ATII Cells Research Articles

ABSTRACT 485

Background and aims: The production of reactive oxygen species (ROS) during hyperoxia is thought to contribute to alveolar epithelial apoptosis. Aims: To explore the molecular mechanisms of oxidative stress-induced alveolar epithelial cell apoptosis and assess the possible cytoprotective effects of N-acetylcysteine (NAC). Methods: 500 μM H2O2 treatment induced primary alveolar type II epithelial cells apoptosis, during this course a rapid increase of phosphorylation levels of JNK activated by H2O2 was found. The apoptosis of ATII cells can be prevented by SP600125, a specific inhibitor of JNK. SP600125 also attenuated Bax expression and p53 nuclear accumulation induced by H2O2. Results: Pretreatment of ATII cells with NAC (5 mM) 0.5 h before H2O2 would markedly decreased cell apoptosis and the activation of JNK. The high level of intracellular ROS induced by H2O2 was also significantly attenuated by NAC pretreatment. Conclusions: Taken together, these findings suggest JNK is the upstream of Bax and p53 in ATII cells apoptosis induced by oxidative stress, and NAC attenuated cells apoptosis through suppressing intracellular ROS generation and JNK activation.

Inter-α-Inhibitor Blocks Epithelial Sodium Channel Activation and Decreases Nasal Potential Differences in ΔF508 Mice

Increased activity of lung epithelial sodium channels (ENaCs) contributes to the pathophysiology of cystic fibrosis (CF) by increasing the rate of epithelial lining fluid reabsorption. Inter-α-inhibitor (IαI), a serum protease inhibitor, may decrease ENaC activity by preventing its cleavage by serine proteases. High concentrations of IαI were detected in the bronchoalveolar lavage fluid (BALF) of children with CF and lower airway diseases. IαI decreased amiloride-sensitive (IENaC) but not cAMP-activated Cl(-) currents across confluent monolayers of rat ATII, and mouse nasal epithelial cells grew in primary culture by 45 and 25%, respectively. Changes in IENaC by IαI in ATII cells were accompanied by increased levels of uncleaved (immature) surface α-ENaC. IαI increased airway surface liquid depth overlying murine nasal epithelial cells to the same extent as amiloride, consistent with ENaC inhibition. Incubation of lung slices from C57BL/6, those lacking phenylalanine at position 508 (∆F508), or CF transmembrane conductance regulator knockout mice with IαI for 3 hours decreased the open probability of their ENaC channels by 50%. ∆F508 mice had considerably higher levels the amiloride-sensitive fractions of ENaC nasal potential difference (ENaC-NPD) than wild-type littermates and only background levels of IαI in their BALF. A single intranasal instillation of IαI decreased their ENaC-NPD 24 hours later by 25%. In conclusion, we show that IαI is present in the BALF of children with CF, is an effective inhibitor of ENaC proteolysis, and decreases ENaC activity in lung epithelial cells of ∆F508 mice.

Breaking the In Vitro Alveolar Type II Cell Proliferation Barrier while Retaining Ion Transport Properties

Alveolar type (AT)I and ATII cells are central to maintaining normal alveolar fluid homeostasis. When disrupted, they contribute to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome. Research on ATII cells has been limited by the inability to propagate primary cells in vitro to study their specific functional properties. Moreover, primary ATII cells in vitro quickly transdifferentiate into nonproliferative "ATI-like" cells under traditional culture conditions. Recent studies have demonstrated that normal and tumor cells grown in culture with a combination of fibroblast (feeder cells) and a pharmacological Rho kinase inhibitor (Y-27632) exhibit indefinite cell proliferation that resembled a "conditionally reprogrammed cell" phenotype. Using this coculture system, we found that primary human ATII cells (1) proliferated at an exponential rate, (2) established epithelial colonies expressing ATII-specific and "ATI-like" mRNA and proteins after serial passage, (3) up-regulated genes important in cell proliferation and migration, and (4) on removal of feeder cells and Rho kinase inhibitor under air-liquid interface conditions, exhibited bioelectric and volume transport characteristics similar to freshly cultured ATII cells. Collectively, our results demonstrate that this novel coculture technique breaks the in vitro ATII cell proliferation barrier while retaining cell-specific functional properties. This work will allow for a significant increase in studies designed to elucidate ATII cell function with the goal of accelerating the development of novel therapies for alveolar diseases.

