Clara Cell Secretory Protein Promoter Research Articles

Role of hepatocyte nuclear factor-3 alpha and hepatocyte nuclear factor-3 beta in Clara cell secretory protein gene expression in the bronchiolar epithelium.

The 5' flanking region of the Clara cell secretory protein (CCSP) gene contains two cis-acting elements which bind hepatocyte nuclear factor (HNF)-3 alpha and HNF-3 beta in vitro. To determine the role of these proteins in mediating CCSP gene expression in the bronchiolar epithelium, chimeric CCSP-reporter gene constructs containing various regions of the CCSP 5' flanking region were co-transfected into H-441 cells with HNF-3 alpha or HNF-3 beta expression plasmids. These studies indicate that each of these transcription factors positively regulates CCSP gene expression and revealed that CCSP region I (-132 to -76) is sufficient to mediate this effect. Gel-mobility-shift assays with oligonucleotides corresponding to CCSP region I, nuclear extract from bronchiolar epithelial cells and HNF-3-specific antibodies indicate that HNF-3 alpha and HNF-3 beta are the only proteins in bronchiolar epithelial cells which directly interact with this region. Consistent with these observations, HNF-3 alpha and HNF-3 beta transcripts were found to be enriched in this cell population and in situ hybridization of adult lung revealed HNF-3 gene expression in non-ciliated bronchiolar epithelial cells expressing the CCSP gene. Finally, experiments with CCSP region I and a heterologous promoter indicate that this region acts in a promoter-specific context, suggesting that additional factors interacting via the minimal CCSP promoter region are essential in determining the effects of HNF-3 on cell-specific CCSP gene expression in the bronchiolar epithelium.

Two members of the HNF-3 family have opposite effects on a lung transcriptional element; HNF-3 alpha stimulates and HNF-3 beta inhibits activity of region I from the Clara cell secretory protein (CCSP) promoter.

Region I of the Clara cell secretory protein (CCSP; also called CC10) promoter contains at least three functional factor binding sites, an upstream HNF-3 site and a downstream overlapping AP-1/HNF-3 site (Sawaya, P.L., Stripp, B. R., Whitsett, J.A., and Luse, D. S. Mol. Cell. Biol. (1993) 13, 3860-3871). Fragments containing -320/+58 of the rat CCSP promoter were mutagenized to eliminate one or two factor binding sites in region I, cloned into a luciferase reporter cassette, and assayed for activity by transfection into cultured lung (H441) cells. We found that the HNF-3 sites alone can account for the activity of region I in H441 cells. The activity of the two HNF-3 sites is synergistic; this effect depends on the presence of an upstream factor binding site. We had shown previously that H441 cells contain exclusively the HNF-3 alpha form of HNF-3, whereas HeLa cells have essentially no HNF-3. Co-transfection of an HNF-3 alpha expression plasmid with a CCSP reporter containing four copies of region I in HeLa cells stimulated CCSP activity 4-fold, whereas co-expression of HNF-3 beta inhibited activity 8-fold. HNF-3 beta was also inhibitory to region I expression in H441 cells, but to a lesser extent than in HeLa cells, presumably because of the high levels of HNF-3 alpha already present in H441 cells. We have thus identified a gene regulatory element through which two members of the HNF-3 transcription factor family, HNF-3 alpha and HNF-3 beta, exert opposite effects.