C/EBPα and C/EBPδ activate the Clara cell secretory protein gene through interaction with two adjacent C/EBP-binding sites

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)alpha is involved in controlling differentiation-dependent gene expression in the distal lung. In this study, immunofluorescence studies demonstrated high level expression of C/EBP delta in the bronchiolar epithelium as well as lower levels of C/EBP alpha. Cotransfection studies in the lung epithelial cell line A549 showed that both C/EBP alpha and C/EBP delta activate the murine CCSP gene and that a C/EBP-response element resides in the proximal CCSP promoter. C/EBP delta exhibits an approximately 2-fold higher transactivation potential than does C/EBP alpha. 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 alpha and C/EBP delta, 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 alpha and C/EBP delta can bind to both C/EBP sites, whereas in DNase 1 footprint analyses, the interaction of C/EBP alpha with the proximal site was weak. Furthermore, electrophoretic mobility shift assays demonstrated that C/EBP alpha and C/EBP delta preferentially form heterodimers at both binding sites. Cotransfections with C/EBP alpha and C/EBP delta together resulted in a superinduction of the CCSP promoter, indicating a regulatory role for the C/EBP alpha-C/EBP delta heterodimers, Our findings demonstrate that C/EBP alpha and C/EBP delta regulate the CCSP gene through a compound response element and suggest that these factors are important for the differentiation-dependent expression of CCSP.