Characterization of the angiotensin (AT1b) receptor promoter and its regulation by glucocorticoids.
Angiotensin II acts through two pharmacologically distinct receptors known as AT1 and AT2. Duplication of the AT1 receptor in rodents into At1a and b subtypes allows tissue-specific expression of the AT1b in adrenal and pituitary tissue. Adrenal expression of this receptor is increased in the offspring of rat mothers exposed to a low-protein diet and this is associated with the undermethylation of its promoter. This phenomenon is blocked by the inhibition of maternal glucocorticoid synthesis by metyrapone. We have mapped the transcriptional start site of the promoter and demonstrated that a 1.2 kbp fragment upsteam of this site is effective in driving luciferase expression in mouse Y1 cells. A combination of bioinformatic analysis, electrophoretic mobility shift analysis (EMSA), and mutagenesis studies demonstrates: i) the presence of a putative TATA box and CAAT box; ii) the presence of three Sp1 response elements, capable of binding SP1; mutation of any pair of these sites effectively disables this promoter; iii) the presence of four potential glucocorticoid response elements which each bind glucocorticoid receptor in EMSA, although only two confer dexamethasone inhibition on the promoter; iv) the presence of two AP1 sites. Mutagenesis of the distal AP1 site greatly diminishes promoter function but this is also associated with the loss of dexamethasone inhibition. These studies will facilitate an understanding of the mechanisms by which fetal programming leads to long term alterations in gene expression and the development of adult disease.
Bogdarina
Irina G
IG
Centre for Endocrinology, Queen Mary University of London, Barts and the London School of Medicine and Dentistry, John Vane Science Centre, London, UK.
King
Peter J
PJ
Clark
Adrian J L
AJ
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002874:Chromosome Mapping; D019175:DNA Methylation / Q000187:drug effects; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology*; D008156:Luciferases / Q000032:analysis; D051379:Mice; D011401:Promoter Regions, Genetic; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics*; D014162:Transfection
D005938:Glucocorticoids; D044140:Receptor, Angiotensin, Type 1; D008156:Luciferases