Background
Vitamin D has roles in both skeletal and non-skeletal health, and vitamin D deficiency is recognized as a prevalent health problem [
1,
2]. A plethora of studies have shown cross-sectional associations between vitamin D deficiency and chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis [
3‐
5]. However, these associations are confounded by the effect of chronic disease on physical activity levels [
6] which are highly correlated with sun exposure and, therefore, vitamin D synthesis [
7]. In line with this, while studies in animal models have suggested vitamin D supplementation may ameliorate markers of chronic lung disease [
8,
9], clinical trials using vitamin D supplementation have been disappointing [
10,
11].
While clinical studies have shown no benefit of vitamin D supplementation in established disease, other studies have suggested that vitamin D deficiency may be a precursor to the development of respiratory disease [
12]. For example, maternal vitamin D deficiency is associated with impairments in postnatal lung function [
13,
14] and an increased risk of developing asthma [
15,
16]. In support of this, we have recently demonstrated that
in utero vitamin D deficiency is sufficient to cause deficits in lung structure and function using a mouse model [
17,
18]. Genomic analyses of lung tissue from mice and humans have identified a range of genes that are involved in lung development [
19] that contain vitamin D response elements (VDREs) [
20,
21] suggesting a wide range of roles for vitamin D in normal lung growth. In addition, in vitro and in vivo studies have suggested that vitamin D may be involved in epithelial-mesenchymal interactions [
22,
23], calcium regulation in alveolar type II cells [
24] and surfactant metabolism [
25] during lung maturation. Taken together, these observations suggest that vitamin D is critical during lung development; however, no study has assessed the pathways that are directly altered by vitamin D deficiency during fetal lung development in vivo.
The aim of this study was to identify lung development pathways that are sensitive to vitamin D deficiency. Specifically, using an established mouse model of vitamin D deficiency, we aimed to determine if maternal vitamin D deficiency 1) has an influence on the protein expression in embryonic and neonatal lungs and 2) results in alterations in lung morphology during early lung development.
Discussion
E14.5, E17.5 and P7 are three key lung development time-points that represent the pseudoglandular, late canalicular/early saccular and alveolar development stages respectively [
35]. This study has clearly shown that maternal vitamin D deficiency alters protein expression in the neonatal lung; particularly during the alveolarization stage of lung development. In addition, maternal vitamin D deficiency had no observable impact on lung morphology at these three time-points. The differential expression of proteins SP-B, COL1A1, and PRDX5 in the P7 vitamin D deficient lungs suggests that the expression of vitamin D sensitive proteins is not evident until later stages of neonatal lung development. Importantly, SP-B and COL1A1 have biologically plausible associations with lung structure and function [
36,
37], which are consistent with the postnatal lung function changes associated with maternal vitamin D deficiency [
17,
18]. The reduction in expression of PRDX5, a cytoprotective antioxidant enzyme [
38], in the vitamin D deficient lung may impair defence against oxidative damage induced by secondary insults. Taken together, these data suggest that maternal vitamin D deficiency has the potential to impact lung development through reduced surfactant synthesis, increased collagen deposition, and impaired anti-oxidative stress response. The potential role of these proteins in the alteration of lung function is discussed below.
SP-B is a lipid-associated protein found in lung surfactant which is produced by type 2 alveolar cells and plays a critical role in the function of pulmonary surfactant [
39]. SP-B rearranges lipid molecules to form a thin monolayer on the surface of the fluid lining at the gas-fluid interface which reduces the surface tension and promotes alveolar inflation [
40]. Previous studies have shown that vitamin D stimulates phosphatidylcholine (PC) and phosphatidylglycerol (PG) biosynthesis and lamellar body release, which are associated with surfactant synthesis, in fetal rat lungs [
25]. Our observations have shown decreased SP-B protein expression in the P7 lungs of vitamin D deficient mice. Collectively, these data suggest a critical role for vitamin D in facilitating normal surfactant function during lung development. Given the importance of surfactant in reducing compliance and maintaining airway patency [
41] and the association between impaired surfactant function and respiratory disease [
42], these observations highlight the importance of maintaining vitamin D levels during lung development. Impairments in surfactant synthesis are entirely consistent with the deficits in lung function that are associated with maternal vitamin D deficiency in animal [
25] and human studies [
43]. Previous studies using mouse models have shown that one of the key effects of early life vitamin D deficiency is a reduction in lung volume and increased lung stiffness in P14 mice [
17]. In a retrospective analysis of a longitudinal birth cohort we also demonstrated that maternal vitamin D deficiency at 16–18 weeks gestation was associated with a reduction in forced expiratory volume (FVC) [
13]. One possible explanation for these observations is a reduction in pulmonary surfactant, which is consistent with data from the present study. Interestingly, in previous birth cohort study, maternal vitamin D deficiency was measured at the canalicular stage of human lung development while in the present study we found that vitamin D deficiency was primarily associated with changes in protein expression at early alveolar stages of mouse lung development. This may suggest that the sensitivity of the lung to vitamin D deficiency varies between mice and humans; although we only measured vitamin D levels at one time-point during gestation in the human study and it is entirely possible that the mothers were deficient throughout gestation. While reduced surfactant production is one explanation for the reduction in lung volume and decreased compliance in early postnatal lung development [
17], it could also be explained by changes in tissue structural proteins, such as collagen, which is other major observation in our study.
