RESUMO
C/EBP delta, a member of the leucine zipper transcription factor family, is expressed at higher levels in the lung than in any other tissue. We detected C/EBP delta mRNA and protein in NCI-H441 cells, a cell line derived from a human adenocarcinoma that produces surfactant protein A (SP-A). NCI-H441 cells were exposed to phosphorothioate-substituted antisense oligonucleotides directed against C/EBP delta. After exposure to the oligonucleotides, cells were harvested, total RNA prepared, and levels of mRNA for C/EBP delta, SP-A and a control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were quantified from Northern blots. An oligonucleotide that overlapped the translational start was effective in reducing C/EBP delta mRNA. Oligonucleotides that corresponded to regions upstream and downstream from the translational start were not as effective. The loss of C/EBP delta was accompanied by a decrease in the level of SP-A mRNA. The overlapping oligonucleotide was tested more extensively. After 72 h, antisense oligonucleotide at 3 and 5 microM reduced the level of C/EBP delta mRNA and protein by 50% or more as compared with sense and scrambled controls. The SP-A mRNA level was reduced even more, by about 75%. GAPDH mRNA was not affected. We conclude that C/EBP delta plays a role in the regulation of SP-A gene expression.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Regulação da Expressão Gênica , Proteolipídeos/genética , Surfactantes Pulmonares/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT , Humanos , Camundongos , Camundongos Knockout , Oligonucleotídeos Antissenso/genética , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes Pulmonares , Fatores de Transcrição/genéticaRESUMO
Members of the CCAAT enhancer binding protein (C/EBP) transcription factor family were detected in fetal lung of both human and rat. In rat lung, the level of C/EBPs increased with time of gestation, peaking around birth. In adult rat lung, C/EBPs were localized to the alveolar type II cells. The effect of C/EBPs on pulmonary surfactant protein A (SP-A), which is also expressed late in gestation, was investigated. In contrast to control plasmids, C/EBP delta expressing plasmids reversed the action of a transcriptional silencer just upstream of the rat SP-A promoter. In order to test the effect of C/EBPs on endogenous SP-A gene expression, cells that express SP-A were exposed to a phosphorothioate-substituted, double-stranded oligonucleotide matching the consensus C/EBP binding site (decoy oligonucleotide) at concentrations from 0.5 to 10 microM for 72 h. A mutant oligonucleotide with an 8-base pair (bp) substitution served as a control. The decoy oligonucleotide reduced SP-A mRNA as much as 75% compared to a mutant oligonucleotide both in the human lung cell line, NCI-H441, and in primary human fetal alveolar type II cells. The data indicate that C/EBPs facilitate SP-A gene expression, possibly by overcoming transcriptional silencing.