Abstract
Secretoglobin (SCGB) 3A2 is a small molecular weight secreted protein predominantly expressed in lung airways. We previously demonstrated that the expression of SCGB3A2 is regulated by homeodomain transcription factor NKX2-1. Here we show that CCAAT/enhancer-binding proteins, C/EBPalpha and C/EBPdelta, regulate mouse Scgb3a2 gene transcription in vivo and in vitro by binding to specific sites located in the Scgb3a2 promoter and the activity is synergistically enhanced through cooperative interaction with NKX2-1. Six C/EBP binding sites lie within 500 bp of the Scgb3a2 gene promoter, of which two sites, located at -44 to -54 bp and -192 to -201 bp, appear to be critical for the synergistic activation of Scgb3a2 gene transcription with NKX2-1. All three transcription factors, C/EBPalpha, C/EBPdelta, and NKX2-1, are expressed in the epithelial cells of airways, particularly the bronchus, where high expression of SCGB3A2 is found. The expression of these transcription factors markedly increases toward the end of gestation, which coincides with the marked increase of SCGB3A2, suggesting the importance of C/EBPalpha and C/EBPdelta, and their synergistic interaction with NKX2-1 in mouse Scgb3a2 gene transcription and lung development.
Highlights
Secretoglobin (SCGB) 3A2 is a small molecular weight secreted protein predominantly expressed in lung airways
Expression plasmids for these NKX2-1 interacting proteins were prepared in plasmid pcDNA3.1, which were subjected to transfection analysis with the mouse Scgb3a2-promoter (Ϫ907 bp) luciferase reporter construct using mtCC [32], MLE 15 [33], TC-1, and/or COS-1 cells
Among 11 putative C/EBP binding sites present within Ϫ506 bp of the promoter of mouse Scgb3a2 gene, six binding sites appear to play a role in transcription of the gene, of which two binding sites located at Ϫ44 to Ϫ54 bp and Ϫ192 to Ϫ201 bp may be the most critical for the synergistic activation of Scgb3a2 gene promoter by C/EBPs and NKX2-1 (Fig. 10)
Summary
Construction of C/EBP Expression Plasmids—Total RNA was isolated from mouse embryonic lungs (embryonic day (E) 17.5) by using TRIzol (Invitrogen) and treated with DNase I (Ambion, Austin, TX). A transfection mixture contained 20 l of serum-free Dulbecco’s modified Eagle’s medium, 1 l of FuGENE 6 (Roche Applied Science), 250 ng of pGL4.11-based reporter construct (Promega), 5 ng of pGL4.74 having the Renilla luciferase gene connected to the herpes simplex virusthymidine kinase promoter as an internal control (Promega), and a total 50 ng of pcDNA3.1-C/EBP␣ or C/EBP␦ mammalian expression vector (Invitrogen). Western Blot—COS-1 cells after transfection of C/EBP␣, C/EBP␦, or NKX2-1 expression plasmids (50 ng each/well of 24 well plate) were washed with PBS and lysed in RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 10 mM sodium fluoride, 1 mM sodium orthovanadate) with Protease Inhibitor Mixture tablets (Complete Mini; Roche Applied Science). The sections were counterstained with Hematoxylin QS (Vector Laboratories), dehydrated, and mounted in permanent mounting medium
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