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

Tumor necrosis factor-alpha (TNF) induces apoptosis in confluent LLC-PK1 ep ithelial cells, but also activates KF-kappa B, a negative regulator of apop tosis, The presence of increased TNF-induced apoptosis causes a transient i ncrease in epithelial permeability; but the epithelial barrier function rec overs, as assessed by measuring the transepithelial electrical resistance, the paracellular flux of mannitol and by the electron microscopic evaluatio n of the penetration of the electron-dense dye ruthenium red across the tig ht junctions. The integrity of the epithelial cell layer is maintained by r earrangement of non-apoptotic cells in the monolayer and by: the phagocytos is of apoptotic fragments, To study the role of NF-kappa B in an epithelium exposed to TNF, NF-kappa B was inhibited in LLC-PK1 epithelial cells with either the dietary compound, curcumin, or by transfection with a dominant n egative mutant inhibitor I kappa B alpha. Replacement of serine 32 and 36 b y alanine has been shown to prevent its phosphorylation and degradation, bl ocking NF-kappa B activation. Inhibition of NF-kappa B altered the morpholo gy of TNF-induced apoptotic cells, which showed lack of fragmentation and m embrane blebbings, and absence of phagocytosis by neighboring cells. TNF tr eatment of NF-kappa B inhibited cells also caused altered distribution of t he tight junction-associated protein ZO-1, increased epithelial leakiness, and impaired the recovery of the epithelial barrier function, which normall y occurs 6 hours after TNF treatment of LLC-PK1 cells. These data demonstra te that NF-kappa B activation is required for the maintenance of the barrie r function of an epithelium undergoing TNF-induced apoptosis.