Genomic elements involved in transcriptional regulation of the rabbit surfactant protein-A gene

Expression of the surfactant protein-A (SP-A) gene is lung specific and is developmentally and hormonally regulated in fetal lung tissue. Cyclic AMP analogs and glucocorticoids stimulate transcriptional activity of the SP-A gene in fetal rabbit lung tissue in culture; an additive effect is observed when the agents are added in combination. To analyze the genomic regions that regulate SP-A promoter activity, fusion genes comprised of -1766, -991, - 378, and -47 basepairs (bp) of DNA flanking the 5'-end of the SP-A gene, the transcription initiation site, and 20 bp of exon I linked to the human GH (hGH) structural gene were subcloned into a replication-defective human adenovirus vector and transfected into differentiated rat type II cells in primary culture. SP-A promoter activity was analyzed by RIA of hGH protein in the culture medium. In type II cells transfected with SP-A_-1766:hGH and SP-A_-991:hGH fusion genes, hGH production was induced 30- to 40-fold by (Bu)₂AMP (Bt₂cAMP; 1 mM). When type II cells were transfected with the SP- A_-378:hGH fusion gene, basal levels of expression were reduced by more than 50%; however, Bt₂cAMP caused an 11-fold increase in hGH production. In type II cells transfected with the SP-A_-47:hGH fusion gene, basal levels of hGH production were essentially undetectable, and no stimulatory effect of Bt₂cAMP was apparent. Cyclic AMP stimulation of expression of the SPA-A_{- 1766}:hGH, SP-A_-991:hGH, and SP-A_-378:hGH fusion genes was limited to type II pneumonocytes in primary culture and was absent in two lung adenocarcinoma cell lines (NCI-H358 and A549), which do not express SP-A, and in cAMP-responsive adrenal Y1 cells. Mutations of a putative cAMP-responsive element (TGACCTCA) at -261 bp revealed its functional importance in mediating cAMP regulation of SP-A gene expression. Unexpectedly, dexamethasone (Dex; 10^-7 M) antagonized the stimulatory effect of Bt₂cAMP on expression of SP- A:Hgh fusion genes containing from -378 to -1766 bp of 5'-flanking DNA as well as that of a fusion gene construct containing -991 bp of 5'-flanking DNA, the first exon, the first intron, and 20 bp of the second exon (SP-A_{- 991+670}:hGH). The inhibitory effect of Dex was dose dependent, with half-maximal inhibition occurring at a Dex concentration of 8 x 10^-10 M. The inhibitory effect of Dex was prevented by the glucocorticoid receptor antagonist RU486. Our findings suggest that our fusion gene constructs lack a functional glucocorticoid-responsive element and that the glucocorticoid receptor antagonizes the cAMP-mediated induction of SP-A promoter activity.