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1.  Effect of Polarized Release of CXC-Chemokines from Wild-Type and Cystic Fibrosis Murine Airway Epithelial Cells 
The respiratory epithelium lining the airway relies on mucociliary clearance and a complex network of inflammatory mediators to protect the lung. Alterations in the composition and volume of the periciliary liquid layer, as occur in cystic fibrosis (CF), lead to impaired mucociliary clearance and persistent airway infection. Moreover, the respiratory epithelium releases chemoattractants after infection, inciting airway inflammation. However, characterizing the inflammatory response of primary human airway epithelial cells to infection can be challenging because of genetic heterogeneity. Using well-characterized, differentiated, primary murine tracheal cells grown at an air–liquid interface, which provides an in vitro polarized epithelial model, we compared inflammatory gene expression and secretion in wild-type and ΔF508 CF airway cells after infection with Pseudomonas aeruginosa. The expression of several CXC-chemokines, including macrophage inflammatory protein–2, small inducible cytokine subfamily member 2, lipopolysaccharide-induced chemokine, and interferon-inducible cytokine–10, was markedly increased after infection, and these proinflammatory mediators were asymmetrically released from the airway epithelium, predominantly from the basolateral surface. Equal amounts of CXC-chemokines were released from wild-type and CF cells. Secreted mediators were concentrated in the thin, periciliary fluid layer, and the dehydrated apical microenvironment of CF airway epithelial cells amplified the inflammatory signal, potentially resulting in high chemokine concentration gradients across the epithelium. Consistent with this observation, the enhanced chemotaxis of wild-type neutrophils was detected in CF airway epithelial cultures, compared with wild-type cells. These data suggest that P. aeruginosa infection of the airway epithelium induces the expression and polarized secretion of CXC-chemokines, and the increased concentration gradient across the CF airway leads to an exaggerated inflammatory response.
doi:10.1165/rcmb.2009-0249OC
PMCID: PMC3266059  PMID: 20639462
inflammation; chemotaxis; chemokine; airway; epithelium
2.  Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains▿ †  
Molecular and Cellular Biology  2009;29(10):2570-2581.
While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy, enhances cell death under hypoxic conditions. In addition, the ablation of BNIP3 and/or BNIP3L triggers cell death, and BNIP3 and BNIP3L are crucial for hypoxia-induced autophagy. First, while the small interfering RNA-mediated ablation of either BNIP3 or BNIP3L has little effect on autophagy, the combined silencing of these two HIF targets suppresses hypoxia-mediated autophagy. Second, the ectopic expression of both BNIP3 and BNIP3L in normoxia activates autophagy. Third, 20-mer BH3 peptides of BNIP3 or BNIP3L are sufficient in initiating autophagy in normoxia. Herein, we propose a model in which the atypical BH3 domains of hypoxia-induced BNIP3/BNIP3L have been designed to induce autophagy by disrupting the Bcl-2-Beclin1 complex without inducing cell death. Hypoxia-induced autophagy via BNIP3 and BNIP3L is clearly a survival mechanism that promotes tumor progression.
doi:10.1128/MCB.00166-09
PMCID: PMC2682037  PMID: 19273585
3.  A synthetic, chloride-selective channel that alters chloride transport in epithelial cells 
An Ussing chamber was used to demonstrate that synthetic amphiphilic anion transporters function as chloride transporters in mammalian airway epithelial cells.
doi:10.1039/b513940f
PMCID: PMC2711530  PMID: 16391750
4.  Smurf1 regulates tumor cell plasticity and motility through degradation of RhoA leading to localized inhibition of contractility 
The Journal of Cell Biology  2007;176(1):35-42.
Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal–amoeboid–like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.
doi:10.1083/jcb.200605135
PMCID: PMC2063621  PMID: 17190792
5.  Pseudomonas aeruginosa Acquires Biofilm-Like Properties within Airway Epithelial Cells  
Infection and Immunity  2005;73(12):8298-8305.
Pseudomonas aeruginosa can notably cause both acute and chronic infection. While several virulence factors are implicated in the acute phase of infection, advances in understanding bacterial pathogenesis suggest that chronic P. aeruginosa infection is related to biofilm formation. However, the relationship between these two forms of disease is not well understood. Accumulating evidence indicates that, during acute infection, P. aeruginosa enters epithelial cells, a process viewed as either a host-mediated defense response or a pathogenic mechanism to avoid host-mediated killing. We investigated the possibility that epithelial cell entry during early P. aeruginosa-epithelial cell contact favors bacterial survival and is linked to chronic infection. Using electron microscopy and confocal microscopy to analyze primary culture airway epithelial cells infected with P. aeruginosa, we found that epithelial cells developed pod-like clusters of intracellular bacteria with regional variation in protein expression. Extracellular gentamicin added to the medium after acute infection led to the persistence of intracellular P. aeruginosa for at least 3 days. Importantly, compared to bacterial culture under planktonic conditions, the intracellular bacteria were insensitive to growth inhibition or killing by antibiotics that were capable of intraepithelial cell penetration. These findings suggest that P. aeruginosa can use airway epithelial cells as a sanctuary for persistence and develop a reversible antibiotic resistance phenotype characteristic of biofilm physiology that can contribute to development of chronic infection.
doi:10.1128/IAI.73.12.8298-8305.2005
PMCID: PMC1307054  PMID: 16299327

Results 1-5 (5)