Transcriptional activation and protein binding by two regions of the rat surfactant protein A promoter.

Surfactant protein A (SP-A) is expressed in lung alveolar type II cells and bronchiolar Clara cells. We have identified two active regions in the promoter of the rat SP-A gene by deletion analysis of a plasmid containing 163 bp before the start of transcription (-163 bp), linked to a reporter gene. Constructs were transfected into lung cell lines derived from each of the cell types that produces SP-A. We found a novel region of promoter activity at approximately 90 bp before the transcriptional start (SP-A(-90)). Mutation of four nucleotides in SP-A(-90) that are highly conserved among species (-92 to -89 bp) decreased expression of the SP-A construct by approximately 50% in both cell lines. Electrophoretic mobility shift analysis showed specific binding to SP-A(-90) by nuclear proteins from the cell lines, as well as from rat lung and liver. The electrophoretic mobility of the bands shifted by lung nuclear proteins changed late in fetal development. Although in the Clara cell line no reduction of promoter activity was seen on deletion of the region upstream of SP-A(-90), in the type II cell line, deletion of residues -163 to -133 did reduce activity by approximately 50%. This region contains a recognition element for thyroid transcription factor-1 (TTF-1). Endogenous TTF-1 binding activity was substantially higher in the type II cell line than in the Clara cell line, but cotransfection of a TTF-1 expression plasmid enhanced expression of the SP-A construct better in the Clara cell line than in the type II cell line. These results suggest that the recognition element for TTF-1 has varying activity in the lung cell lines of different origin due to the availability of TTF-1.

Influence of epidermal growth factor and transforming growth factor beta-1 on patterns of fetal mouse lung branching morphogenesis in organ culture.

Transforming growth factor-beta (TGF-beta), a potent inhibitor of epithelial cell proliferation, and epidermal growth factor (EGF), a mitogenic polypeptide that binds to cell surface receptors, are important regulators of cell differentiation; however, their distinct role(s) in lung development and their mechanisms of action are not well understood. We evaluated the effects of these factors on lung morphogenesis in murine fetal lungs at gestational day 14 (time:zero) and again after 7 days in culture. Baseline controls were cultured after tracheal transection in supplemented BGJb medium, and other tracheally transected lungs were cultured following addition of EGF (10 ng/ml BGJb), TGF-beta1 (2 ng/ml BFJb), or with both in combination added to the medium. The control lungs in culture had poorly developed airways and an absence of defined acinar structures. The addition of EGF resulted in hyperplasia of primary airways with stunted outgrowths, monopodial branching, and absence of distinct acinar structures. Addition of TGF-beta1 alone, led to significant elongation of primary airways, without normal airway branching; however, terminal dipodial branching was seen and the prospective pulmonary acini were well defined. Combination of these growth factors (GF) resulted in a more normal branching pattern and differentiation, suggesting their epigenetic role in lung morphogenesis and mutual interactive mechanisms that regulate lung development. These lungs had more abundant and larger lamellar bodies than those after other treatments. Control lungs remained immature with prominent glycogen aggregates with occasional dense lamellar bodies. The total protein and DNA contents were highest with EGF treatment, followed by combination treatment; these observations were supported by immunohistochemical localization of proliferating cell nuclear antigen, an indication of the proliferative state of tissues. All the surfactant proteins were relatively unaltered and their messages were up-regulated for SP-A, but down-regulated for SP-B and SP-C in the lungs treated with growth factors. In conclusion, we have demonstrated enhanced biochemical and structural development of lungs treated in vitro with GF, and propose that further research in this area may lead to therapeutic uses of GF alone or in combination with other agents for the treatment of newborn respiratory distress due to lung immaturity or hypoplastic lung development.

Sequence of rat surfactant protein A gene and functional mapping of its upstream region.

Surfactant protein A (SP-A) is the major pulmonary surfactant protein. We have isolated a rat SP-A genomic clone and determined the sequence from 2.9 kilobases upstream of the transcriptional start through the termination of translation. The exon-intron structure of the rat gene has been determined and compared with the mouse, rabbit, and human genes. We have localized the major transcriptional start site in adult rat lung to nucleotide 30 downstream from the start of the TATA box. Functional mapping indicates that a DNA fragment containing 163 nucleotides upstream of the transcriptional start (-163) can function as a promoter of transcription of a reporter gene in both lung and nonlung derived cell lines. However, the function of this element is weaker in cells of nonlung origin. DNA elements located between -2902 and -163 silence the promoter activity in both lung and nonlung cells. Because the SP-A gene promoter region exhibits limited tissue specificity, the results suggest the existence of other DNA elements which overcome the silencer and confer further lung specificity.

Transcriptional regulation of the apolipoprotein B-100 gene: identification of cis-acting elements in the first nontranslated exon of the human apolipoprotein B-100 gene.