Collagens are present in major structures of the lung, and any alterations in quantity, structure, or geometry of their distribution would be likely to alter the lung function. Particularly, changes in the interstitium would have dramatic effects on lung function, where the distance between air and blood may be as little as 50 nm [
44]. COL1A1 encodes collagen type Ι which is a major structural protein in the lung interstitium and is produced in large quantities during lung development and during pathological fibrotic processes [
45]. In vitro studies have shown that the addition of vitamin D reduces the expression of collagen type Ι [
46], and induces an anti-fibrotic phenotype in various types of cells [
23]. In our study, COL1A1 was increased in lungs of vitamin D deficient mice suggesting that collagen synthesis during lung development is also sensitive to vitamin D. Again, this is consistent with the functional defects associated with maternal vitamin D deficiency that have been described previously [
17,
18]. It was interesting to note that differences in COL1A1 were only evident at the P7 time-point from the LC-MS/MS assay, whereas the ELISA analysis showed that vitamin D deficiency increased the expression of COL1A1 at E14.5, E17.5 and P7. Notwithstanding this discrepancy, our data suggest that vitamin D deficiency increases the synthesis of collagen type Ι during early lung development.
Using two differential analysis approaches to analyse the LC-MS/MS assay data we have identified differential expression of a range of proteins including peroxidredoxin (PRDX) 1, 5, and 6, which were decreased in P7 lungs of vitamin D deficient mice compared to vitamin D replete mice and myosin (MYH) 9, 11, and 14 were increased in P7 lungs with vitamin D deficiency. While we were unable to validate the differential expression of PRDX6 of MYH11 in independent samples, we are able to confirm that the expression of PRDX5 was reduced in the lungs of vitamin D deficient mice at P7. PRDXs are a family of peroxidases which are present in aerobic organisms and have an important role in protection of human tissue from endogenous and exogenous oxidative damage [
47]. PRDXs are classified into typical 2-Cys (PRDX1-4), atypical 2-Cys (PPXD5), and 1-Cys (PRDX6) PRDX subfamilies. These enzymes degrade hydrogen peroxide (H
2O
2) using their thiol groups of cysteines (Cys) as catalytic centres [
47,
48]. Various studies have shown the overexpression of PRDXs providing significant protection against hyperoxia and apoptosis, and the deficiency of these enzymes can enhance lipid peroxidation [
49‐
52]. PRDX1, 3, 5, and 6 have prominent and cell specific expression in human lung tissue [
53,
54]. PRDX5 plays a protective role in against oxidative stress by reducing apoptosis in human tendon cells and lung carcinoma cells [
51,
55]. Furthermore, studies have indicated that PRDX5 has function in maintaining collagen synthesis and protecting against fibrosis [
51,
56], which is consistent with our finding that overexpression of collagen in lungs of maternal vitamin D deficient mice in early life may be one of the driven factors of impaired lung function in later life.
We were unable to confirm the differential expression of smooth muscle heavy chain MYH11I which we identified by LC-MS/MS. Given that we did not independently quantify MYH4 and 9, it is possible that these proteins do play a role in vitamin D deficiency induced alterations in lung function. In particular, MYH has been linked to airway smooth muscle contractility [
57] which is consistent with our previous observation that maternal vitamin D deficiency causes increased responsiveness of the airways to bronchoconstricting agents [
18]. The potential role of MYH in vitamin D deficiency induced impairments in lung function requires further study.
The lack of difference in lung morphology between vitamin D deficient and replete mice across these three developmental time-points that we have shown in this study suggests that vitamin D deficiency has little impact on gross lung structure during the early stages of lung development. In contrast, our previous findings have shown that lung volume is reduced in P14 mice that were vitamin D deficient
in utero [
17] while parenchymal volume and the volume of the air in the alveolar ducts are reduced in adult vitamin D deficient mice [
18]. This is, however, consistent with the observation that differences in protein expression were not evident until later in lung development and it may be that these differences in protein expression take time to manifest into structural differences at the whole organ level.
This study had some limitations that should be acknowledged. Firstly, as with any complex tissue, the depth of proteome coverage is limited to the most abundant proteins. However, when working with small quantities of embryonic tissue there are practical limitations to sample fractionation for lower abundance protein detection. Secondly, because we used whole lung homogenates we are unable to comment on the cell specific differences in protein expression. Thirdly, in this study we only used lungs from female offspring due to the fact that females are more sensitive to the effect of vitamin D deficiency on lung development [
17,
58]. Thus, it is unclear whether the same effects of vitamin D deficiency on protein expression are evident in male lungs.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LC, EB, and RW carried out the experiments of the study. LC and RW analysed the data and interpreted the results. LC and GRZ conceived of the study, and participated in its design. LC coordinated and helped to draft the manuscript. All authors read and approved the final manuscript.