Surfactant protein D gene regulation. Interactions among the conserved CCAAT/enhancer-binding protein elements.
Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following acute lung injury. Given the importance of CCAAT/enhancer-binding protein (C/EBP)-binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoforms by lung epithelial cells, we hypothesized that conserved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expression by C/EBPs. Five SP-D motifs (-432, -340, -319, -140, and -90) homologous to the C/EBP consensus sequence specifically bound to C/EBPs in gel shift assays, and four of the five sites (-432, -340, -319, and -90) efficiently competed for the binding of C/EBPalpha, C/EBPbeta, or C/EBPdelta to consensus oligomers. Cotransfection of C/EBPalpha, C/EBPbeta, or C/EBPdelta cDNA in H441 lung adenocarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containing 698 bp of upstream sequence (SS698). Transfection of C/EBP also increased the level of endogenous SP-D mRNA in H441 cells. Transactivation of the reporter construct was abrogated by deletion of sequences upstream of -205. Independent site-directed mutagenesis of the sites at -432, -340, and -319 reduced C/EBP-mediated activation by approximately 50%, and mutagenesis of the site at -432 in combination with either of the tandem sites at -340 and -319 blocked activation. The conserved AP-1 element at -109 was required for maximal promoter activity, but not for the transactivation of SS698 by C/EBPs. Thus, interactions among C/EBP elements in the near-distal promoter can modulate the promoter activity of SP-D.
He
Yanchun
Y
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Crouch
Erika
E
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D020033:Protein Isoforms; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants / Q000096:biosynthesis* / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D022762:CCAAT-Enhancer-Binding Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D020033:Protein Isoforms; D037663:Pulmonary Surfactant-Associated Protein D; D011663:Pulmonary Surfactants; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase