The lung alveolus is lined with alveolar type 1 (AT1) and type 2 (AT2) epithelial cells. During alveologenesis, increasing demand associated with expanding alveolar numbers is met by proliferating ...progenitor AT2s (pAT2). Little information exists regarding the identity of this population and their niche microenvironment. We show that during alveologenesis, Hedgehog-responsive PDGFRa(+) progenitors (also known as SCMFs) are a source of secreted trophic molecules that maintain a unique pAT2 population. SCMFs are in turn maintained by TGFβ signaling. Compound inactivation of Alk5 TβR2 in SCMFs reduced their numbers and depleted the pAT2 pool without impacting differentiation of daughter cells. In lungs of preterm infants who died with bronchopulmonary dysplasia, PDGFRa is reduced and the number of proliferative AT2s is diminished, indicating that an evolutionarily conserved mechanism governs pAT2 behavior during alveologenesis. SCMFs are a transient cell population, active only during alveologenesis, making them a unique stage-specific niche mesodermal cell type in mammalian organs.
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•Lung alveolar type 2 progenitors (pAT2s) are heterogeneous during alveologenesis•Blocking TGFβ in SCMF fibroblasts reduces their number and blocks alveologenesis•SCMF signaling regulates proliferation of unique pAT2 population in alveologenesis•An evolutionarily conserved mechanism appears to govern pAT2s in neonatal human lungs
Much of the information regarding heterogeneity of alveolar epithelial cells in the lung is from adult mice. Gao et al. analyze the complexity of the alveolar epithelial cell population during alveologenesis, a process interrupted in the pathogenesis of the neonatal chronic lung disease bronchopulmonary dysplasia.
Lung development is precisely controlled by underlying gene regulatory networks (GRN). Disruption of genes in the network can interrupt normal development and cause diseases such as bronchopulmonary ...dysplasia (BPD) - a chronic lung disease in preterm infants with morbid and sometimes lethal consequences characterized by lung immaturity and reduced alveolarization. Here, we generated a transgenic mouse exhibiting a moderate severity BPD phenotype by blocking IGF1 signaling in secondary crest myofibroblasts (SCMF) at the onset of alveologenesis. Using approaches mirroring the construction of the model GRN in sea urchin's development, we constructed the IGF1 signaling network underlying alveologenesis using this mouse model that phenocopies BPD. The constructed GRN, consisting of 43 genes, provides a bird's eye view of how the genes downstream of IGF1 are regulatorily connected. The GRN also reveals a mechanistic interpretation of how the effects of IGF1 signaling are transduced within SCMF from its specification genes to its effector genes and then from SCMF to its neighboring alveolar epithelial cells with WNT5A and FGF10 signaling as the bridge. Consistently, blocking WNT5A signaling in mice phenocopies BPD as inferred by the network. A comparative study on human samples suggests that a GRN of similar components and wiring underlies human BPD. Our network view of alveologenesis is transforming our perspective to understand and treat BPD. This new perspective calls for the construction of the full signaling GRN underlying alveologenesis, upon which targeted therapies for this neonatal chronic lung disease can be viably developed.
WNT5a is a mainly "non-canonical" WNT ligand whose dysregulation is observed in lung diseases such as idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and asthma. ...Germline deletion of Wnt5a disrupts embryonic lung development. However, the temporal-specific function of WNT5a remains unknown. In this study, we generated a conditional loss-of-function mouse model (Wnt5a
) and examined the specific role of Wnt5a during the saccular and alveolar phases of lung development. The lack of Wnt5a in the saccular phase blocked distal airway expansion and attenuated differentiation of endothelial and alveolar epithelial type I (AT1) cells and myofibroblasts. Postnatal Wnt5a inactivation disrupted alveologenesis, producing a phenotype resembling human bronchopulmonary dysplasia (BPD). Mutant lungs showed hypoalveolization, but endothelial and epithelial differentiation was unaffected. The major impact of Wnt5a inactivation on alveologenesis was on myofibroblast differentiation and migration, with reduced expression of key regulatory genes. These findings were validated in vitro using isolated lung fibroblasts. Conditional inactivation of the WNT5a receptors Ror1 and Ror2 in alveolar myofibroblasts recapitulated the Wnt5a
phenotype, demonstrating that myofibroblast defects are the major cause of arrested alveologenesis in Wnt5a
lungs. Finally, we show that WNT5a is reduced in human BPD lung samples, indicating the clinical relevance and potential role for WNT5a in pathogenesis of BPD.
Postnatal alveolar formation is the most important and the least understood phase of lung development. Alveolar pathologies are prominent in neonatal and adult lung diseases. The mechanisms of ...alveologenesis remain largely unknown. We inactivated
postnatally in secondary crest myofibroblasts (SCMF), a subpopulation of lung mesenchymal cells. Lack of
arrested alveologenesis akin to bronchopulmonary dysplasia (BPD), a neonatal chronic lung disease. The transcriptome of mutant SCMF revealed 1808 altered genes encoding transcription factors, signaling and extracellular matrix molecules. Elastin mRNA was reduced, and its distribution was abnormal. Absence of
disrupted expression of elastogenic genes, including members of the Lox, Fbn and Fbln families. Expression of EGF family members increased when
was repressed in mouse. Similar, but not identical, results were found in human BPD lung samples.
