Cell confluency-induced Stat3 activation regulates NHE3 expression by recruiting Sp1 and Sp3 to the proximal NHE3 promoter region during epithelial dome formation.
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.
Su
Hsiao-Wen
HW
Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, 1 Univ. Road, Tainan 70101, Taiwan.
Wang
Shainn-Wei
SW
Ghishan
Fayez K
FK
Kiela
Pawel R
PR
Tang
Ming-Jer
MJ
D000818:Animals; D018938:Caco-2 Cells; D049109:Cell Proliferation; D004247:DNA / Q000378:metabolism; D004285:Dogs; D024202:Electrophoretic Mobility Shift Assay; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D050796:STAT3 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D034741:RNA, Small Interfering; C000613385:SLC9A3 protein, human; C497121:SP3 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D016329:Sp1 Transcription Factor; D051705:Sp3 Transcription Factor; D004247:DNA