Using gene expression profiling across the pseudoglandular and canalicular transition of human fetal airway development, we demonstrate enrichment of differentially expressed genes regulating the Wnt signaling pathway during the critical stages of airway branching morphogenesis. Although murine models of lung development have demonstrated a complex role for the Wnt pathway in airway branching morphogenesis, the precise role of the Wnt genes in human lung development has yet to be defined. Using gene expression patterns of early human fetal lung development and genetic association testing in childhood asthma cohorts, we have identified two genes in the Wnt pathway that harbor polymorphisms associated with impaired lung function in two well-characterized cohorts of childhood asthma. To our knowledge, this is the first report demonstrating both differential expression of genes in the Wnt signaling pathway during in utero airway development using human fetal lung tissue and an association between variants within the WISP1 and WIF1 genes with lung function in two asthma populations. Based on these results, we demonstrate that gene expression profiling in early human fetal lung development may help elucidate the molecular processes underlying lung function impairment in patients with asthma.
The Wnt signaling pathway constitutes a large family of highly conserved, secreted glycoproteins, which function as growth factors that are essential to organogenesis. The canonical Wnt proteins bind to frizzled receptors, causing β-catenin inhibition and subsequent transcription of TCF/LEF target genes (47
). Genes in the Wnt signaling pathway regulate cell fate and differentiation during embryogenesis, modulate cell proliferation, and are involved in homeostatic functions in adult tissues (48
). To date, 19 Wnt proteins have been identified in humans with a vast array of biologic functions allowing for redundancy in the pathway with compensation for the loss of certain Wnt ligands by other genes in the pathway (51
). Furthermore, the Wnt signaling pathway is highly regulated by Wnt inhibitors, including the sFRPs and WIF. Branching morphogenesis in fetal lung development is mediated by a complex interaction between the epithelium and its surrounding mesenchyme during early lung development (49
). Genes in the Wnt signaling pathway are expressed in the developing lung at sites of critical epithelial–mesenchymal interactions that involve cell–cell and cell–matrix interactions that are essential for normal lung development (28
). Murine models have shown that deletion of β-catenin signaling in the epithelium during early lung development results in profound perturbations of the normal epithelial and mesenchymal compartments resulting in decreased secondary and tertiary branching (54
). Furthermore, studies have shown that Wnt signaling is critical to the proliferation and survival of airway submucosal gland progenitor cells. Alterations of Wnt signaling have been associated with pathologic alterations, including hypertrophy and hyperplasia of submucosal glands (55
), which are common pathologic changes found in the airway wall of individuals with asthma. Thus, murine models suggest a plausible role for the Wnt genes in the pathogenesis of lung function impairment in the susceptible host.
Differential expression in human fetal lung tissue during the period of branching morphogenesis was a prerequisite for our considering a gene for genetic association testing. We were thus able to identify two genes (WISP1 and WIF1) that are associated with both intrauterine airway development and lung function impairment in susceptible populations. Neither WISP1 nor WIF1 has been previously implicated in the pathogenesis of obstructive airway diseases. However, recent work has demonstrated a role for the Wnt signaling pathway at sites of tissue injury and repair. Colston and colleagues demonstrated a role for WISP1 in postinfarction cardiac remodeling, by demonstrating the proliferative effect of WISP1 on fibroblasts (56
). Aberrant Wnt signaling has also been implicated as a profibrotic mechanism underlying the pathogenesis idiopathic pulmonary fibrosis (IPF) (57
). Specifically, WISP1 expression was increased in alveolar epithelial cells in both in vitro
models and in vivo
). In addition, WISP1 showed the greatest difference in expression between lung tissue of normal control subjects and in patients with IPF (60
). Treatment with exogenous WISP1 resulted in increased type II cell proliferation, increased extracellular matrix deposition from lung fibroblasts, and increased fibrosis. Furthermore, depletion of WISP1 using neutralizing antibodies in a bleomycin-induced murine model of IPF resulted in a marked attenuation of lung fibrosis, decreased extracellular matrix deposition, partial restoration of pulmonary function assessed by lung compliance measures, and improved survival (60
). Although WISP1 has not been specifically studied in asthma, the profibrotic effect of WISP1 may explain some of the characteristic changes of airway remodeling that occur in asthma, which includes subepithelial fibrosis, neovascularization, and increased smooth muscle deposition. These histopathologic changes may explain why variants in this gene are associated with lower levels of lung function in children with asthma. Further investigation of the functional role of this gene in asthma is warranted.
