Abstract
Collecting duct-derived ET-1 regulates salt excretion and blood pressure. We have reported the presence of an inner medullary collecting duct (IMCD)-specific enhancer region in the 5'-upstream ET-1 promoter (Strait, K. A., Stricklett, P. K., Kohan, J. L., Miller, M. B., and Kohan, D. E. (2007) Am. J. Physiol. Renal Physiol. 293, F601-F606). 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.
Highlights
Most work on ET-1 regulation has been confined to endothelial cells of the vasculature, in the past several years, Grant DK96392
As anticipated from the previous studies cited above, we showed that maximal transcriptional activity of the rat ET-1 promoter in primary cultures of aortic endothelial cells was confined to the first Ϫ366 bp of 5Ј-upstream sequence; additional sequences up to 3.0 kb 5Ј-upstream produced no further enhancement of promoter activity
Characterization of the 5Ј-Upstream ET-1 Promoter—In our previous studies, we demonstrated that Ϫ366 bp of the 5Ј-upstream ET-1 promoter reporter construct produced similar transcriptional activity to a much larger Ϫ3048-bp construct when transfected into primary cultures of rat aorta endothelial cells
Summary
Materials—Type 1 collagenase was obtained from Worthington; penicillin, streptomycin, and glutamine were from Invitrogen; pGL3 and pGL4 vectors were from Promega (Madison, WI); and all other reagents and materials were obtained from Sigma unless stated otherwise. Our 3.2-kb construct (containing Ϫ3048 bp of 5Ј-flanking sequence) was generated using high fidelity Platinum Taq (Invitrogen) PCR from rat genomic DNA and ligated into the XhoI/NheI sites in the pGL3 basic vector (Ϫ3048 ET-1 pGL3). The co-transfected control vector, pRL-TK (Promega), contained the promoter for the herpes simplex virus thymidine kinase (TK) gene linked to the Renilla luciferase reporter. EMSA—Nuclear extracts were prepared from rat IMCD cell primary cultures or kidney papilla using the NE-PER nuclear protein isolation kit (Pierce Biotechnology). DNA-protein binding reactions were performed by incubating 5–10 g of nuclear extract with 15 fmol of 32P-end-labeled doublestranded oligonucleotides in 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM EDTA (pH 8), 1 mM DTT, 100 ng/l poly(dI-dC), 1 mg/ml BSA, 0.05% Nonidet P-40, and 5% (v/v) glycerol. All data are expressed as means Ϯ S.E. of the mean
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