Mesenchymal nuclear transcription factors in nitrofen-induced hypoplastic lung

Nitrofen-induced pulmonary hypoplasia (PH) with or without coexistent congenital diaphragmatic hernia (CDH) in animals mimics the human condition. We have demonstrated reduced steroid-thyroid-retinoid receptors in hypoplastic lungs. Therefore, we hypothesize that expression of two additional mesenchymally derived nuclear transcription factors, retinoid X receptor alpha (RXR-alpha) and homeobox b-5 (Hoxb-5), would also be altered in hypoplastic lungs.

We used timed-pregnant CD-1 mice either untreated or gavaged with nitrofen on gestational day (Gd) 8. Normal lungs were compared with hypoplastic lungs with severe left PH and CDH at Gd 14, 16, and 19 and from neonates. We performed immunoblotting for RXR-alpha and Hoxb-5 proteins and immunohistochemistry for Hoxb-5 protein.

Almost 70-80% of nitrofen-exposed mice had no apparent external phenotypic abnormalities, such as craniofacial defects. Fetal body and lung weights were reduced. RXR-alpha and Hoxb-5 proteins were highest at Gd 14 and decreased as development progressed. Densitometric analysis of RXR-alpha or Hoxb-5 proteins in normal and hypoplastic lungs showed no significant difference; however, the immunolocalization pattern of Hoxb-5 protein differed. Hoxb-5 protein was primarily in mesenchymal cells of normal lungs on Gd 14; however, by Gd 19, it appeared to be mainly in the epithelial cells of prealveolar structures and in adjacent subepithelial mesenchymal cells. In hypoplastic lungs no significant changes were observed in Hoxb-5 staining in mesenchymal cells at Gd 14 nor at Gd 16; however, Hoxb-5 expression persisted in mesenchyme and epithelium at Gd 19 and in neonatal hypoplastic lungs, unlike normal lungs.

(1) Unaltered RXR-alpha protein implies that despite altered retinoic acid receptors (RARs) in hypoplastic lungs, the mechanisms of nitrofen-induced PH may be independent of RXR-alpha pathways. (2) In hypoplastic lungs, the persistent mesenchymal expression of Hoxb-5, in later stages of development and at birth, suggests delayed development and maturation compared to normal lungs. We speculate that nitrofen induces PH via RAR-dependent but RXR-independent interactions, which may be downstream of the Hoxb-5 gene or may involve other more anteriorly expressed Hox genes.