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 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.
Bai
L
L
Department of Pediatrics, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724, USA.
Collins
J F
JF
Muller
Y L
YL
Xu
H
H
Kiela
P R
PR
Ghishan
F K
FK
D000818:Animals; D002460:Cell Line; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000502:physiology; D009994:Osmolar Concentration; D009995:Osmosis / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020218:Response Elements / Q000502:physiology; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics*; D013237:Stereoisomerism
D009687:Nuclear Proteins; D012333:RNA, Messenger; C513111:Slc9a2 protein, mouse; C513110:Slc9a2 protein, rat; D017923:Sodium-Hydrogen Exchangers