Identification of Two Nuclear Factor of Activated T-cells (NFAT)-response Elements in the 5′-Upstream Regulatory Region of the ET-1 Promoter

Abstract

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)....

Role of collecting duct endothelin in control of renal function and blood pressure

“…Rat ET-1 promoter-reporter analysis revealed a calmodulin-sensitive region between 0.56 and 1.9 kb 5= to the transcription start site in IMCD but not endothelial cells (79). Two consensus nuclear factor of activated T-cells (NFAT) binding sites were identified at Ϫ1263 and Ϫ1563 5= to the transcriptional start site which, upon mobility shift analysis, revealed that ET-1263 bound NFATc1 while ET-1563 bound NFATc3 (80). Site-directed mutagenesis of either the ET-1263 or ET-1563 sites prevented NFAT binding and reduced ET-1 promoter activity.…”

Osmolar regulation of endothelin-1 production by the inner medullary collecting duct

“…Inhibition of calcineurin with cyclosporine A prevented the flow stimulated ET-1 mRNA increase (Figure 5), however it had no effect on hyperosmolality or hyperosmolality + flow stimulated ET-1 mRNA. Since nuclear factor of activated T-cells (NFAT) 1-4 (NFATc) has been implicated in ET-1 regulation in IMCD cells (21), the effect of inhibition of NFATc (VIVIT peptide) was examined; VIVIT peptide had no effect on flow and/or hyperosmolality stimulation of ET-1 mRNA (Figure 6). The effect of blockade of two Ca 2+ cellular entry pathways (polycystin-2 and purinergic receptors) was examined since both pathways have been implicated in modulation of IMCD ET-1 production (14).…”

Aldosterone alters the chromatin structure of the murine endothelin-1 gene

“…Four regions that are known to be important in the transcriptional regulation of the gene in renal collecting duct cells were chosen for study (Stow et al, 2009, Stow et al, 2012, Strait et al, 2010). These were the two HREs located at −551 bp and −671 bp and the two putative nuclear factor of activated T-cells (NFAT) elements located at approximately −1263 bp and −1563 bp upstream of the transcription start site in the murine gene (Strait et al, 2010). Primers were carefully designed to avoid inclusion of both HREs in the same amplicon to unambiguously discern differences in chromatin structure between the two elements (Figure 1).…”

“…In the kidney, ET-1 is known to be regulated by calcium, aldosterone, and in a time-dependent manner by Per1. In the upstream distal region of the Edn1 promoter there are two calcium/calmodulin regulatory elements, nuclear factor of activated T-cells (NFATs), that appear to be important for regulation of the gene in the kidney (Strait et al , 2010). The more distal NFAT element was found to bind NFATc3-4 and the proximal NFAT element preferentially bound NFATc1-2.…”