Differential regulation of immediate early gene expression in preadipocyte cells through multiple signaling pathways

Using digoxigenin (DIG)-based differential hybridization, a series of immediate early genes (IEG) was identified following the adipogenic stimulation in 3T3-L1 preadipocyte cells. Most of the known IEGs were identified as well as new members such as zf9 and Stra13. To delineate possible signaling pathways accounting for these gene expression, a subset of specific kinase inhibitors, SB203580, PD98059, rapamycin, LY294002, and Ro-32-0432, which inhibit p38 (HOG), MEK (MAPKK), S6 kinase, PI3 kinase, and protein kinase C (PKC), respectively, were employed. The IEGs were classified into three categories according to their susceptibility to the inhibitors. Expression of the first group (c-fos, jun-B, egr-1, tis11, tis21, thrombospondin-1, erp, thyroid hormone receptor [N-10], cyr61, and zf9) was mainly dependent on PKC and MEK pathways, while that of the second class (gene33 and tis10) exhibited an additional dependence on PI3 kinase pathways. The third one (Id-3, gly96, and Stra13) was characterized in that none of these inhibitors interfered with gene expression. Our results suggest that the induction of IEGs by the adipogenic stimuli is mediated by common as well as distinct signaling pathways.     

In vivo immediate early gene expression induced in intestinal and colonic mucosa by feeding.

Since the gut responds rapidly to food intake, the levels of expression of several immediate early genes were measured in mucosa from small and large intestine of rats starved for 3 days or refed. Within 1 h of refeeding, jejunal and ileal c-fos, jun B and zif/268 mRNA and colonic zif/268 dramatically increased. The zif/268 gene in jejunum corresponded in size to the full-length cDNA but, in ileum, an RNA band of about 1.2 kb in size increased greatly after feeding. This represents a physiologic in vivo model for the study of gene regulation associated with intestinal epithelial cellular responses to feeding.     

Transcriptional regulation of a herpes simplex virus immediate early gene is mediated through an enhancer-type sequence

An enhancer-type sequence has been identified in the promoter region for the herpes simplex virus type 1 (HSV-1) immediate early (IE) mRNA 3. The enhancer-type activity is host cell dependent, being greater in human cells and Syrian hamster cells (the usual host cell for in vitro propagation of HSV) than in Chinese hamster or mouse cells. Enhancer activity is stimulated up to 10-fold after superinfection by tsK, a temperature-sensitive mutant of HSV-1. The induction of enhancer activity is independent of de novo protein synthesis, showing that trans-activation is effected by a component of the virion. We propose that trans-regulation of IE mRNA 3 is mediated through an enhancer-type sequence, and that this provides one explanation for previously described regulation of HSV IE mRNAs by virion components.     

Unique signal transduction of Eyk: constitutive stimulation of the JAK-STAT pathway by an oncogenic receptor-type tyrosine kinase.

The proto-oncogene c-eyk, the cellular counterpart of a transforming oncogene, v-eyk, encodes a receptor protein tyrosine kinase with a distinctive extracellular region. We now demonstrate that c-Eyk can be constitutively activated through dimerization, and that the active Eyk displays a unique signaling pattern. When the kinase domain of c-Eyk was fused to the extracellular and transmembrane domains of CD8, the resulting chimera showed elevated kinase activity and caused cellular transformation. We found that the activated Eyk kinases, both v- and c-Eyk, constitutively stimulate the JAK-STAT pathway, while exerting little effect on other signaling routes such as the Ras-MAP kinase and the JNK pathways. The activated Eyk kinases specifically stimulate tyrosine phosphorylation of STAT1, STAT3 and JAK1. These downstream molecules also co-immunoprecipitate with the constitutively dimerized form of Eyk. The Eyk kinase activity is required for STAT1 stimulation. We found that the activation of STAT1 but not STAT3 correlates well with cellular transformation. In constitutively stimulating the JAK-STAT pathway, particularly STAT1, Eyk is unique in its downstream signaling and may be dependent on this pathway for cellular transformation.     

Abrogation of the antiproliferative activity of oncostatin M by a monoclonal antibody.

