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 osmolyt e betaine in hypertonic Madin-Darby canine kidney (MDCK) cells and pre sumably in the hypertonic renal medulla. In MDCK cells, the increase i n activity of the betaine transporter is preceded by an increase in tr anscription 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 trans fection 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 -6 9 to -50 5' to the transcription initiation site (-69/-50) has hyperto nicity-responsive enhancer activity. A double-stranded -69/-50 concate mer cloned 5' to an SV40 basal promoter and luciferase reporter gene i n hypertonic cells exhibited more than 11-fold the activity in isotoni c cells. Expression assays and electrophoretic mobility shift assays o f mutants of -69/-50 identified a smaller region that is required for hypertonicity to induce increased expression and a slowly migrating ba nd on mobility shift assays.