title: GATA-6 mediates transcriptional activation of aquaporin-5 through interactions with Sp1.
abstract: 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 approximately 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.
authors.affiliation: Will Rogers Institute Pulmonary Research Center, Los Angeles, CA, USA.
authors.forename: Beiyun
authors.initials: B
authors.lastname: Zhou
authors.forename: Tricia A
authors.initials: TA
authors.lastname: Francis
authors.forename: Hui
authors.initials: H
authors.lastname: Yang
authors.forename: Wanru
authors.initials: W
authors.lastname: Tseng
authors.forename: Qian
authors.initials: Q
authors.lastname: Zhong
authors.forename: Baruch
authors.initials: B
authors.lastname: Frenkel
authors.forename: Edward E
authors.initials: EE
authors.lastname: Morrisey
authors.forename: David K
authors.initials: DK
authors.lastname: Ann
authors.forename: Parviz
authors.initials: P
authors.lastname: Minoo
authors.forename: Edward D
authors.initials: ED
authors.lastname: Crandall
authors.forename: Zea
authors.initials: Z
authors.lastname: Borok