-tolerant cells, whereas activation of p42 MAP kinase was relatively constant. The specific p38 MAP kinase inhibitor, SB202190, potently inhibited LPS-induced junB mRNA expression. These results suggest that the LPS-induced desensitization of junB gene expression occurs at or upstream of the level of gene transcription and may be involved in a defective LPS-induced p38 MAP kinase pathway.

Receptors for the anaphylatoxin C5a (CD88) on human mesangial cells.

In these studies, we determined whether there are receptors for the anaphylatoxin C5a (C5aR, CD88) on human mesangial cells (HMC). To prepare Abs to C5aR, we first synthesized an immunogenic peptide spanning residues 8-32 of the molecule, and this peptide was used to immunize rabbits. Anti-C5aR antiserum, but not preimmune serum, stained fixed and unfixed HMC in culture. By Western blotting anti-C5aR, Abs identified a 49.6-kDa protein in HMC. By reverse-transcription PCR, a cDNA product of 558 bp was amplified corresponding to the expected size of C5aR cDNA. A cDNA of the same size was amplified simultaneously from human PBL. Restriction mapping of the products amplified from HMC and from PBL gave restriction fragments of the same size. Incubation of HMC with increasing doses of C5a caused a progressive increase in the levels of the transcription factors activator protein-1 (AP-1) and cAMP response element binding protein (CREB), but C5a had no effect on the level of nuclear factor-kappaB (NF-kappaB). The effects of C5a on AP-1 were concentration and time dependent and peaked after 60 min. In contrast, the C5a metabolite C5adesArg had no significant effect on AP-1 levels. Preincubation of HMC with rabbit anti-C5aR antiserum inhibited partially the effect of C5a on AP-1. However, anti-C5aR Abs alone had no appreciable effects on AP-1. C5a caused a significant up-regulation of mRNA for the early response genes c-jun and c-fos on HMC. These results provide evidence for the presence of C5aR in adult HMC in culture and indicate that, after binding to C5aR, the anaphylatoxin C5a causes significant up-regulation of certain transcription factors and early response genes.

Transcriptional regulation of the junB gene in B lymphocytes: role of protein kinase A and a membrane Ig-regulated protein phosphatase.

We have examined herein whether membrane Ig (mIg) stimulates junB transcription through a protein kinase A (PKA)-dependent or PKA-independent pathway. PKA phosphotransferase activity was not increased following mIg cross-linking of Bal17 B cells. However, junB transcriptional activation was dependent upon PKA activity, as evidenced by inhibition of goat anti-mouse IgM-stimulated junB promoter-chloramphenicol acetyltransferase reporter gene activity in transfected Bal17 B cells treated with the PKA inhibitor H-89. mIg-stimulated junB promoter-chloramphenicol acetyltransferase activity was also blocked in B cells expressing a specific PKA inhibitor peptide, whereas in vivo expression of an inactive PKA inhibitor peptide variant was not inhibitory. Expression of a mutant cAMP response element binding protein (CREB) containing an inactivated kinase A phosphoacceptor site at Ser133 reduced mIg-stimulated junB transcription. Okadaic acid increased CREB1 phosphorylation at Ser133 and junB transcriptional activation, suggesting the action of protein phosphatase-1 (PP-1) or -2A (PP-2A). Extracts from unstimulated B cells exhibited phosphatase activity against an in vitro PKA-phosphorylated peptide containing the Ser133 phosphoacceptor site. The involvement of a phosphatase activity in regulating mIg-stimulated junB transcription is supported by our finding that extracts from goat anti-mouse IgM-stimulated B cells exhibited a significantly reduced level of Ser133 phosphatase activity. Hence, the level of CREB1 phosphorylation is governed by the balance between PKA and phosphatase activities. junB transcriptional activation results in part from mIg signals that negatively regulate a CREB1-targeted PP-1 or PP-2A activity.

Interferon-gamma induces the expression of immediate early genes c-fos and c-jun in astrocytes.

The expression of the proto-oncogenes, c-fos and c-jun, in cultured mouse astrocytes and its induction by the potent astrocyte activator interferon-gamma (IFN-gamma), were examined by Northern blot and flow cytometry. Both proto-oncogenes were induced in a dose-dependent manner, peaking around 100 U/ml of IFN-gamma. The kinetics of expression is very transient for c-fos, reaching a maximum at 30 min and decreasing rapidly thereafter. The c-jun remained high throughout the stages analysed. Cycloheximide superinduced c-fos and c-jun induction by IFN-gamma, thus indicating that both act as immediate early genes. The products of c-fos and c-jun, proteins FOS and JUN, that act in conjunction forming the regulatory factor AP-1, were detected 1 hr after stimulation in virtually all cells, using flow cytometry. The induction in astrocytes of both proto-oncogenes could be the first stage of immunological activation of these central nervous system cells by immune interferon.

Terminal complement complexes induce cell cycle entry in oligodendrocytes through mitogen activated protein kinase pathway.

