Kevin Yu

Functional and Trafficking Defects in ATP Binding Cassette A3 Mutants Associated with Respiratory Distress Syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. 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.

Dextran and polymer polyethylene glycol (PEG) coating reduce both 5 and 30 nm iron oxide nanoparticle cytotoxicity in 2D and 3D cell culture.

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.

ABCA3 Is Critical for Lamellar Body Biogenesis in Vivo

Mutations in ATP-binding cassette transporter A3 (human ABCA3) protein are associated with fatal respiratory distress syndrome in newborns. We therefore characterized mice with targeted disruption of the ABCA3 gene. Homozygous Abca3–/– knock-out mice died soon after birth, whereas most of the wild type, Abca3+/+, and heterozygous, Abca3+/–, neonates survived. The lungs from E18.5 and E19.5 Abca3–/– mice were less mature than wild type. Alveolar type 2 cells from Abca3–/– embryos contained no lamellar bodies, and expression of mature SP-B protein was disrupted when compared with the normal lung surfactant system of wild type embryos. Small structural and functional differences in the surfactant system were seen in adult Abca3+/– compared with Abca3+/+ mice. The heterozygotes had fewer lamellar bodies, and the incorporation of radiolabeled substrates into newly synthesized disaturated phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylserine in both lamellar bodies and surfactant was lower than in Abca3+/+ mouse lungs. In addition, since the fraction of near term Abca3–/– embryos was significantly lower than expected from Mendelian inheritance ABCA3 probably plays roles in development unrelated to surfactant. Collectively, these findings strongly suggest that ABCA3 is necessary for lamellar body biogenesis, surfactant protein-B processing, and lung development late in gestation.

Interaction of Surfactant Protein A with Peroxiredoxin 6 Regulates Phospholipase A2 Activity

Peroxiredoxin 6 (Prdx6) is a “moonlighting” protein with both GSH peroxidase and phospholipase A2 (PLA2) activities. This protein is responsible for degradation of internalized dipalmitoylphosphatidylcholine, the major phospholipid component of lung surfactant. The PLA2 activity is inhibited by surfactant protein A (SP-A). We postulate that SP-A regulates the PLA2 activity of Prdx6 through direct protein-protein interaction. Recombinant human Prdx6 and SP-A isolated from human alveolar proteinosis fluid were studied. Measurement of kinetic constants at pH 4.0 (maximal PLA2 activity) showed Km0.35 mm and Vmax 138 nmol/min/mg of protein. SP-A inhibited PLA2 activity non-competitively with Ki 10 μg/ml and was Ca2+ -independent. Activity at pH 7.4 was ∼50% less, and inhibition by SP-A was partially dependent on Ca2+. Interaction of SP-A and Prdx6 at pH 7.4 was shown by Prdx6-mediated inhibition of SP-A binding to agarose beads, a pull-down assay using His-tagged Prdx6 and Ni2 -chelating beads, co-immunoprecipitation from lung epithelial cells and from a binary mixture of the two proteins, binding after treatment with a trifunctional cross-linker, and size-exclusion chromatography. Analysis by static light scattering and surface plasmon resonance showed calcium-independent SP-A binding to Prdx6 at pH 4.0 and partial Ca2+ dependence of binding at pH 7.4. These results indicate a direct interaction between SP-A and Prdx6, which provides a mechanism for regulation of the PLA2 activity of Prdx6 by SP-A.

PECAM-targeted delivery of SOD inhibits endothelial inflammatory response

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, H2O2, 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 endo‐somes may have anti‐inflammatory effects.—Shuvaev, V. V., Han, J., Yu, K. J., Huang, S., Hawkins, B. J., Madesh, M., Nakada, M., and Muzykantov, V. R. PECAM‐targeted delivery of SOD inhibits endothelial inflammatory response. FASEB J. 25, 348–357 (2011). www.fasebj.org

Comparison of glutathione peroxidase 1 and peroxiredoxin 6 in protection against oxidative stress in the mouse lung

Peroxiredoxin 6 (Prdx6) and cytosolic GSH peroxidase (GPx1), both GSH-dependent peroxidases, were compared for the effects of their knockout on injury and lipid peroxidation in: (a) lungs of mice exposed to 0.85 or 1.0atm O(2), (b) isolated perfused mouse lungs exposed to 5mM tert-butylhydroperoxide (t-BOOH) or 1mM paraquat, and (c) primary mouse pulmonary microvascular endothelial cells exposed to 50muM t-BOOH. Derangements in GPx1 null were similar or slightly greater than in wild type for all parameters in the various models of oxidant stress, whereas Prdx6 null showed markedly increased effects. GSH peroxidase activity with phosphatidylcholine hydroperoxide as substrate in GPx1-null lung homogenate was decreased only slightly vs wild type, whereas activity in Prdx6-null lungs was decreased by ~95%, indicating that Prdx6 is the major enzyme for reduction of oxidized lung phospholipids. Expression levels of oxidant-related genes measured with a PCR-based gene array indicated no significant differences between the Prdx6 and the GPx1 null except for the target genes and IL-19. Thus, Prdx6-null mice are significantly more sensitive to oxidant stress compared to GPx1 null, suggesting that scavenging of phospholipid hydroperoxides by Prdx6 plays a major role in lung antioxidant defense.