Long-chain Acyl-CoA Dehydrogenase Deficiency as a Cause of Pulmonary Surfactant Dysfunction

Long-chain acyl-CoA dehydrogenase (LCAD) is a mitochondrial fatty acid oxidation enzyme whose expression in humans is low or absent in organs known to utilize fatty acids for energy such as heart, muscle, and liver. This study demonstrates localization of LCAD to human alveolar type II pneumocytes, which synthesize and secrete pulmonary surfactant. The physiological role of LCAD and the fatty acid oxidation pathway in lung was subsequently studied using LCAD knock-out mice. Lung fatty acid oxidation was reduced in LCAD(-/-) mice. LCAD(-/-) mice demonstrated reduced pulmonary compliance, but histological examination of lung tissue revealed no obvious signs of inflammation or pathology. The changes in lung mechanics were found to be due to pulmonary surfactant dysfunction. Large aggregate surfactant isolated from LCAD(-/-) mouse lavage fluid had significantly reduced phospholipid content as well as alterations in the acyl chain composition of phosphatidylcholine and phosphatidylglycerol. LCAD(-/-) surfactant demonstrated functional abnormalities when subjected to dynamic compression-expansion cycling on a constrained drop surfactometer. Serum albumin, which has been shown to degrade and inactivate pulmonary surfactant, was significantly increased in LCAD(-/-) lavage fluid, suggesting increased epithelial permeability. Finally, we identified two cases of sudden unexplained infant death where no lung LCAD antigen was detectable. Both infants were homozygous for an amino acid changing polymorphism (K333Q). These findings for the first time identify the fatty acid oxidation pathway and LCAD in particular as factors contributing to the pathophysiology of pulmonary disease.

Importance of Both Innate Immunity and Acquired Immunity for Rapid Expulsion of S. venezuelensis

In the first part of this review, we described the relevant roles of endogenous IL-33 for accumulation of ILC2 and eosinophils even in the lungs of Rag2−/− mice. Type II alveolar epithelial (ATII) cells express IL-33 in their nucleus and infection with Strongyloides venezuelensis induces IL-33 production by increasing the number of ATII cells possibly by the action of chitin. IL-33 from ATII cells induces ILC2 proliferation and at the same time activates them to produce IL-5 and IL-13, which in combination induce lung eosinophilic inflammation, aiding to expel infected worms in the lungs. In the second part, we showed that, although AID−/− mice normally develop Th2 cells and intestinal mastocytosis after infection with S. venezuelensis, they need adoptive transfers of immune sera from S. venezuelensis infected mice to obtain the capacity to promptly expel S. venezuelensis. Thus, intestinal nematode infection induces various Th2 immune responses (e.g., Th2 cell, ILC2, goblet cell hyperplasia, intestinal mastocytosis, smooth muscle cell contraction, local and systemic eosinophilia, and high serum level of IgE and IgG1). However, all of them are not necessary for rapid expulsion of intestinal nematodes. Instead, some combinations of Th2 immune responses are essentially required.

Isolation and characterization of lung resident mesenchymal stem cells capable of differentiating into alveolar epithelial type II cells

AbstractControversies and risks continue to be reported about exogenous mesenchymal stem cell‐based therapies. In contrast with employing exogenous stem cells, making use of lung resident mesenchymal stem cells (LR‐MSCs) could be advantageous. Our study sought to isolate the LR‐MSCs and explore their potential to differentiate into alveolar epithelial type II cells (ATII cells). Total lung cells were first precultured, from which the Sca‐1+CD45−CD31− population was purified using fluorescence activated cell sorting (FACS). By these methods, it would seem that the Sca‐1+CD45−CD31− cells were LR‐MSCs. Similar to bone marrow derived mesenchymal stem cells (BM‐MSCs), these cells express Sca‐1, CD29, CD90, CD44 and CD106, but not CD31 or CD45. They share the same gene expression file with the BM‐MSCs and have a similar DNA content during long‐term culturing. Furthermore, they could be serially passaged with all these properties being sustained. Above all, LR‐MSCs could differentiate into ATII cells when co‐cultured with ATII cells in a trans‐well system. These findings demonstrated that the Sca‐1+CD45−CD31− cells appear to be LR‐MSCs that can differentiate into ATII cells. This approach may hold promise for their use in the treatment of lung disease.

