Am. J. Respir. Cell Mol. Biol., Volume 22, Number 4, April, 2000 469-480

C/EBP and C/EBP Activate the Clara Cell Secretory Protein Gene through Interaction with Two Adjacent C/EBP-Binding Sites

Tobias N. Cassel, Lena Nordlund-Möller, Olof Andersson, Jan-Åke Gustafsson, and Magnus Nord

Department of Medical Nutrition, Karolinska Institutet, Huddinge; and Department of Lung Medicine, Huddinge University Hospital, Huddinge, Sweden

The Clara cell secretory protein (CCSP) gene is a cell-specific differentiation marker for the bronchiolar Clara cell. Previous studies suggest that CCAAT/enhancer binding protein (C/EBP) is involved in controlling differentiation-dependent gene expression in the distal lung. In this study, immunofluorescence studies demonstrated high level expression of C/EBP in the bronchiolar epithelium as well as lower levels of C/EBP. Cotransfection studies in the lung epithelial cell line A549 showed that both C/EBP and C/EBP activate the murine CCSP gene and that a C/EBP-response element resides in the proximal CCSP promoter. C/EBP exhibits an approximately 2-fold higher transactivation potential than does C/EBP. DNase I footprint analyses revealed a footprint region located at 100 to 62 bp, corresponding to two C/EBP-binding sites. Mutation of either site resulted in abolished or strikingly reduced transactivation of the CCSP promoter by C/EBP and C/EBP, as well as impaired binding of both factors, indicating that the two C/EBP-binding sites form a compound response element. In electrophoretic mobility shift assays, it was shown that C/EBP and C/EBP can bind to both C/EBP sites, whereas in DNase I footprint analyses, the interaction of C/EBP with the proximal site was weak. Furthermore, electrophoretic mobility shift assays demonstrated that C/EBP and C/EBP preferentially form heterodimers at both binding sites. Cotransfections with C/EBP and C/EBP together resulted in a superinduction of the CCSP promoter, indicating a regulatory role for the C/EBP-C/EBP heterodimers. Our findings demonstrate that C/EBP and C/EBP regulate the CCSP gene through a compound response element and suggest that these factors are important for the differentiation-dependent expression of CCSP.

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