WIF1 acts as an inhibitor of Wnt signaling by directly binding to Wnt ligands, thus disrupting their ability to bind to the Wnt/frizzled receptor (48
). Decreased WIF1 expression in human disease states, including lung cancer, has been related to hypermethylation of the promoter region of the gene (61
). Sun and colleagues have previously shown that abrogation of Alk3-mediated bone morphogenetic protein (BMP) signaling in lung epithelial cells during early lung development (E16.5 in the murine model) disrupts cell differentiation and proliferation resulting in abnormal lung branching morphogenesis (62
). Their murine model demonstrates that changes in BMP signaling subsequently cause respiratory distress syndrome in the early postnatal period. Analysis of the lung tissue of the Alk3 conditional knock-out mouse demonstrate that decreased BMP signaling is associated with increased canonical Wnt signaling activity as demonstrated by increased phosphorylation of the Wnt coreceptor LRP6 and activation of downstream β-catenin (62
). Of note, WIF1 expression in the Alk-3 conditional knock-out mouse was reduced in the perinatal lung tissue, suggesting both a role for WIF1 in abnormal airway branching and the development of impaired lung function in later life. Although increased WIF1 expression may explain why variants in the WIF1 gene are associated with increased lung function in patients with childhood asthma, functional validation of these variants is necessary to further elucidate their mechanistic function. Furthermore, investigation of genotype-specific differences in the expression of Wnt signaling genes in lung tissue specimens from patients with asthma would help to further elucidate the role of both WISP1 and WIF1 in asthma pathogenesis.
We performed an extensive search of the medical literature for abstracts involving each of the 1,776 differentially expressed genes to identify those genes that were previously associated with abnormal lung pathology, as evidenced by (1) murine models demonstrating an association with abnormal pulmonary phenotypes, (2) implication of the gene in lung development, or (3) human data demonstrating gene associations with lung disease. Of the 1,776 differentially expressed genes, only 102 met at least one of these criteria, suggesting the usefulness of gene expression profiling in early human lung development to identify genes and molecular pathways that may have a novel biologic role in the pathogenesis of airflow obstruction in susceptible individuals. Furthermore, although the Wnt signaling pathway has been implicated in murine lung development, the role for the WISP1 and WIF1 genes in the pathobiology of asthma in human populations has not been identified to date. The current data suggest an association of genes within this pathway with lung function in human populations. However, further molecular characterization of the genetic variants within these genes is required to determine their precise role in the pathogenesis of impaired lung function.
Several limitations of our genomic analysis must be addressed. Our genome-wide gene expression analysis of lung development was limited to fetal lung tissue samples from the pseudoglandular and canalicular stages of development. Therefore, we are not able to determine the changes in gene expression profile that occur during the later stages of gestation. Although we acknowledge that investigation of gene expression patterns during the later stages of development during which the airway size increases may allow us to gain further insights into the pathogenesis of lung function impairment, the use of early developmental expression patterns is scientifically justified based on what we have already learned from genetic manipulation of mice wherein interval sacrifice throughout development is possible. It is quite well established that the same genes that are identified as having key functions in early embryonic lung development are reused during later fetal development, postnatal alveolarization, and response to injury (28
). In addition to its scientific merit, using the early fetal lung to do a genome-wide expression screen is also feasible because of the limits imposed by our ability to obtain fetal tissue during the later stages of gestation.
Like other intermediate asthma phenotypes, lung function is likely to be a complex process, resulting from the interplay of both genetic and environmental factors. We have previously shown that of early life exposures (including intrauterine tobacco smoke exposure) influence early postnatal lung function (14
). Because our fetal lung tissue samples are de-identified samples obtained from a national tissue repository, we do not have access to maternal information. Therefore, we are unable to assess the effect that in utero
exposures may have on gene expression patterns, which would result in a source of misclassification that would bias our results toward the null hypothesis. Despite this limitation, our results suggest that we can use gene expression profiling to investigate the genes and pathways that influence in utero
lung development, which may help define the molecular processes involved in the pathogenesis of impaired lung function in the susceptible host.
We also recognize that the use of gene expression profiling of early lung development to identify genes associated with lung function may have broader implications in terms of lung function in both health and disease. Recent work has suggested a role for Wnt signaling genes in the pathogenesis of idiopathic pulmonary fibrosis and pulmonary arterial hypertension (59
); genetic association testing of variants in Wnt genes in these diseases should be considered.
Genetic association studies of lung function in obstructive airways diseases have yielded inconsistent findings. In addition to differences in linkage disequilibrium (LD) patterns, gene-by-gene, or gene-by-environment interactions among study populations, potential explanations for these discrepant results include differences in statistical power, failure to control for multiple testing, and (for case-control studies) population stratification. Our study had adequate statistical power to detect associations of relatively large magnitude, and we have reduced the potential for false-positive results by replicating our findings in two family-based studies, which are not susceptible to population stratification. We recognize that some of our nonreplicated results at the SNP level may be due to underlying differences between our study populations (e.g., ancestral history, LD, and environmental exposures). However, based on recent work from Rogers and colleagues, the Illumina genome-wide genotyping platform also provides variable coverage across genetic loci (64
). Studies with sample sizes similar to ours can detect associations with relatively large effect size (odds ratios ≥1.5), but are underpowered to detect genes with small effect sizes, which may be expected for complex traits like lung function. Furthermore, important genetic variation (functional or regulatory) may not have been genotyped as part of the HumanHap550 platform. Therefore, sequencing and functional validation studies for the lung development candidate gene set identified in this study are still required.
In summary, genes in the Wnt signaling pathway are associated with impaired lung function in two ethnically distinct cohorts of childhood asthma. Furthermore, in concordance with previous experimental animal data, we have demonstrated that genes in critical lung development pathways influence lung function later in life. Our findings provide evidence that genome-wide gene expression profiles of lung development using human fetal lung tissue can be used to elucidate candidate genes involved in the pathogenesis of impaired lung function in children with asthma.