Dexamethasone Mediated Stabilization of Insulin Receptor mRNA

Abstract

To determine the mechanism of glucocorticoid mediated enhancement of insulin receptor (IR) gene expression, we cotransfected a glucocorticoid receptor expression vector and a plasmid containing a reporter gene driven by an MMTV or IR promoter into COS 7 cells. Dexamethasone (Dex) increased MMTV promoter activity by 100% but had no effect on IR promoter activity. In the glucocorticoid responsive breast cancer cell line, MCF-7, Dex increased IR mRNA by 60%, and increased the IR mRNA half-life from approximately 6hrs to >24hrs. No glucocorticoid responsive element could be located in the insulin receptor 3′ untranslated region. Glucocorticoids stabilize IR mRNA.     

A quantitative comparison of dual control of a hormone response element by progestins and glucocorticoids in the same cell line

Progesterone receptor-containing T47D human breast cancer cells are responsive to progestins but fail to respond to other steroid hormones, in particular dexamethasone, because they have no measurable levels of receptors for estrogens, androgens, or glucocorticoids. To quantitatively study dual responsiveness of the mouse mammary tumor virus (MMTV) promoter to progestins and glucocorticoids, we have stably transfected T47D cells with a glucocorticoid receptor (GR) expression vector. A cloned derivative (A1-2) was isolated that expresses a normal, full length GR, as assessed by steroid binding and Western immunoblot with a monoclonal anti-GR antibody. Moreover, GR is expressed at levels (80,000-100,000 molecules per cell) comparable to the high levels of endogenous progesterone receptor (200,000 molecules per cell). In A1-2 cells transiently transfected with an MMTV-chloramphenicol acetyl transferase reporter gene, induction by glucocorticoid was substantially greater (5-fold) than induction mediated by progestins. These results suggest that glucocorticoids may be the primary regulator of MMTV.     

Introduction of the glucocorticoid binding sequences into the expression vector p delta SVE-CAT and its effect on the CAT gene expression in mammalian cells.

We have demonstrated that synthetic oligonucleotide representing glucocorticoid responsive element (GRE I) of MMTV inserted into the enhancerless early promoter of SV40 in p delta SVE-CAT expression vector, enhances transient expression of chloramphenicol acetyltransferase gene in HeLa and hepatoma cells cultivated in the presence of dexamethasone. The following changes in the structure of the core sequences (GTTACAAACTGTTCT) of the synthesized GRE eliminated its enhancing ability: i, changes in the left end of the core sequences from GTTACAAAATGTTCT to TCTTCAAACTGTTCT or to TACTCAAACTGTTCT; ii, the increase of gap between TGTTCT and the inverted repeat of this sequence. The above changes did not eliminate specific binding of glucocorticoid receptor to the synthetic oligonucleotides studied.     

A novel promoter sequence is involved in the oxidative stress-induced expression of the adult T-cell leukemia-derived factor (ADF)/human thioredoxin (Trx) gene.

Adult T cell leukemia-derived factor (ADF) is a human thioredoxin (Trx) and is a disulfide reducing protein with various biological functions. We found that expression of the ADF/Trx gene was increased by oxidative agents such as hydrogen peroxide, diamide and menadione in Jurkat cells. Analysis using a CAT expression vector plasmid under the control of the ADF/Trx gene promoter revealed that CAT gene expression in Jurkat cells was increased after exposure to oxidative agents. A series of deletion analyses showed that a region from -976 to -890 of the 5' flanking sequence was required for enhancement of ADF/Trx promoter activity against the oxidative agents. Gel mobility shift assay revealed the specific DNA binding activities to the sequences from -953 to -930 in the nuclear extracts from the Jurkat cells. The sequences in this region showed no homology with any known consensus sequences for DNA binding factors. It is suggested that ADF/Trx gene expression is enhanced through a novel cis-acting regulatory element responsive for the oxidative stress and a new factor(s) is involved in this oxidative stress responsive element.     

Glucocorticoid hormone interactions with cloned proviral DNA of mouse mammary tumor virus

The molecular details of glucocorticoid hormone regulation of expression of the mouse mammary tumor virus (MMTV) proviral gene have been investigated. Cloned proviral DNA was introduced into cultured cells by a gene transfer procedure. DNA acquired by transfection was shown to be expressed in a hormone regulated fashion. The proviral DNA was fragmented and recombined in vitro with an indicator gene to delimit the hormone response sequence. Inducibility of the indicator gene (thymidine kinase gene from Herpes Simplex Virus, tk) was observed upon recombination with the long terminal repeat (LTR) sequence of MMTV. Further delimitation of the LTR DNA demonstrated that 202 nucleotides located 5' of the RNA initiation site are sufficient to confer glucocorticoid regulation. In vitro interaction of LTR DNA with glucocorticoid hormone receptor complex, showed a preferential affinity to the same sequence which mediated hormonal regulation in transfected cells. Evidence for a direct receptor gene interaction in the process of gene induction was gained by the measurement of the kinetics of induction and the use of a glucocorticoid antagonist (RU 486). The induction of the transfected gene is very rapid, independent of simultaneous protein synthesis and requires a functional glucocorticoid receptor hormone complex.