Oncostatin M (OM) is a novel cytokine which exhibits pleiotropic effects on a wide variety of normal and transformed cell lines. To determine some of the physiological functions of OM we have characterized several monoclonal antibodies to the recombinant molecule. Antibodies OM1 and OM2 bound native, but not denatured OM, suggesting they recognize non-contiguous epitopes. A third antibody, OM6, bound predominantly denatured OM. Of the two antibodies which detect discontinuous epitopes, OM2, but not OM1, was identified as a neutralizing antibody based on its ability to abrogate OM activity in the growth inhibition assay (GIA) and to inhibit OM binding in the radioreceptor assay (RRA). OM2 was equally effective in abrogating the functional effects of either natural or recombinant OM, thereby demonstrating that the active sites of these molecules are structurally similar, if not identical.     

Interleukin-10 receptor signaling through the JAK-STAT pathway. Requirement for two distinct receptor-derived signals for anti-inflammatory action.

Interleukin-10 (IL-10) is a cytokine that has pleiotropic effects on a variety of different cell types. Although many of the biologic responses induced by IL-10 are also induced by other cytokines, such as IL-6, IL-10 is relatively unique in its ability to potently inhibit production of pro-inflammatory cytokines in macrophages. In this study, we have used gain-of-function and loss-of-function genetic approaches to define the intracellular components involved in the different biologic actions of IL-10. Herein, we demonstrate that the ability of IL-10 to inhibit tumor necrosis factor alpha (TNFalpha) production in lipopolysaccharide-stimulated macrophages requires the presence of Stat3, Jak1, and two distinct regions of the IL-10 receptor intracellular domain. Macrophages deficient in Stat3 or Jak1 were unable to inhibit lipopolysaccharide-induced TNFalpha production following treatment with murine IL-10. Structure-function analysis of the intracellular domain of the IL-10 receptor alpha chain showed that whereas two redundant Stat3 recruitment sites (427YQKQ430 and 477YLKQ480) were required for all IL-10-dependent effects on either B cells or macrophages, expression of IL-10-dependent anti-inflammatory function required the presence on the intracellular domain of the IL-10 receptor of a carboxyl-terminal sequence containing at least one functionally critical serine. These results thus demonstrate that IL-10-induced inhibition of TNFalpha production requires two distinct regions of the IL-10 receptor intracellular domain and thereby establish a distinctive molecular basis for the developmental versus the anti-inflammatory actions of IL-10.     

Diptericin-like protein: an immune response gene regulated by the anti-bacterial gene induction pathway in Drosophila.

Insects produce various anti-microbial peptides in response to injury and infection. In Drosophila, diptericin has previously been studied as an anti-bacterial immune response gene. Here, we report the cloning of the diptericin-like protein (dptlp) gene as a paralog of Drosophila diptericin. By comparison of their sequences, we found that the dptlp gene has all of the functional domains conserved in the diptericin gene and other anti-bacterial proteins. The dptlp gene was rapidly induced by bacterial infections and showed different time-dependent gene expression patterns from those of diptericin. Like diptericin, dptlp was specifically produced from the fat body, and its expression was strictly dependent on bacterial infections. In addition, the dptlp gene expression was almost completely abolished in the imd mutant, which implicates that its expression is regulated by the anti-bacterial arm of the Drosophila innate immune regulatory pathways. In support of this, we found GATA, interferon consensus responding element, and kappa B binding sites, which is known to be important for the proper expression of anti-bacterial genes, in the proximal promoter region of the dptlp gene. Taken together, our findings support that dptlp is a novel anti-bacterial peptide whose expression is regulated by the anti-bacterial immune response mechanism.     

A novel type II membrane receptor up-regulated by IFN-alpha in fibroblasts functions in cell proliferation through the JAK-STAT signalling pathway

A type II membrane protein similar to CD69 (TIIMPSC) has been isolated in human embryo fibroblasts treated with IFN-alpha. Structural analysis and immunofluorescence detection has suggested that this protein is located on the surface of fibroblasts, generally considered, a receptor. Cell proliferation assay has revealed that activation of TIIMPSC elevates the level of fibroblast proliferation. Further, examination of signal transduction has indicated that expression of this protein is up-regulated by IFN-alpha stimulation, and that it is involved in the regulation of fibroblast growth through the JAK-STAT signalling pathway.