Alveolar epithelial cell (AEC) trans-differentiation is a process where type II alveolar epithelial cells (AEC II) trans-differentiate into type I alveolar epithelial cells (AEC I) during lung recovery after various injuries, in which AEC I are damaged. This process is critical for lung tissue repair. MicroRNAs are a group of small RNAs that regulate gene expression at the post-transcriptional level. They have the potential to regulate almost every aspect of cell physiology. However, whether AEC trans-differentiation is regulated by microRNAs is completely unknown. In this study, we found that miR-375 was downregulated during AEC trans-differentiation. The overexpression of miR-375 with an adenoviral vector inhibited alveolar epithelial trans-differentiation as indicated by an increase in the AEC II marker, surfactant protein C, and decreases in the AEC I markers, T1α and advanced glycosylation end product-specific receptor. miR-375 also inhibited the Wnt/β-catenin pathway. The constitutively activation of Wnt/β-catenin signaling with a stabilized form of β-catenin blocked the miR-375 effects. Frizzled 8 was identified as a target of miR-375. In summary, our results demonstrate that miR-375 regulates AEC trans-differentiation through the Wnt/β-catenin pathway. This discovery may provide new targets for therapeutic intervention to benefit lung recovery from injuries.

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 ...

Type II alveolar epithelial cells (AEC II) proliferate and transdifferentiate into type I alveolar epithelial cells (AEC I) when the normal AEC I population is damaged in the lung alveoli. We hypothesized that signaling by transforming growth factor ␤1 (TGF ␤1), through its downstream Smad proteins, is involved in keeping AEC II quiescent in normal cells and its altered signaling may be involved in the trans-differentiation of AEC II to AEC I. In the normal lung, TGF ␤1 and Smad4 were highly expressed in AEC II. Using an in vitro cell culture model, we demonstrated that the trans-differentiation of AEC II into AEC I-like cells began with a proliferative phase, followed by a differentiation phase. The expression of TGF ␤1, Smad2, and Samd3 and their phosphorylated protein forms, and cell cycle inhibitors, p15Ink4b and p21 Cip1 , was lower during the proliferative phase but higher during the differentiation phase. Furthermore, cyclin-dependent kinases 2, 4, and 6 showed an opposite trend of expression. TGF ␤1 secretion into the media increased during the differentiation phase, indicating an autocrine regulation. The addition of TGF ␤1 neutralizing antibody after the proliferative phase and silencing of Smad4 by RNA interference inhibited the trans-differentiation process. In summary, our results suggest that the trans-differentiation of AEC II to AEC I is modulated by signaling through the Smad-dependent TGF ␤1 pathway by altering the expression of proteins that control the G 1 to S phase entry in the cell cycle. Alveolar epithelial cells (AEC)2 II have a multifunctional role in the lung including secretory, synthetic, and progenitor capacities. AEC II serve as remodeling reservoirs for lung epithelium (1). They are the progenitors for AEC I. By virtue of their squamous shape, AEC I cover the major surface area of alveoli and thus are the main epithelial component of the thin air-blood barrier. In contrast, AEC II are limited to the corners of the alveoli. The progenitor function of AEC II is activated when the lung epithelium encounters a variety of disease conditions including acute lung injury (2). AEC II cell proliferation and hyperplasia, followed by trans-differentiation into AEC I is a hallmark of alveolar epithelial injury. This helps to restore the normal air-blood barrier. However, the molecular mechanisms involved in the trans-differentiation process are not clear. Questions regarding the causes of AEC II proliferation and trans-differentiation into AEC I during lung injury and prevention of this in normal alveoli largely remain unanswered.The transforming growth factor ␤ (TGF ␤) superfamily consists of cytokines that are implicated in the regulation of a variety of biological responses (3-6). Its members include isoforms of TGF ␤, bone morphogenetic proteins, Mullerian inhibiting substance, inhibins, and activins. A member of this family, TGF ␤1, regulates cell proliferation, differentiation, and migration in many cell and tissue systems. This cytokine is secreted by various cell types and acts both in...

BackgroundAcute 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.MethodsRat 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.ResultsThe 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.ConclusionThe 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.

SummaryAlveolar epithelium is composed of alveolar epithelial cells of type I (AEC I) and type II (AEC II). AEC II secrete lung surfactant by means of exocytosis. P2X 7 receptor (P2X 7 R), a P2 purinergic receptor, has been implicated in the regulation of synaptic transmission and inflammation. Here, we report that P2X 7 R, which is expressed in AEC I but not AEC II, is a novel mediator for the paracrine regulation of surfactant secretion in AEC II. In primary co-cultures of AEC I and AEC II benzoyl ATP (BzATP; an agonist of P2X 7 R) increased surfactant secretion, which was blocked by the P2X 7 R antagonist Brilliant Blue G. This effect was observed in AEC II cocultured with human embryonic kidney HEK-293 cells stably expressing rat P2X 7 R, but not when co-cultured with AEC I in which P2X 7 R was knocked down or in co-cultures of AEC I and AEC II isolated from P2X 7 R -/-mice. BzATP-mediated secretion involved P2Y 2 receptor signaling because it was reduced by the addition of the ATP scavengers apyrase and adenosine deaminase and the P2Y 2 receptor antagonist suramin. However, the stimulation with BzATP might also release other substances that potentially increase surfactant secretion as a greater stimulation of secretion was observed in AEC II incubated with BzATP when co-cultured with E10 or HEK-293-P2X 7 R cells than with ATP alone. P2X 7 R -/-mice failed to increase surfactant secretion in response to hyperventilation, pointing to the physiological relevance of P2X 7 R in maintaining surfactant homeostasis in the lung. These results suggest that the activation of P2X 7 R increases surfactant secretion by releasing ATP from AEC I and subsequently stimulating P2Y 2 receptors in AEC II.