IκBα Nuclear Export Enables 4-1BB-Induced cRel Activation and IL-2 Production to Promote CD8 T Cell Immunity.

Optimal CD8 T cell immunity is orchestrated by signaling events initiated by TCR recognition of peptide Ag in concert with signals from molecules such as CD28 and 4-1BB. The molecular mechanisms underlying the temporal and spatial signaling dynamics in CD8 T cells remain incompletely understood. In this study, we show that stimulation of naive CD8 T cells with agonistic CD3 and CD28 Abs, mimicking TCR and costimulatory signals, coordinately induces 4-1BB and cRel to enable elevated cytosolic cRel:IκBα complex formation and subsequent 4-1BB-induced IκBα degradation, sustained cRel activation, heightened IL-2 production and T cell expansion. NfkbiaNES/NES CD8 T cells harboring a mutated IκBα nuclear export sequence abnormally accumulate inactive cRel:IκBα complexes in the nucleus following stimulation with agonistic anti-CD3 and anti-CD28 Abs, rendering them resistant to 4-1BB induced signaling and a disrupted chain of events necessary for efficient T cell expansion. Consequently, CD8 T cells in NfkbiaNES/NES mice poorly expand during viral infection, and this can be overcome by exogenous IL-2 administration. Consistent with cell-based data, adoptive transfer experiments demonstrated that the antiviral CD8 T cell defect in NfkbiaNES/NES mice was cell intrinsic. Thus, these results reveal that IκBα, via its unique nuclear export function, enables, rather than inhibits 4-1BB-induced cRel activation and IL-2 production to facilitate optimal CD8 T cell immunity.

Investigation of the genetic overlap between rheumatoid arthritis and psoriatic arthritis in a Greek population.

OBJECTIVES: Several rheumatoid arthritis (RA) susceptibility loci have also been found to be associated with psoriatic arthritis (PsA), demonstrating that there is a degree of genetic overlap between various autoimmune diseases. We sought to investigate whether single nucleotide polymorphisms (SNPs) mapping to previously reported RA and/or PsA susceptibility loci, including PLCL2, CCL21, REL, STAT4, CD226, PTPN22, and TYK2, are associated with risk for the two diseases in a genetically homogeneous Greek population. METHOD: This study included 392 RA patients, 126 PsA patients, and 521 healthy age- and sex-matched controls from Greece. Genotyping of the SNPs was performed with Taqman primer/probe sets. Bioinformatic analysis was performed using BlastP, PyMOL, and Maestro and Desmond. RESULTS: A significant association was detected between the GC genotype of rs34536443 (TYK2) in both the PsA and RA cohorts. The C allele of this SNP was associated with PsA only. Evidence for association with PsA was also found for the GG genotype and G allele of the rs10181656 SNP of STAT4. The TC genotype of the rs763361 SNP of CD226 was associated with PsA only. CONCLUSIONS: Genetic overlap between PsA and RA was detected for the rs34536443 SNP of the TYK2 gene within a Greek population. An association of STAT4 (rs10181656) with PsA was confirmed whereas CD226 (rs763361) was associated with PsA but not with RA, in contrast to previous reports. The different findings of this study compared to previous ones highlights the importance of comparative studies that include various ethnic or racial populations.

Glucosamine inhibits lipopolysaccharide-stimulated inducible nitric oxide synthase induction by inhibiting expression of NF-kappaB/Rel proteins at the mRNA and protein levels.

Expression of inducible nitric oxide synthase (iNOS) protein by lipopolysaccharide (LPS) in BV2 microglia cells increased in a biphasic manner. Glucosamine (GlcN) selectively suppressed the late- but not early-stage iNOS response to LPS. Prolonged induction of iNOS expression by LPS was inhibited by cycloheximide, suggesting that de novo protein synthesis was required. Late-phase activation of nuclear factor-kappaB (NF-κB) activity required for sustained iNOS induction. Nuclear translocation and DNA binding of NF-κB, and Rel proteins expressions were inhibited by GlcN at later time points but not upon immediate early-stage activation by LPS. We show that GlcN selectively inhibits sustained iNOS induction by inhibiting Rel protein expression at both the mRNA and protein levels; such expression is required for prolonged iNOS induction by LPS. Our results provide mechanistic evidence that GlcN regulates inflammation, represented by iNOS. The implication of these results is that GlcN may be a potent transcriptional regulator of iNOS and other genes involved in the general inflammation process.

