Molecular cloning and characterization of the rat NHE-2 gene promoter

To understand the molecular mechanisms underlying NHE-2 regulation in the mammalian kidney and intestine, we cloned and sequenced 5.6 kb of the 5'-flanking region of the rat NHE-2 gene. DNA sequence analysis revealed multiple putative cis-acting regulatory elements including SP1, CK, NFY-CBF, Tant, GCN4, and one progesterone and several retinoic acid response elements. The upstream sequence lacked TATA and CAAT boxes, but contained a high G/C rich region within the first 300 bp. A single transcriptional initiation site was identified by primer extension in rat kidney and small intestine, approximately 103 bp upstream of the previously identified 5'-end of the rat NHE-2 cDNA. Various regions of the promoter (from [-]5567 to [+]105 bp) were tested for their ability to drive expression of the luciferase reporter gene in transiently transfected murine Inner Medullary Collecting Duct (mIMCD-3) cells. Results demonstrated that [-]289, [-]1271 and [-]2630 bp constructs showed promoter activity that was significantly higher than the negative control construct (20-fold). These results also demonstrated that basal cis-acting elements are contained within [-]289 bp of the transcriptional start site. However, the functional activity of the [-]5567 bp construct was not significantly different from the negative control, suggesting that a negative regulatory element may be present between [-]2630 and [-]5567 bp of the promoter region.