Comparison of effects of epidermal and insulin-like growth factors, gastrin releasing peptide and retinoic acid on fetal lung cell growth and maturation in vitro.

The role in cell multiplication and maturation of several factors present in the late fetal lung was explored on isolated fetal rat pulmonary fibroblasts and alveolar epithelial type II cells cultivated in serum-free medium. The low degree of reciprocal contamination of each cell population was assessed by immunocytochemistry. Epidermal Growth Factor (EGF) stimulated thymidine incorporation and DNA accumulation in both cell types. In type II cells, it increased labeled-choline incorporation into surfactant phosphatidylcholine (PC), consistently with previous data obtained with lung explant cultures, but not into non-surfactant PC. Insulin-like growth factor (IGF)-I slightly stimulated DNA accumulation in fibroblasts although it did not significantly stimulate thymidine incorporation, contrary to IGF-II which presented a dose-dependent stimulating activity of thymidine incorporation. Neither IGF-I nor IGF-II stimulated type II cell growth. IGFs thus appear to primarily control the growth of lung mesenchyme. In type II cells, they stimulated the most non-surfactant PC biosynthesis. Gastrin releasing peptide (GRP) which was recently reported to promote fetal lung growth in vivo and to stimulate surfactant biosynthesis in lung organ culture revealed as a growth factor for type II cells only, at concentrations below 10(-9) M. At concentration 10(-8) M, although it did not affect DNA synthesis, GRP tended to increase surfactant and non-surfactant-PC biosynthesis. Retinoic acid inhibited thymidine incorporation into type II cells on a dose-dependent manner but nevertheless enhanced surfactant-PC biosynthesis to a similar extent as EGF. It is suggested that retinoic acid may represent a differentiation or maturation factor for the alveolar epithelium.

Pre-translational regulation of lipid synthesizing enzymes and surfactant proteins in fetal rat lung in explant culture.

In hormone-free explant cultures of 18-day fetal rat lung the levels of the mRNAs for fatty acid synthase, ATP citrate lyase and surfactant proteins A, B, and C, increased as they do in vivo. Also CTP:phosphocholine cytidylyltransferase mRNA increased spontaneously. Unlike in vivo, malic enzyme mRNA increased drastically in cultured explants. Culture with dexamethasone increased the abundance of fatty acid synthase and surfactant protein mRNAs, but considerably depressed that of malic enzyme mRNA. Dexamethasone had little effect on CTP:phosphocholine cytidylyltransferase mRNA, supporting the concept that the increased activity of this enzyme caused by glucocorticoid is due to increased fatty acid synthesis.

Retinoids control surfactant phospholipid biosynthesis in fetal rat lung.

Vitamin A (retinol) may play an important role in lung maturation: 1) premature delivery is simultaneously a source of vitamin A deficiency and increased risk of neonatal respiratory distress syndrome and subsequent bronchopulmonary dysplasia (BPD), due to deficit in pulmonary surfactant; 2) neonatal supplementation with retinol reduces the risk of BPD; and 3) fetal rat lung stores retinol in late gestation just before the onset of surfactant synthesis. To test the hypothesis of an implication of retinoids in the control of pulmonary surfactant synthesis, experiments were designed in the pregnant rat, aiming either at enhancing fetal lung vitamin A stores, bringing the active metabolite of vitamin A, retinoic acid (RA), or inhibiting the conversion of retinol to RA with aid of citral. Maternal administration of a single dose of 50,000 IU of retinyl palmitate on day 16 (term 22 days) increased 22 and 29%, respectively, the total phospholipid (TPL) and disaturated fraction of phosphatidylcholine (PC) in extracted fetal surfactant on day 19 but did not change surfactant protein (SP) A concentration. Chronic administration of retinyl palmitate to the mother from day 16 through 20 increased disaturated PC content on day 21 but decreased SP-A concentration. Fetal lung surfactant phospholipids were increased by chronic administration of RA and considerably reduced by citral (-31 and -35% for TPL and PC concns, respectively). RA also enhanced labeled choline incorporation into fetal lung PC on day 20. Given once on day 17, it accelerated the appearance of surfactant precursors on day 18.(ABSTRACT TRUNCATED AT 250 WORDS)

Fetal rat lung type II cell differentiation in serum-free isolated cell culture: modulation and inhibition.

