Expression profile of nuclear receptors upon Epstein -- Barr virus induced B cell transformation.

BACKGROUND: Infection of human B cells with Epstein - Barr virus (EBV) induces metabolic activation, morphological transformation, cell proliferation and eventual immortalization. AIM: To identify the nuclear receptors, which are the cellular interaction partners of EBNAs, that will help to elucidate the mechanism of B cell transformation. METHODS: We have compared the nuclear receptor profile in the naïve and EBV-transformed B-lymphocytes, using TaqMan LDA microfluidic card technology. RESULTS: Out of 48 nuclear receptor, 17 showed differential expression at the mRNA level. The expression of 5 genes was elevated in EBV-transformed cells, whereas 12 genes were downregulated in lymphoblastoid cells (LCLs). 7 genes were studied at the protein level; 2 genes were up regulated (Nr2F2 and RARA) and 4 genes were down regulated (ERB, NUR77, PPARG, and VDR) in LCLs. CONCLUSION: The nuclear receptor profiling on EBV infected B cells showed alterations of nuclear receptors expression at both mRNA and protein levels compared with non infected peripheral blood cells. Further analysis on a possible role of each nuclear receptor in EBV induced cell transformation should be performed.

Gastrointestinal inflammation: lessons from metabolic modulators.

The link between inflammation and metabolism was apparent already early last century, but has recently been revitalized following molecular studies of atherosclerosis, obesity and insulin resistance. A growing list of nuclear receptors, pivotal players in lipid, xenobiotic and energy metabolism has been identified as having immunomodulatory functions. These receptors might hold the key to some of the questions pertinent to chronic inflammation, and can lend themselves to be manipulated as therapeutic agents. This review will attempt to appraise the importance of such mediators in the pathophysiology of chronic inflammation in the colon.

A completely foreign receptor can mediate an interferon-gamma-like response.

A tripartite receptor comprising the external region of the erythropoietin (Epo) receptor, the transmembrane and JAK-binding domains of the gp130 subunit of the interleukin-6 (IL-6) receptor, and a seven amino acid STAT1 recruitment motif (Y440) from the interferon (IFN)-gamma receptor, efficiently mediates an IFN-gamma-like response. An analogous completely foreign chimeric receptor in which the Y440 motif is replaced with the Y905 motif from gp130 also mediates an IFN-gamma-like response, but less efficiently. The IFNGR1 signal-transducing subunit of the IFN-gamma receptor is tyrosine phosphorylated through the chimeric receptors and the endogenous IL-6 and OSM receptors. Cross phosphorylation of IFNGR1 is not, however, required for the IFN-gamma-like response through the chimeric receptors, nor does it mediate an IFN-gamma-like response to IL-6 or OSM. The data argue strongly for modular JAK/STAT signalling and against any rigid structural organization for the "pathways" involved. They emphasize the likely high degree of overlap between the signals generated from disparate JAK-receptor complexes and show that relatively minor changes in such complexes can profoundly affect the response.

Inhibition of activated/memory (CD45RO(+)) T cells by oxidative stress associated with block of NF-kappaB activation.

Impaired immune responses in cancer patients have been associated with oxidative stress. Increased levels of reactive oxygen species released from activated, tumor-infiltrating macrophages or granulocytes may therefore constitute a hurdle for effective immunotherapy against cancer. In this study, we investigated functional consequences and molecular events in T cells exposed to low levels of oxidative stress. We observed that cytokine production of human PBMC, upon stimulation with an HLA-A*0201-restricted influenza peptide and nonspecific receptor cross-linking, was reduced after exposure to micromolar levels of H2O2. Functional impairment as measured by IFN-gamma release occurred earlier and at lower doses of exogenously added H2O2 than required to induce apoptosis. This suggests that there is a dose window of oxidative stress leading to T cell unresponsiveness in the absence of apoptosis. The reduction of Th1 cytokines, induced by H2O2, was predominantly observed in memory/effector (CD45RO(+)) T cells and correlated with a block in NF-kappaB activation. IL-10 production was more profoundly influenced by low doses of H2O2 than IFN-gamma, TNF-alpha, and IL-2. The influence of H2O2 on production of IL-10 was not significantly different between memory/activated and naive T cells. These observations suggest that Th1 and Th2 cytokines are differently regulated under conditions of oxidative stress. Taken together, these findings may explain why Ag-experienced, CD45RO(+), T cells found in the tumor milieu are functionally suppressed.

The enhancer shift: a model to explain the developmental control of IgH gene expression in B-lineage cells.

The roles attributed to the E mu enhancer and the downstream 3' regions acting as locus control regions (LCRs) have allowed comparisons to be drawn between regulation of the IgH locus in early and late B-cell development and other systems governed by LCR activity. Here, the importance of the IgH 3'LCR and its putative functional role in the control of IgH gene expression is evaluated and compared with the 5'LCR regulatory region of the human beta-globin locus.