Apolipoprotein B-100, produced primarily in the human liver, is the sole protein component of low-density lipoprotein and serves as a ligand for the LDL receptor. Two cis-acting positive elements between -128 and -70 control hepatic cell-specific expression of the human apoB gene (H. K. Das, T. Leff, and J. L. Breslow, J. Biol. Chem. 263: 11452-11458, 1988). In this study, two apoB cis-acting elements (+20 to +40; +43 to +53) have been identified using DNase I footprint analysis. Through in vitro mutagenesis and transient transfection experiments in Hep G2 and HeLa cells, the element (+20 to +40) was observed to have a negative effect on transcription of the apoB gene. The element (+43 to +53) was found to have a strong positive effect on apoB gene transcription in Hep G2 cells and mild positive effect in HeLa cells. Therefore these two cis-acting elements mediate hepatic-cell specific expression of the apolipoprotein gene by interacting with trans-acting protein factors.

Correlation of expression of transcription factor C/EBP alpha and surfactant protein genes in lung cells.

C/EBP alpha is a transcription factor which can stimulate expression of genes in lipid-metabolizing epithelial cells. We have detected an increase in mRNA for C/EBP alpha in lungs of fetal rats between days 18 and 20 of gestation, correlating with events occurring during the maturation of the surfactant system, such as an increase in the amount of surfactant protein A mRNA. We have found that C/EBP alpha mRNA levels are substantially enriched in type II alveolar epithelial cells purified from adult lung and that the C/EBP alpha protein is present in type II cell nuclei. When the type II cells are removed from the lung and purified, the protein is rapidly lost. However, both surfactant protein gene expression and C/EBP alpha reappear when cells are plated on Matrigel. Levels of C/EBP alpha mRNA from purified cells decline much more slowly than the protein and are still detectable 48 h after cells have been plated on standard tissue culture plastic. We have also detected the C/EBP alpha protein in nuclear extracts of NCI-H441, a lung-derived cell line that expresses surfactant proteins A and B, but not in A549, a lung-derived cell line which does not express the surfactant proteins. Our data suggest that C/EBP alpha is involved in the development and maintenance of the surfactant system in lung type II cells.

Rabbit and rat liver nuclei both contain proteins which bind to the regions controlling apolipoprotein A-I gene expression.

We have tested the 5' flanking region of the apolipoprotein A-I (apo A-I) gene, which controls its expression in hepatic cells, for the ability to bind protein factors from rat and rabbit liver nuclei. We found that nuclear extracts from each species contain proteins which bind to three sites in the region which have been shown to be important for control of apo A-I gene transcription. These results contrast with a previous report [Dai, P. H., Lan, S. S. F., Ding, X. H. & Chao, Y. S. (1990) Eur. J. Biochem. 190, 305-310] that no rabbit liver nuclear protein could be detected which binds to the rat apo A-I upstream region and that this lack of binding could explain the failure of the rabbit liver to express the apo A-I gene. We have also shown that the low levels of apo A-I mRNA in the rabbit liver correlate with decreased transcription. Our data suggest that the lack of apo A-I gene expression in liver is a result of transcriptional control but cannot be due to simple lack of protein binding to this region of DNA.

Increased expression of apolipoprotein genes accompanies differentiation in the intestinal cell line Caco-2.

We have analyzed determinants of the synthesis and secretion of apolipoproteins including mRNA for apolipoproteins, in the human colon carcinoma cell line Caco-2 during differentiation in continuous culture. Significant increases in both cellular and secreted apolipoprotein A-I were observed early in the differentiation process. Increases in apolipoprotein B were limited to secreted protein and started later in the differentiation process. Levels of mRNA for apolipoproteins A-I, A-IV, B, C-III, and E increased significantly between the time cells reached confluence and 1 week postconfluence. The kinetics of mRNA accumulation were influenced by culture conditions. Nuclear extracts from postconfluent Caco-2 cells contained increased amounts of protein that bound to oligonucleotides containing the control regions of the apolipoprotein A-I and B genes. A competition experiment suggested that this protein recognized the control regions of both genes. We propose to name this protein DRIFT-1 (differentiation-related intestinal factor for transcription 1).

Rat strain differences in cyclic-AMP levels in liver and epididymal fat pad tissue as influenced by glucagon.

A glucagon-saline solution (0.1 ml, 10(7) mole/100 g body weight) was injected via the portal vein into nonfasted Wistar and carbohydrate-sensitive BHE rats. Levels of liver and epididymal fat pad cyclic-AMP were observed after 6 and 24 minutes. When compared to sham injected rats at 6 minutes, glucagon injected rats of both strains had twice the level of cyclie-AMP in liver and fat pad tissue. By 24 minutes, the cyclic-AMP levels of the Wistar rats had decreased to those observed in their sham injected counterparts, and the concentration of liver cyclic-AMP in both sham injected and glucagon injected BHE rats had decreased to levels significantly below those observed in the Wistar rats. This observation suggests that a lipolytic-lipogenic imbalance may reside in the livers of rats of the BHE strain.

Phosphorylation of DNA polymerase.

Both E. coli and calf thymus DNA polymerase can be phosphorylated by cAMP-dependent protein kinase and phosphorylation appears to stimulate the DNA polymerase reaction. Conversely, dephosphorylation of the polymerase molecule, by a protein phosphatase, inhibits the polymerase reaction.