Modulation of cell signaling pathways can enhance or impair glucocorticoid-induced gene expression without altering the state of receptor phosphorylation

We have stably introduced expression vectors for the glucocorticoid receptor and a sensitive, hormone-responsive reporter (mouse mammary tumor virus-luciferase) into a human breast carcinoma-derived cell line. Employing this cell line, we have conducted a detailed examination of the induction of glu...

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Bibliographic Details
Published in:	The Journal of biological chemistry Vol. 268; no. 30; pp. 22933 - 22940
Main Authors:	Moyer, M.L., Borror, K.C., Bona, B.J., DeFranco, D.B., Nordeen, S.K.
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 25-10-1993 American Society for Biochemistry and Molecular Biology
Subjects:	1-Methyl-3-isobutylxanthine - pharmacology 8-Bromo Cyclic Adenosine Monophosphate - pharmacology Breast Neoplasms Chloramphenicol O-Acetyltransferase - metabolism Colforsin - pharmacology Dexamethasone - pharmacology Ethers, Cyclic - pharmacology Female Gene Expression - drug effects Gene Expression Regulation, Neoplastic - drug effects Genetic Vectors Humans Luciferases - metabolism Mammary Tumor Virus, Mouse - genetics Okadaic Acid Phosphopeptides - isolation & purification Phosphorylation Promoter Regions, Genetic Protein Tyrosine Phosphatases - antagonists & inhibitors Receptors, Glucocorticoid - metabolism Recombinant Proteins - metabolism Signal Transduction - drug effects Tetradecanoylphorbol Acetate - pharmacology Transfection Tumor Cells, Cultured
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Summary:	We have stably introduced expression vectors for the glucocorticoid receptor and a sensitive, hormone-responsive reporter (mouse mammary tumor virus-luciferase) into a human breast carcinoma-derived cell line. Employing this cell line, we have conducted a detailed examination of the induction of glucocorticoid-regulated genes and the phosphorylation of glucocorticoid receptor following pharmacologic manipulation of cell signaling pathways. The hormone response can be enhanced from 2 to 10-fold by activators of protein kinase A, protein kinase C, and inhibitors of protein phosphatase. Forskolin and 8-bromoadenosine 3‘:5‘-cyclic monophosphate (BrcAMP), but not BrcGMP, enhance the hormone effect, yet surprisingly, phosphodiesterase inhibitors, isobutylmethylxanthine and Ro20-1724, strongly inhibit hormone-mediated induction of the reporter gene. These treatments do not alter cellular receptor content, dexamethasone binding, nor hormone-mediated receptor down-regulation. Tryptic peptide analysis of 32P-labeled receptor reveals that neither BrcAMP, isobutylmethylxanthine, nor the tumor promoter and protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate, detectably alter the state of glucocorticoid receptor phosphorylation. The only agent which alters receptor phosphorylation is the protein phosphatase inhibitor okadaic acid, but only at concentrations higher than required for maximum effects on glucocorticoid receptor transactivation. We propose that these effectors do not modify receptor directly but alter its interaction with transcription complexes.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0021-9258 1083-351X
DOI:	10.1016/S0021-9258(18)41616-X