Anti-apoptotic effects of Z alpha-1 antitrypsin in human bronchial epithelial cells.

α1-antitrypsin (α1-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of α1-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z α1-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved.

Control, M variant α1-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-κB activation and induced expression of a selection of pro- and anti-apoptotic genes.

Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-κB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-κB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies.

The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Cytopathogenesis of Sendai virus in well-differentiated primary pediatric bronchial epithelial cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.

Jagged/Notch signalling is required for a subset of TGFβ1 responses in human kidney epithelial cells.

The Jagged/Notch pathway has been implicated in TGFß1 responses in epithelial cells in diabetic nephropathy and other fibrotic conditions in vivo. Here, we identify that Jagged/Notch signalling is required for a subset of TGFß1-stimulated gene responses in human kidney epithelial cells in vitro. TGFß1 treatment of HK-2 and RPTEC cells for 24 h increased Jagged1 (a Notch ligand) and Hes1 (a Notch target) mRNA. This response was inhibited by co-incubation with Compound E, an inhibitor of ?-secretase (GSI), an enzyme required for Notch receptor cleavage and transcription regulation. In both cell types, TGFß1-responsive genes associated with epithelial–mesenchymal transition such as E-cadherin and vimentin were also affected by ?-secretase inhibition, but other TGFß1 targets such as connective tissue growth factor (CTGF) and thrombospondin-1 (THBS1) were not. TGFß1-induced changes in Jagged1 expression preceded EMT-associated gene changes, and co-incubation with GSI altered TGFß1-induced changes in cell shape and cytoskeleton. Transfection of cells with the activated, cleaved form of Notch (NICD) triggered decreased expression of E-cadherin in the absence of TGFß1, but did not affect a-smooth muscle actin expression, suggesting differential requirements for Notch signalling within the TGFß1-responsive gene subset. Increased Jagged1 expression upon TGFß1 exposure required Smad3 signalling, and was also regulated by PI3K and ERK. These data suggest that Jagged/Notch signalling is required for a subset of TGFß1-responsive genes, and that complex signalling pathways are involved in the crosstalk between TGFß1 and Notch cascades in kidney epithelia.


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SGK1 activity in Na+ absorbing airway epithelial cells monitored by assaying NDRG1-Thr(346/356/366) phosphorylation

Studies of HeLa cells and serum- and glucocorticoid-regulated kinase 1 (SGK1) knockout mice identified threonine residues in the n-myc downstream-regulated gene 1 protein (NDRG1-Thr(346/356/366)) that are phosphorylated by SGK1 but not by related kinases (Murray et al., Biochem J 385:1-12, 2005). We have, therefore, monitored the phosphorylation of NDRG1-Thr(346/356/366) in order to explore the changes in SGK1 activity associated with the induction and regulation of the glucocorticoid-dependent Na+ conductance (G (Na)) in human airway epithelial cells. Transient expression of active (SGK1-S422D) and inactive (SGK1-K127A) SGK1 mutants confirmed that activating SGK1 stimulates NDRG1-Thr(346/356/366) phosphorylation. Although G (Na) is negligible in hormone-deprived cells, these cells displayed basal SGK1 activity that was sensitive to LY294002, an inhibitor of 3-phosphatidylinositol phosphate kinase (PI3K). Dexamethasone (0.2 mu M) acutely activated SGK1 and the peak of this response (2-3 h) coincided with the induction of G (Na), and both responses were PI3K-dependent. While these data suggest that SGK1 might mediate the rise in G (Na), transient expression of the inactive SGK1-K127A mutant did not affect the hormonal induction of G (Na) but did suppress the activation of SGK1. Dexamethasone-treated cells grown on permeable supports formed confluent epithelial sheets that generated short circuit current due to electrogenic Na+ absorption. Forskolin and insulin both stimulated this current and the response to insulin, but not forskolin, was LY294002-sensitive and associated with the activation of SGK1. While these data suggest that SGK1 is involved in the control of G (Na), its role may be minor, which could explain why sgk1 knockout has different effects upon different tissues.

Effects of IL-13 on Mucociliary Differentiation of Pediatric Asthmatic Bronchial Epithelial Cells

Goblet cell hyperplasia (GCH) and decreased ciliated cells are characteristic of asthma. We examined the effects of IL-13 (2 and 20 ng/mL) on in vitro mucociliary differentiation in pediatric bronchial epithelial cells (PBECs) of normal PBEC [PBEC(N)] and asthmatic PBEC [PBEC(A)] children. Markers of differentiation, real-time PCR for MUC5AC, MUC5AC ELISA, and transepithelial electrical resistance (TEER) were assessed. Stimulation with 20 ng/mL IL-13 in PBEC(N) resulted in GCH [20 ng/mL IL-13: mean, 33.8% (SD, 7.2) versus unstimulated: mean, 18.9% (SD, 5.0); p < 0.0001] and decreased ciliated cell number [20 ng/mL IL-13: mean, 8% (SD, 5.6) versus unstimulated: mean, 22.7% (SD,7.6); p < 0.01]. PBEC(N) stimulated with 20 ng/mL IL-13 resulted in >5-fold (SD, 3.2) increase in MUC5AC mRNA expression, p < 0.001, compared with unstimulated PBEC(N). In PBEC(A), GCH was also seen [20 ng/mL IL-13: mean, 44.7% (SD, 16.4) versus unstimulated: mean, 30.4% (SD, 13.9); p < 0.05] with a decreased ciliated cell number [20 ng/mL IL-13: mean, 8.8% (SD, 7.5) versus unstimulated: mean, 16.3% (SD, 4.2); p < 0.001]. We also observed an increase in MUC5AC mRNA expression with 20 ng/mL IL-13 in PBEC(A), p < 0.05. IL-13 drives PBEC(N) toward an asthmatic phenotype and worsens the phenotype in PBEC(A) with reduced ciliated cell numbers and increased goblet cells.

