Maternal smoke exposure decreases mesenchymal proliferation and modulates Rho-GTPase-dependent actin cytoskeletal signaling in fetal lungs

Abstract

The present study tested the hypothesis that maternal smoke exposure results in fetal lung growth retardation due to dysregulation in various signaling pathways, including the Wnt (wingless-related integration site)/β-catenin pathway. Pregnant female C57BL/6J mice were exposed to cigarette smoke (100-150 mg/m3) or room air, and offspring were humanely killed on 12.5,14.5,16.5, and 18.5 d post coitum (dpc). We assessed lung stereology with Cavalieri estimation; apoptosis with proliferating cell nuclear antigen, TUNEL, and caspase assays; and gene expression with quantitative PCR (qPCR) and RNA sequencing on lung epithelium and mesenchyme retrieved by laser capture microdissection. Results demonstrated a significant decrease in body weight and lung volume of smoke-exposed embryos. At 16.5 dpc, the reduction in lung volume was due to loss of lung mesenchymal tissue correlating with a decrease in cell proliferation (n = 10; air: 61.65% vs. smoke: 44.21%, P < 0.05). RNA sequence analysis demonstrated an alteration in the Wnt pathway, and qPCR confirmed an increased expression of secreted frizzled-related protein 1 (sFRP-1) [n = 12; relative quantification (RQ) 1 vs. 2.33, P < 0.05] and down-regulation of Cyclin D1 (n = 7; RQ1 vs. 0.61, P < 0.05) in mesenchymal tissue. Furthermore, genome expression studies revealed a smoke-induced up-regulation of Rho-GTPasedependent actin cytoskeletal signaling that can lead to loss of tissue integrity.