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14: Whole transcriptome shotgun sequencing (RNA-SEQ) in a murine model of antenatal glucocorticoid-rescued pulmonary immaturity reveals essential roles for surfactant protein B (SPB) and corticotropin releasing hormone (CRH)

      Objective

      Despite over three decades of use, the mechanisms by which in utero glucocorticoids promote fetal lung maturity are not well understood. We reasoned that whole transcriptome shotgun sequencing (RNA-Seq) in biologically relevant models would reveal potential gene signature pathways by which glucocorticoids antenatally function.

      Study Design

      We previously reported that in our Erk3−/− MAP kinase family knockout (ko) mouse model, 0.4mg/kg dexamethasone administered at E16.5 and E17.5 rescued pulmonary immaturity (decreased sacculation and increased type II pneumocyte glycogen; Figure A PNAS 106:16710, 2009). To significantly expand this work, high integrity (>8.5, Agilent bioanalyzer) coding 3' polyadenylated mRNA was extracted from flash-frozen lung E18.5, and sequencing libraries were generated from random-hexamer synthesized cDNA. Fragmented cDNAs <400bp served as sequencing templates (Illumina GAII). Transcriptome alignment was against the murine reference genome (NCBI 37), and Cuffdiff software was used to infer expression levels. IPA analysis deciphered novel pathways, and significant findings were validated with gene-specific QPCR.

      Results

      IUGR Erk3−/− neonatal mice died within 24 hours from acute respiratory failure; in utero dexamethasone rescued histologic lung maturation and differentiation of type II cells but not neonatal lethality. Employing RNA-Seq, 114 million sequence reads were mapped to the reference genome. Following in silico subtractive hybridization and extensive data mining, two significant glucocorticoid-induced signature pathways in the neonatal lung emerged (B): corticotropin (CRH, CRH receptor 2, urocortin) and surfactants (SPB but not SPA). Biologic replicates validated these observations (Table).

      Conclusions

      Antenatal glucocorticoids decrease pulmonary CRH in an Erk3-independent pathway (i.e., histologic type II maturation), and increase Erk3-dependent SPB (i.e., functional pulmonary maturation). Our unbiased RNA shotgun sequencing of fetal signature pathways reveals a previously undescribed role for Erk3, CRH and SPB in fetal pulmonary maturation.