293: Transcriptomic analysis reveals potential roles for ERK3 in fetal lung maturation via surfactant protein B and corticotropin releasing hormone

ObjectiveThe mechanism by which prenatal glucocorticoid treatment promotes fetal lung maturation remains poorly understood despite decades of clinical use. In an Erk3-/- murine model of neonatal respiratory distress syndrome (RDS), we showed partial abrogation of the pulmonary immaturity phenotype through antenatal dexamethasone (dex) treatment. Using transcriptomic analysis, we have previously identified key molecular pathways in lung maturation. In this study, we aimed to determine the interaction of antenatal glucocorticoids with regulatory genes at crucial intervals in lung development employing IPA analysis & immunohistochemistry validation.Study DesignDexamethasone (0.4 mg/kg) or saline was administered on E16.5 and 17.5, and lungs were harvested at E18.5. RNA-Seq was performed and analyzed for differential expression, then tested by Ingenuity Pathway Analysis (IPA) to determine gene/dex interaction. CRH and SFTPB protein expression was determined by immunohistochemistry of fetal lungs (n=5) examined by two reviewers. Statistical analysis was performed using Student's t test.ResultsOf 596 differentailly expressed genes, IPA revealed 36 genes that interact with dex, including several with roles in lung development (Fig 1), such as corticotropin releasing hormone (Crh) and surfactant protein B (Sftpb). Antenatal dex was associated with significantly attenuated CRH levels at E18.5 in both wildtype (WT) and Erk3-/- lungs (0.56-fold and 0.67-fold, p<0.001) (Fig 2). As in humans, lungs of WT mice demonstrated increased SFTPB production in response to dex (p=0.003). However, Erk3-/- mice exhibited decreased pulmonary SFTPB when treated with dexamethasone (p=0.012).ConclusionView Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3. CRH and SFTPB are expressed in lung tissue of both Erk3+/+ and Erk3-/- mice. Embryos were collected at E18.5 from dex and saline treated animals. Immunohistochemistry was performed on lung sections for (A) corticotropin releasing hormone (CRH) or (B) surfactant protein B (SFTPB). Quantitation of DAB chromogen staining in lung sections (n=5) illustrates CRH down-regulation with dexa treatment (Erk3+/+ (p<0.001) and Erk3-/- (p<0.00.1). Quantitation of staining in lung sections (n=5) illustrates SFTPB production increases after dex treatment in Erk3+/+ (p=0.0038), but no increase of SFTPB despite dexamethasone in Erk3-/- (p=0.012). ObjectiveThe mechanism by which prenatal glucocorticoid treatment promotes fetal lung maturation remains poorly understood despite decades of clinical use. In an Erk3-/- murine model of neonatal respiratory distress syndrome (RDS), we showed partial abrogation of the pulmonary immaturity phenotype through antenatal dexamethasone (dex) treatment. Using transcriptomic analysis, we have previously identified key molecular pathways in lung maturation. In this study, we aimed to determine the interaction of antenatal glucocorticoids with regulatory genes at crucial intervals in lung development employing IPA analysis & immunohistochemistry validation. The mechanism by which prenatal glucocorticoid treatment promotes fetal lung maturation remains poorly understood despite decades of clinical use. In an Erk3-/- murine model of neonatal respiratory distress syndrome (RDS), we showed partial abrogation of the pulmonary immaturity phenotype through antenatal dexamethasone (dex) treatment. Using transcriptomic analysis, we have previously identified key molecular pathways in lung maturation. In this study, we aimed to determine the interaction of antenatal glucocorticoids with regulatory genes at crucial intervals in lung development employing IPA analysis & immunohistochemistry validation. Study DesignDexamethasone (0.4 mg/kg) or saline was administered on E16.5 and 17.5, and lungs were harvested at E18.5. RNA-Seq was performed and analyzed for differential expression, then tested by Ingenuity Pathway Analysis (IPA) to determine gene/dex interaction. CRH and SFTPB protein expression was determined by immunohistochemistry of fetal lungs (n=5) examined by two reviewers. Statistical analysis was performed using Student's t test. Dexamethasone (0.4 mg/kg) or saline was administered on E16.5 and 17.5, and lungs were harvested at E18.5. RNA-Seq was performed and analyzed for differential expression, then tested by Ingenuity Pathway Analysis (IPA) to determine gene/dex interaction. CRH and SFTPB protein expression was determined by immunohistochemistry of fetal lungs (n=5) examined by two reviewers. Statistical analysis was performed using Student's t test. ResultsOf 596 differentailly expressed genes, IPA revealed 36 genes that interact with dex, including several with roles in lung development (Fig 1), such as corticotropin releasing hormone (Crh) and surfactant protein B (Sftpb). Antenatal dex was associated with significantly attenuated CRH levels at E18.5 in both wildtype (WT) and Erk3-/- lungs (0.56-fold and 0.67-fold, p<0.001) (Fig 2). As in humans, lungs of WT mice demonstrated increased SFTPB production in response to dex (p=0.003). However, Erk3-/- mice exhibited decreased pulmonary SFTPB when treated with dexamethasone (p=0.012). Of 596 differentailly expressed genes, IPA revealed 36 genes that interact with dex, including several with roles in lung development (Fig 1), such as corticotropin releasing hormone (Crh) and surfactant protein B (Sftpb). Antenatal dex was associated with significantly attenuated CRH levels at E18.5 in both wildtype (WT) and Erk3-/- lungs (0.56-fold and 0.67-fold, p<0.001) (Fig 2). As in humans, lungs of WT mice demonstrated increased SFTPB production in response to dex (p=0.003). However, Erk3-/- mice exhibited decreased pulmonary SFTPB when treated with dexamethasone (p=0.012). ConclusionFig. 3. CRH and SFTPB are expressed in lung tissue of both Erk3+/+ and Erk3-/- mice. Embryos were collected at E18.5 from dex and saline treated animals. Immunohistochemistry was performed on lung sections for (A) corticotropin releasing hormone (CRH) or (B) surfactant protein B (SFTPB). Quantitation of DAB chromogen staining in lung sections (n=5) illustrates CRH down-regulation with dexa treatment (Erk3+/+ (p<0.001) and Erk3-/- (p<0.00.1). Quantitation of staining in lung sections (n=5) illustrates SFTPB production increases after dex treatment in Erk3+/+ (p=0.0038), but no increase of SFTPB despite dexamethasone in Erk3-/- (p=0.012). Fig. 3. CRH and SFTPB are expressed in lung tissue of both Erk3+/+ and Erk3-/- mice. Embryos were collected at E18.5 from dex and saline treated animals. Immunohistochemistry was performed on lung sections for (A) corticotropin releasing hormone (CRH) or (B) surfactant protein B (SFTPB). Quantitation of DAB chromogen staining in lung sections (n=5) illustrates CRH down-regulation with dexa treatment (Erk3+/+ (p<0.001) and Erk3-/- (p<0.00.1). Quantitation of staining in lung sections (n=5) illustrates SFTPB production increases after dex treatment in Erk3+/+ (p=0.0038), but no increase of SFTPB despite dexamethasone in Erk3-/- (p=0.012).