Lipid Rafts and Lung Surfactant Secretion

The present study was initiated to elucidate the role of lipid rafts in lung surfactant secretion. Lipid rafts were isolated and probed for the association of SNARE proteins. Cholesterol depletion was done to study its role in modulating the surfactant secretion and membrane fusion. Flotillin proteins were silenced to examine their role in surfactant secretion. Finally, we determined the proteomic profile of type II cell lipid rafts to identify novel proteins in this unique subproteome and study the roles of one of the identified proteins, namely, v-ATPases, in surfactant secretion. We have used a number of techniques including RT-PCR, immunoblotting, immunoflorescence, secretion assay, membrane fusion assay, isolation of plasma membrane and lamellar bodies, and RNA interference to address the biological phenomenon. 1. SNARE proteins were differentially associated with lipid rafts in a cholesterol-dependent manner. 2. Cholesterol depletion drastically reduced surfactant secretion, membrane fusion and fusion pVeterinary Pathobiolog

Identification of rat lung-specific microRNAs by micoRNA microarray: valuable discoveries for the facilitation of lung research

BACKGROUND: An important mechanism for gene regulation utilizes small non-coding RNAs called microRNAs (miRNAs). These small RNAs play important roles in tissue development, cell differentiation and proliferation, lipid and fat metabolism, stem cells, exocytosis, diseases and cancers. To date, relatively little is known about functions of miRNAs in the lung except lung cancer. RESULTS: In this study, we utilized a rat miRNA microarray containing 216 miRNA probes, printed in-house, to detect the expression of miRNAs in the rat lung compared to the rat heart, brain, liver, kidney and spleen. Statistical analysis using Significant Analysis of Microarray (SAM) and Tukey Honestly Significant Difference (HSD) revealed 2 miRNAs (miR-195 and miR-200c) expressed specifically in the lung and 9 miRNAs co-expressed in the lung and another organ. 12 selected miRNAs were verified by Northern blot analysis. CONCLUSION: The identified lung-specific miRNAs from this work will facilitate functional studies of miRNAs during normal physiological and pathophysiological processes of the lung

MicroRNA-206 regulates surfactant secretion by targeting VAMP-2

AbstractLung surfactant secretion is a highly regulated process. Our previous studies have shown that VAMP-2 is essential for surfactant secretion. In the present study we investigated the role of miR-206 in surfactant secretion through VAMP-2. VAMP-2 was confirmed to be a target of miR-206 by 3′-untranslational region (3′-UTR) luciferase assay. Mutations in the predicated miR-206 binding sites reduced the binding of miR-206 to the 3′-UTR of VAMP-2. miR-206 decreased the expression of VAMP-2 protein and decreased the lung surfactant secretion in alveolar type II cells. In conclusion, miR-206 regulates lung surfactant secretion by limiting the availability of VAMP-2 protein

Role of GABA Receptors in Fetal Lung Development in Rats

Fluid accumulation is critical for lung distension and normal development. The multi-subunit γ-amino butyric acid type A receptors (GABAA) mainly act by mediating chloride ion (Cl−) fluxes. Since fetal lung actively secretes Cl−-rich fluid, we investigated the role of GABAA receptors in fetal lung development. The physiological ligand, GABA, and its synthesizing enzyme, glutamic acid decarboxylase, were predominantly localized to saccular epithelium. To examine the effect of activating GABAA receptors in fetal lung development in vivo, timed-pregnant rats of day 18 gestation underwent an in utero surgery for the administration of GABAA receptor modulators into the fetuses. The fetal lungs were isolated on day 21 of gestation and analyzed for changes in fetal lung development. Fetuses injected with GABA had a significantly higher body weight and lung weight when compared to phosphate-buffered saline (control)-injected fetuses. GABA-injected fetal lungs had a higher number of saccules than the control. GABA increased the number of alveolar epithelial type II cells as indicated by surfactant protein C-positive cells. However, GABA decreased the number of α-smooth muscle actin-positive myofibroblasts, but did not affect the number of Clara cells or alveolar type I cells. GABA-mediated effects were blocked by the GABAA receptor antagonist, bicuculline. GABA also increased cell proliferation and Cl− efflux in fetal distal lung epithelial cells. In conclusion, our results indicate that GABAA receptors accelerate fetal lung development, likely through an enhanced cell proliferation and/or fluid secretion

Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca2+ chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca2+/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion

MicroRNA and mRNA expression profiling in rat acute respiratory distress syndrome

