Characterization of cis-elements required for osmotic response of rat Na+/H+ exchanger-2 (NHE-2) gene

The Na+/H+ exchanger (NHE-2) has been implicated in osmoregulation in the k idney, because it transports Na+ across the cell membrane and efficiently a lters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increase s in abundance in mouse inner medullary collecting duct (mIMCD-3) cells, su ggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolari ty, we have functionally characterized the 5'-flanking region of the gene i n mIMCD-3 cells. Transient transfection of luciferase reporter gene constru cts revealed a novel cis-acting element, which we call OsmoE (osmotic-respo nsive element, bp -808 to -791, GGGCCAGTTGGCGCTGGG), and a TonE-like elemen t (tonicity-responsive element, bp - 1201 to - 1189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE- 2 gene. Electrophoretic mobility shift assays suggest that different DNA-pr otein interactions occur between these two osmotic response elements. Howev er, both DNA sequences were shown to specifically bind nuclear proteins tha t dramatically increase in abundance under hyperosmotic conditions. Isolati on of transacting factors and characterization of their specific interactio n with these osmotic response elements will further elucidate the transcrip tional mechanisms controlling NHE-2 gene expression under hyperosmolar cond itions.