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Originally published In Press as doi:10.1074/jbc.M209473200 on December 2, 2002

J. Biol. Chem., Vol. 278, Issue 8, 5659-5668, February 21, 2003
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Transcriptional Regulation of the Rat NHE3 Gene
FUNCTIONAL INTERACTIONS BETWEEN GATA-5 AND Sp FAMILY TRANSCRIPTION FACTORS*

Pawel R. Kiela, Jeffrey LeSueur, James F. Collins, and Fayez K. GhishanDagger

From the Departments of Pediatrics and Physiology, Steele Memorial Children's Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724

Expression of sodium-hydrogen exchanger isoform 3 (NHE3) in the intestinal and renal epithelium plays a critical role in sodium absorption and acid/base homeostasis. To decipher rat NHE3 gene regulation, its cis-acting regulatory elements and associated transcription factors were characterized by transient transfection of Caco-2, IEC-6, Qt6, and Drosophila SL2 cells. Deletion and mutational analyses demonstrated that the atypical TATA box located at bp -26/-31 was not necessary for promoter activity, and that a -20/+8-bp fragment represents a functional initiator. Within the 81-bp upstream region, three Sp transcription factor binding sites were critical because their mutation drastically reduced promoter activity. The roles of Sp1 and Sp3 were further demonstrated by electromobility shift assay and by transactivation of the NHE3 promoter in SL2 cells by forced expression of Sp1 and Sp3. Both of these transcription factors were found to act synergistically with GATA-5 bound to a GATA box in exon 1 (+20/+23 bp). These studies demonstrate that rat NHE3 promoter is initiator-driven and controlled mainly by Sp1 and Sp3, which functionally interact with GATA-5. This interaction represents a novel regulatory mechanism, which is likely to participate in a gradient of intestinal gene expression along the crypt-villus axis.

* This work was supported by National Institutes of Health Grant 2R01DK041274-15.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Arizona Health Sciences Center, Dept. of Pediatrics, 1501 N. Campbell Ave., Tucson AZ 85724. Tel.: 520-626-5170; Fax: 520-626-4141; E-mail: fghishan@ peds.arizona.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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