NF-kappaB down-regulates expression of the B-lymphoma marker CD10 through a miR-155/PU.1 pathway.

Cell-surface protein CD10 is a prognostic marker for diffuse large B-cell lymphoma (DLBCL), where high expression of CD10 is found in the germinal center B-cell (GCB) subtype and CD10 expression is low or absent in the activated B-cell (ABC) subtype. As compared with the GCB subtype, patients with ABC DLBCL have a poorer prognosis after standard treatment, and ABC tumor cells have higher NF-κB activity. Herein, we show that increased expression of the NF-κB target micro-RNA miR-155 is correlated with reduced expression of transcription factor PU.1 and CD10 in several B-lymphoma cell lines. Moreover, electromobility shift assays and luciferase reporter assays indicate that PU.1 can directly activate expression from the CD10 promoter. Expression of a DLBCL-derived mutant of the adaptor CARD11 (a constitutive activator of NF-κB) in the GCB-like human BJAB cell line or v-Rel in the chicken DT40 B-lymphoma cell line causes reduced expression of PU.1. The CARD11 mutant also causes a decrease in CD10 levels in BJAB cells. Similarly, overexpression of miR-155, which is known to down-regulate PU.1, leads to reduced expression of CD10 in BJAB cells. Finally, we show that CD10 expression is reduced in BJAB cells after treatment with the NF-κB inducer lipopolysaccharide (LPS). Additionally, miR-155 is induced by LPS treatment or expression of the CARD11 mutant in BJAB cells. These results point to an NF-κB-dependent mechanism for down-regulation of CD10 in B-cell lymphoma: namely, that increased NF-κB activity leads to increased miR-155, which results in decreased PU.1, and consequently reduced CD10 mRNA and protein.

Coordinated regulation of Rel expression by MAP3K4, CBP, and HDAC6 controls phenotypic switching.

Coordinated gene expression is required for phenotypic switching between epithelial and mesenchymal phenotypes during normal development and in disease states. Trophoblast stem (TS) cells undergo epithelial-mesenchymal transition (EMT) during implantation and placentation. Mechanisms coordinating gene expression during these processes are poorly understood. We have previously demonstrated that MAP3K4-regulated chromatin modifiers CBP and HDAC6 each regulate thousands of genes during EMT in TS cells. Here we show that CBP and HDAC6 coordinate expression of only 183 genes predicted to be critical regulators of phenotypic switching. The highest-ranking co-regulated gene is the NF-κB family member Rel. Although NF-κB is primarily regulated post-transcriptionally, CBP and HDAC6 control Rel transcript levels by binding Rel regulatory regions and controlling histone acetylation. REL re-expression in mesenchymal-like TS cells induces a mesenchymal-epithelial transition. Importantly, REL forms a feedback loop, blocking HDAC6 expression and nuclear localization. Together, our work defines a developmental program coordinating phenotypic switching.

Mitochondria to nucleus stress signaling: a distinctive mechanism of NFkappaB/Rel activation through calcineurin-mediated inactivation of IkappaBbeta.

Mitochondrial genetic and metabolic stress causes activation of calcineurin (Cn), NFAT, ATF2, and NFkappaB/Rel factors, which collectively alter the expression of an array of nuclear genes. We demonstrate here that mitochondrial stress-induced activation of NFkappaB/Rel factors involves inactivation of IkappaBbeta through Cn-mediated dephosphorylation. Phosphorylated IkappaBbeta is a substrate for Cn phosphatase, which was inhibited by FK506 and RII peptide. Chemical cross-linking and coimmunoprecipitation show that NFkappaB/Rel factor-bound IkappaBbeta forms a ternary complex with Cn under in vitro and in vivo conditions that was sensitive to FK506. Results show that phosphorylation at S313 and S315 from the COOH-terminal PEST domain of IkappaBbeta is critical for binding to Cn. Mutations at S313/S315 of IkappaBbeta abolished Cn binding, inhibited Cn-mediated increase of Rel proteins in the nucleus, and had a dominant-negative effect on the mitochondrial stress-induced expression of RyR1 and cathepsin L genes. Our results show the distinctive nature of mitochondrial stress-induced NFkappaB/Rel activation, which is independent of IKKalpha and IKKbeta kinases and affects gene target(s) that are different from cytokine and TNFalpha-induced stress signaling. The results provide new insights into the role of Cn as a critical link between Ca2+ signaling and NFkappaB/Rel activation.

