Up-regulation of the phosphoinositide 3-kinase pathway in human lamina propria T lymphocytes.

Human intestinal lamina propria T lymphocytes (LPT), when investigated ex vivo, exhibit functional properties profoundly different from those of peripheral blood T lymphocytes (PBT). One prominent feature represents their enhanced sensitivity to CD2 stimulation when compared to PBT. Given that LPT are hyporesponsive to T cell receptor (TCR)/CD3 stimulation, an alternative activation mode, as mimicked by CD2 triggering in vitro, may be functional in mucosal inflammation in vivo. This study provides insight into signalling events associated with the high CD2 responsiveness of LPT. When compared to PBT, LPT show an increased activation of the phosphoinositide 3/protein kinase B/glycogen synthase kinase 3beta (PI3-kinase/AKT/GSK-3beta) pathway in response to CD2 stimulation. Evidence is provided that up-regulation of this pathway contributes to the enhanced CD2-induced cytokine production in LPT. Given the importance of TCR-independent stimulation for the initiation of intestinal immune responses analysis of signalling pathways induced by 'co-stimulatory' receptors may provide valuable information for therapeutic drug design.

Cyclosporin A-resistant transactivation of the IL-2 promoter requires activity of okadaic acid-sensitive serine/threonine phosphatases.

Expression of the IL-2 gene requires activation of T cells through stimulation of the TCR and costimulation through accessory receptors. We have found recently that okadaic acid-sensitive Ser/Thr phosphatases are involved in a cyclosporin A-insensitive pathway that selectively transmits costimulatory signals. In this study, we analyzed whether activities of these phosphatases are necessary for the expression of the IL-2 gene. In both activated peripheral blood T lymphocytes and activated tumorigenic T cell lines, IL-2 gene expression was blocked at the transcriptional level by okadaic acid. The transcription factors active at the IL-2 promoter were differentially influenced: upon down-modulation of okadaic acid-sensitive phosphatases, transactivation by octamer, NF-kappa B, and NF of activated T cells proteins was abrogated, while transactivation by AP-1 proteins was even enhanced.

A dominant mechanism coordinately suppresses the expression of Th2 lymphokines.

This study, we present evidence for a negatively acting control mechanism that coordinately suppresses the synthesis of the Th2 lymphokines IL-4, IL-5 and IL-6. This control mechanism operates in the murine thymoma cell line BW 5147. When cells of this line were fused to four independently established, well-defined Th2 cell clones, all resulting 74 lymphokine-secreting hybridomas secreted IL-2 which was not secreted by any of the parental Th2 cell clones. Most interestingly, however, none of the 74 hybridomas retained the capacity of the parental Th2 cells to express IL-4. Likewise, the secretion of IL-5 and IL-6 was also suppressed. Obviously, BW 5147 cells dominated the pattern of lymphokines produced, although the lymphokine pattern of Th2 cells was previously considered to be irreversibly fixed due to terminal differentiation of these cells. Suppression of IL-4 production was also observed at the mRNA level, as tested in Northern blot assays. Putative DNA target sequences for suppression of IL-4 gene transcription were not part of the proximal IL-4 promotor regions. Remote DNA control sequences may exist which coordinately regulate the proper, stage-specific expression of the Th2 lymphokines IL-4, IL-5 and IL-6.

Dephosphorylation of serine 3 regulates nuclear translocation of cofilin.

Signal transduction processes in T-cells and other cell types alter the phosphorylation state of cofilin, an actin-binding phosphoprotein. Whether reversible phosphorylation is responsible for the regulation of the functional activities of cofilin is not clear at present. Here we have identified the phosphoacceptor site of cofilin and analyzed the role of cofilin phosphorylation with respect to its subcellular localization. Site-directed mutagenesis studies show that phosphorylation occurs exclusively on Ser-3. Expression of non-phosphorylatable mutant cofilin proteins in NIH3T3 cells and determination of their subcellular localization by confocal laser scanning microscopy reveal that non-phosphorylated cofilin accumulates within nuclei. This analysis shows that the subcellular localization of cofilin depends on the phosphorylation state of Ser-3.

NFI/X proteins: a class of NFI family of transcription factors with positive and negative regulatory domains.

NFI is a family of transcription factors that binds discrete nucleotide sequence and regulates transcriptional activity of genes. Transactivation occurs through proline-rich sequences. Among the genes whose transcriptional activity is regulated are those expressed in a cell-type specific manner. This suggests the possible existence of NFI proteins with distinct properties. With the use of the polymerase chain reaction technique, we have cloned and analyzed the activity of three spliced variants of NFI (class X) genes in murine cells. Expression vectors containing the regulatory regions of these proteins fused to the DNA binding domain of the yeast transcription factor GAL4 helped identify both negative and positive regulatory domains. In the context of the whole NFI/X proteins, spliced variants lacking the positive regulatory sequences functioned as repressors, whereas those containing both regulatory domains functioned as weak activators. With the identification of the negative domain in the NFI/X proteins, we demonstrate here a novel regulatory feature of these proteins in positive and negative modulation of gene expression.

Post-transcriptional down-regulation of expression of transcription factor NF1 by Ha-ras oncogene.

NF1 is a family of polypeptides that binds to discrete DNA motifs and plays varying roles in the regulation of gene expression. These polypeptides are also thought to mediate the expression of differentiation-specific markers such as adipocyte and mammary cell type-specific genes. The expression of a number of cellular differentiation-specific markers is down-regulated during neoplastic transformation. We therefore investigated whether oncogenic transformation interferes with the action of NF1. Stable transfection of activated Ha-ras into a number of murine cells correlated with a down-regulation of the expression of the NF1 genes NF1/CTF and NF1/X. The down-regulation was not at the transcriptional level but at the level of stability of the NF1 mRNAs. The level of the DNA binding activity of the NF1 proteins was also reduced in Ha-v-ras-transformed cells, and the expression of a gene that depends on this family of transcription factors was specifically repressed. These results demonstrate that an activated Ha-ras-induced pathway destabilizes the half-life of mRNAs encoding specific members in the NF1 family of transcription factors, which leads to a decrease in NF1-dependent gene expression.

Regulation of expression of mouse mammary tumor virus through sequences located in the hormone response element: involvement of cell-cell contact and a negative regulatory factor.

Mouse mammary tumor virus (MMTV) is a latently oncogenic retrovirus responsible for the neoplastic transformation of mammary epithelial cells. Its expression is regulated by steroids, polypeptide growth factors, and cell-type-specific factors. Using GR mouse mammary cells and NIH 3T3 fibroblasts stably transfected with chimeric constructs of the long terminal repeat region of MMTV, we have demonstrated a novel mechanism of cell-type-specific expression of this virus. In confluent monolayer cultures that permit cell-cell interaction, MMTV long terminal repeat expression is positively regulated by sequences within the hormone response element (HRE) that bind the transcription factors CTF/NFI and OTFI. Although these factors are present in NIH 3T3 cells, MMTV expression in these cells is not regulated by cell density. This is partially due to a negative regulatory factor that binds sequences between -164 and -151 in the HRE. Mutations that destroy the binding site for this factor restored in part the cell density-regulated expression of MMTV to NIH 3T3 fibroblasts. The HRE is thus a central coordinator of regulatory pathways that positively or negatively influence the expression of MMTV.