Induction of AP-1 binding to intron 1 of SP-A1 and SP-A2 is implicated in the phorbol ester inhibition of human SP-A promoter activity

A deletional analysis of the SP-A1 promoter in NCI-H441 cells was performed to identify potential cis-acting elements involved in phorbol ester-mediated repression of human SP-A transcription. The phorbol ester TPA reduced SP-A1 and SP-A2 promoter activity to approximately 35% to 45% compared to that of control cells. The inhibitory effect of TPA was significantly reduced upon removal of the region +64/+394 relative to the SP- A1 transcription start site. Using NCI-H441 nuclear proteins, electromobility shift assay analysis showed that the intron region +309/+329 of SP-A1 and the corresponding region of SP-A2 formed sequence-specific DNA/protein complexes that were induced by TPA exposure. The region +318/+324 of SP-A1 contains sequences similar to a consensus AP-1 binding site, TGACTGA (TCACTGA for SP- A2), which when mutated (TGAGAGT) prevented the formation of the TPA-induced DNA/protein complex. The TPA-induced complex was supershifted in the presence of antibody against the Fun family of proteins, but not the Fos family of proteins. These results suggest that the binding of AP-1 or an AP-1-like factor to the first intron of SP-A1 and SP-A2 may be involved in the phorbol ester inhibition of human SP-A gene expression.