Developmental Changes of Tropoelastin Synthesis by Rat Pulmonary Fibroblasts and Effects of Dexamethasone

Abstract

ABSTRACT. Lung elastin is an important extracellular structural protein and it has been postulated that it plays a regulatory role in alveolar formation. To study the developmental regulation of elastin gene expression, we examined the tropoelastin (TE) production in primary culture of rat pulmonary fibroblasts (RPF). We found that developmental changes in elastin production as assessed by T E synthesis and 3.6-kb TE mRNA levels were similar for RPF and whole tissue except those results from late gestation animals, with peak elastin expression occurring 7 d postnatally with a decline out to 21 d. At late gestation (20 d), TE mRNA was barely detectable in RPF but clearly detectable TE mRNA in the whole tissue, indicating that there are elastogenic cells other than RPF in the tissue at this age. When TE-producing cells were treated with dexamethasone, there was a dose-dependent stimulation of T E synthesis with the maximum response at lo-' to lo-" M. Interestingly, dexamethasone had no stimulatory effect on cells from late gestation animals. The developmental window of elastin synthesis in this RPF model between late gestation and 21 d postnatal seems to correlate with the reported period of secondary alveolar formation, and thus we speculate that RPF elastogenic activity reflects that of the alveolar wall. (Pediatr Res 28: 379-382, 1990) Abbreviations RPF, rat pulmonary fibroblast TE, tropoelastin SSC, sodium chloride sodium citrate buffer RT, room temperature TCA, trichloroacetic acid Elastin is an important extracellular protein component that forms a resilient fibrous network in the lung. It gives structural compliance to the tissues and may play an important role in the formation of terminal airsacs/alveoli during perinatal stages in various animals, including humans (1-8). This hypothesis is largely based on morphologic observations of elastin deposition in the lung parenchyma and its timely appearance associated with terminal airsac formation.Elastin is a cross-linked mature form of the protein and proform is secreted as TE, a soluble protein of approximately 72 000-75 000 mol wt (9, 10). Regulation of TE synthesis is poorly understood, partly because the regulatory elements of the gene have not been clearly identified. Recent reports indicate that cultured RPF are elastogenic in contrast to lung fibroblasts from larger animals and thus provide a good model for studying lung elastin metabolism (10). However, ontogeny of TE expression in vitro in relation to the original lung tissues has not been described. It is important, therefore, to better characterize the in vitro phenotype so that RPF can be used to investigate the TE gene expression in the lung.In our study, RPF were isolated from rat tissues of various ages and TE expression was examined at the mRNA and protein levels and compared with the steady-state TE mRNA levels of the original tissues to determine if the TE phenotype is maintained in vitro. We also examined whether dexamethasone modulates TE synthesis of the cultured rat lung cell, inasmuch...

IGF-I Stimulates Tropoelastin Synthesis in Neonatal Rat Pulmonary Fibroblasts

“…The project was approved by the Animal Care Committee of St. Louis University. Postnatal d 1 was counted as the birth date, and RPF were isolated as described before (12). Selection of the ages and response to DEX stimulation was dependent on our previous finding that TE expression is maximum at 7 d (12).…”

“…At this FCS concentration, the results were more consistent than with 0.1 % FCS. Twelve h after the medium switch, cells were treated with increasing concentrations of IGF-I for another 48 h, harvested by 0.25% trypsin and 0.0 1 % EDTA treatment, and processed for extraction of total RNA (12). Cell counts were determined simultaneously using a hematocytometer.…”

“…The 3H-leucine incorporation increased progressively, and was 35 + 5% greater than control at 500 ng/mL. Because DEX is known to stimulate TE synthesis in RPF (12), the effect of IGF-I plus DEX was studied. At this lower FCS concentration at 0.5%, DEX did not stimulate TE gene expression and did not increase the effects of IGF-I (Figs.…”

“…We have previously demonstrated that cultured RPF are elastogenic in early passaged cells and that TE expression is stimulated by DEX (12). To further understand the humoral factors that could stimulate TE synthesis in the developing lung, we have examined IGF-I in the present study.…”

“…The common ultrastructurd characteristics in all sodium ,-itrate buffer, 100 m~ N~ phosphate, PH 6.5, 10 x of the cells examined were prominent rough endoplasmic retic~~~h~~d~'~ solution (17), and 1 m g / m~ salmon sperm DNA. ulum cisternae and lack of basal lamina around the plasma washing conditions were as described before (12). ~h~ dried membranes.…”