Sublytic complement attack through C5b-9 assembly induces oligodendrocytes (OLG) to express proto-oncogenes and to enter the cell cycle from resting G0/G1 phase to S phase. We have investigated whether cell cycle induction by C5b-9 is mediated by mitogen activated protein kinase (MAPK) pathway in OLG. C5b-9 but not C5b6 induced activation of both ERK1 and c-jun NH2 terminal kinases 1 (JNK1) in OLG. The increased ERK1 and JNK1 activities are transient, reaching a maximum around 20 min following exposure to C5b-9. Activation of Raf-1 and MEK1, upstream kinases of ERK1, was shown by increased Raf-1 kinase activity in anti-Raf-1 immunoprecipitates of OLG treated with C5b-9 and ERK1 activity that can be inhibited by PD098,059, a specific MEK1 inhibitor. Requirement for the ERK1 pathway in DNA synthesis was then evaluated using PD098,059. Enhanced DNA synthesis induced by serum complement was completely abolished when OLG were pretreated with PD098,059. On the other hand, c-fos mRNA expression induced by complement was inhibited only 50% by PD098,059, while the c-jun mRNA level was not affected by this MEK1 inhibitor. Interestingly, p70 S6 kinase, an important ribosomal kinase in mitogenesis, was also activated by C5b-9. These findings indicated that the MAPK pathways appears to play a major role in inducing OLG to enter the S phase of the cell cycle from the resting G1/G0 phase.

Transcriptional regulation of the junB promoter in mature B lymphocytes. Activation through a cyclic adenosine 3',5'-monophosphate-like binding site.

The experiments presented herein were designed to understand the molecular mechanism(s) by which membrane Ig (mIg)-dependent signals are integrated at the level of the junB promoter to induce gene transcription. Functional studies using chloramphenicol acetyltransferase reporter gene constructs that contained deleted 5' flanking region junB sequences identified a region located between -194 and -87 that contains an Ets binding site and a putative cAMP response element binding site (CRE-like). Point mutagenesis of the CRE-like site blocked junB promoter activation in response to mIg cross-linking in mature Bal17 B cells. Nuclear extract binding activity to a synthetic oligonucleotide containing the junB CRE-like site was detected in unstimulated B cells and was increased in response to mIg cross-linking. Binding activity was competed with unlabeled oligonucleotides that contained the junB CRE-like site or the somatostatin CRE consensus motif, the latter observation suggests that members of the activating transcription factor/CRE binding protein (CREB) family may mediate mIg-dependent junB transcription. Consistent with this interpretation, recombinant CREB and activating transcription factor proteins bound the junB CRE-like site, but did not interact with a mutant CRE-like site. Expression of a dominant negative CREB protein blocked mIg-mediated transcription from a junB CRE-like site-chloramphenicol acetyltransferase reporter gene. CRE-like nucleoprotein complexes from Bal17 B cells contained constitutively bound CREB-1, which was phosphorylated on serine 133 in response to mIg cross-linking. Activating transcription factor-1 protein was also constitutively expressed in CRE-like nucleoprotein complexes. Collectively, these results suggest that components of the protein kinase A signaling pathway are recruited by mIg to induce junB transcription.

Sublethal levels of oxidative stress stimulate transcriptional activation of c-jun and suppress IL-2 promoter activation in Jurkat T cells.

Sublethal levels of oxidative stress are well known to alter T cell functional responses, but the underlying mechanisms are unknown. The current study examined the effects of oxidative stress on transcriptional activities mediated by c-Fos/c-Jun AP-1 and the nuclear factor of activated T cells (NF-AT). The present results show that Jurkat T cells acutely exposed to micromolar concentrations of H2O2 exhibit substantial increases in AP-1 binding activity and the expression of c-jun but not c-fos mRNA. The preferential induction of c-jun by H2O2 did not represent redox stabilization of mRNA transcripts, and oxidative signals closely resembled PHA/PMA stimulation by effectively transactivating the full length c-jun promoter via the proximal jun1 tumor promoter-responsive element (TRE)-like promoter element. Similarly, the complexes binding the consensus AP-1 TRE and jun TRE-like motifs in cells exposed to oxidative signals or PHA/PMA were indistinguishable, being composed of c-Fos, c-Jun, and JunD. However, PHA/PMA but not oxidative signals induced the coordinate activation of reporter constructs containing the AP-1-TRE, NF-AT, and IL-2 promoter regions along with IL-2 mRNA expression. Furthermore, sublethal levels of H2O2 actively suppressed the transcriptional activation of NF-AT and IL-2 reporters as well as the expression of IL-2 mRNA in cells stimulated with PHA/PMA. Gel shift analysis revealed that oxidative suppression of NF-AT represented inhibition in the early generation of NFAT complexes rather than the binding of preformed NF-AT complexes. These results suggest that oxidative signals can positively and negatively regulate T cell transcriptional events and that changes in cellular redox can uncouple AP-1 regulation of c-jun from transcriptional up-regulation of IL-2 via NF-AT.