Microfluidic shear stress-regulated surfactant secretion in alveolar epithelial type II cells in vitro

We investigated the role of flow-induced shear stress on the mechanisms regulating surfactant secretion in type II alveolar epithelial cells (ATII) using microfluidic models. Following flow stimulation spanning a range of wall shear stress (WSS) magnitudes, monolayers of ATII (MLE-12 and A549) cells were examined for surfactant secretion by evaluating essential steps of the process, including relative changes in the number of fusion events of lamellar bodies (LBs) with the plasma membrane (PM) and intracellular redistribution of LBs. F-actin cytoskeleton and calcium levels were analyzed in A549 cells subjected to WSS spanning 4-20 dyn/cm(2). Results reveal an enhancement in LB fusion events with the PM in MLE-12 cells upon flow stimulation, whereas A549 cells exhibit no foreseeable changes in the monitored number of fusion events for WSS levels ranging up to a threshold of ∼8 dyn/cm(2); above this threshold, we witness instead a decrease in LB fusion events in A549 cells. However, patterns of LB redistribution suggest that WSS can potentially serve as a stimulus for A549 cells to trigger the intracellular transport of LBs toward the cell periphery. This observation is accompanied by a fragmentation of F-actin, indicating that disorganization of the F-actin cytoskeleton might act as a limiting factor for LB fusion events. Moreover, we note a rise in cytosolic calcium ([Ca(2+)]c) levels following stimulation of A549 cells with WSS magnitudes ranging near or above the experimental threshold. Overall, WSS stimulation can influence key components of molecular machinery for regulated surfactant secretion in ATII cells in vitro.

Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of Integrin Receptor Subunits Beta 2/6 and WNT Signaling

Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarray-based transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2(-/-) mice showed that Itgb2 is required for proper WNT signaling regulation in the lung.

Aflatoxin G1-induced oxidative stress causes DNA damage and triggers apoptosis through MAPK signaling pathway in A549 cells

Our previous studies showed that Aflatoxin G1 (AFG1) could induce lung adenocarcinoma, and that the cancer cells originated from alveolar type II cells (AT-II cells). Recently, we found AFG1 induced structural impairment in rat AT-II cells, which may account for an early event in lung tumorigenesis. However, the mechanism of AFG1-induced AT-II cell damage remains unclear. DNA damage and apoptosis induced by oxidative stress are well accepted causes of cell damage. Thus, we explore whether AFG1 activates the reactive oxygen species (ROS)/MAPK/apoptosis pathway to cause cell damage in human AT-II cells like the cell line (A549). We found AFG1 induced oxidative stress by increasing ROS generation and caused DNA double-strand breaks (DSBs) by up-regulating γH2AX expression. AFG1 also triggered apoptosis in A549 cells by regulating Fas/FasL, caspase-8, Bax, Bcl-2, and activating caspase-3. Pre-treatment with antioxidant n-acetyl-l-cysteine (NAC) reduced ROS generation and DNA DSBs, inhibited apoptosis, and increased cell viability in AFG1-treated cells. Furthermore, we found AFG1 activated ROS-mediated JNK and p38 pathways to induce cell apoptosis in A549 cells. In conclusion, our results indicate that AFG1 induces oxidative DNA damage and triggers apoptosis through ROS-mediated JNK and p38 signaling pathways in A549 cells, which may contribute to AFG1-induced AT-II cell damage.

Regulation of Lung Injury and Fibrosis by p53-Mediated Changes in Urokinase and Plasminogen Activator Inhibitor-1

Alveolar type II epithelial cell (ATII) apoptosis and proliferation of mesenchymal cells are the hallmarks of idiopathic pulmonary fibrosis, a devastating disease of unknown cause characterized by alveolar epithelial injury and progressive fibrosis. We used a mouse model of bleomycin (BLM)-induced lung injury to understand the involvement of p53-mediated changes in urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) levels in the regulation of alveolar epithelial injury. We found marked induction of p53 in ATII cells from mice exposed to BLM. Transgenic mice expressing transcriptionally inactive dominant negative p53 in ATII cells showed augmented apoptosis, whereas those deficient in p53 resisted BLM-induced ATII cell apoptosis. Inhibition of p53 transcription failed to suppress PAI-1 or induce uPA mRNA in BLM-treated ATII cells. ATII cells from mice with BLM injury showed augmented binding of p53 to uPA, uPA receptor (uPAR), and PAI-1 mRNA. p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions neither interfered with p53 DNA binding activity nor p53-mediated promoter transactivation. However, increased expression of p53-binding sequences from uPA, uPAR, and PAI-1 mRNA 3' untranslated regions in ATII cells suppressed PAI-1 and induced uPA after BLM treatment, leading to inhibition of ATII cell apoptosis and pulmonary fibrosis. Our findings indicate that disruption of p53-fibrinolytic system cross talk may serve as a novel intervention strategy to prevent lung injury and pulmonary fibrosis.

