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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 rat Na+/H+ exchanger
isoform-2 (NHE-2) gene promoter lacks a TATA box and is very
GC rich. A minimal promoter extending from bp 
36 to +116 directs
high-level expression of NHE-2 in mouse inner medullary
collecting duct (mIMCD-3) cells. Four Sp1 consensus elements were found
in this region. The introduction of mutations within these Sp1
consensus elements and DNA footprinting revealed that only two of them
were utilized and are critical for basal transcriptional activation in
mIMCD-3 cells. The use of Sp1, Sp3, and Sp4 antisera in electrophoretic
mobility shift assays demonstrated that Sp1, Sp3, and Sp4 bound to this
minimal promoter. We further analyzed the transcriptional regulation of NHE-2 by members of the Sp1 multigene family. In
Drosophila SL2 cells, which lack endogenous Sp1, the minimal
promoter cannot drive transcription. Introduction of Sp1 activated
transcription over 100-fold, suggesting that Sp1 is critical for
transcriptional regulation. However, neither Sp3 nor Sp4 was able to
activate transcription in these cells. Furthermore, in mIMCD-3 cells,
Sp1-mediated transcriptional activation was repressed by expression of
Sp3 and Sp4. These data suggest that Sp1 is critical for the basal promoter function of rat NHE-2 and that Sp3 and Sp4 may
repress transcriptional activation by competing with Sp1 for binding to core cis-elements.
Sp3; Sp4; Drosophila SL2 cells; sodium-hydrogen exchanger; mIMCD-3 cells
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