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.

Keywords:

• alveolus
• claudin
• lung barrier
• tight junction

Abbreviations:

• ARDS, Acute Respiratory Distress Syndrome
• GM-CSF, Granulocyte Macrophage Colony Stimulating Factor
• IκB, Inhibitor of κB
• IL, interleukin
• 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

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Samuel Molina and William Koval for critical reading of the manuscript.

Additional information

Funding

Supported by NIH R01-HL116958, R01-HL070891, P50-AA013757, and T32-AA013528.