The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression.
The rat renal urea transporter UT-A includes four isoforms. UT-A1, UT-A3, and UT-A4 are transcribed from a single initiation site at the 5'-end of the gene; a distinct internal initiation site is used for UT-A2 transcription. We cloned 1.3 kilobases (kb) of the 5'-flanking region upstream of the transcription start site of UT-A1, UT-A3, and UT-A4. This region contains three CCAAT sequences but lacks a TATA motif. A tonicity-responsive enhancer (TonE) was identified at -377bp. The 1.3-kb full fragment subcloned into pGL3 vector induced luciferase activity in Madin-Darby canine kidney cells and in mouse inner medullary collecting duct cells in isotonic medium. Luciferase activity was increased significantly in hypertonic medium, whereas deletion or mutation of the TonE sequence abolished this response. Electrophoretic mobility shift assay using the 5' UT-A TonE sequence as DNA probe showed formation of a specific DNA-protein complex with nuclear extracts from cells exposed to hypertonic medium and was weakly detectable in isotonic controls. A supershift in the mobility of the DNA-protein complex was observed with antiserum targeted to the TonE-binding protein (TonEBP). Co-transfection with dominant-negative TonEBP abolished the luciferase activity induced by the UT-A 1.3-kb construct under hypertonic and isotonic conditions. These data suggest that the TonE/TonEBP pathway mediates tonicity-responsive transcriptional regulation of UT-A1, UT-A3, and UT-A4 expression.
Nakayama
Y
Y
Renal Division, Department of Medicine and the Department of Pathology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Peng
T
T
Sands
J M
JM
Bagnasco
S M
SM
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D004285:Dogs; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D007679:Kidney Medulla / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D008562:Membrane Glycoproteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D000096982:Urea Transporters
D002352:Carrier Proteins; D008156:Luciferases; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000096982:Urea Transporters; C492200:Nfat5 protein, mouse; C492199:Nfat5 protein, rat