TY - JOUR
T1 - The TonE/TonEBP pathway mediates tonicity-responsive regulation of UT-A urea transporter expression
AU - Nakayama, Yushi
AU - Peng, Tao
AU - Sands, Jeff M.
AU - Bagnasco, Serena M.
PY - 2000/12/8
Y1 - 2000/12/8
N2 - 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). Cotransfection 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.
AB - 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). Cotransfection 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.
UR - http://www.scopus.com/inward/record.url?scp=0034623952&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034623952&partnerID=8YFLogxK
U2 - 10.1074/jbc.M004678200
DO - 10.1074/jbc.M004678200
M3 - Article
C2 - 10995747
AN - SCOPUS:0034623952
SN - 0021-9258
VL - 275
SP - 38275
EP - 38280
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 49
ER -