The current studies provide further characterization of the ET-1 5-upstream distal promoter to identify the IMCD-specific enhancer elements. Deletion studies identified two regions of the 5-upstream ET-1 promoter, ؊1725 to ؊1319 bp and ؊1319 to ؊1026 bp, which were required for maximal promoter activity in transfected rat IMCD cells. Transcription factor binding site analysis of these regions identified two consensus nuclear factor of activated T-cells (NFAT) binding sites at ؊1263 and ؊1563. EMSA analysis using nuclear extracts from IMCD cells showed that both the ؊1263 and the ؊1563 NFAT sites in the ET-1 distal promoter competed for NFAT binding to previously identified NFAT sites in the IL-2 and TNF genes. Gel supershift analysis showed that each of the NFAT binding sites in the ET-1 promoter bound NFAT proteins derived from IMCD nuclear extracts, but they selectively bound different NFAT isoforms; ET-1263 bound NFATc1, whereas ET-1563 bound NFATc3. Site-directed mutagenesis of either the ET-1263 or the ET-1563 sites prevented NFAT binding and reduced ET-1 promoter activity. Thus, NFAT appears to be an important regulator of ET-1 transcription in IMCD cells, and thus, it may play a role in controlling blood pressure through ET-1 regulation of renal salt excretion.Endothelin-1 (ET-1) is a 21-amino acid peptide initially isolated from endothelial cells that functions as a potent vasoconstrictor (1). Since its discovery, it has become apparent that besides vasoconstriction, ET-1 exerts multiple effects, including regulation of mitogenesis, hypertrophy, synthesis of extracellular matrix, ion transport, and many others (2). In the vast majority of instances, ET-1 exerts its effects via autocrine or paracrine mechanisms, producing localized regulation of cellular functions.Although most work on ET-1 regulation has been confined to endothelial cells of the vasculature, in the past several years, the kidney has also emerged as a major site of ET-1 actions (3). In the kidney, ET-1 has been shown to modulate a number of important physiological processes, including blood flow, glomerular filtration rate, salt and water excretion, and acid/base handling. Given the critical role ET-1 plays in renal physiology, it was not surprising to discover that almost every cell type in the kidney synthesizes ET-1 and/or contains ET-1 receptors. The kidney is also, by some accounts, 10 times more sensitive to ET-1 actions than the vasculature (4). Finally, within kidney, the inner medullary collecting duct cells may have the highest concentrations of ET-1 immunoreactivity of any cell type in the body (5).In the nephron, collecting duct cells produce more ET-1 than any other cell type (6). In the collecting duct, ET-1 has been shown to inhibit both sodium (7,8) and water reabsorption (9). The actions of ET-1 on sodium and water reabsorption make it a target in the study of renal-induced hypertension. Mice containing a collecting duct-specific deletion of the ET-1 gene are hypertensive and have impaired water and sodium excretion (10)....
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