An optimal range of transcription potency is necessary for efficient cell transformation by c-Rel to ensure optimal nuclear localization and gene-specific activation.

c-Rel is overexpressed in several B-cell lymphomas and c-rel gene overexpression can transform primary chicken lymphoid cells and induce tumors in animals. Although c-Rel is generally a stronger transcriptional activator than its viral derivative v-Rel, its oncogenic activity is significantly weaker. Among the mutations acquired during c-Rel's evolution into v-Rel are deletion of c-Rel's transactivation domain 2 (cTAD2) and mutations in cTAD1. Given the critical role of the Rel TADs in cell transformation, we investigated how mutations in c-Rel's cTAD1 and cTAD2 contribute to its oncogenicity and that of v-Rel. Mutations in cTAD2 noticeably increased c-Rel's transforming activity by promoting its nuclear localization and gene-specific transactivation, despite an overall decrease in kappaB site-dependent transactivation potency. Conversely, substitution of vTAD by cTAD1 increased v-Rel's transactivation and transforming efficiencies, whereas its substitution by the stronger cTAD2 compromised activation of mip-1beta but not irf-4 and was detrimental to cell transformation. These results suggest that the Rel TADs differentially contribute to gene-specific activation and that an optimal range of transcription potency is necessary for efficient transformation. These findings may have important implications for understanding how Rel TAD mutations can lead to a more oncogenic phenotype.

Inhibition of v-rel-Induced Oncogenesis through microRNA Targeting.

Several studies have shown that microRNA-targeting is an effective strategy for the selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. However, the exploitation of microRNA-targeting for the inhibition of transformation by oncogenic viruses has not been studied. The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transforming primary chicken spleen and bone marrow cells. Here, by engineering the target sequence of endogenous microRNA miR-142 downstream of the v-rel gene in a Replication-Competent ALV (avian leukosis virus) long terminal repeat (LTR) with a splice acceptor (RCAS) vector and using a v-rel-induced transformation model of chicken embryonic splenocyte cultures, we show that hematopoietic-specific miR-142 can inhibit the v-rel-induced transformation, and that this inhibition effect is due to the silencing of v-rel expression. The data supports the idea that microRNA-targeting can be used to inhibit viral oncogene-induced oncogenesis.

Cytoplasmic sequestration of rel proteins by IkappaBalpha requires CRM1-dependent nuclear export.

Rel and IkappaB protein families form a complex cellular regulatory network. A major regulatory function of IkappaB proteins is to retain Rel proteins in the cell cytoplasm. In addition, IkappaB proteins have also been postulated to serve nuclear functions. These include the maintenance of inducible NF-kappaB-dependent gene transcription, as well as termination of inducible transcription. We show that IkappaBalpha shuttles between the nucleus and the cytoplasm, utilizing the nuclear export receptor CRM1. A CRM1-binding export sequence was identified in the N-terminal domain of IkappaBalpha but not in that of IkappaBbeta or IkappaBepsilon. By reconstituting major aspects of NF-kappaB-IkappaB sequestration in yeast, we demonstrate that cytoplasmic retention of p65 (also called RelA) by IkappaBalpha requires Crm1p-dependent nuclear export. In mammalian cells, inhibition of CRM1 by leptomycin B resulted in nuclear localization of cotransfected p65 and IkappaBalpha in COS cells and enhanced nuclear relocation of endogenous p65 in T cells. These observations suggest that the main function of IkappaBalpha is that of a nuclear export chaperone rather than a cytoplasmic tether. We propose that the nucleus is the major site of p65-IkappaBalpha association, from where these complexes must be exported in order to create the cytoplasmic pool.

Mutations in the v-Rel transactivation domain indicate altered phosphorylation and identify a subset of NF-kappaB-regulated cell death inhibitors important for v-Rel transforming activity.

Consistent with the constitutive activation of Rel/NF-kappaB in human hematopoietic tumors, the v-Rel oncoprotein induces aggressive leukemia/lymphomas in animal models. v-Rel is thus a valuable tool to characterize the role of Rel/NF-kappaB in cancer and the mechanisms involved. Prior studies by our group identified a serine-rich domain in v-Rel that was required for biological activity. Here, we investigated the molecular basis for the transformation defect of specific serine mutants. We show that the transforming efficiency of these mutants in primary lymphoid cells is correlated with their ability to mediate kappaB site-dependent transactivation and with specific changes in phosphorylation profiles. Interestingly, coexpression of the death antagonists Bcl-xL and Bcl-2 significantly increased their oncogenicity, whereas other NF-kappaB-regulated death inhibitors showed little or no effect. The fact that a subset of apoptosis inhibitors could rescue v-Rel transactivation mutants suggests that their reduced transcriptional activity may critically affect expression of defined death antagonists essential for oncogenesis. Consistent with this hypothesis, we observed selection for high endogenous expression of Bcl-2-related death antagonists in cells transformed by weakly transforming v-Rel mutants. These results emphasize the need for Rel/NF-kappaB to efficiently activate expression of a subset of antiapoptotic genes from the Bcl-2 family to manifest its oncogenic phenotype.

