A GT Box Element Is Essential for Basal and Cyclic Adenosine 3′,5′-Monophosphate Regulation of the Human Surfactant Protein A2 Gene in Alveolar Type II Cells: Evidence for the Binding of Lung Nuclear Factors Distinct from Sp1

The gene encoding surfactant protein-A (SP-A) is developmentally regulated in type II cells of the fetal lung. In humans there are two SP-A genes, SP-A1 and SP-A2. The SP-A2 gene is more highly regulated by cAMP and during fetal development than SP-A1. In earlier studies we determined that 296 bp of sequence flanking the 5'-end of the SP-A2 gene is sufficient to mediate high basal and cAMP-inducible reporter gene expression in primary cultures of transfected type II cells, suggesting that this region contains important cis-acting elements involved in tissue-specific and hormonal regulation of SP-A2 promoter activity. We also observed that mutagenesis of a cAMP response element (CRE)-like sequence at -242 bp (CRE(SP-A2)) greatly reduced basal and cAMP-stimulated expression in transfected type II cells. In the present study, we identified a GT box (GGGGTGGGG) at -61 bp of SP-A2 5'-flanking sequence that is highly conserved among the SP-A genes of different species. In type II cell transfection studies, we found that mutagenesis of the GT box of SP-A2 markedly reduced basal and abolished cAMP-induced reporter gene expression. Thus, CRE(SP-A2) and the GT box cooperatively interact to mediate basal and cAMP induction of SP-A2 promoter activity in type II cells. By electrophoretic mobility shift assays (EMSA), it was observed that nuclear proteins isolated from primary cultures of type II cells bound the GT box as five specific complexes. By contrast, nuclear proteins isolated from lung fibroblasts displayed notably reduced binding activity. Competition and supershift EMSA indicate that the ubiquitously expressed transcription factor Sp1, a GC box-binding protein of approximately 100 kDa, is a component of the complex of proteins that bind the GT box of SP-A2. The finding that only two of the five GT box-binding complexes were supershifted by incubation with Sp1 antibody suggests that a factor(s) in type II cell nuclear extracts that is distinct from Sp1 also interacts with the GT box. By UV cross-linking and SDS-PAGE/EMSA analysis, we have identified a approximately 55-kDa GT box-binding factor in type II cell nuclear proteins that preferentially binds the GT box of SP-A2 over the consensus Sp1 GC box sequence. This 55-kDa factor was able to bind the GT box independently of Sp1.