Characterization of the Cyclic Adenosine 3′,5′-Monophosphate Response Element of the Rabbit Surfactant Protein-A Gene: Evidence for Transactivators Distinct from CREB/ATF Family Members

Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5′-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression. This element, termed CRE_sp-a (TGACCTCA), differs by one nucleotide from a palindromic CRE (CRE_pal, TGACGTCA), which is known to bind CREB as a homodimer. In the present study, we found that mutagenesis of CRE_sp-a to CRE_pal also caused a marked decrease in basal and cAMP-induced fusion gene expression. The findings of competitive electrophoretic mobility shift assays (EMSA) using fetal rabbit lung nuclear extracts suggest that different protein complexes bind CRE_sp-a and CRE_pal. By UV cross-linking analysis, an ∼43-kilodalton protein complex was found to interact both with CRE_sp-a and CRE_pal; however, purified CREB was ineffective in binding CRE_sp-a but did bind CRE_pal. In EMSA using fetal rabbit lung nuclear proteins, antibodies directed against CREB, CRE modulator (CREM), and activating transcription factor-1 (ATF-1) failed to supershift the complex of proteins bound to CRE_sp-a; whereas, a supershift was evident using CRE_sp-a, as a probe. Moreover, in competition EMSA using radiolabeled CRE_sp-a and fetal rabbit lung nuclear proteins, a purified basic leucine zipper (bLZ) polypeptide failed to compete for binding. By contrast, the bLZ polypeptide competed effectively with CRE_pal for lung nuclear protein binding. This finding suggests that leucine zipper transcription factors do not bind CRE_sp-a. Additionally, expression of a CRE_sp-a:HIS3 fusion gene in yeast was unaffected either by CREB or bLZ polypeptides fused to the GAL4 activation domain. By contrast, HIS3 expression was markedly induced both by CREB and bLZ fusion proteins in a CRE_pal:HIS3 yeast strain. By competition EMSA using mutagenized CRE_sp-a oligonucleotides, the critical protein-binding nucleotides in CRE_sp-a were found to constitute a hexameric element, TGACCT, which corresponds to a binding site for members of the steroid receptor superfamily. Since the TGACCT motif is present in the SP-A gene as a single site, we propose that a unique orphan member of the steroid receptor superfamily may bind to this element.