Lung cancer remains the leading cause of cancer death. Genome sequencing of lung tumors from patients with squamous cell carcinoma has identified SMAD4 to be frequently mutated. Here, we use a mouse model to determine the molecular mechanisms by which Smad4 loss leads to lung cancer progression. Mice with ablation of Pten and Smad4 in airway epithelium develop metastatic adenosquamous tumors. Comparative transcriptomic and in vivo cistromic analyses determine that loss of PTEN and SMAD4 results in ELF3 and ErbB2 pathway activation due to decreased expression of ERRFI1, a negative regulator of ERBB2 in mouse and human cells. The combinatorial inhibition of ErbB2 and Akt signaling attenuate tumor progression and cell invasion, respectively. Expression profile analysis of human lung tumors substantiated the importance of the ErbB2/Akt/ELF3 signaling pathway as both a prognostic biomarker and a therapeutic drug target for treating lung cancer.

There are no ideal cell lines available for alveolar epithelial type I and II cells (AEC I and II) at the present time. The current methods for isolating AEC I and II give limited purities. Here, we reported improved and reproducible methods for the isolation of highly pure AEC I and II from rat lungs. AEC I and II were released from lung tissues using different concentrations of elastase digestion. Macrophages and leukocytes were removed by rat IgG 'panning' and anti-rat leukocyte common antigen antibodies. For AEC II isolation, polyclonal rabbit anti-T1a (an AEC I apical membrane protein) antibodies were used to remove AEC I contamination. For AEC I isolation, positive immunomagnetic selection by polyclonal anti-T1a antibodies was used. The purities of AEC I and II were 9174 and 9771%, respectively. The yield per rat was B2 Â 10 6 for AEC I and B33 Â 10 6 for AEC II. The viabilities of these cell preparations were more than 96%. The protocol for AEC II isolation is also suitable to obtain pure AEC II (93-95%) from hyperoxia-injured and recovering lungs. The purified AEC I and II can be used for gene expression profiling and functional studies. It also offers an important tool to the field of lung biology. The alveolar epithelium is composed of type I and II pneumocytes (AEC I and II). AEC I and II are morphologically and functionally different. AEC I are squamous in shape, with a diameter of B50-100 mm and a volume of B2000-3000 mm 3 . 1 AEC I cover B95% of the surface area of the lung and are important for gas exchange. Recent studies indicated that AEC I play active roles in water permeability and the regulation of alveolar fluid homeostasis.2,3 AEC II are cuboidal, with a diameter of B10 mm and a volume of B450-900 mm 3 . 1 AEC II occupy only B5% of the surface area. They produce, secrete, and recycle lung surfactant. AEC II can be also converted to AEC I to repair damaged epithelium after lung injury or during fetal lung development.Given the importance of AEC I and II in lung functions, it is necessary to isolate enriched populations of AEC I and II with sufficient viability and purity for functional studies. The method for AEC II isolation developed by Dobbs et al 4 has been used by most investigators. However, the purity of the isolated AEC II is only 80-89%. Although those cell preparations may be appropriate for studying lung surfactant metabolism, they are not pure enough for gene expression profiling. A few reports attempted to obtain higher purities of AEC II. Weller and Karnovsky 5 reported a 90% pure AEC II from rats using Percoll gradient centrifugation, while Abraham et al 6 isolated 90-95% pure AEC II by using rat IgG panning and rabbit IgG coated BioMag beads. AEC I have been studied to a lesser extent. Recently, a few laboratories have isolated AEC I with limited purities or yields using AEC I-specific monoclonal antibodies produced in their laboratories. The typical purities were 60 B86%. 2,3,7 In order to isolate a specific type of cells, reliable methods to identify the cells are needed....

