An emerging global health problem, COPD is rapidly becoming the third most common cause of death and is the only major disease whose contribution to morbidity and mortality continues to increase (31
). Emphysema, a key component of COPD, is characterized structurally by progressive loss of alveolar architecture and increased alveolar size, and functionally by a concomitant loss of gas exchange capacity in the lung (32
). Thus, airspace enlargement and reduced lung compliance constitute a major cause of declining lung function, dyspnea, hypoxemia, and decreased quality of life in patients. Because no effective therapy exists to attenuate loss of lung function in patients with emphysema, insights into the cell biology of tissue loss could contribute to novel pharmacological interventions that could improve the prognosis of patients with COPD.
In our previous genetic analysis of lung function of age-matched offspring derived from the JF1/Ms mouse strain crossed with the C3H/HeJ mouse strain, we observed a QTL on mouse chromosome 5 that was associated with lung compliance. This analysis was based on an initial assessment of nine mouse strains, of which JF1/Ms and C3H/HeJ demonstrated the greatest phenotypic difference. In the present study, we found that CZECH/EiJ and NZW/LacJ had a slightly greater difference in lung compliance, but were quite similar to JF1/Ms and C3H/HeJ, respectively. On the basis of the likely candidate genes contained in the QTL region on chromosome 5, we selected Kit for additional analysis. By expanding the phenotyping of mouse strains by an additional 10 strains, we developed additional information useful in associating genotype with phenotypes.
On the basis of the results with these 19 mouse strains, an analysis of Kit
genetic variants revealed several SNPs with possible functional consequences. Two promoter SNPs (rs37967821 and rs3799853) were of special interest. The first, rs37967821, alters a conserved sequence of an HOXB4-binding site such that it is converted to an MYB1 site. In the mouse lung, HOXB4 is expressed in mesenchyme and epithelial cells throughout development (33
). In HSCs, HOXB4 treatment can produce HSC expansion (34
), whereas HOXB4 expression in HSCs can reduce long-term engraftment (35
). The other promoter SNP, rs3799853, could eliminate a putative C/EBPα-binding site. Lung-specific gene-targeted Cepba
mice have mildly altered fetal lung development, but at 90 days these mice have abnormal alveolar histology (diminished alveolar septa, increased mean linear intercept), mucous cell hyperplasia, and inflammation, features consistent with COPD (36
). Previously, Didon and colleagues (37
) reported that C/EBP-binding activity is increased in the airway epithelial cells of healthy smokers compared with never-smokers, whereas C/EBP-binding activity was not increased in the epithelium of smokers with COPD. Thus, these investigators proposed that inappropriate C/EBP activity could render the epithelium incompetent for efficient regeneration.
Both of the promoter SNPs have relatively high allelic frequency (~20%), which could explain much (~50%) of the phenotypic difference noted between the polar mouse strains. It is noteworthy that the CZECHII/EiJ and JF1/Ms mouse strains share the same genotype for these SNPs whereas the NZW/LacJ and C3H/HeJ mouse strains share the opposing genotype for these SNPs. Thus, the genotypes are consistent and serve to link our previous genome-wide analysis with this candidate gene.
In addition to the promoter differences, SNPs associated with Cl were identified in other regions of the Kit gene. A nonsynonymous SNP (rs36972615) could produce an aspartic acid-to-glutamic acid conversion at amino acid position 927. This amino acid substitution is likely to have minor consequence to protein structure because these amino acids share identical side chain polarity (polar), side chain charge (negative), and hydropathy index (−3.5). Three of the 3′ UTR SNPs (rs33755426, rs36266306, and rs36248044) involved G-from-A conversions that could disrupt segments of the polyadenosine mRNA region, a region that often alters mRNA stability. Together, our SNP interrogation suggests that regulation of Kit gene expression (transcription or message stabilization) may be more important than amino acid substitutions in the mouse. It should be noted that as with any study of genetic variants, the association may be not be due to the exact SNP that we examined but rather to another SNP in linkage disequilibrium with the SNPs identified in Kit. Furthermore, we do not know the functional consequences of each individual SNP identified to be associated with changes in Cl. Thus, more direct functional evidence that Kit has a role in determining lung compliance was needed.
Further supporting the possible role of this candidate gene, lung function was examined in SASH mice that harbor an insertion in the 5′ UTR of Kit
. Importantly, the KitW-sh
mutation diminishes KIT mRNA expression in mast cells and mesenchyme cells of the lung on E13 and in adult lung, whereas expression in other tissues is normal (25
). We observed altered lung histology in the SASH mouse that was accompanied by alterations in lung volumes and both static and dynamic compliance, which are indicative of an emphysema-like phenotype. These observations suggest that signaling through the KIT receptor tyrosine kinase is essential for maintenance of normal lung parenchymal homeostasis in mice. The specific molecular mechanism by which the presence of KIT spares the lung from developing spontaneous airspace enlargement during normal growth is worthy of additional investigation. Nonetheless, the SASH mouse provides an additional model of emphysema in which delayed-onset alveolar enlargement occurs in the absence of differences in inflammation. This suggests that dysregulation of Kit
expression may be a more proximal cause of emphysema and possibly an event elicited by inflammation or inflammatory mediators.
