Glucocorticoid Receptor Mutants That Define a Small Region Sufficient for Enhancer Activation

Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that is subject to regulation by hormonal and nutritional signals. In mammalian tissues such as skeletal muscle, glucocorticoid hormones specify a catabolic response that influences both protein synthetic and protein degradative pathways. With regard to the former, glucocorticoids attenuate mRNA translation at two levels: translational efficiency, i.e. translation initiation, and translational capacity, i.e. ribosome biogenesis. Glucocorticoids may impair translational capacity through the ribosomal S6 protein kinase (p70 S6K), a recognized glucocorticoid target and an effector of ribosomal protein synthesis. We demonstrate here that the reduction in growth factor-activated p70 S6K activity by glucocorticoids depends upon a functional glucocorticoid receptor (GR) and that the GR is both necessary and sufficient to render p70 S6K subject to glucocorticoid regulation. Furthermore, the DNA binding and transcriptional activation but not repression properties of the GR are indispensable for p70 S6K regulation. Finally, a mutational analysis of the p70 S6K carboxyl terminus indicates that this region confers glucocorticoid sensitivity, and thus glucocorticoids may facilitate autoinhibition of the enzyme ultimately reducing the efficiency with which T389 is phosphorylated.

Section: Transcriptional Scenarios Involved In Regulation Of P70 S6ksupporting

confidence: 51%

The Activated Glucocorticoid Receptor Modulates Presumptive Autoregulation of Ribosomal Protein S6 Protein Kinase, p70 S6K

Shah

Iñiguez‐Lluhí

Romanelli

et al. 2002

“…Several cultures were tested from each stage of the tumorigenic process: normal fibroblasts (23784, 40950); mild fibromatosis (14249, 39614, 27877); aggressive fibromatosis (BPV3, BPV7, BPV21); fibrosarcomas (BPV1, BPV22, BPV2, BPV11). For experiments involving steroid hormone treatments, cells were maintained in medium containing charcoal-stripped serum (24). COS-7 cells were also maintained as above.…”

Section: Methodsmentioning

confidence: 99%

Glucocorticoids Inhibit Apoptosis during Fibrosarcoma Development by Transcriptionally Activating Bcl-xL

Gascoyne

Kypta

Vivanco

2003

Glucocorticoids influence many physiological processes, and in particular apoptosis, often with opposite effects depending on the cell type examined. We found that during fibrosarcoma development there is a strong increase in apoptosis at the tumor stage, which is repressed by dexamethasone to levels observed in normal fibroblasts. The anti-apoptotic Bcl-2 family protein Bcl-x L is induced by dexamethasone at the transcriptional level at all stages of fibrosarcoma development. The ligand-activated glucocorticoid receptor (GR) activates the Bcl-x promoter in transient transfection experiments, and GR binds to specific Bcl-x promoter sequences in vitro and in vivo. Furthermore, a GR antagonist abolishes this effect, indicating that Bcl-x L induction is mediated by GR. Importantly, exogenous Bcl-x L inhibits apoptosis and caspase-3 activity in fibrosarcoma cells to levels found in dexamethasone-treated fibrosarcoma cells. We conclude that Bcl-x L is a key target mediating the anti-apoptotic effects of glucocorticoids during fibrosarcoma development. These observations provide further understanding of the molecular basis of glucocorticoid regulation of cell death during tumorigenesis.

“…The XbaI and XhoI restriction sites were filled in by incubation with Klenow fragment before digestion with SalI. pSVL407-795 was made by removing the 407-795 sequence as a BamHI fragment from VARO407-795 (23) and inserting it into the empty pSVLT vector obtained by BamHI digestion of pSVLT407-556 (24). pSVLGR(R732Q) was constructed by replacing the BstBI/PflMI fragment from pSVLGR with the same restriction fragment from dhfr550C(R732Q) (21).…”

Section: Chemicals-chemicalsmentioning

confidence: 99%

Induction Properties of a Transiently Transfected Glucocorticoid-responsive Gene Vary with Glucocorticoid Receptor Concentration

Szapary

Simons

1996

Transient transfections of steroid receptors have yielded much of the data used to construct the current models of steroid hormone action. These experiments invariably examine the ability of receptors to regulate transcription when occupied by saturating concentrations of steroid. We now report that other induction properties of a transiently transfected gene are not constant but vary with the concentration of transiently transfected glucocorticoid receptors. Thus, the percentage of maximal induction seen with subsaturating concentrations of glucocorticoid could be dramatically increased, and an antiglucocorticoid could be converted into a partial glucocorticoid, simply by increasing the concentration of glucocorticoid receptors. This behavior was observed in HeLa cells, containing endogenous receptors, or in CV-1 cells, containing almost no endogenous receptor, with either homologous or heterologous receptors. These increases were relatively insensitive to the concentration of reporter gene, suggesting the titration of some transcription factor(s) involved in regulating the position of the glucocorticoid dose-response curve and the agonist activity of an antiglucocorticoid. This property of transfected glucocorticoid receptors required a full-length, functionally active receptor but was retained, albeit reduced in magnitude, in the absence of binding to a glucocorticoid response element. Furthermore, this phenomenon was specific in that the A form of the human progesterone receptor had no effect under the same conditions. These variations in induction properties of antiglucocorticoids and of subsaturating concentrations of glucocorticoid, in a manner that was proportional to the amount of transfected receptor, reveal processes that are not operative with saturating concentrations of glucocorticoid. These variations also demonstrate that caution should be exercised in making mechanistic conclusions based solely on experiments conducted with saturating concentrations of glucocorticoid.The overriding experimental advantage of transient transfections is time. Thus, the biological consequences of altered nucleotide compositions in the cDNAs encoding active proteins, and in genomic sequences, can be examined in a fraction of the time required to establish cell lines with the same sequences stably integrated into the cellular genome. In the field of steroid receptors, most of the recent advances have emerged from transient transfection experiments, including the contributions of cis-acting elements (1, 2), of different nucleotides in receptor binding to the hormone-responsive element (3), of various regions of receptors in steroid binding and biological activity (reviewed in Ref. 4), of promoter structure and cell type as determinants for the activity of antisteroid activity (5), and of overlapping signaling systems such as dopamine (6), epidermal growth factor (7), and protein kinase A inducers (8, 9). The utility of transient transfections has been further enhanced by the development of the "two-hybrid" (10, 11) and...