The transcription factor Relish controls Anaplasma marginale infection in the bovine tick Rhipicephalus microplus.

Rhipicephalus microplus is an important biological vector of Anaplasma marginale, the etiological agent of bovine anaplasmosis. The knowledge of tick immune responses to control bacterial infections remains limited. In this study, we demonstrate that transcription factor Relish from the IMD signaling pathway has an important role in the control of A. marginale infection in ticks. We found that RNA-mediated silencing of Relish caused a significant increase in the number of A. marginale in the midgut and salivary glands of R. microplus. In addition, the IMD pathway regulates the expression of the gene that encodes the antimicrobial peptide (AMP) microplusin. Moreover, microplusin expression was up-regulated in the midgut (2×) and salivary glands (8×) of A. marginale infected R. microplus. Therefore, it is plausible to hypothesize that microplusin may be involved in the A. marginale control. This study provides the first evidence of IMD signaling pathway participation on the A. marginale control in R. microplus.

Downregulation of microRNA­574 in cancer stem cells causes recurrence of prostate cancer via targeting REL.

miR­574­5p has been reported involved in the pathogenesis of numerous human malignancies such as colorectal and lung cancer. In this study, we aimed to explore the roles of REL and miR­574 in the recurrence of prostate cancer (PCa) and to identify the underlying molecular mechanisms. Our literature search found that miR­574 is regulated in cancer stem cells (CSCs), and next we used the microRNA (miRNA) database (www.mirdb.org) to find REL as a target of miR­574. Luciferase assay was performed to verify the miRNA/target relationship. Oligo-transfection, real­time PCR and western blot analysis were used to support the conclusions. We validated REL to be the direct gene via luciferase reporter assay system, and real­time PCR and western blot analysis were also conducted to study the mRNA and protein expression level of REL between different groups (recurrence and non­recurrence) or cells treated with scramble control, miR­574 mimics, REL siRNA and miR­574 inhibitors, indicating the negative regulatory relationship between miR­574 and REL. We also investigated the relative viability of prostate CSCs when transfected with scramble control, miR­574 mimics, REL siRNA and miR­574 inhibitors to validate miR­574 to be positively interfering with the viability of prostate CSCs. We then investigated the relative apoptosis of prostate CSCs when transfected with scramble control, miR­574 mimics, REL siRNA and miR­574 inhibitors. The results showed miR­574 inhibited apoptosis. In conclusion, miR­574 might be a novel prognostic and therapeutic target in the management of PCa recurrence.

Characterization of a human REL-estrogen receptor fusion protein with a reverse conditional transforming activity in chicken spleen cells.

Overexpression of the human REL transcription factor can malignantly transform chicken spleen cells in vitro. In this report, we have created and characterized a cDNA encoding a chimeric protein (RELDelta424-490-ER) in which sequences of a highly transforming REL mutant (RELDelta424-490) are fused to the ligand-binding domain of the human estrogen receptor (ER). Surprisingly, RELDelta424-490-ER is constitutively nuclear in A293 cells, and RELDelta424-490-ER activates transcription in the absence, but not in the presence, of estrogen in kappaB-site reporter gene assays. Furthermore, RELDelta424-490-ER transforms chicken spleen cells in the absence of estrogen, but the addition of estrogen blocks the ability of RELDelta424-490-ER-transformed cells to form colonies in soft agar, even though estrogen induces increased nuclear translocation of RELDelta424-490-ER in these cells. ERalpha can also inhibit REL-dependent transactivation in trans in an estrogen-dependent manner, and ERalpha can interact with REL in vitro. Thus, the RELDelta424-490-ER fusion protein shows an unusual, reverse hormone regulation, in that its most prominent biological activities (transformation and transactivation) are inhibited by estrogen, probably due to an estrogen-induced interaction between the ER sequences and sequences in the Rel homology domain. Nevertheless, these results indicate that the continual activity of REL is required to sustain the transformed state of chicken spleen cells in culture, suggesting that direct and specific inhibitors of REL may have therapeutic efficacy in certain human lymphoid cancers.

Envelope-dependent transactivation by the retroviral oncoprotein v-Rel is required for efficient malignant transformation of chicken spleen cells.

The retroviral oncoprotein v-Rel is a chimeric protein that has 11 helper virus-derived Envelope (Env) amino acids (aa) at its N terminus. Within these N-terminal Env aa of v-Rel there are three aa substitutions compared to the Rev-A helper virus Env. These aa substitutions have previously been shown to impart a number of unique properties onto v-Rel, including increased transforming and transactivating ability. In this study, we have analysed the sequence requirements for the Env aa to influence several properties of v-Rel. Phe residues at aa 3 and 9 are critical for an N-terminal transactivation function of v-Rel, and the analysis of several Env mutants demonstrates that transactivation ability parallels the transforming ability of v-Rel. Substitutions of conservative aa, such as leucine and tyrosine, for Phe 3 and 9 are tolerated for transactivation in chicken embryo fibroblasts and for transformation of chicken spleen cells. In contrast, the substitution of 10 Phe residues at the N terminus of v-Rel does not enable transactivation, indicating that a distinct structure surrounding Phe-3 and Phe-9 is essential for v-Rel function. We also show that the addition of the v-Rel Env aa to the N terminus of human c-Rel can enable it to activate transcription. Taken together, these results indicate that Phe residues at positions 3 and 9 have been selected for their ability to enhance the oncogenicity of v-Rel by increasing its ability to activate transcription.

Discreet mutations from c-Rel to v-Rel alter kappaB DNA recognition, IkappaBalpha binding, and dimerization: implications for v-Rel oncogenicity.

The avian Rev-T retrovirus encodes the oncoprotein v-Rel, a member of the Rel/nuclear factor (NF)-kappaB transcription factor family. The aggressive oncogenic potential of v-Rel has arisen from multiple mutations within the coding sequence of the avian cellular protein c-Rel. In this study, using quantitative biochemical experiments, we have tested the role of a limited set of alterations between v-Rel and c-Rel located within the Rel homology region (RHR) of the family that might confer functional differences. Our results show that only a set of six mutations within the RHR of v-Rel are responsible for its ability to bind to a broad spectrum of kappaB-DNA that are normally regulated by distinct NF-kappaB dimers. We also observe that both v-Rel homodimer and p50/v-Rel heterodimer bind IkappaBalpha weakly compared to other cellular Rel/NF-kappaB dimers with transcription activation potential. We suggest that the ability of v-Rel homodimer to deregulate subunit-specific gene expression and its ability to evade IkappaB inhibition are crucial to its strong oncogenic potential.

Inhibition of Rel/Nuclear Factor-kappaB signaling in skin results in defective DNA damage-induced cell cycle arrest and Ha-ras- and p53-independent tumor development.

In recent years a growth inhibitory role in skin for the Rel/NF-kappaB transcription factors has been established, and the block of Rel/NF-kappaB signaling results in rapid development of spontaneous skin cancer. The molecular mechanism underlying tumor development is however unknown. In the present study, we show that inhibition of NF-kappaB signaling in mouse skin by targeted expression of degradation resistant IkappaB-alpha generates transgenic keratinocytes unable to arrest the cell cycle in response to DNA damage induced by gamma-radiation. The results indicate that transgenic keratinocytes have a defect at the G1-S checkpoint whereas the G2-M checkpoint response was found to be intact. However, transgenic keratinocytes still respond by induction of the cyclin dependent kinase inhibitor p21(Cip1/Waf) after exposure to gamma-radiation. In the spontaneous skin tumors that develop in transgenic mice no mutations were found in the Ha-ras or p53 gene, suggesting that inhibition of NF-kappaB signaling in skin can induce cancer development independently of initiating mutations in the Ha-ras gene or additional mutations in the p53 gene. These findings demonstrate an involvement of NF-kappaB signaling in the DNA damage response and cell cycle checkpoint control in the skin.

Three mutations in v-Rel render it resistant to cleavage by cell-death protease caspase-3.