, blocking PDGF signaling decreased elastogenic gene expression associated with increased
and decreased Tgfb family mRNAs. The effect was reversible by inhibiting EGF or activating TGFβ signaling. These observations demonstrate the previously unappreciated postnatal role of PDGFA/PDGFRα in controlling elastogenic gene expression via a secondary tier of signaling networks composed of EGF and TGFβ.
Lung maturation is not limited to proper structural development but also includes differentiation and functionality of various highly specialized alveolar cell types. Alveolar type 1 (AT1s) cells ...occupy nearly 95% of the alveolar surface and are critical for establishing efficient gas exchange in the mature lung. AT1 cells arise from progenitors specified during the embryonic stage as well as alveolar epithelial progenitors expressing surfactant protein C (Sftpcpos cells) during postnatal and adult stages. Previously, we found that Wnt5a, a non-canonical Wnt ligand, is required for differentiation of AT1 cells during the saccular phase of lung development. To further investigate the role of Wnt5a in AT1 cell differentiation, we generated and characterized a conditional Wnt5a gain-of-function mouse model. Neonatal Wnt5a gain-of-function disrupted alveologenesis through inhibition of cell proliferation. In this setting Wnt5a downregulated β-catenin-dependent canonical Wnt signaling, repressed AT2 (anti-AT2) and promoted AT1 (pro-AT1) lineage-specific gene expression. In addition, we identified 2 subpopulations of Sftpchigh and Sftpclow alveolar epithelial cells. In Sftpclow cells, Wnt5a exhibits pro-AT1 and anti-AT2 effects, concurrent with inhibition of canonical Wnt signaling. Interestingly, in the Sftpchigh subpopulation, although increasing AT1 lineage-specific gene expression, Wnt5a gain-of-function did not change AT2 gene expression, nor inhibit canonical Wnt signaling. Using primary epithelial cells isolated from human fetal lungs, we demonstrate that this property of Wnt5a is evolutionarily conserved. Wnt5a therefore serves as a selective regulator that ensures proper AT1/AT2 balance in the developing lung.
O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair protein that removes mutagenic and cytotoxic adducts from the O6 position of guanine. O6-methylguanine mispairs with thymine during ...replication, and if the adduct is not removed, this results in conversion from a guanine-cytosine pair to an adenine-thymine pair. In vitro assays show that MGMT expression avoids G to A mutations and MGMT transgenic mice are protected against G to A transitions at ras genes. We have recently demonstrated that the MGMT gene is silenced by promoter methylation in many human tumors, including colorectal carcinomas. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of K-ras mutations, we studied 244 colorectal tumor samples for MGMT promoter hypermethylation and K-ras mutational status. Our results show a clear association between the inactivation of MGMT by promoter hypermethylation and the appearance of G to A mutations at K-ras: 71% (36 of 51) of the tumors displaying this particular type of mutation had abnormal MGMT methylation, whereas only 32% (12 of 37) of those with other K-ras mutations not involving G to A transitions and 35% (55 of 156) of the tumors without K-ras mutations demonstrated MGMT methylation (P = 0.002). In addition, MGMT loss associated with hypermethylation was observed in the small adenomas, including those that do not yet contain K-ras mutations. Hypermethylation of other genes such as p16INK4a and p14ARF was not associated with either MGMT hypermethylation or K-ras mutation. Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to a particular genetic change in human cancer, specifically G to A transitions in the K-ras oncogene.
The INK4a/ARF locus encodes two distinct tumor suppressors, p16INK4a and p14ARF. Although the contribution of p16INK4a to human tumorigenesis through point mutation, deletion, and hypermethylation ...has been widely documented, little is known about specific p14ARF lesions and their consequences. Recent data indicate that p14ARF suffers inactivation by promoter hypermethylation in colorectal cancer cells. Because it is known that p14ARF prevents MDM2 nucleocytoplasmic shuttling and thus stabilizes p53 by attenuating MDM2-mediated degradation, we studied the relationship of p14ARF epigenetic silencing to the expression and localization of MDM2 and p53. Cancer cell lines with an unmethylated p14ARF promoter showed strong nuclear expression of MDM2, whereas in a colorectal cell line with p14ARF hypermethylation-associated inactivation, MDM2 protein was also seen in the cytosol. Treatment with the demethylating agent 5-aza-2'-deoxycytidine was able to reinternalize MDM2 to the nucleus, and p53 expression was restored. No apparent changes in retinoblastoma localization were observed. We also studied the profile of p14ARF promoter hypermethylation in an extensive collection of 559 human primary tumors of different cell types, observing that in colorectal, gastric, renal, esophageal, and endometrial neoplasms and gliomas, aberrant methylation of p14ARF was a relatively common epigenetic event. MDM2 expression patterns revealed that lack of p14ARF promoter hypermethylation was associated with tumors showing exclusive nuclear MDM2 staining, whereas MDM2 cytosolic staining was frequently observed in neoplasms with aberrant p14ARF methylation. Taken together, these data support that epigenetic silencing of p14ARF by promoter hypermethylation is a key mechanism in the disturbance of the MDM2 nuclear localization in human cancer.
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