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.

The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families.

We have shown that a large fragment (-2339 to +57) from the rat CC10 gene directed lung-specific expression of a reporter construct in transgenic animals. Upon transfection, a smaller fragment (-165 to +57) supported reporter gene expression exclusively in the Clara cell-like NCI-H441 cell line, suggesting that a Clara cell-specific transcriptional element resided on this fragment (B. R. Stripp, P. L. Sawaya, D. S. Luse, K. A. Wikenheiser, S. E. Wert, J. A. Huffman, D. L. Lattier, G. Singh, S. L. Katyal, and J. A. Whitsett, J. Biol. Chem. 267:14703-14712, 1992). The interactions of nuclear proteins with a particular segment of the CC10 promoter which extends from 79 to 128 bp upstream of the CC10 transcription initiation site (CC10 region I) have now been studied. This sequence can stimulate both in vitro transcription in H441 nuclear extract and transient expression of reporter constructs in H441 cells. Electrophoretic mobility shift assays using extracts from H441, HeLa, rat liver, and fetal sheep lung cells were used to demonstrate that members of the AP-1, octamer, and HNF-3 families bind to CC10 region I. Transcription factors from H441 cells which are capable of binding to CC10 region I are either absent in HeLa, rat liver, and fetal sheep lung extracts or enriched in H441 extracts relative to extracts from non-Clara cells.

cis-acting elements that confer lung epithelial cell expression of the CC10 gene.

To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.

The action of parotoid venom on the heart of the toad (Bufo ictericus ictericus Spix 1824) and its effects on the inhibition caused by vagal stimulation.

1. The administration of crude venom of the parotoid glands of the toad Bufo ictericus ictericus to the in situ (via abdominal vein) or isolated heart of this anuran causes both chronotropic and inotropic effects. 2. While under action of parotoid venom, the heart of the animal is insensitive to vagus nerve stimulation. 3. This blocking of vagal action is dose dependent and it is suggested that it results from a functional antagonism between the venom constituents and the acetylcholine liberated by the nerve endings on stimulation. 4. The venom constituents probably involved in this antagonism are catecholamines (adrenaline and noradrenaline), tryptamine derivatives (serotonin and bufotenidin) and genins (bufagin and bufotoxin), possibly also ATP. 5. Adrenaline, noradrenaline and serotonin, or a mixture of the three, mimic, at least partially, the blocking of vagal action caused by crude venom. 6. The blocking action of crude venom can be prevented by previously or simultaneously adding acetylcholine to the infused crude venom. This prevention is dose dependent. 7. The blocking action persists in the boiled venom and in the material dialysed from crude venom.