Identification of a novel DNA regulatory element in the rabbit surfactant protein B (SP-B) promoter that is a target for ATF/CREB and AP-1 transcription factors

Abstract

Surfactant protein B (SP-B) is required for the maintenance of biophysical properties and physiological function of pulmonary surfactant. SP-B is expressed in a cell/tissue-specific manner by the alveolar type II and bronchiolar (Clara) epithelial cells of the lung and is developmentally and hormonally regulated. We previously identified a minimal promoter region containing −236/+39 base pairs (bp) of rabbit SP-B gene that is necessary and sufficient for high level promoter activity in NCI-H441 cells, a cell line with characteristics of Clara cells. In this study, we have characterized the functional importance of a novel DNA regulatory element, termed SP-B CRE, with the sequence TGAGGTCA in the SP-B minimal promoter. The SP-B CRE sequence shared homology to cyclic AMP responsive element (CRE) binding sequence and contained an overlapping nuclear receptor element binding half-site. Mutation of SP-B CRE into a scrambled sequence reduced promoter activity by greater than 70%, whereas mutation into a palindromic consensus CRE increased the promoter activity by 100%. Electrophoretic mobility shift assay (EMSA) and Western immunoblot analysis of affinity purified proteins interacting with SP-B CRE showed that it is a target for binding of members of the activating transcription factor (ATF)/cyclic AMP response element binding protein (CREB) family of transcription factors, such as CREB, CREM, ATF-1, ATF-2 as well as c-Jun and TTF-1. Overexpression of CREB, ATF-2 and c-Jun inhibited SP-B promoter activity in NCI-H441 cells. These data have shown that members of the ATF/CREB family of transcription factors and c-Jun play important roles in mediating the transcriptional regulation of the SP-B gene.