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Glucocorticoid inhibition of human SP-A1 promoter activity in NCI-H441 cells
R R Hoover1, K H Thomas, J Floros
1Department of Cellular and Molecular Physiology, P.O. Box 850, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Abstract:
Glucocorticoids have complex effects on human surfactant protein (SP) SP-A1 and SP-A2 gene expression that occur at both transcriptional and post-transcriptional levels. In the lung adenocarcinoma cell line NCI-H441, dexamethasone causes a dose-dependent decrease in total SP-A mRNA levels and inhibits SP-A gene transcription. In this study, a deletional analysis of the SP-A1 promoter was performed in order to identify cis-acting elements that mediate dexamethasone responsiveness in NCI-H441 cells. The region -32/+63 relative to the start of SP-A1 transcription mediated both basal promoter activity and dexamethasone repression of transcription. Removal of the region +18/+63 abolished dexamethasone responsiveness, indicating that sequences within this region are necessary for the inhibitory effect. Furthermore, the region -32/+63 formed a sequence-specific DNA-protein complex with NCI-H441 nuclear extract. This DNA-protein complex was induced by dexamethasone exposure and its formation was mediated partially by sequences within the region +26/+63.
Insights
Dexamethasone inhibits surfactant protein A1 (SP-A1) gene transcription by affecting specific promoter regions. These findings reveal key DNA elements mediating glucocorticoid repression in lung cells.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Glucocorticoids modulate human surfactant protein (SP) gene expression at multiple levels.
- Dexamethasone, a synthetic glucocorticoid, reduces SP-A mRNA and inhibits SP-A gene transcription in NCI-H441 lung adenocarcinoma cells.
Purpose of the Study:
- To identify cis-acting elements in the SP-A1 promoter responsible for dexamethasone-induced transcriptional repression.
- To investigate the molecular mechanisms underlying glucocorticoid regulation of SP-A1 gene expression.
Main Methods:
- Deletional analysis of the SP-A1 promoter region in NCI-H441 cells.
- Reporter gene assays to assess promoter activity and transcriptional regulation.
- Electrophoretic mobility shift assays (EMSAs) to study DNA-protein interactions.
Main Results:
- The SP-A1 promoter region -32/+63 was essential for basal activity and dexamethasone-mediated repression.
- Deletion of the region +18/+63 abolished dexamethasone responsiveness, highlighting its critical role.
- Dexamethasone exposure induced a sequence-specific DNA-protein complex within the -32/+63 region, with sequences in +26/+63 partially mediating this induction.
Conclusions:
- Specific sequences within the SP-A1 promoter, particularly the +18/+63 region, are crucial for mediating dexamethasone's inhibitory effect on transcription.
- Glucocorticoid regulation of SP-A1 involves the formation of dexamethasone-inducible DNA-protein complexes on the promoter.