Environmentally persistent free radicals (EPFRs) in combustiongenerated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustionderived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 mm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 mg/cm 2 ) caused substantial necrosis. At low doses (20 mg/cm 2 ), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased a-smooth muscle actin (a-SMA) and collagen I production. Similar results were observed in neonatal air-liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal a-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma.Keywords: particulate matter; epithelial-mesenchymal transition; environmental asthma; pediatric Combustion-generated particulate matter (PM) from industrial processes and burning of biomass and fossil fuels has been linked with adverse pulmonary health effects (1). Environmental PM, both fine and ultrafine, is capable of airway deposition, alveolar penetration, respiratory distress, and exacerbation of preexisting pulmonary conditions. Previous studies highlight the potential roles of PM exposure in predisposing to asthma and pulmonary fibrosis (2-4). Additionally, PM has adjuvant effects when combined with innocuous antigen (5-7) and induces cellular damage, stimulating fibrotic remodeling in adult rodent exposure models (2). The developing pulmonary and immune systems are particularly vulnerable (8). We have developed a model for studying particulate exposures in neonatal rodents (, 7 d of age) (9), which we apply here to understand the effects of combustiongenerated environmentally persistent free radicals (EPFRs) on pulmonary airway remodeling.Delineation of the influences of particulate burden from the reactive chemical species complexed with the particulate has proven difficult. The nature of the chemical species drastically influences ...

Alveolar epithelial type II cells secrete lung surfactant via exocytosis. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) are implicated in this process. Lipid rafts, the cholesterol-and sphingolipid-rich microdomains, may offer a platform for protein organization on the cell membrane. We tested the hypothesis that lipid rafts organize exocytotic proteins in type II cells and are essential for the fusion of lamellar bodies, the secretory granules of type II cells, with the plasma membrane. The lipid rafts, isolated from type II cells using 1% Triton X-100 and a sucrose gradient centrifugation, contained the lipid raft markers, flotillin-1 and -2, whereas they excluded the nonraft marker, Na ϩ -K ϩ ATPase. SNAP-23, syntaxin 2, and VAMP-2 were enriched in lipid rafts. When type II cells were depleted of cholesterol, the association of SNAREs with the lipid rafts was disrupted and the formation of fusion pore was inhibited. Furthermore, the cholesterol-depleted plasma membrane had less ability to fuse with lamellar bodies, a process mediated by annexin A2. The secretagogue-stimulated secretion of lung surfactant from type II cells was also reduced by methyl-␤-cyclodextrin. When the raft-associated cell surface protein, CD44, was cross-linked using anti-CD44 antibodies, the CD44 clusters were observed. Syntaxin 2, SNAP-23, and annexin A2 co-localized with the CD44 clusters, which were cholesterol dependent. Our results suggested that lipid rafts may form a functional platform for surfactant secretion in alveolar type II cells, and raft integrity was essential for the fusion between lamellar bodies with the plasma membrane.Keywords: alveolar type II cells; lipid rafts; membrane fusion; SNARE proteins; surfactant secretion Lung alveolar epithelium consists of two different types of cells, the cuboidal type II cells and squamous type I cells. Type II cells synthesize, store, and secrete a surface-active lipid-rich substance, the lung surfactant. The released surfactant lines the alveolar epithelium, lowers the surface tension, and thus prevents the collapse of alveoli at end-expiration. Lung surfactant deficiency causes respiratory distress syndrome (RDS) in infants. Type II cells are also involved in defense, injury and repair, and trans-differentiation into type I cells.Lung surfactant, stored in lamellar bodies, is released upon its fusion with the plasma membrane via exocytosis. The formation of fusion pore precedes the release of lamellar body contents. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) hypothesis was proposed to elucidate the mechanisms of membrane fusion during exocytosis. During fusion, the proteins on the plasma membrane (target or t-SNAREs) and vesicles (vesicular or v-SNARE) form a highly stable, hetero-tetrameric SNARE complex. The two coiled-coil domains are contributed by soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-25/23 and one each from syntaxin and VAMP (vesicle-associated membrane protein) (1). NSF ...

RNA interference (RNAi) provides a powerful tool to silence genes in a sequence-speci¢c manner in a variety of systems. However, not all sequences are e¡ective in the RNAimediated gene silencing. In this study, we developed a polymerase chain reaction (PCR)-based RNAi strategy for a quick screening of small interfering RNA (siRNA) e⁄ciency. This method utilized a two-step PCR to generate a chimeric DNA template containing the U6 promoter or cytomegalovirus promoter and short hairpin DNA. We demonstrated that the transfection of the PCR products into mammalian cells resulted in speci¢c depressions of exogenous (luciferase, green £uorescent protein and L L-galactosidase) and endogenous (annexin II) gene expressions. This PCR strategy provides a rapid, easy and cheap approach for testing candidates siRNA sequences and is an attractive alternative to subcloning. ß