Identification of a mineralocorticoid/glucocorticoid response element in the human Na/K ATPase alpha1 gene promoter.
Sodium-potassium ATPase (Na/K ATPase) is a major target of mineralocorticoids. Both aldosterone and glucocorticoids activate the human Na/K ATPase alpha1 and beta1 genes transcriptionally. The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) have been shown to bind the glucocorticoid response element (GRE); however, a specific element responsible for the activation of the MR is not known. Sequence analysis of the putative regulatory region of the Na/K ATPase alpha1 gene revealed the presence of a hormone response element that allows the MR to interact with it, at least as well as if not better than the GR. This response element is designated MRE/GRE. In this investigation, we demonstrated the MR and GR induced gene expression in COS-1 cells by cotransfecting with respective expression plasmids (RshMR and RshGR) along with a luciferase reporter. The synthetic MRE/GRE linked to a neutral promoter was activated by MR (6-fold); however, the GR induced a lower level of expression (3.8-fold), suggesting that the element may be preferably MR responsive. Mutations in the synthetic MRE/GRE could not induce the expression with MR, whereas GR had a small effect. Electrophoretic mobility shift analyses demonstrated a direct interaction of MR and GR with the MRE/GRE that was supershifted by an antiMR antibody and the complex was partially cleared by an antiGR antibody, respectively, whereas nonimmune serum had no effect. Footprinting analyses of the promoter region showed that a portion of the DNA containing this element is protected by recombinant MR and GR. Thus these data confirm that this MRE/GRE interacts with both MR and GR but interaction with receptors may be more MR-responsive than response elements previously described.
Kolla
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Department of Biochemistry and Molecular Pharmacology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107, USA.
Robertson
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Litwack
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D000305:Adrenal Cortex Hormones / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D018983:DNA Footprinting; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008024:Ligands; D009154:Mutation / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D018161:Receptors, Mineralocorticoid / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000235:genetics*; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D013379:Substrate Specificity; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
D000305:Adrenal Cortex Hormones; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011965:Receptors, Glucocorticoid; D018161:Receptors, Mineralocorticoid; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase