Transcriptional regulation of differentiation markers in the distal lung epithelium : a role for C/EBP factors

Abstract

Late lung development is characterised by extensive cellular differentiation, resulting in a variety of specialised epithelial cell types. Although much is known of the extracellular signalling involved in lung morphogenesis, less is currently known of the intracellular developmental mechanisms that govern cellular differentiation.

In this thesis, to begin unravel the molecular mechanism underlying lung cellular differentiation we have analysed the transcriptional regulation of two differentiation markers of the distal lung epithelium, the Clara cell secretory protein (CCSP) and the P450 enzyme CYP21B1. Specifically, we have examined the role of transcription factors from the basic region-leucine zipper CCAAT/enhancer binding protein (C/EBP) family. These factors are potentially involved in differentiation-dependent processes in the lung epithelium.

In Paper I, it was demonstrated that C/EBPalpha expression correlates with CCSP expression in cultured primary rat Clara cells. Furthermore, C/EBPalpha was demonstrated to transactivate the proximal CCSP promoter in lung epithelial cell lines. In Paper II, a more detailed study was performed to elucidate the role of C/EBPalpha and C/EBPdelta in CCSP gene expression. Immunohistochemical studies demonstrated the expression of both C/EBPalpha and C/EBPdelta in the distal lung epithelium. It was demonstrated that C/EBPalpha and C/EBPdelta, as well as C/EBPalpha-C/EBPdelta heterodimers, activate the CCSP gene through two adjacent C/EBP-binding sites in the proximal promoter. Mutation of either C/EBP-binding site resulted in markedly reduced transactivation suggesting that these sites form a compound response element. In paper III, cooperative action of transcription factors known to be important for CCSP gene regulation was investigated. The roles of C/EBP factors, the homeodomain transcription factor Nkx2.1 and the Forkhead box transcription factor HNF-3alpha were tested in a system based on Drosophila SL-2 cells. It was demonstrated that C/EBPalpha acts synergistically with Nkx2.1 to transactivate the CCSP promoter. The synergistic activity was mapped to transactivation domains of C/EBPalpha and Nkx2.1 that are known to be important for interaction with members of the basal transcription machinery. These results suggest that recruitment of general transcription factors is involved in the synergism between C/EBPalpha and Nkx2.1. In paper IV, a potential role of C/EBP factors in lung-specific gene expression was further supported. It was demonstrated that C/EBPalpha and C/EBPdelta control the expression of the CYP2B1 gene in a similar fashion to that of the CCSP gene.

In summary, the data presented in this thesis support a role for C/EBP factors in the regulation of differentiation markers in the distal lung epithelium. The involvement of C/EBP factors in the regulation of CCSP and CYP2B1 supports the hypothesis that C/EBP factors have an overall important role in differentiation and maturation of the lung epithelium.