MECHANISM FOR BIPHASIC REL A-CENTER-DOT-NF-KAPPA-B1 NUCLEAR TRANSLOCATION IN TUMOR-NECROSIS-FACTOR ALPHA-STIMULATED HEPATOCYTES

The proinflammatory cytokine, tumor necrosis factor alpha (TNF alpha), is a potent activator of angiotensinogen gene transcription in hepato cytes by activation of latent nuclear factor-kappa B (NF-kappa B) DNA binding activity. In this study, we examine the kinetics of TNF alpha- activated translocation of the 65-kDa (Rel A) and 50-kDa (NF-kappa B1) NF-kappa B subunits mediated by inhibitor (I kappa B) proteolysis in HepG2 hepatoblastoma cells. HepG2 cells express the I kappa B members I kappa B alpha, I kappa B beta, and I kappa B gamma. In response to T NF alpha, Rel A.NF-kappa B1 translocation and DNA binding activity fol lows a biphasic profile, with an ''early'' induction (15-30 min), foll owed by a nadir to control levels at 60 min, and a ''late'' induction (>120 min). The early phase of Rel A NF-kappa B1 translocation depends on simultaneous proteolysis of both I kappa B alpha and I kappa B bet a isoforms; I kappa B gamma is inert to TNF alpha treatment. The 60-mi n nadir is due to a rapid I kappa B alpha resynthesis that reassociate s with Rel A and completely inhibits its DNA binding activity; the 60- min nadir is not observed when I kappa B alpha resynthesis is prevente d by cycloheximide treatment. By contrast, selective inhibition of I k appa B beta proteolysis by pretreatment of HepG2 cells with the peptid e aldehyde N-acetyl-Leu-Leu-nor-leucinal completely blocks the late ph ase of Rel A.NF-kappa B1 translocation. These studies indicate the pre sence of inducible and constitutive cytoplasmic NF-kappa B pools in he patocytes. TNF alpha induces a coordinated proteolysis and resynthesis of I kappa B isoforms to produce dynamic changes in NF-kappa B nuclea r abundance.