Characterization of cis-elements required for osmotic response of rat Na1/H1 exchanger-2 (NHE-2) gene

Bai, Liqun, James F. Collins, Yunhua L. Muller, Hua Xu, Pawel R. Kiela, and Fayez K. Ghishan. Characterization of cis-elements required for osmotic response of rat Na1/H1 exchanger-2 (NHE-2) gene. Am. J. Physiol. 277 (Regulatory Integrative Comp. Physiol. 46): R1112–R1119, 1999.—The Na1/H1 exchanger (NHE-2) has been implicated in osmoregulation in the kidney, because it transports Na1 across the cell membrane and efficiently alters intracellular osmolarity. On hyperosmotic stress, NHE-2 mRNA increases in abundance in mouse inner medullary collecting duct (mIMCD-3) cells, suggesting possible transcriptional regulation. To investigate the molecular mechanism of potential transcriptional regulation of NHE-2 by hyperosmolarity, we have functionally characterized the 58-flanking region of the gene in mIMCD-3 cells. Transient transfection of luciferase reporter gene constructs revealed a novel cis-acting element, which we call OsmoE (osmotic-responsive element, bp 2808 to 2791, GGGCCAGTTGGCGCTGGG), and a TonE-like element (tonicity-responsive element, bp 21201 to 21189, GCTGGAAAACCGA), which together are shown to be responsible for hyperosmotic induction of the NHE-2 gene. Electrophoretic mobility shift assays suggest that different DNA-protein interactions occur between these two osmotic response elements. However, both DNA sequences were shown to specifically bind nuclear proteins that dramatically increase in abundance under hyperosmotic conditions. Isolation of transacting factors and characterization of their specific interaction with these osmotic response elements will further elucidate the transcriptional mechanisms controlling NHE-2 gene expression under hyperosmolar conditions.