Identification of enhancers in the 5′-flanking region of the rabbit surfactant protein a (SP-A) gene and characterization of their binding proteins

The gene encoding surfactant protein A (SP-A) is expressed in type II pneumonocytes and is developmentally and hormonally regulated in fetal lung. In the present study, rabbit lung nuclear proteins were found to bind to two genomic elements within the 5′-flanking region of the rabbit SP-A gene, termed distal binding element (DBE; -986 to -977 base pairs) and proximal binding element (PBE; -87 to -70 base pairs). Binding activity was enriched in type II pneumonocytes as compared with whole lung tissue. Although binding activity was undetectable in nuclear proteins from rabbit liver and kidney, low levels of binding activity were detected in nuclear proteins from cardiac and skeletal muscle. DNase I footprinting indicated that lung nuclear proteins protected the palindromic sequence CCCACGTGGG in the DBE. The underlined core sequence is an E box motif; a similar sequence (CCCTCGTG) is present within the PBE. Both elements competed for binding to the same size species of nuclear proteins of M_r ≈ 69,000, 45,000, and 22,000. In type II cells transfected with fusion genes containing SP-A 5′-flanking DNA linked to the human growth hormone structural gene, mutagenesis of the DBE or PBE resulted in a marked reduction of basal and cyclic AMP-stimulated fusion gene expression. These findings suggest that the DBE and PBE act as enhancers that interact with the same or related trans-acting proteins and serve an important role in type II cell-specific, cyclic AMP-mediated regulation of SP-A gene expression.