Structural and transcriptomic response to antenatal corticosteroids in an Erk3-null mouse model of respiratory distress

Abstract

Background: Neonatal respiratory distress syndrome in preterm infants is a leading cause of neonatal death. Pulmonary insufficiency-related infant mortality rates have improved with antenatal glucocorticoid treatment and neonatal surfactant replacement. However, the mechanism of glucocorticoid-promoted fetal lung maturation is not understood fully, despite decades of clinical use. We previously have shown that genetic deletion of Erk3 in mice results in growth restriction, cyanosis, and early neonatal lethality because of pulmonary immaturity and respiratory distress. Recently, we demonstrated that the addition of postnatal surfactant administration to antenatal dexamethasone treatment resulted in enhanced survival of neonatal Erk3-null mice.

Objective: To better understand the molecular underpinnings of corticosteroid-mediated lung maturation, we used high-throughput transcriptomic and high-resolution morphologic analysis of the murine fetal lung. We sought to examine the alterations in fetal lung structure and function that are associated with neonatal respiratory distress and antenatal glucocorticoid treatment.

Study design: Dexamethasone (0.4 mg/kg) or saline solution was administered to pregnant dams on embryonic days 16.5 and 17.5. Fetal lungs were collected and analyzed by microCT and RNA-seq for differential gene expression and pathway interactions with genotype and treatment. Results from transcriptomic analysis guided further investigation of candidate genes with the use of immunostaining in murine and human fetal lung tissue.

Results: Erk3(-/-) mice exhibited atelectasis with decreased overall porosity and saccular space relative to wild type, which was ameliorated by glucocorticoid treatment. Of 596 differentially expressed genes (q < 0.05) that were detected by RNA-seq, pathway analysis revealed 36 genes (q < 0.05) interacting with dexamethasone, several with roles in lung development, which included corticotropin-releasing hormone and surfactant protein B. Corticotropin-releasing hormone protein was detected in wild-type and Erk3(-/-) lungs at E14.5, with significantly temporally altered expression through embryonic day 18.5. Antenatal dexamethasone attenuated corticotropin-releasing hormone at embryonic day 18.5 in both wild-type and Erk3(-/-) lungs (0.56-fold and 0.67-fold; P < .001). Wild type mice responded to glucocorticoid administration with increased pulmonary surfactant protein B (P = .003). In contrast, dexamethasone treatment in Erk3(-/-) mice resulted in decreased surfactant protein B (P = .012). In human validation studies, we confirmed that corticotropin-releasing hormone protein is present in the fetal lung at 18 weeks of gestation and increases in expression with progression towards viability (22 weeks of gestation; P < .01).

Conclusion: Characterization of whole transcriptome gene expression revealed glucocorticoid-mediated regulation of corticotropin-releasing hormone and surfactant protein B via Erk3-independent and -dependent mechanisms, respectively. We demonstrated for the first time the expression and temporal regulation of corticotropin-releasing hormone protein in midtrimester human fetal lung. This unique model allows the effects of corticosteroids on fetal pulmonary morphologic condition to be distinguished from functional gene pathway regulation. These findings implicate Erk3 as a potentially important molecular mediator of antenatal glucocorticoid action in promoting surfactant protein production in the preterm neonatal lung and expanding our understanding of key mechanisms of clinical therapy to improve neonatal survival.

Keywords: CRH; ERK3; SFTPB; antenatal glucocorticoid; fetal lung maturation.

FIGURE 1. Micro computed tomography analysis of fetal mouse lungs

A, Representative images of the right lobe show normal pulmonary structure in wild-type lungs and atelectasis in lungs of Erk3^−/− mice that were treated with saline solution. Gross structural defects were reversed by dexamethasone treatment. B, Histogram of voxel density from micro computed tomography scans. Higher greyscale values indicate greater density. Lungs from Erk3^−/− mice exhibited higher relative density because of lower total airspace and collapse of lung structures. Treatment with dexamethasone partially reversed this phenotype. Pew et al. Regulation of CRH and SFTPB in fetal lung maturation. Am J Obstet Gynecol 2016.

