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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

GATA-6 mediates transcriptional activation of aquaporin-5 through interactions with Sp1

Will Rogers Institute Pulmonary Research Center and Division of Pulmonary and Critical Care Medicine, Departments of ¹Medicine, ²Pediatrics, ³Pharmacology and Pharmaceutical Sciences, and ⁴Orthopedics, University of Southern California, Los Angeles; ⁵Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and ⁶Department of Clinical and Molecular Pharmacology, City of Hope Medical Center, Duarte, California

Submitted 25 February 2008 ; accepted in final form 30 August 2008

We investigated mechanisms underlying GATA-6-mediated transcriptional activation of the alveolar epithelial type I cell-enriched gene aquaporin-5 (AQP5). GATA-6 expression increases in alveolar epithelial cells in primary culture, concurrent with upregulation of AQP5 and transition to a type I cell-like phenotype. Cotransfections in MLE-15 and NIH 3T3 cells demonstrated trans-activation by GATA-6 of a rat 1,716-bp-AQP5-luciferase (–1716-AQP5-Luc) reporter. Electrophoretic mobility shift assay and chromatin immunoprecipitation identified an interaction between GATA-6 and putative binding sites in the AQP5 promoter. However, mutation of these sites did not reduce GATA-6-mediated activation, implicating mechanisms in addition to direct binding of GATA-6 to DNA. A 5'-deletion construct, –358-AQP5-Luc, that does not encompass GATA motifs was still activated by GATA-6 by as much as 50% relative to –1716-AQP5-Luc. Internal deletion of the –358/–173 GC-rich domain, which includes several putative Sp1 consensus sites, reduced trans-activation by 60%, suggesting importance of this region for GATA-mediated activity. –358-AQP5-Luc was similarly activated by both GATA-6 and a GATA DNA-binding defective mutant, whereas cotransfections in Schneider S2 cells demonstrated dose-dependent trans-activation of –358-AQP5-Luc by Sp1. Activation of –358-AQP5-Luc by GATA-6 was dramatically reduced by Sp1 small-interfering RNA, and –358-AQP5-Luc was activated synergistically by GATA-6 and Sp1 in NIH 3T3 cells. Furthermore, association between endogenous GATA-6 and Sp1 was demonstrated by coimmunoprecipitation. These results suggest that transcriptional activation of AQP5 by GATA-6 is mediated at least in part through cooperative interactions with Sp1 occurring at the proximal promoter.

transcription; alveolar epithelium; gene regulation; transfection; protein-protein interaction