Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures. ATP binding cassette (ABC)2 transporters are a superfamily of highly conserved membrane proteins that transport a wide variety of substrates across cell membranes (1). Among the several subfamilies, the ABCA subclass has received considerable attention, because mutations of the ABCA1 gene cause Tangier disease and mutations of the ABCA4 gene cause Stargardt macular dystrophy in humans (2-5). ABCA1 and ABCA4 are proposed to be transmembrane transporters for intracellular cholesterol/phospholipids and N-retinylidene phosphatidylethanolamine, respectively (3-5). ABCA3, a member of the ABCA subfamily with unknown function (6 -10), is predominantly expressed in the lung and localized to the limiting membrane of lamellar bodies in alveolar epithelial type II cells (ATII) in both humans and rats (7,8).In the lung, development of structures for effective pulmonary gas exchange and production of pulmonary surfactant are necessary for successful adaptation to extrauterine life in the newborn infant. These key processes in lung maturation require differentiation of epithelium into ATII cells, the cellular source for surfactant. Pulmonary surfactant is a complex mixture of lipids, primarily phosphatidylcholine (60 -70% of which is dipalmitoylphosphatidylcholine) and specific proteins that line the alveolar surface of the lung, reducing surface tension at the air-liquid interface and preventing collapse of the lung on expiration (11). Surfactant is assembled and stored in lamellar bodies, the secretory organelles of ATII cells (11-13). Two other members of the ABCA subfamily, ABCA1 and ABCA4, have been implicated in lipid transport leading to the hypothesis that ABCA3 transports lipid into the lamellar bodies of ATII cells (7-9). Recently, it has been reported that mutations in ABCA3 are associated with defective assembly of lamellar bodies and fatal respiratory distress syndrome (RDS) in the newborn infant and interstitial lung disease (6, 10).To study the potential role of ABCA3 in RDS, we examined the subcellular trafficking and substrate specificity of ABCA3 in hATII cells and mammalian cell lines using green fluorescent protein (GFP)-tagged protein and fluorescent lipid analogs. Morphological and functional changes secondary to both loss-and gain-of-function experiments demonstrate that ABCA3 selectively transports phosphatidylcholine, sphingomyelin, and cholesterol to lamellar bodies in hATII cells. Our findings indicate that lipid trafficking by ABCA3 across lamellar body membranes is necessary for lamellar body biogenesis as a key step in assembly of lung surfactant in hATII cells.

Superparamagnetic iron oxide nanoparticles are widely used in biomedical applications, yet questions remain regarding the effect of nanoparticle size and coating on nanoparticle cytotoxicity. In this study, porcine aortic endothelial cells were exposed to 5 and 30 nm diameter iron oxide nanoparticles coated with either the polysaccharide, dextran, or the polymer polyethylene glycol (PEG). Nanoparticle uptake, cytotoxicity, reactive oxygen species (ROS) formation, and cell morphology changes were measured. Endothelial cells took up nanoparticles of all sizes and coatings in a dose dependent manner, and intracellular nanoparticles remained clustered in cytoplasmic vacuoles. Bare nanoparticles in both sizes induced a more than 6 fold increase in cell death at the highest concentration (0.5 mg/mL) and led to significant cell elongation, whereas cell viability and morphology remained constant with coated nanoparticles. While bare 30 nm nanoparticles induced significant ROS formation, neither 5 nm nanoparticles (bare or coated) nor 30 nm coated nanoparticles changed ROS levels. Furthermore, nanoparticles were more toxic at lower concentrations when cells were cultured within 3D gels. These results indicate that both dextran and PEG coatings reduce nanoparticle cytotoxicity, however different mechanisms may be important for different size nanoparticles.

Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH-oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule-1 (VCAM-1). Catalase and superoxide dismutase (SOD) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM-1) bind specifically to endothelium and inhibit effects of corresponding ROS, H(2)O(2), and superoxide anion. In this study, anti-PECAM/SOD, but not anti-PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)-SOD, inhibited 2- to 3-fold VCAM expression caused by tumor necrosis factor (TNF), interleukin-1β, and lipopolysaccharide. Anti- PECAM/SOD, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide-caused VCAM-1 expression in mice. Anti-PECAM/SOD colocalized with EEA-1-positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC3 blocked TNF-induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti-PECAM/SOD abolished VCAM expression caused by poly(I:C)-induced activation of toll-like receptor 3 localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site-specific interception of this signal attained by targeted delivery of anti-PECAM/SOD into endothelial endosomes may have anti-inflammatory effects.

The biological functions of N6-methyladenosine (m 6 A) RNA methylation are mainly dependent on the reader; however, its role in lung tumorigenesis remains unclear. Here, we have demonstrated that the m 6 A reader YT521-B homology domain containing 2 (YTHDC2) is frequently suppressed in lung adenocarcinoma (LUAD). Downregulation of YTHDC2 was associated with poor clinical outcome of LUAD. YTHDC2 decreased tumorigenesis in a spontaneous LUAD mouse model. Moreover, YTHDC2 exhibited antitumor activity in human LUAD cells. Mechanistically, YTHDC2, via its m 6 A-recognizing YTH domain, suppressed cystine uptake and blocked the downstream antioxidant program. Administration of cystine downstream antioxidants to pulmonary YTHDC2-overexpressing mice rescued lung tumorigenesis. Furthermore, solute carrier 7A11 (SLC7A11), the catalytic subunit of system X C − , was identified to be the direct target of YTHDC2. YTHDC2 destabilized SLC7A1 1 mRNA in an m 6 A-dependent manner because YTHDC2 preferentially bound to m 6 A-modified SLC7A1 1 mRNA and thereafter promoted its decay. Clinically, a large proportion of acinar LUAD subtype cases exhibited simultaneous YTHDC2 downregulation and SLC7A11 elevation. Patient-derived xenograft (PDX) mouse models generated from acinar LUAD showed sensitivity to system X C − inhibitors. Collectively, the promotion of cystine uptake via the suppression of YTHDC2 is critical for LUAD tumorigenesis, and blocking this process may benefit future treatment.