The pathogenesis of COPD remains an area of considerable interest (38
). Two hypotheses have been proposed for the etiology of emphysema in COPD, namely the proteinase–antiproteinase imbalance hypothesis (40
) and the oxidant–antioxidant hypothesis (42
). Accordingly, mouse models of spontaneous emphysema have features consistent with one of these two hypotheses (43
). In the pallid
mouse, for example, spontaneous emphysema is associated with an antiproteinase deficiency (45
) that is presumed to increase the burden of active neutrophil elastase in the lung (46
). Mice deficient in Toll-like receptor-4, the receptor for bacterial LPS, develop emphysema spontaneously that is associated with an imbalance in antioxidant capacity (44
). It remains controversial whether spontaneous airspace enlargement observed in murine models is homologous to human emphysematous lung disease (47
Human studies of COPD have focused mainly on chromosomes 2 and 12 and on a distal region of chromosome 4 (~145 cM). Human KIT
is located on chromosome 4 (55 Mbp) and near/in a region (~55–70 Mbp) found to have suggestive linkage to both moderate airflow obstruction (48
) and postbronchodilator flow limitation (49
). In addition, KIT
is near a region (48
cM) associated with the ratio of FEV1
to FVC in the Framingham Heart Study (51
). To our knowledge, there are no human genome-wide association studies of lung compliance. Our studies using divergent strains of mice provide a candidate gene on human chromosome 4, which could contribute to lung compliance. Studies in animal models could additionally provide insight into the molecular and cellular mechanisms that regulate lung homeostasis.
Here, we have reported that many pathognomonic features of emphysema are evident in the SASH mouse. c-KIT protein is a recognized distinguishing marker for HSCs and is a receptor for c-KIT ligand, also known as stem cell factor. Given the association with stem cell function, we consider the possibility that Kit
dysregulation may limit normal stem cell function and thereby contribute to the pathogenesis of airspace enlargement due to an inappropriate maintenance of alveolar structure, rather than loss of lung tissue due to dysregulation of response to injury. The role of stem cells in the maintenance of lung architecture remains an area of substantial controversy (52
), with conflicting reports regarding whether cells derived from bone marrow can function as lung epithelial progenitor cells (17
). However, previous work suggests a role for bone marrow–derived cells in lung repair after injury with combined radiation and elastase (19
), and bone marrow transplantation can salvage spontaneous emphysema observed in the tight skin
(Tsk) mouse that harbors a spontaneous fibrillin 1
). Furthermore, in a murine model of elastase-induced emphysema, intranasal hepatocyte growth factor causes a reduction in airspace enlargement, a return of static lung compliance to control levels, and repair of alveolar wall destruction that was associated with an increase in the expression of bone marrow–derived c-KIT–positive cells (55
). Overall, previous studies suggest a potential role for bone marrow–derived cells in protection against the development of airspace enlargement in mice in the context of lung injury. However, we did not use a model of lung injury in our studies. Our findings suggest that c-KIT derived from either epithelia or fibroblasts contributes to normal lung homeostasis.
To determine whether KIT protein expression varied in mouse lung epithelial cells, we performed flow cytometric analysis (56
) on dissociated lung cells to quantify alveolar and airway epithelial cell populations. No detectible difference was noted between the SASH and C57BL/6J mouse strains among either EpCAMpos
(alveolar epithelial cells) or EpCAMpos
(conducting airway epithelial cells) populations within total lung cell preparations (Figures E2 and E3). However, we did observe differences in the capacity of epithelial progenitor cells to undergo in vitro
clonal expansion in a manner dependent on c-Kit. Because of the limitations of our assay, it remains unclear whether these effects are either a direct or indirect effect of fibroblast-derived c-KIT on epithelia homeostasis. Evidence supports that bone marrow stromal cells can both attenuate parenchymal injury in a paracrine manner (57
) and prevent arrested alveolar growth through paracrine activity (58
). Furthermore, lung stromal cells can regulate the growth of lung epithelial cells (59
). Interestingly, KIT mRNA is highly expressed in the developing lungs of mice (E14.5) in stromal cells that are in close proximity to epithelia (Figure E4). In humans, KIT-immunoreactive interstitial cells have been observed in the alveolar septa of 12 normal newborn lungs, but were absent in lungs from 2 cases of congenital alveolar capillary dysplasia (18
). On the basis of these observations, we hypothesize that Kit
expression in interstitial mesenchyme or bone marrow stromal cells facilitates an appropriate alveolar septa microenvironment, which helps maintain alveolar structure possibly through local paracrine activity.
Our study has a limitation in that at this time we cannot identify or assign the specific cell types that are required to express Kit to maintain adult lung structure, but rather highlights the temporal consequences of Kit deficiency to the development of lung structure and function. Although the specific role of Kit in bone marrow–derived stem cells in this model remains unknown, our study supports a role for Kit expression in lung development and maintenance of normal lung architecture in a monopodial branched lung, as exists in rodent models, and suggests that a better understanding of strategies to control Kit-dependent signaling in lung injury and repair may be of value to restore normal lung homeostasis.