Multiple promoters exist in the human GR gene, one of which is activated by glucocorticoids.

Department of Biochemistry and Molecular Biology and the Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Molecular Endocrinology (Impact Factor: 4.2). 09/2001; 15(8):1381-95.
Source: PubMed

ABSTRACT A new human GR gene sequence (hGR 1Ap/e), which is distinct from the previously identified human GR promoter and coding sequences, has been isolated and characterized. The hGR 1Ap/e sequence is approximately 31 kbp upstream of the human GR coding sequence. This sequence (2,056 bp) contains a novel promoter (the hGR 1A promoter; 1,075 bp) and untranslated exon sequence (hGR exon 1A sequence; 981 bp). Alternative splicing produces three different hGR 1A-containing transcripts, 1A1, 1A2, and 1A3. GR transcripts containing exon 1A1, 1A2, 1B, and 1C are expressed at various levels in many cancer cell lines, while the exon 1A3-containing GR transcript is expressed most abundantly in blood cell cancer cell lines. Glucocorticoid hormone treatment causes an up-regulation of exon 1A3-containing GR transcripts in CEM-C7 T-lymphoblast cells and a down-regulation of exon 1A3-containing transcripts in IM-9 B-lymphoma cells. Deoxyribonuclease I footprinting using CEM-C7 cell nuclear extract reveals four footprints in the promoter region and two intraexonic footprints. Much of the basal promoter-activating function is found in the +41/+269 sequence, which contains two deoxyribonuclease I footprints (FP5 and FP6). When this sequence is cloned into the pXP-1 luciferase reporter gene, hormone treatment causes a significant increase in luciferase activity in Jurkat T cells that are cotransfected with a GR expression vector. FP5 is an interferon regulatory factor-binding element, and it contributes significantly to basal transcription rate, but it is not activated by steroid. FP6 resembles a glucocorticoid response element and can bind GRbeta. This novel hGR 1Ap/e sequence may have future applications for the diagnosis, prognosis, and treatment of T-cell leukemia and lymphoma.

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    ABSTRACT: Glucocorticoids (GC) play essential role in the regulation of the maturation and activation processes in T-cells, regarding especially apoptosis mechanisms. The mutual antagonism between GC signals and T-cell receptor (TcR) derived signals concerned to be crucial in thymocyte selection. The non-genomic actions of GCs are less characterised yet, but based on literature data the existence of cross-talk mechanisms between GC and TcR signal transduction pathways are feasible. We aimed in our work to reveal rapid, non-genomic GC signal transduction mechanisms in T-cells using in vitro cultured Jurkat T-cell leukaemia as a model cell line. Our results show that glucocorticoid receptor (GR) agonist Dexamethasone (DX) causes changes in tyrosine phosphorylation pattern of resting and activated Jurkat cells. These GC effects occurred rapidly, within 5 minutes. ZAP-70 kinase has a crucial role in signal transduction originating from the TcR-CD3 complex. ZAP-70 deficient T-cells fail to respond antigenic stimuli in vitro, and ZAP-70 deficiency in humans and mice results in Severe Combined Immunodeficiency (SCID syndrome). We demonstrate that DX treatment induces rapid ZAP-70 phosphorylation in both resting and activated Jurkat cells. DX induced ZAP-70 phosphorylation is inhibited by GR antagonist (RU486) pre-treatment suggesting the process is GR dependent. However, DX fails to trigger ZAP-70 phosphorylation in p56-lck deficient JCaM-1 cells proposing the involvement of the upstream src-family kinase p56-lck. We investigated the close physical relation of GR and ZAP-70 by coimmunoprecipitation and confocal microscopy. We show, that the ligand-bound GR associates with ZAP-70 in both Jurkat cells and HeLa/trZAP-70 cells stably expressing transgenic ZAP-70. Examining the role of Hsp-90 we found, that a presumably inactive ZAP-70 fraction is associated with Hsp-90 which is most likely excluded from this signal transduction process. The association of ZAP-70 with phosphorylated ITAM tyrosines of the CD3 complex is indispensable for the appropriate transmission of signals derived from the TcR. Our results demonstrate that the association of ZAP-70 with the CD3 complex is inhibited in the DX treated samples. Based on our experimental data we suggest the following signal transduction model: GR, in the presence of its ligand, associates with ZAP-70, triggering tyrosine phosphorylation events. The alteration of tyrosine phosphorylation may influence the kinase activity of ZAP-70 or affect the clustering of other signal transduction molecules with ZAP-70. The GR association with ZAP-70 also inhibits its binding to the ITAMs of the CD3 complex. Recently GCs are widely used as immunosuppressive drugs although their sideeffects are often serious. We revealed here a new non-genomic signal transduction mechanism which may have a crucial role in GC mediated immunosuppression. The characterisation of this new non-genomic pathway may make possible the design and synthesis of new molecules which targets exclusively this immunosuppressive process. In the future this may contribute to the application of new immunosuppressive agents with more favourable side-effect spectra.
    09/2007, Degree: PhD, Supervisor: Timea Berki

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