Claudin-1 Has Tumor Suppressive Activity and Is a Direct Target of RUNX3 in Gastric Epithelial Cells

BACKGROUND & AIMS: The transcription Factor RUNX3 is a gastric tumor suppressor. Tumorigenic Runx3(-/-) gastric epithelial cells attach weakly to each other, compared with nontumorigenic Runx3(+/+) cells. We alined to identify RUNX3 target genes that promote cell-cell contact to Improve our understanding of RUNX3's role in Suppressing gastric carcinogenesis. METHODS: We compared gene expression profiles of Runx3(+/+) and Runx3(-/-) cells and observed down-regulation of genes associated with cell-cell adhesion in Runx3(-/-) cells. Reporter, mobility shift, and chromatin immunoprecipitation assays were used to examine the regulation of these genes by RUNX3. Tumorigenesis assays and immunohistologic, analyses of human gastric tumors were performed to confirm the role of the candidate genes ill gastric tumor development. RESULTS: Mobility shift and chromatin immunoprecipitation assays revealed that the promoter activity of the gene that encodes the tight Junction protein claudin-1 was up-regulated via the binding of RUNX3 to the RUNX consensus sites. The tumorigenicity of gastric epithelial cells From Runx3(-/-) mice was significantly reduced by restoration of claudin-1 expression, whereas knockdown of claudin-1. increased the tumorigenicity of human gastric cancer cells. Concomitant expression of RUNX3 and claudin-1 was observed in human normal gastric epithelium and cancers. CONCLUSIONS: The tight junction protein claudin-1 has gastric tumor suppressive activity and is a direct transcriptional target of RUNX3. Claudin-1 is down-regulated during the epithelial-mesenchymal transition; RUNX3 might therefore act as a tumor suppressor to antagonize the epithelial-mesenchymal transition.

C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis

The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior.

Differential cytopathogenesis of respiratory syncytial virus prototypic and clinical isolates in primary pediatric bronchial epithelial cells.

Background Human respiratory syncytial virus (RSV) causes severe respiratory disease in infants. Airway epithelial cells are the principle targets of RSV infection. However, the mechanisms by which it causes disease are poorly understood. Most RSV pathogenesis data are derived using laboratory-adapted prototypic strains. We hypothesized that such strains may be poorly representative of recent clinical isolates in terms of virus/host interactions in primary human bronchial epithelial cells (PBECs). Methods To address this hypothesis, we isolated three RSV strains from infants hospitalized with bronchiolitis and compared them with the prototypic RSV A2 in terms of cytopathology, virus growth kinetics and chemokine secretion in infected PBEC monolayers. Results RSV A2 rapidly obliterated the PBECs, whereas the clinical isolates caused much less cytopathology. Concomitantly, RSV A2 also grew faster and to higher titers in PBECs. Furthermore, dramatically increased secretion of IP-10 and RANTES was evident following A2 infection compared with the clinical isolates. Conclusions The prototypic RSV strain A2 is poorly representative of recent clinical isolates in terms of cytopathogenicity, viral growth kinetics and pro-inflammatory responses induced following infection of PBEC monolayers. Thus, the choice of RSV strain may have important implications for future RSV pathogenesis studies.

Wild-type measles virus infection of primary epithelial cells occurs via the basolateral surface without syncytium formation or release of infectious virus

The lymphotropic and myelotropic nature of wild-type measles virus (wt-MV) is well recognized, with dendritic cells and lymphocytes expressing the MV receptor CD150 mediating systemic spread of the virus. Infection of respiratory epithelial cells has long been considered crucial for entry of MV into the body. However, the lack of detectable CD150 on these cells raises the issue of their importance in the pathogenesis of measles. This study utilized a combination of in vitro, ex vivo and in vivo model systems to characterize the susceptibility of epithelial cells to wt-MV of proven pathogenicity. Low numbers of MV-infected epithelial cells in close proximity to underlying infected lymphocytes or myeloid cells suggested infection via the basolateral side of the epithelium in the macaque model. In primary cultures of human bronchial epithelial cells, foci of MV-infected cells were only observed following infection via the basolateral cell surface. The extent of infection in primary cells was enhanced both in vitro and in ex vivo cornea rim tissue by disrupting the integrity of the cells prior to the application of virus. This demonstrated that, whilst epithelial cells may not be the primary target cells for wt-MV, areas of epithelium in which tight junctions are disrupted can become infected using high m.o.i. The low numbers of MV-infected epithelial cells observed in vivo in conjunction with the absence of infectious virus release from infected primary cell cultures suggest that epithelial cells have a peripheral role in MV transmission.