Surfactant protein A (SP-A) is a member of the collectin family of innate host defense molecules expressed primarily in respiratory epithelial cells of the lung. SP-A concentrations are influenced by both cell-specific and ubiquitous nuclear proteins that regulate SP-A gene transcription in a cell-selective and temporally regulated manner. In this work, a consensus GATA-binding site (GBS) was identified at positions −69 to −64 of the mouse SP-A gene. The transcriptional activity of wild-type SP-A reporter constructs in HeLa cells was increased 5–10-fold when cotransfected with a GATA-6 expression plasmid. Deletion of the GBS completely blocked transactivation by GATA-6. Transfection of a construct expressing GATA-6-engrailed fusion protein inhibited basal expression of the SP-A/chloramphenicol acetyltransferase construct in MLE-15 cells. Nuclear extract proteins from MLE-15 cells bound to the GBS in the mouse SP-A gene, and a supershifted band was detected with a GATA-6-specific antibody. Transactivation of the wild-type SP-A constructs by GATA-6 increased transcriptional activity 7–10-fold, whereas thyroid transcription factor-1 (TTF-1) increased the activity of these constructs 12–18-fold. The effects of cotransactivating with both GATA-6 and TTF-1 expression constructs were additive. However, mutation of the TTF-1-binding sites alone or in combination decreased GATA-6 transactivation. Likewise, mutation of the GBS blocked TTF-1 activation of the SP-A promoter. In situ hybridization demonstrated GATA-6 mRNA in the peripheral epithelial cells of fetal mouse lung, consistent with the sites of SP-A expression. GATA-6 is expressed in respiratory epithelial cells and binds to acis-acting element in the SP-A gene promoter, activating the transcriptional activity of the gene.
This work was supported in part by National Institutes of Health Grants HL28623 (to J. A. W. and T. R. K.) and HL58795 (to T. R. K.). This work was presented in part at the Annual Meeting of the American Thoracic Society, San Diego, CA, April 23–28, 1999.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.