The retroviral oncoprotein v-Rel is a transcriptional activator in the Rel/NF-kappa B family. v-Rel causes rapidly fatal lymphomas in young chickens, and transforms and immortalizes chicken lymphoid cells in vitro. Several mutations that have enhanced the oncogenicity of v-Rel have been selected during in vitro and in vivo passage of v-Rel-containing retroviruses. In this report, we show that the C-terminal deletion and two point mutations (Asp-->Gly at residue 91 and Asp-->Asn at residue 437) in v-Rel make it resistant to cleavage by the cell-death protease caspase-3. In contrast, c-Rel, which has Asp residues at these sites, can be cleaved by caspase-3 in vitro as well as in vivo in cells induced to undergo apoptosis. We have characterized activities of v-Rel mutants with recreated single caspase-3 cleavage sites, two cleavage sites, or an introduced artificial cleavage site. All of these mutant v-Rel proteins are sensitive to caspase-3 cleavage in vitro, and show wild-type activity in terms of nuclear localization in chicken fibroblasts and DNA binding in vitro. Moreover, all caspase-3-sensitive v-Rel mutants transform chicken spleen cells in vitro and induce fatal lymphoid tumors in vivo to approximately the same extent as wild-type v-Rel. As with v-Rel mutants, caspase-3-resistant c-Rel mutants behave similarly to caspase-3-sensitive wild-type c-Rel in terms of DNA binding, transcriptional activation, in vitro transformation, and tumorigenicity. Mammalian c-Rel proteins can also be cleaved by caspase-3 in vitro, and a c-Rel mutant from a human pre-T lymphoma cell line is less sensitive than wild-type human c-Rel to cleavage by caspase-3. Taken together, these results demonstrate that specific mutations render oncogenic forms of Rel proteins resistant to cleavage by a cell-death caspase; however, the biological relevance of this resistance remains unclear. Nevertheless, to our knowledge, this is the first demonstration of mutations in caspase-3 recognition sites occurring during the evolution of an oncogenic protein.

The early stages of the immune response of the European abalone Haliotis tuberculata to a Vibrio harveyi infection.

Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone mortalities in France, Japan and Australia. In the European abalone, V. harveyi invades the circulatory system in a few hours after exposure and is lethal after 2 days of infection. In this study, we investigated the responses of European abalone immune cells over the first 24 h of infection. Results revealed an initial induction of immune gene expression including Rel/NF-kB, Mpeg and Clathrin. It is rapidly followed by a significant immuno-suppression characterized by reduced cellular hemocyte parameters, immune response gene expressions and enzymatic activities. Interestingly, Ferritin was overexpressed after 24 h of infection suggesting that abalone attempt to counter V. harveyi infection using soluble effectors. Immune function alteration was positively correlated with V. harveyi concentration. This study provides the evidence that V. harveyi has a hemolytic activity and an immuno-suppressive effect in the European abalone.

Dendritic cells conditionally transformed by v-relER oncogene express lymphoid marker genes.

The initiation of primary immune responses is the key function of specialized antigen presenting cells, the dendritic cells (DC). DC of myeloid origin capture antigens in tissues, migrate to lymphoid organs and stimulate T cell responses. A subset of DC has been described which expresses lymphoid determinants and has potential regulatory functions. Conditional transformation of chicken bone marrow progenitors with v-relER, a v-rel estrogen receptor (ER) fusion gene, allows expansion of progenitors that can be induced to differentiate into DC in vitro. In this paper we describe that v-relER cells exhibit both myeloid and lymphoid surface markers, while B cell, T cell and NK (natural killer)-specific surface markers are absent. v-relER DC express, however, cytoplasmic CD3 protein and mRNA for CD8alpha and the lymphoid transcription factor GATA-3. These data suggest that v-relER DC might be related to the lymphoid subset of DC described in mammals.

Participation of transcription factors from the Rel/NF-kappa B family in the circadian system in hamsters.

We have studied the presence and activity of components of the nuclear factor-kappaB (NF-kappaB) transcription factor in the hamster circadian system analyzing wheel-running activity, protein expression and DNA binding activity by electrophoresis mobility shift assays (EMSA). Non-rhythmic specific immunoreactive bands corresponding to a NF-kappaB subunit (p65) were found in hamster suprachiasmatic nuclei (SCN) homogenates. The active form of NF-kappaB evidenced by EMSA was clear and specific in SCN nuclear extracts. The administration of the NF-kappaB inhibitor pyrrolidine-dithiocharbamate (PDTC) blocked the light-induced phase advance at circadian time 18 (vehicle+light pulse: 2.08+/-0.46 h, PDTC+light: 0.36+/-0.35 h). These results demonstrate the presence and activity of Rel/NF-kappaB family proteins in the hamster SCN and suggest that these proteins may be related to the entrainment and regulation of circadian rhythms.