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-H441nuclear 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 comple. The TPA-induced complex was supershifted in the presence of antibody against the Jun 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 rst intron of SP-A1 and SP-A2 may be involved in the phorbol ester inhibition of human SP-A gene expression.