Concomitant downregulation of IgH 3' enhancer activity and c-myc expression in a plasmacytoma x fibroblast environment: implications for dysregulation of translocated c-myc.

Regulation of immunoglobulin heavy chain (IgH) gene expression is controlled by a B cell-specific promoter, intronic enhancer and additional B cell-specific enhancer elements identified recently in the 3' end of the IgH locus. One of the latter elements, the IgH 3' enhancer, is of particular interest: (1) it is B cell-specific and active only in late B cell development; (2) in rodent plasmacytomas and in some human Burkitt's lymphomas it is part of a locus control region (LCR) that is involved in deregulation of the c-myc oncogene as a result of translocation into the IgH locus; and (3) it has been implicated in the mechanisms that control Ig gene class switch recombination. We have used a somatic cell hybridization approach to genetically analyse regulation of the activity of the IgH 3' enhancer. When mouse MPC11 plasmacytoma cells, in which the IgH 3' enhancer is active, are fused with fibroblasts, Ig expression is extinguished at the level of transcription. Here we show that in a MPC11 plasmacytoma x fibroblast environment, the IgH 3' enhancer is transcriptionally inactive. Furthermore, we demonstrate that binding of several B cell-specific transcription factors, essential for IgH 3' enhancer activity, is lacking, which may explain 3' enhancer inactivity, although the binding of repressors cannot be excluded. Moreover, the high expression level of c-myc, characteristic of the parental MPC11 cells carrying the t(12;15) translocation, is down-regulated in the hybrids to that in unfused fibroblasts. Therefore, inactivation of the IgH 3' enhancer is a multifactorial process affecting several transcription factors that control the cell-specific and developmental activity of the enhancer.

Temporal control of IgH gene expression in developing B cells by the 3' locus control region.

The suggested roles of the downstream 3' regions acting as a Locus Control Region (LCR), have allowed comparisons to be made between the regulation of the IgH locus with other model systems whose gene expression is governed by LCR activity. Here we summarize the importance of the IgH 3'LCR and its putative functional role in IgH gene expression and compare it with the 5'LCR regulatory region of the human beta-globin locus.

A T cell controlled molecular pathway regulating the IgH locus: CD40-mediated activation of the IgH 3' enhancer.

Immunoglobulin heavy chain (IgH) class switch recombination and regulation of IgH expression levels are processes suggested to be controlled by the IgH 3' enhancer. Here we demonstrate that CD40 or IgM receptor stimulation of primary B cells results in transactivation of this enhancer. 4-Hydroxy-3-nitrophenylacetyl (NIP)-BSA induction of a K46 B cell line expressing a chimeric NIP-specific CD40 single chain receptor results in a ligand receptor-dependent response of a 3' enhancer ETS/AP-1 minimal promoter construct. Gel retardation analysis and genomic footprinting experiments reveal that CD40 or IgM induction recruits NFAB (nuclear factors of activated B cells) to the ETS/AP-1 motif. While IgM signalling recruits c-Fos, JunB and Elf-1 (NFAB-I), only JunB and Elf-1 were observed following CD40 signalling (NFAB-II). CD40 signalling, however, induces a Fos family-related partner for JunB, which may account for the transcriptional activity observed by NFAB-II in K46 cells. We propose a model whereby CD40 and IgM receptor-mediated signalling converge in the process of 3' enhancer activation in B lymphocytes. Our data provide a putative molecular explanation as to why CD40L-deficient mice, and possibly patients with hyper-IgM syndrome, are unable to undergo T cell-dependent class switch recombination but respond properly upon lipopolysaccharide-induced switch recombination.

Elevated expression levels of an Ig transgene in mice links the IgH 3' enhancer to the regulation of IgH expression.

To delineate the role of the IgH 3' enhancer in the regulation of Ig heavy (IgH) chain gene expression, mice harbouring rearranged IgH transgenes, with (PSVmu3) and without (PSVmu1) this element, were produced. RNA and protein analysis from the different transgenic lines revealed a 5- to 7-fold increase in the expression level of the transgene containing the IgH 3' enhancer. This difference is also reflected at the protein level in hybridomas generated from the two transgenic lines. The elevation of transgene Ig expression in the PSVmu3 lines is restricted to activated B lymphocytes, an observation which is further supported by the ability of this transgene to be reactivated upon immunization. Interestingly, although the up-regulation of transgene expression in PSVmu3 animals is considerably higher in comparison to the PSVmu1 animals, a significant response is still observed in the PSVmu1 mice. We speculate therefore that the IgH locus is subject to transcriptional modification in late B cell development. Our data suggest that both the Emu enhancer and the IgH 3' enhancer can up-regulate transgene Ig expression, but the presence of the 3' enhancer results in elevated levels of transgene Ig production. It therefore appears that the expression level of IgH genes is subject to transcriptional modification during B cell development. Additional control elements are most likely required for optimal Ig expression, since our expression data from the transgene in PSVmu3 animals are incompatible with endogenous Ig levels. The recent identification of additional enhancer elements in the far 3' end of the IgH locus supports this possibility. The data presented here provides a sound basis for the production of high levels of mAb, possibly tailored to suit the needs of the researcher.

