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Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724
The
Na+/H+
exchanger (NHE-2) has
been implicated in osmoregulation in the kidney, because it transports
Na+ 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 5'-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 
808 to 791, GGGCCAGTTGGCGCTGGG), and a TonE-like element
(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-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
trans-acting 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.
mouse inner medullary collecting duct cells; gel mobility shift; transcriptional regulation; kidney; OsmoE
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