Since the initial observations of Liggins demonstrating dexamethasone-induced inflation of fetal lungs, antenatal glucocorticoids have become the mainstay clinical management for the promotion of fetal lung maturation. The molecular mechanisms by which this prevalent therapy manifests functionally are poorly understood. We previously reported that in our Erk3-/- MAP kinase family knockout mouse model, antenatal dexamethasone (dex) rescued histologic evidence of pulmonary immaturity but failed to restore neonatal lung function (PNAS 106:16710, 2009). To further advance this work we used whole transcriptome RNA shotgun sequencing, where we identified corticotropin releasing hormone (CRH) and surfactant protein B (SFTPB) as molecular mediators in fetal pulmonary maturation. We hypothesized that attenuation of CRH is the key molecular mediator involved in this glucocorticoid induced functional pulmonary maturation.
In a double crossover design, we administered antenatal dex (0.4mg/kg) or saline subcutaneously to pregnant mothers at E17.5 and E18.5, followed by postnatal surfactant (50mg/kg) or saline via inhalation intubation to newborn pups. Confirmation of pulmonary surfactant was visualized by adding indigo carmine stain to the surfactant. Analysis of CRH gene and protein expression were performed on postnatal lung sections.
Quantitation of gene expression (mRNA) showed that antenatal dex accelerated Crh downregulation in both Erk3+/+ (p<0.001) and Erk3-/- (p<0.001) when compared to saline alone. Immunohistochemistry analysis confirmed these findings, and further demonstrated that antenatal dex rescued pulmonary immaturity in Erk3 deficient mice (Figure A). At birth Erk3-/- pups treated with antenatal dex and postnatal surfactant demonstrated persistent attenuation of CRH production when compared with controls (saline/saline (p<0.01)) and relative to dex alone (p<0.049, Figure B). Moreover, administration of antenatal dex and postnatal surfactant in Erk3-/- pups persistently maintained CRH attenuation up to 6 hrs of life when compared to controls (saline/saline (p<0.01)).
These findings confirm and validate our whole-transcriptome findings, demonstrating that the normal attenuation of CRH production in the fetal lung with advancing gestation is accelerated with administration of antenatal glucocorticoids in an Erk3-independent manner. These investigations are relevant to public health as they describe how a prevalent a meaningful intervention (antenatal glucocorticoids) activate key signature pathways and may advance our understanding of the specific molecular pathways and the mechanisms of action by which antenatal steroids promotes fetal lung maturity.