Essential Regulation of Lung Surfactant Homeostasis by the Orphan G Protein-Coupled Receptor GPR116

GPR116 is an orphan seven-pass transmembrane receptor whose function has been unclear. Global disruption of the Gpr116 gene in mice revealed an unexpected, critical role for this receptor in lung surfactant homeostasis, resulting in progressive accumulation of surfactant lipids and proteins in the alveolar space, labored breathing, and a reduced lifespan. GPR116 expression analysis, bone marrow transplantation studies, and characterization of conditional knockout mice revealed that GPR116 expression in ATII cells is required for maintaining normal surfactant levels. Aberrant packaging of surfactant proteins with lipids in the Gpr116 mutant mice resulted in compromised surfactant structure, function, uptake, and processing. Thus, GPR116 plays an indispensable role in lung surfactant homeostasis with important ramifications for the understanding and treatment of lung surfactant disorders.

Differentiated phenotypes of primary murine alveolar epithelial cells and their susceptibility to infection by respiratory viruses

Severe respiratory viral infections are associated with spread to the alveoli of the lungs. There are multiple murine models of severe respiratory viral infections that have been used to identify viral and host factors that contribute to disease severity. Primary cultures of murine alveolar epithelial cells provide a robust in vitro model to perform mechanistic studies that can be correlated with in vivo studies to identify cell type-specific factors that contribute to pathology within the alveoli of the lung during viral infection. In this study, we established an in vitro model to compare the responses of type I (ATI) and type II (ATII) alveolar epithelial cells to infection by respiratory viruses used in murine models: mouse-adapted severe acute respiratory syndrome-associated coronavirus (SARS-CoV, v2163), murine coronavirus MHV-1, and influenza A (H1N1) virus, strain PR8. Murine alveolar cells cultured to maintain an ATII cell phenotype, determined by expression of LBP180, were susceptible to infection by all three viruses. In contrast, ATII cells that were cultured to trans-differentiate into an ATI-like cell phenotype were susceptible to MHV-1 and PR8, but not mouse-adapted SARS-CoV. Epithelial cells produce cytokines in response to viral infections, thereby activating immune responses. Thus, virus-induced cytokine expression was quantified in ATI and ATII cells. Both cell types had increased expression of IL-1β mRNA upon viral infection, though at different levels. While MHV-1 and PR8 induced expression of a number of shared cytokines in ATI cells, there were several cytokines whose expression was induced uniquely by MHV-1 infection. In summary, ATI and ATII cells exhibited differential susceptibilities and cytokine responses to infection by respiratory viruses. This in vitro model will be critical for future studies to determine the roles of these specialized cell types in the pathogenesis of respiratory viral infection.

Why more attentions to fetus in cases of intrahepatic cholestasis of pregnancy?

Intrahepatic cholestasis of pregnancy (ICP) is a peculiar disease in middle-late pregnancy with the pathological characteristics of hepatic capillary bile duct silts and is accompanied by clinical presentations of pruritus and bile acid (BA) elevation in serum. Maternal outcomes for patients diagnosed with ICP are usually good. However, fetal outcomes can be devastating with high frequencies of perinatal complications. Patients with ICP generally have an early delivery due to fetal complications. The current hypothesis is that ICP has higher frequencies of fetal complications due to high concentrations of BA which has toxic cellular effects to many organs. In lungs, it destroys the AT-II cells, decreasing phospholipids synthesis leading to the alveolar capillary permeability to increase and pulmonary surfactant to decrease. In heart, cholate can cross into the fetal compartment and causing fetal arrhythmias and decreased contractility. In the nervous system, high BAs can cause nerve cell denaturation and necrosis, mitochondria edema and membrane dissolve. In the placenta, high BA concentration can cause edema of the villous, decrease number of villous, intervillous thickening and balloon formation. EDITORIAL

S67 Wnt11 Inhibits TGFb-Induced Epithelial-Mesenchymal Transition in a 3-Dimensional Human Lung Tissue Model

TGFβ1 has been implicated as a key effector in mediating Epithelial-Mesenchymal Transition (EMT), my ofibroblastic transdifferentiation and fibroproliferation in the fibrotic diseases of the lung. ATII cells show high differentiational...

