Persistent activation of nuclear factor-kappa B in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of I kappa B family proteins

Rat hepatic stellate cells (HSC) cultured in serum-containing medium underw ent a rapid (3-hour) classical induction of p50:p65 and p65:p65 nuclear fac tor-kappa B (NF-kappa B) dimers, Subsequent culturing was associated with p rolonged expression of active p50:p65 and persistent induction of a high-mo bility NF-kappa B DNA binding complex consisting of potentially novel Rel-l ike protein(s). Formation of the latter complex was competed for by specifi c double-stranded oligonucleotides, was up-regulated by treatment of HSCs w ith tumor necrosis factor alpha (TNF-alpha), and was maintained at basal le vels of expression by a soluble HSC-derived factor. An NF-kappa B-responsiv e CAT reporter gene was highly active in early cultured HSCs but was also t rans-activated at a lower but significant level in longer-term cultured cel ls and could be completely suppressed by expression of dominant negative I kappa B-alpha. Physiological significance of the lower persistent NF-kappa B activities was also demonstrated by the ability of long-term cultured HSC s to support the activity of the NF-kappa B-dependent human intercellular a dhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of I kappa B-alpha and I kappa B-beta. Culture activation was accomp anied by a long-term reduction in levels of I kappa B-alpha With no detecta ble expression in the nuclear fraction of cells, under these conditions p50 :p65 was detected in the nucleus. I kappa B-beta expression was transiently reduced and, upon replenishment, was associated with appearance of a lower -mobility I kappa B-beta antibody-reactive species. Bcl3 expression was abs ent in freshly isolated HSC but was induced during culturing and became a p ersistent feature of the activated HSC, Inhibition of NF-kappa B DNA bindin g activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-kappa B in cultured HSC is required for expression of specific genes associated with the activated phenotype s uch as ICAM-1 and may be antiapoptotic for rat HSCs.