Altered regulation of retinoic acid synthesis in nitrofen-induced hypoplastic lung

Abstract

Retinoids are a group of molecules derived from vitamin A, which play an important role in lung development. Within the cell, retinol can either be oxidized to retinal or esterified to retinyl esters by lecithin : retinol acyltransferase (LRAT) for storage. Retinal is then oxidized to an active metabolite of vitamin A, retinoic acid (RA) by retinal dehydrogenase (RALDH). RA is the active metabolite of vitamin A. Cyp26 (a1,b1, and c1), which is a member of the cytochrome P450 family, acts by reducing the activity of RA. Cyp26 type b1 is the predominant subtype expressed in the murine lung. Several studies have suggested that nitrofen may interfere with the retinoid pathway resulting in congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia. Recently, it was reported that nitrofen may act by inhibiting RALDH2. The aim of this study was to examine the pulmonary expression of Cyp26b1, LRAT, and RALDH2, the key enzymes involved in the synthesis of RA, in order to understand the mechanisms underlying pulmonary hypoplasia in the nitrofen CDH model. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9 of gestation (D9). Fetal lungs were harvested at D15, D17, D19, and D21. D17, D19, and D21 lungs were divided into three groups: control, nitrofen without CDH and nitrofen with CDH, whereas D15 lungs were divided into only two groups; control and nitrofen as the diaphragm is not fully formed yet at this stage. Real- time PCR was performed to evaluate the relative level of Cyp26b1, LRAT, and RALDH2 expression in the lung. Relative levels of Cyp26b1 mRNA were significantly decreased in the lungs of nitrofen with CDH (D17;0.19 +/- 0.09, D19;0.70 +/- 0.20, D21;0.40 +/- 0.36) and nitrofen without CDH (D17;0.14 +/- 0.06, D19;0.54 +/- 0.42, D21;0.51 +/- 0.56) compared to controls (D17;0.35 +/- 0.16, D19;1.15 +/- 0.48, D21;1.28 +/- 0.78) (P < 0.05). LRAT expression was also significantly decreased in nitrofen with CDH (D17; 19.3 +/- 7.8, D19; 4.3 +/- 1.1, D21; 3.3 +/- 1.6) and nitrofen without CDH (D17; 21.2 +/- 11.1, D19; 4.5 +/- 3.6, D21; 4.1 +/- 1.6) compared to controls (D17; 153.7 +/- 29.8, D19; 26.8 +/- 16.8 D21; 10.1 +/- 3.8) (P < 0.05). There was no significant difference in the relative levels of Cyp26b1 and LRAT between nitrofen with CDH and nitrofen without CDH. There were no significant differences in RALDH2 expression among the groups at any stages. Down-regulation of Cryp26b1 and LRAT demonstrates that RA content is decreased in nitrofen induced hypoplastic lungs compared to controls. The finding that RALDH2 expression in the hypoplastic lung is not altered suggests that nitrofen may act by interfering with the retinoid metabolism during the early stage of the retinoid signaling pathway.

Retinoid Pathway and Congenital Diaphragmatic Hernia: Hypothesis from the Analysis of Chromosomal Abnormalities

“…Recently, Nakazawa et al [79] studied the effects of nitrofen on the retinoid-signaling pathway in hypoplastic lungs. They demonstrated that LRAT was downregulated, causing a shift of retinol from storage to conversion in RA.…”

Gene expression analysis in hypoplastic lungs in the nitrofen model of congenital diaphragmatic hernia

“…In the nitrofen model, coadministration of large doses of vitamin A reduces the incidence of CDH and attenuates lung hypoplasia [26]. Furthermore, nitrofen has been reported to inhibit retinal dehydrogenase (RALDH-2) [42], the major retinoic acid synthesizing enzyme, whereas other studies have shown a downregulation of Cyp26b1 (a cytochrome P450 that reduces the activity of retinoic acid) and LRAT (lecithinretinol transferase that esterifies retinol to retinyl esters for storage) [27,28]. In the current study, we have found a decrease of RARα after nitrofen.…”

“…An increased expression of matrix metalloproteinase 9 (MMP-9) has been reported [24], as well as an alteration of the retinoid acid pathway at different levels [25][26][27][28]. The lungs of rodents with nitrofen-induced CDH have also been shown to have increased expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 [29], increased angiotensin-converting enzyme activity [30], decreased endothelial isoform of nitric oxide synthase (eNOS) expression [31], increased endothelin-1 and endothelin A vasoconstrictor receptor [32], and altered K + channel expression [33], which might contribute to increased pulmonary vascular tone in the teratogenic model of CDH.…”

Spatiotemporal alteration in phosphatidylinositide 3-kinase–serine/threonine protein kinase B signaling in the nitrofen-induced hypoplastic lung

“…Previous study from our laboratory has shown that gene expression of FGF-7 and FGF-10 is downregulated in the nitrofeninduced hypoplastic lung [13]. Furthermore, retinoids, which can also activate PI3K, has been reported to be involved in pathogenesis of CDH and associated pulmonary hypoplasia, and recent studies have also suggested that the retinoid signaling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung [14][15][16][17]. However, the relationship between PI3K-AKT signaling pathway and lung hypoplasia associated with CDH has not been elucidated.…”