The tonicity-sensitive element that mediates increased transcription of the betaine transporter gene in response to hypertonic stress.
BGT1, the Na(+)-and Cl- coupled betaine transporter, is responsible for the accumulation of high concentrations of the non-perturbing osmolyte betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and presumably in the hypertonic renal medulla. In MDCK cells, the increase in activity of the betaine transporter is preceded by an increase in transcription of BGT1 and in the abundance of BGT1 mRNA. To investigate the molecular mechanism of transcriptional regulation by tonicity, we have characterized the 5'-flanking region of the gene. Transient transfection assays in MDCK cells cultured in isotonic or hypertonic medium using luciferase reporter constructs containing various fragments of the 5'-flanking region revealed that the region spanning base pairs -69 to -50 5' to the transcription initiation site (-69/-50) has hypertonicity-responsive enhancer activity. A double-stranded -69/-50 concatemer cloned 5' to an SV40 basal promoter and luciferase reporter gene in hypertonic cells exhibited more than 11-fold the activity in isotonic cells. Expression assays and electrophoretic mobility shift assays of mutants of -69/-50 identified a smaller region that is required for hypertonicity to induce increased expression and a slowly migrating band on mobility shift assays.
Takenaka
M
M
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Preston
A S
AS
Kwon
H M
HM
Handler
J S
JS
D000818:Animals; D001483:Base Sequence; D001622:Betaine / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA; D004285:Dogs; D050485:GABA Plasma Membrane Transport Proteins; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D013539:Simian virus 40 / Q000235:genetics
D002352:Carrier Proteins; D050485:GABA Plasma Membrane Transport Proteins; D012333:RNA, Messenger; C072207:betaine plasma membrane transport proteins; D001622:Betaine; D004247:DNA; D008156:Luciferases