title: NF-κB inhibitors impair lung epithelial tight junctions in the absence of inflammation.
abstract: NF-κB (p50/p65) is the best characterized transcription factor known to regulate cell responses to inflammation. However, NF-κB is also constitutively expressed. We used inhibitors of the classical NF-κB signaling pathway to determine whether this transcription factor has a role in regulating alveolar epithelial tight junctions. Primary rat type II alveolar epithelial cells were isolated and cultured on Transwell permeable supports coated with collagen for 5 d to generate a model type I cell monolayer. Treatment of alveolar epithelial monolayers overnight with one of 2 different IκB kinase inhibitors (BAY 11-7082 or BMS-345541) resulted in a dose-dependent decrease in TER at concentrations that did not affect cell viability. In response to BMS-345541 treatment there was an increase in total claudin-4 and claudin-5 along with a decrease in claudin-18, as determined by immunoblot. However, there was little effect on the total amount of cell-associated claudin-7, occludin, junctional adhesion molecule A (JAM-A), zonula occludens (ZO)-1 or ZO-2. Moreover, treatment with BMS-345541 resulted in altered tight junction morphology as assessed by immunofluorescence microscopy. Cells treated with BMS-345541 had an increase in claudin-18 containing projections emanating from tight junctions ("spikes") that were less prominent in control cells. There also were several areas of cell-cell contact which lacked ZO-1 and ZO-2 localization as well as rearrangements to the actin cytoskeleton in response to BMS-345541. Consistent with an anti-inflammatory effect, BMS-345541 antagonized the deleterious effects of lipopolysaccharide (LPS) on alveolar epithelial barrier function. However, BMS-345541 also inhibited the ability of GM-CSF to increase alveolar epithelial TER. These data suggest a dual role for NF-κB in regulating alveolar barrier function and that constitutive NF-κB function is required for the integrity of alveolar epithelial tight junctions.
authors.affiliation: Pulmonary Division; Department of Medicine; Emory University School of Medicine ; Atlanta, GA USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Ward
authors.affiliation: Pulmonary Division; Department of Medicine; Emory University School of Medicine ; Atlanta, GA USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Schlingmann
authors.affiliation: Department of Pediatrics; Emory University School of Medicine ; Atlanta, GA USA.
authors.forename: Arlene A
authors.initials: AA
authors.lastname: Stecenko
authors.affiliation: Pulmonary Division; Department of Medicine; Emory University School of Medicine ; Atlanta, GA USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Guidot
authors.affiliation: Pulmonary Division; Department of Medicine; Emory University School of Medicine ; Atlanta, GA USA ; Department of Cell Biology; Emory University School of Medicine ; Atlanta, GA USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Koval
keywords: ARDS, Acute Respiratory Distress Syndrome; GM-CSF, Granulocyte Macrophage Colony Stimulating Factor; IL, interleukin; IκB, Inhibitor of κB; JAM-A, junctional adhesion molecule A; LPS, lipolysaccharide; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PBS, phosphate buffered saline; TER, transepithelial resistance; TNF, Tumor Necrosis Factor; ZO, zonula occludens; alveolus; claudin; lung barrier; tight junction
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