Restoration of alveolar type II cell function contributes to simvastatin-induced attenuation of lung ischemia-reperfusion injury

Alveolar type (AT) II cells transdifferentiate into ATI cells and as such represent a promising source for regenerating lung epithelium following lung injury. ATII cells are characterized by the presence of lamellar bodies (LBs), which store and secrete the surfactant protein-C (SP-C). Lung ischemia-reperfusion injury (LIRI) causes a distinct impairment of the ATII cell function, subsequently hindering lung repair by loss of ATI transdifferentiation. In this study, we provide new evidence that the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin may restore the function of impaired ATII cells in vitro and in vivo. ATII cell lines, A549 (human) and MLE-12 (mouse), were subjected to hypoxia-reoxygenation (H/R) injury. Simvastatin pretreatment at low (5-20 µM), but not high (50-100 µM) doses markedly reduced apoptosis and increased proliferation and SP-C expression. In a rat lung ischemia-reperfusion (I/R) model, simvastatin treatment also increased ATII cell proliferation in vivo, as demonstrated by proliferating cell nuclear antigen/SP-C double staining. Transmission electron microscopy revealed that the number and volume density of LBs were significantly increased in the simvastatin-treated rat lungs. The protective effects of simvastatin were reversed in vitro by PI3-kinase (PI3K) inhibitors wortmannin and L-mevalonate, indicating that the PI3K/Akt and mevalonate pathways may be involved in simvastatin-induced ATII cell function restoration. These data demonstrate that an appropriate dose of simvastatin has a protective effect on LIRI in vitro and in vivo, due at least partially to restored ATII cell function via the HMG-CoA reductase pathway-dependent activation of PI3K/Akt signaling in a mevalonate pathway-dependent manner.

Expression and activity of epithelial sodium channel in hyperoxia‐induced bronchopulmonary dysplasia in neonatal rats

The aim of the present study was to investigate the expression and activity of epithelial sodium channel (ENaC) in hyperoxia-induced bronchopulmonary dysplasia (BPD) in neonatal rats. Neonatal rats were exposed to hyperoxia to establish BPD models (control group was exposed to air), lung water was measured and Western blot was applied to detect the expression of three homologous subunits: α-, β- and γ-ENaC in the lung tissues. Furthermore, ATII cells were isolated from neonatal rats, and primarily cultured under normoxic or hyperoxic conditions. The ENaC expression was also examined in these cells. In addition, the amiloride-sensitive Na(+) currents induced by hyperoxia were recorded using the whole-cell patch clamp technique. The α-ENaC expression was increased after 5 days of hyperoxia in rat lung tissues, whereas not after 1, 3 and 7 days. ATII cells showed α-ENaC expression was reduced after 1 and 2 days' hyperoxia, but no change after 3 days. In contrast, β- and γ-ENaC expression was increased after hyperoxia in both in vivo and in vitro experiments. The amiloride-sensitive Na(+) currents in hyperoxia-exposed ATII cells were also increased, which was consistent with the upregulated expression of β- and γ-ENaC. Hyperoxia upregulates the expression of ENaC, especially β- and γ-ENaC subunits, in both neonatal rat lung tissues and ATII cells. Hyperoxia also enhanced the activity of ENaC in neonatal rat ATII cells. Dysfunctional transport of Na(+) may not be a key factor involving pulmonary edema at the early stage of BPD.

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

The role of CFTR in transepithelial liquid transport in pig alveolar epithelia

Combined Atomic Force Microscopy–Fluorescence Microscopy: Analyzing Exocytosis in Alveolar Type II Cells

