Identification of two nuclear factor of activated T-cells (NFAT)-response elements in the 5'-upstream regulatory region of the ET-1 promoter.
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.
Strait
Kevin A
KA
Division of Nephrology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA.
Stricklett
Peter K
PK
Kohan
Rachel M
RM
Kohan
Donald E
DE
D000818:Animals; D001794:Blood Pressure; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000096:biosynthesis* / Q000235:genetics; D007376:Interleukin-2 / Q000235:genetics / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D020218:Response Elements / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
D019332:Endothelin-1; D007376:Interleukin-2; D050778:NFATC Transcription Factors; D014409:Tumor Necrosis Factor-alpha; C445503:transcription factor NF-AT c3