PECAM-1 and caveolae form the mechanosensing complex necessary for NOX2 activation and angiogenic signaling with stopped flow in pulmonary endothelium

We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however, a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1), and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule-1 (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1(-/-) lungs compared with lungs from wild-type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1(-/-) PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in WT mice was significantly lowered in PECAM-1(-/-) mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker VEGF compared with untreated (sham) and PECAM-1(-/-) lungs. Thus PECAM-1 (and caveolae) are parts of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal.

Rab38 targets to lamellar bodies and normalizes their sizes in lung alveolar type II epithelial cells.

Rab38 is a rat Hermansky-Pudlak syndrome gene that plays an important role in surfactant homeostasis in alveolar type II (ATII) pneumocytes. We examined Rab38 function in regulating lamellar body (LB) morphology in ATII cells. Quantitative electron microscopy revealed that LBs in ATII cells were ∼77% larger in Rab38-null fawn-hooded hypertension (FHH) than control Sprague-Dawley (SD) rats. Rab38 protein expression was restricted in lung epithelial cells but was not found in primary endothelial cells. In SD ATII cells, Rab38 protein level gradually declined during 5 days in culture. Importantly, endogenous Rab38 was present in LB fractions purified from SD rat lungs, and transiently expressed enhanced green fluorescent protein (EGFP)-tagged Rab38 labeled only the limiting membranes of a subpopulation (∼30%) of LBs in cultured ATII cells. This selective targeting was abolished by point mutations to EGFP-Rab38 and was not shared by Rab7 and Rab4b, which also function in the ATII cells. Using confocal microscopy, we established a method for quantitative evaluation of the enlarged LB phenotype temporally preserved in cultured FHH ATII cells. A direct causal relationship was established when the enlarged LB phenotype was reserved and then rescued by transiently reexpressed EGFP-Rab38 in cultured FHH ATII cells. This rescuing effect was associated with dynamic EGFP-Rab38 targeting to and on LB limiting membranes. We conclude that Rab38 plays an indispensible role in maintaining LB morphology and surfactant homeostasis in ATII pneumocytes.

Role of P63 (CKAP4) in binding of surfactant protein-A to type II pneumocytes.

We have recently described a putative receptor for lung surfactant protein-A (SP-A) on rat type II pneumocytes. The receptor, P63, is a 63-kDa type II transmembrane protein. Coincubation of type II cells with P63 antibody (Ab) reversed the inhibitory effect of SP-A on secretagogue-stimulated surfactant secretion from type II cells. To further characterize SP-A interactions with P63, we expressed recombinant P63 protein in Escherichia coli and generated antibodies to P63. Immunogold electron microscopy confirmed endoplasmic reticulum and plasma membrane localization of P63 in type II cells with prominent labeling of microvilli. Binding characteristics of iodinated SP-A to type II cells in the presence of P63 Ab were determined. Binding (4 degrees C, 1 h) of (125)I-SP-A to type II cells demonstrated both specific (calcium-dependent) and nonspecific (calcium-independent) components. Ab to P63 protein blocked the specific binding of (125)I-SP-A to type II cells and did not change the nonspecific SP-A association. A549 cells, a pneumocyte model cell line, expressed substantial levels of P63 and demonstrated specific binding of (125)I-SP-A that was inhibited by the P63 Ab. The secretagogue (cAMP)-stimulated increase in calcium-dependent binding of SP-A to type II cells was blocked by the presence of P63 Ab. Transfection of type II cells with small interfering RNA to P63 reduced P63 protein expression, attenuated P63-specific SP-A binding, and reversed the ability of SP-A to prevent surfactant secretion from the cells. Our results further substantiate the role of P63 as an SP-A receptor protein localized on the surface of lung type II cells.

Characterization of the Niemann-Pick C pathway in alveolar type II cells and lamellar bodies of the lung

The Niemann-Pick C (NPC) pathway plays an essential role in the intracellular trafficking of cholesterol by facilitating the release of lipoprotein-derived sterol from the lumen of lysosomes. Regulation of cellular cholesterol homeostasis is of particular importance to lung alveolar type II cells because of the need for production of surfactant with an appropriate lipid composition. We performed microscopic and biochemical analysis of NPC proteins in isolated rat type II pneumocytes. NPC1 and NPC2 proteins were present in the lung, isolated type II cells in culture, and alveolar macrophages. The glycosylated and nonglycosylated forms of NPC1 were prominent in the lung and the lamellar body organelles. Immunocytochemical analysis of isolated type II pneumocytes showed localization of NPC1 to the limiting membrane of lamellar bodies. NPC2 and lysosomal acid lipase were found within these organelles, as confirmed by z-stack analysis of confocal images. All three proteins also were identified in small, lysosome-like vesicles. In the presence of serum, pharmacological inhibition of the NPC pathway with compound U18666A resulted in doubling of the cholesterol content of the type II cells. Filipin staining revealed a striking accumulation of cholesterol within lamellar bodies. Thus the NPC pathway functions to control cholesterol accumulation in lamellar bodies of type II pneumocytes and, thereby, may play a role in the regulation of surfactant cholesterol content.