Background: Acute respiratory distress syndrome (ARDS) is characterized by pulmonary epithelial injury and extensive inflammation of the pulmonary parenchyma. Systematic analyses of microRNA (miRNA) and mRNA expression profiling in ARDS provide insights into understanding of molecular mechanisms of the pathogenesis of ARDS. The objective of this study was to identify miRNA and mRNA interactions in a rat model of ARDS by combining miRNA and mRNA microarray analyses.Methods: Rat model of ARDS was induced by saline lavage and mechanical ventilation. The expression profiles of both mRNAs and miRNAs in rat ARDS model were performed by microarray analyses. Microarray data were further verified by quantitative RT-PCR. Functional annotation on dys-regulated mRNAs and miRNAs was carried out by bioinformatics analysis.Results: The expression of 27 miRNAs and 37 mRNAs were found to be significantly changed. The selected miRNAs and genes were further verified by quantitative real-time PCR. The down-regulated miRNAs included miR-24, miR-26a, miR-126, and Let-7a, b, c, f. The up-regulated miRNAs were composed of miR-344, miR-346, miR-99a, miR-127, miR-128b, miR-135b, and miR-30a/b. Gene ontology and functional annotation analyses indicated that up-regulated mRNAs, such as Apc, Timp1, and Sod2, were involved in the regulation of apoptosis. Bioinformatics analysis showed the inverse correlation of altered miRNAs with the expression of their predicted target mRNAs. While Sod2 was inversely correlated with Let-7a, b, c, f., Ebf1 and Apc were inversely correlated with miR-24 and miR-26a, respectively. miR-26a, miR-346, miR-135b, miR-30a/b, miR-344, and miR-18a targeted multiple altered mRNAs. Gabrb1, Sod2, Eif2ak1, Fbln5, and Tspan8 were targeted by multiple altered miRNAs.Conclusion: The expressions of miRNAs and mRNAs were altered in a rat model of ARDS. The identified miRNA-mRNA pairs may play critical roles in the pathogenesis of ARDS.Peer reviewedPathobiologyOklahoma Center for Respiratory and Infectious DiseasesPhysiological Science

Physical and Functional Interactions of SNAP-23 with Annexin A2

Lung surfactant is secreted through the fusion of lamellar bodies with the plasma membrane of alveolar epithelial type II cells. Annexin A2, a Ca2+- and phospholipid-binding protein, promotes the fusion of lamellar bodies with the plasma membrane. Soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) are known to have an essential role in surfactant secretion. We hypothesized that annexin A2 acts as a Ca2+ sensor and mediates membrane fusion via its interaction with SNAREs. Both purified or endogenous annexin A2 in type II cells specifically bound with SNAP-23 in a Ca2+-dependent manner, as determined by pull-down experiments using recombinant glutathione S-transferase-tagged SNAP-23. A deletion study identified the cysteine-rich region (CRR) of SNAP-23 as the binding site for annexin A2. Mutations of cysteine residues in the CRR dramatically decreased the binding. SNAP-23 also co-immunoprecipitated with annexin A2; however, a SNAP-23 mutant failed to co-immunoprecipitate with annexin A2. Immunofluorescence revealed a co-localization of SNAP-23 and annexin A2 in type II cells. Furthermore, anti–SNAP-23 antibody significantly inhibited annexin A2–mediated fusion between lamellar bodies and the plasma membrane. These data suggest that annexin A2 and SNAP-23 are involved in the same pathway in the regulation of lung surfactant secretion

TRPA1 and CGRP antagonists counteract vesicant-induced skin injury and inflammation

The skin is highly sensitive to the chemical warfare agent in mustard gas, sulfur mustard (SM) that initiates a delayed injury response characterized by erythema, inflammation and severe vesication (blistering). Although SM poses a continuing threat, used as recently as in the Syrian conflict, no mechanism-based antidotes against SM are available. Recent studies demonstrated that Transient Receptor Potential Ankyrin 1 (TRPA1), a chemosensory cation channel in sensory nerves innervating the skin, is activated by SM and 2‐chloroethyl ethyl sulfide (CEES), an SM analog, in vitro, suggesting it may promote vesicant injury. Here, we investigated the effects of TRPA1 inhibitors, and an inhibitor of Calcitonin Gene Related Peptide (CGRP), a neurogenic inflammatory peptide released upon TRPA1 activation, in a CEES-induced mouse ear vesicant model (CEES-MEVM). TRPA1 inhibitors (HC-030031 and A-967079) and a CGRP inhibitor (MK-8825) reduced skin edema, pro-inflammatory cytokines (IL-1β, CXCL1/KC), MMP-9, a protease implicated in skin damage, and improved histopathological outcomes. These findings suggest that TRPA1 and neurogenic inflammation contribute to the deleterious effects of vesicants in vivo, activated either directly by alkylation, or indirectly, by reactive intermediates or pro-inflammatory mediators. TRPA1 and CGRP inhibitors represent new leads that could be considered for validation and further development in other vesicant injury models