Undifferentiated fetal rat lung epithelial cells were isolated on gestational days 15 or 17 (term 22 days) and cultured in a defined medium. On plastic, most of the cells developed structurally abnormal lamellar bodies. On a basement membrane matrix (BMM), they sequentially accumulated glycogen and formed typical lamellar bodies. Biochemical analysis of the latter indicated that they had a phospholipid composition typical of surfactant for cells on BMM but not on plastic and that surfactant protein A appeared on BMM only. Progressing maturation from day 1 to day 6 in culture was demonstrated for 17-day cells on BMM by a sevenfold increase of labeled precursor incorporation into surfactant phospholipids. Exposure to medium conditioned by 21-day fetal fibroblasts enhanced incorporation already after a 1-day culture. The antisteroid RU 486 had no effect on differentiation, whereas transforming growth factor-beta, a factor produced by lung mesenchyme at early fetal stages, inhibited it markedly. Alveolar epithelial type II cells appear to be committed early, but their maturational process would be prevented until a definite gestational stage.

Culture of fetal alveolar epithelial type II cells in serum-free medium.

A serum-free culture medium (defined medium = DM) was elaborated by adding to Eagle's minimum essential medium (MEM), non-essential amino acids, transferrin, putrescine, tripeptide glycyl-histidyl-lysine, somatostatin, sodium selenite, ethanolamine, phosphoethanolamine, sodium pyruvate, and metal trace elements. This medium was tested for its ability to support sustained surfactant biosynthesis in fetal alveolar epithelial type II cells. For up to 8 days, ultrastructure was maintained with persistence of lamellar inclusion bodies. Thymidine incorporation into DNA was enhanced about 50% in DM as compared with MEM, whereas it was enhanced 300% in 10% fetal bovine serum. With DM, the incorporation of tritiated choline into phosphatidylcholine (PC) of isolated surfactant material was about twice that with MEM. Deletion experiments evidenced the prominent role of pyruvate, transferrin, and selenium in the stimulation of surfactant PC biosynthesis. The addition of biotin to DM enhanced surfactant PC biosynthesis slightly and nonsurfactant PC biosynthesis markedly. The presence of nucleosides seemed unfavorable to the synthesis of surfactant PC. Type II cells responded to the addition of epidermal growth factor and insulinlike growth factor-I both by increased thymidine incorporation into DNA and choline incorporation into PC. It is concluded that DM represents a useful tool for cultivating type II cells without loss of their specialized properties and for studying the regulation of cell proliferation and surfactant biosynthesis in a controlled environment.

Characterization of the rat pulmonary surfactant protein A promoter.

The expression of the pulmonary surfactant protein A (SP-A) is developmentally regulated and controlled by several hormones. In an attempt to characterize cis-acting elements involved in the regulation of SP-A expression, we have cloned the 5' flanking sequence of the rat SP-A gene. The promoter region contains a TATA box but no CAAT box. The transcription start site has been identified by anchored polymerase chain reaction and S1 nuclease mapping of the mature and precursor transcripts. S1 mapping of precursor transcripts has confirmed the stimulating effect of glucocorticoids on SP-A rat gene transcription in vivo. This hormonal effect may be mediated by a putative glucocorticoid responsive element located 140 bp upstream from the initiation site and protected against DNase 1 digestion in footprinting experiments. In vitro transcription of a G-free reporter cassette linked to the 212-bp 5' flanking DNA fragment has established that this putative promoter region is functional. Efficient transcription of the G-free reporter cassette was obtained with cell-free fetal lung extracts, whereas no transcript was detectable with cell-free liver extracts. Comparative analysis of the human and rat 5' flanking sequences shows the presence of strongly conserved motifs, unrelated to previously known consensus sequences. Some of these motifs, specifically protected in DNase 1 footprinting studies, could therefore be involved in the regulation of SP-A gene expression.