FIGURE 2. Differential gene expression and pathway interactions detected in fetal lungs

A, Differential expression of Crh and Igf2 as detected by RNA-seq. Expression estimates (fragments per kilobase of transcript per million mapped reads, FPKM) for Crh and Igf2 across each sample group with FDR-corrected q-values for significant expression differences in comparisons between saline solution— and dexamethasone-treated groups. Error bars represent cross-replicate variability and measurement uncertainty, as estimated by the Cuffdiff statistical model. B, Ingenuity Pathway Analysis interactions. Crh, Id1, Pdgfa, and Igf2 are expressed differentially in saline solution— vs dexamethasone-treated lungs. All but Igf2 were identified by DAVID Gene Ontology as being involved in lung development. Surfactant proteins were included in the Ingenuity Pathway Analysis because of their established role in lung function and development. CRH, corticotropin-releasing hormone; Dex, dexamethasone; FPKM, fragments per kilobase of transcript per million mapped reads; ID1, Inhibitor Of DNA Binding 1; IGF2, insulin-like growth factor 2; PDGFA, platelet-derived growth factor alpha; SFTPA1, surfactant protein A1; SFTPA2, surfactant protein A2; SFTPB, surfactant protein B; SFTPC, surfactant protein C. Pew et al. Regulation of CRH and SFTPB in fetal lung maturation. Am J Obstet Gynecol 2016.

FIGURE 3. Crh gene expression in lung tissue of wild type and Erk3^−/− mice

Evaluation of corticotropin-releasing hormone expression in embryonic day 14.5, 16.5, or 18.5 fetuses that were exposed to transplacental administration of dexamethasone or saline solution at embryonic days 16.5 and 17.5 of gestation. A, Immunohistochemistry photomicrographs of lung sections stained for corticotropin-releasing hormone. (Original magnification, ×40). B, Quantitation of staining in lung sections (n = 5) illustrates corticotropin-releasing hormone down-regulation with dexamethasone treatment (Erk3^+/+ [P < .001] and Erk3^−/− [P < .001]). C, Crh expression relative to saline solution—treated Erk3^+/+ mice by quantitative RT-PCR. CRH, corticotropin-releasing hormone; Dex, dexamethasone; E, embryonic day; IHC, immunohistochemistry; mRNA, messenger RNA. Pew et al. Regulation of CRH and SFTPB in fetal lung maturation. Am J Obstet Gynecol 2016.

FIGURE 4. Protein expression of corticotropin-releasing hormone in human fetal lung

A, Corticotropin-releasing hormone staining demonstrated for the first time that corticotropin-releasing hormone is expressed broadly in human lungs through the mid trimester in both epithelial and interstitial cells. (Original magnification, ×40.) B, Protein expression of corticotropin-releasing hormone was evaluated by immunohistochemistry on archived human fetal lung specimens (n = 5). Corticotropin-releasing hormone increased as gestational age advanced from 18−22 weeks, with a significant increased expression from 18−22 weeks (P < .0001), 19−22 weeks (P < .001), and 20−22 weeks (P < .001). Two asterisks indicate P ≤ .001 vs 22 weeks; 3 asterisks indicate P ≤ .0001 vs 22 weeks. CRH, corticotropin-releasing hormone; IHC, immunohistochemistry. Pew et al. Regulation of CRH and SFTPB in fetal lung maturation. Am J Obstet Gynecol 2016.

FIGURE 5. Dexamethasone-induced increase in surfactant protein B is modulated by ERK3 signaling

Pregnant female mice were treated with dexamethasone or saline solution at embryonic days 16.5 and 17.5 of gestation. Fetuses were collected by cesarean delivery at embryonic day 18.5. A, Immunohistochemistry images of lung sections that were stained for surfactant protein. (Original magnification, ×40.) B, Quantitation of staining in lung sections (n = 5) illustrates increased surfactant protein B production with dexamethasone treatment in wild type mice (P = .003) but not in Erk3^−/− mice, which show decreased expression (P = .012). C, Sftpb expression relative to saline solution—treated Erk3^+/+ mice by quantitative RT-PCR. Dex, dexamethasone; IHC, ••••; mRNA, messenger RNA; SFTPB, surfactant protein B. Pew et al. Regulation of CRH and SFTPB in fetal lung maturation. Am J Obstet Gynecol 2016.