Characterization of the glucocorticoid receptor in fetal rat lung during development: influence of proteolytic...

Glucocorticoid receptor (GR) from fetal rat lung cytosol was characterized during development. A gradual increase in receptor concentration without an apparent change in ligand affinity was observed during ontogenesis (16-20 days of gestation). GR was present at least 2 days prior to gestational day 18, from which day maternal betamethasone administration stimulated choline chloride incorporation into phosphatidylcholine, the major phospholipid in surfactant. Gel permeation analysis of lung cytosolic GR from fetuses of different gestational ages showed a gradual disappearance of a 3.6 nm GR seen in day 16 cytosol and to the appearance of a 5.8 nm GR in cytosol from day 19. The differences in Stokes' radii of GR were not due to transcriptional or posttranscriptional modifications of the GR transcript, since both day 16 and day 19 fetal lung contained a 7 kb GR mRNA similar to that in adult rat lung. Mixing experiments showed that the 3.6 nm GR was generated by an increased proteolytic activity in day 16 lung tissue. Preservation of a normal size 5.8 nm in day 16 fetal lung upon extraction could only be achieved by preincubating and homogenizing the lung tissue in the presence of protease inhibitors. No protease activity was found in day 16 cytosol suggesting the presence of a rapidly inactivated protease(s). The protease activity responsible for GR degradation was probably of a serine protease type, since proteolytic activity could be inactivated by diisopropylfluorophosphate alone, a potent inhibitor of serine proteases. From these results we conclude that: (i) the observed differences in Stokes' radii between GR from fetal lung of different developmental stages is attributable to proteolysis following extraction, most likely by a rapidly inactivated serine protease. This activity diminished during fetal lung development. However, in intact lung cells, GR is physicochemically identical throughout development; (ii) the lack of glucocorticoid stimulation or surfactant synthesis on day 16 and 17 in fetal rat lung despite the presence of low concentration of GR is therefore not explained by any differences in GR structure.