Mechanism of SPB gene regulation by cortisol and retinoic acid in a human pulmonary epithelial cell line. † 280

Surfactant protein B (SPB) is required for the normal function of pulmonary surfactant. A deficiency in SPB at birth has been associated with lethal respiratory distress in term infants. H441 cells are a human pulmonary epithelial adenocarcinoma cell line that produces SPB mRNA and protein. We have shown that both cortisol and alltrans retinoic acid (RA) increase the content of SPB mRNA in a dosedependent manner in H441 cells when compared to untreated controls. We have also shown that the combination of cortisol plus RA causes a further stimulation of SPB mRNA content over the levels in cells treated with either hormone alone. We investigated the mechanisms of action of cortisol, RA and the combination of cortisol plus RA by studying their transcriptional and posttranscriptional effects on SPB gene expression. In the first series of experiments, H441 cells were treated for 24 hours in serumfree media with either no additions (control), cortisol (10⁶M), RA(10⁶M), or a combination of cortisol (10⁶M) plus RA(10⁶M). Nuclear runon transcription assays were performed to determine the rate of SPB gene transcription. When compared to controls, the rate of SPB gene transcription was increased 4.2 ± 0.8 times by cortisol, 1.7± 0.3 times by RA and 4.6 ± 1.2 times by the combination of cortisol and RA (all values are mean ± SEM, n=5). We then examined the effects of these hormones and hormone combinations on SPB mRNA stability. H441 cells were treated for 12 hours with cortisol and/or RA, at the concentrations listed above, and then with the same hormones in the presence of actinomycin D(10 μg/ml), a RNA synthesis inhibitor, for up to an additional 24 hours. Northern blot analysis was performed on RNA harvested from the control and treated cells after 0, 4, 8, 12 and 24 hours of incubation in hormone plus actinomycin D. The halflife of SPB mRNA was 21 hours in control cells, 29 hours in cortisoltreated cells, 22 hours in RA treated cells, and 44 hours in cells treated with cortisol plus RA (n = 3). We conclude that cortisol regulates SPB mRNA via increased gene transcription and increased mRNA stability. RA, in contrast, increases SPB gene transcription but has no effect on SPB mRNA stability. We conclude further that the combination of cortisol plus RA increases SPB mRNA levels primarily via an effect on mRNA stability.