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 epithel ium, chimeric CCSP-reporter gene constructs containing various regions of the CCSP 5' flanking region were co-transfected into H-441 cells w ith HNF-3 alpha or HNF-3 beta expression plasmids, These studies indic ate that each of these transcription factors positively regulates CCSP gene expression and revealed that CCSP region I (-132 to -76) is suff icient to mediate this effect. Gel-mobility-shift assays with oligonuc leotides corresponding to CCSP region I, nuclear extract from bronchio lar 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 l ung revealed HNF-3 gene expression in non-ciliated bronchiolar epithel ial cells expressing the CCSP gene. Finally, experiments with CCSP reg ion I and a heterologous promoter indicate that this region acts in a promoter-specific context, suggesting that additional factors interact ing via the minimal CCSP promoter region are essential in determining the effects of HNF-3 on cell-specific CCSP gene expression in the bron chiolar epithelium.