Activation of NF-kappaB is necessary for the restoration of the barrier function of an epithelium undergoing TNF-alpha-induced apoptosis

Eur J Cell Biol. 1999 Jan;78(1):56-66. doi: 10.1016/s0171-9335(99)80007-7.

Abstract

Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 epithelial cells, but also activates NF-kappaB, a negative regulator of apoptosis. The presence of increased TNF-induced apoptosis causes a transient increase in epithelial permeability, but the epithelial barrier function recovers, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluation of the penetration of the electron-dense dye ruthenium red across the tight junctions. The integrity of the epithelial cell layer is maintained by rearrangement of non-apoptotic cells in the monolayer and by the phagocytosis of apoptotic fragments. To study the role of NF-kappaB in an epithelium exposed to TNF, NF-kappaB was inhibited in LLC-PK1 epithelial cells with either the dietary compound, curcumin, or by transfection with a dominant negative mutant inhibitor I kappaB alpha. Replacement of serine 32 and 36 by alanine has been shown to prevent its phosphorylation and degradation, blocking NF-kappaB activation. Inhibition of NF-kappaB altered the morphology of TNF-induced apoptotic cells, which showed lack of fragmentation and membrane blebbings, and absence of phagocytosis by neighboring cells. TNF treatment of NF-kappaB-inhibited cells also caused altered distribution of the tight junction-associated protein ZO-1, increased epithelial leakiness, and impaired the recovery of the epithelial barrier function, which normally occurs 6 hours after TNF treatment of LLC-PK1 cells. These data demonstrate that NF-kappaB activation is required for the maintenance of the barrier function of an epithelium undergoing TNF-induced apoptosis.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis*
  • Blotting, Western
  • Cell Membrane Permeability
  • Curcumin / pharmacology
  • Dose-Response Relationship, Drug
  • Electrophoresis, Polyacrylamide Gel
  • Electrophysiology
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Epithelial Cells / ultrastructure
  • Fluorescent Antibody Technique
  • Kidney / metabolism
  • Membrane Proteins / metabolism
  • Microscopy, Electron
  • Mutagenesis
  • NF-kappa B / metabolism*
  • Phosphoproteins / metabolism
  • Ruthenium Red / metabolism
  • Swine
  • Tight Junctions / metabolism
  • Time Factors
  • Transfection
  • Tumor Necrosis Factor-alpha / metabolism*
  • Tumor Necrosis Factor-alpha / pharmacology
  • Zonula Occludens-1 Protein

Substances

  • Membrane Proteins
  • NF-kappa B
  • Phosphoproteins
  • Tumor Necrosis Factor-alpha
  • Zonula Occludens-1 Protein
  • Ruthenium Red
  • Curcumin