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PERSPECTIVES IN CELL PHYSIOLOGY

Cell confluency-induced Stat3 activation regulates NHE3 expression by recruiting Sp1 and Sp3 to the proximal NHE3 promoter region during epithelial dome formation

¹Institute of Basic Medical Sciences, ²Institute of Molecular Medicine, ³Department of Physiology, and ⁴Center for Gene Regulation and Signal Transduction Research, College of Medicine, National Cheng Kung University, Tainan, Taiwan; ⁵Department of Pediatrics, Steele Children's Research Center; and ⁶Department of Immunobiology, University of Arizona Health Sciences Center, Tucson, Arizona

Submitted 18 May 2008 ; accepted in final form 14 October 2008

ABSTRACT

Activation of signal transducer and activator of transcription-3 (Stat3) during cell confluency is related to its regulatory roles in cell growth arrest- or survival-related physiological or developmental processes. We previously demonstrated that this signaling event triggers epithelial dome formation by transcriptional augmentation of sodium hydrogen exchanger-3 (NHE3) expression. However, the detailed molecular mechanism remained unclear. By using serial deletions, site-directed mutagenesis, and EMSA analysis, we now demonstrate Stat3 binding to an atypical Stat3-response element in the rat proximal NHE3 promoter, located adjacent to a cluster of Sp cis-elements (SpA/B/C), within –77/–36 nt of the gene. SpB (–58/–55 nt) site was more effective than SpA (–72/–69 nt) site for cooperative binding of Sp1/Sp3. Increasing cell density had no effect on Sp1/Sp3 expression but resulted in their increased binding to the SpA/B/C probe along with Stat3 and concurrently with enhanced nuclear pTyr⁷⁰⁵-Stat3 level. Immunoprecipitation performed with the nuclear extracts demonstrated physical interaction of Stat3 and Sp1/Sp3 triggered by cell confluency. Stat3 inhibition by overexpression of dominant-negative Stat3-D mutant in MDCK cells or by small interfering RNA-mediated knockdown in Caco-2 cells resulted in inhibition of the cell density-induced NHE3 expression, Sp1/Sp3 binding, and NHE3 promoter activity and in decreased dome formation. Thus, during confluency, ligand-independent Stat3 activation leads to its interaction with Sp1/Sp3, their recruitment to the SpA/B/C cluster in a Stat3 DNA-binding domain-dependent fashion, increased transcription, and expression of NHE3, to coordinate cell density-mediated epithelial dome formation.

signal transducer and activator of transcription-3; Slc9a3; dome formation; Na⁺/H⁺ exchanger-3

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