Aberrant regulation of the IgH 3' enhancer by c-myc in plasmacytoma cells.

The identification of enhancers at the 3' end of the IgH locus has prompted a re-evaluation of the regulation of Ig gene expression. Moreover, these elements may provide a possible explanation as to how the c-myc oncogene becomes dysregulated upon translocation into the IgH locus with the IgH intragenic enhancer on the reciprocal chromosome. These 3' enhancers have also been shown to redirect promoter utilization on c-myc reporter gene constructs in transient transfection experiments. This region of the locus also contains the B cell specific IgH 3' enhancer. This temporally regulated enhancer has been implicated in the mechanisms that control class switch recombination. Here we demonstrate that overexpression of the c-myc protein in mouse plasmacytoma cells (MPC-11) and HeLa cells can transcriptionally upregulate a reporter gene construct driven by a subregion (domain C) of the IgH 3' enhancer. Domain C contains a functional dual symmetry E-box motif, CACGTG. The DNA binding experiments demonstrate that USF was the major factor interacting with this motif. Based on these observations, we speculate that the c-myc protein may upregulate expression of translocated c-myc in mouse plasmacytomas possibly via an USF-binding E-box motif in the IgH 3' enhancer.

Constitutive function of the basic helix-loop-helix/PAS factor Arnt. Regulation of target promoters via the E box motif.

Arnt is a nuclear basic helix-loop-helix (bHLH) transcription factor that, contiguous with the bHLH motif, contains a region of homology (PAS) with the Drosophila factors Per and Sim. Arnt dimerizes in a ligand-dependent manner with the bHLH dioxin receptor, a process that enables the dioxin-(2,3,7,8-tetrachlorodibenzo-p-dioxin)-activated Arnt-dioxin receptor complex to recognize dioxin response elements of target promoters. In the absence of dioxin, Arnt does not bind to this target sequence motif. The constitutive function of Arnt is presently not understood. Here we demonstrate that Arnt constitutively bound the E box motif CACGTG that is also recognized by a number of distinct bHLH factors, including USF and Max. Importantly, amino acids that have been identified to be critical for E box recognition by Max and USF are conserved in Arnt. Consistent with these observations, full-length Arnt, but not an Arnt deletion mutant lacking its potent C-terminal transactivation domain, constitutively activated CACGTG E box-driven reporter genes in vivo. These results indicate a role of Arnt in regulation of a network of target genes that is distinct from that regulated by the Arnt-dioxin receptor complex in dioxin-stimulated cells.

Lipopolysaccharide-dependent transactivation of the temporally regulated immunoglobulin heavy chain 3' enhancer.

To execute different biological functions, the expression pattern of immunoglobulin heavy chain genes (IgH) is altered during B lymphocyte differentiation. Early in B cell differentiation, it is assumed that the heavy chain promoter and the intragenic enhancer (E mu) ensure VDJ recombination. This leads to the expression of the immunoglobulin receptor on the cell surface. An additional strong enhancer in the far 3' end of the IgH locus has, however, prompted a re-evaluation of the regulation of immunoglobulin gene expression. To define the temporal and spatial regulation of the IgH 3' enhancer, transgenic mice harboring an enhancer-dependent reporter gene construct were generated. Here we demonstrate that IgH 3' enhancer activity is largely restricted to activated immunocompetent B cells. Furthermore, the enhancer can be transactivated following mitogen stimulation with lipopolysaccharide and 12-O-tetradecanoylphorbol 13-acetate. We propose a model whereby 3' enhancer activation is linked to the activation of resting immunocompetent B cells. The implications of the enhancer being active in late B lymphocyte differentiation, when heavy chain class switching occurs, are discussed.

Identification of Ets-like lymphoid specific elements within the immunoglobulin heavy chain 3' enhancer.

Recently we identified an additional enhancer in the 3' end of the immunoglobulin heavy chain (IgH) locus. To identify individual regulatory elements within the rat IgH 3' enhancer, deletion analysis was performed. Transfection experiments using reporter constructs suggest that the enhancer contains three functionally distinct domains, two of which are lymphoid specific and one domain is active in both lymphoid and in nonlymphoid cells. The three domains together contribute to enhancer function and act synergistically. Further analyses suggest that a putative mu E1 site, octanucleotide motif, mu E3 site, and mu B/Ets-like motif are important for the overall transcriptional activity of the IgH 3' enhancer. Moreover, we provide evidence that an additional Ets-like element, micro A, is involved in the tissue specific regulation of enhancer activity and that binding of a protein to this element correlates with the transcriptional activity of one of the lymphoid restricted domains.