Hybrid atomic force microscopy (AFM)-fluorescence microscopy (FM) investigation of exocytosis in lung epithelial cells (ATII cells) allows the detection of individual exocytic events by FM, which can be simultaneously correlated to structural changes in individual cells by AFM. Exocytosis of lamellar bodies (LBs) represents a slow form of exocytosis found in many non-neuronal cells. Exocytosis of LBs, following stimulation with adenosine-5'-triphosphate (ATP) and phorbol 12-myristate 13-acetate (PMA), results in a cation influx via P2X(4) receptors at the site of LB fusion with the plasma membrane (PM), which should induce a temporary increase in cell height/volume. AFM measurements were performed in single-line scans across the cell surface. Five minutes after stimulation, ATII cells revealed a cell height and volume increase of 13.7% ± 4.1% and 15.9 ± 4.8% (N = 9), respectively. These transient changes depend on exocytic LB-PM fusion. Nonstimulated cells and cells lacking LB fusions did not show a significant change in cell height/volume (N = 8). In addition, a cell height decrease was observed in ATII cells stimulated by uridine-5'-triphosphate (UTP) and PMA, agonists inducing LB fusion with the PM, but not activation of P2X(4) receptors. The cell height and volume decreased by -8.6 ± 3.6% and -11.2 ± 3.9% (N = 5), respectively. Additionally, low force contact and dynamic mode AFM imaging of cell areas around the nucleus after stimulation with ATP/PMA was performed. Fused LBs are more pronounced in AFM topography images compared to nonfused LBs, concluding that different "dynamic states" of LBs or locations from the PM are captured during imaging.

Impairment of alveolar type-II cells involved in the toxicity of Aflatoxin G1 in rat lung

Our previous studies showed intragastric administration of Aflatoxin G1 (AFG1) could induce lung adenocarcinoma, which derived from alveolar type II cells (AT-II cells). AT-II cells contribute to Aflatoxin B1 (AFB1) metabolism and also serve as the potential progenitor cells for AFB1-induced tumorigenesis. Thus, AT-II cells may constitute a target for AFG1 exposure and serve as progenitor cells for tumorigenesis induced by AFG1. The current experiment was designed to identify the acute toxicity of AFG1 in AT-II cells following a single intratracheal administration of AFG1. We observed inflammatory changes in the alveolar septum at days 3 and 7 after AFG1 treatment, which resolved by 14days. We also found AFG1 caused lamellar bodies damage in AT-II cells at days 3 and 7 post-treatment. Surfactant protein C (SP-C) expression, an AT-II cell-specific marker, was reduced at day 7 post-treatment. The structural and functional impairment in AT-II cells returned to normal by day 14. Moreover, we found that AFG1 induced a elevation of intracellular calcium concentration [Ca2+]i in AT-II cells in vitro, which may contribute to the decreased SP-C expression. In conclusion, our results show AFG1 induces structural and functional impairment in AT-II cells involved in the acute toxicity of AFG1 in lung.

Regulation of Airway and Alveolar Epithelial Cell Apoptosis by p53-Induced Plasminogen Activator Inhibitor-1 during Cigarette Smoke Exposure Injury

Increased expression of tumor suppressor protein p53 and of plasminogen activator inhibitor (PAI)-1 is associated with cigarette smoke (CS) exposure-induced lung epithelial injury. p53 induces PAI-1 through mRNA stabilization in lung epithelial cells. However, it is unclear how this process affects lung epithelial damage. Here, we show that CS induces p53 and PAI-1 expression and apoptosis in cultured Beas2B and primary alveolar type (AT)II cells. CS exposure augmented binding of p53 protein with PAI-1 mRNA. Inhibition of p53 from binding to PAI-1 mRNA through expression of p53-binding 70 nt PAI-1 mRNA 3'UTR sequences suppressed CS-induced PAI-1 expression. Treatment of Beas2B cells with caveolin-1 scaffolding domain peptide (CSP) suppressed p53 expression and p53-PAI-1 mRNA interaction. These changes were associated with parallel inhibition of CS-induced PAI-1 expression and apoptosis in Beas2B cells. Wild-type mice exposed to passive CS likewise show augmented p53 and PAI-1 with parallel induction of ATII cell apoptosis, whereas mice deficient for p53 or PAI-1 expression resisted apoptosis of ATII cells. CSP suppressed CS-induced ATII cell apoptosis in wild-type mice and abrogated p53-PAI-1 mRNA interaction with parallel inhibition of p53 and PAI-1 expression. The protection against ATII cell apoptosis by CSP involves inhibition of passive CS-induced proapoptotic Bax and Bak expression and restoration of the prosurvival proteins Bcl-X(L). These observations demonstrate that inhibition of p53 binding to PAI-1 mRNA 3'UTR attenuates CS-induced ATII cell apoptosis. This presents a novel link between p53-mediated PAI-1 expression and CS-induced ATII cell apoptosis.