Persistent activation of nuclear factor‐κB in cultured rat hepatic stellate cells involves the induction of potentially novel rel‐like factors and prolonged changes in the expression of IκB family proteins

Rat hepatic stellate cells (HSC) cultured in serum‐containing medium underwent a rapid (3‐hour) classical induction of p50:p65 and p65:p65 nuclear factor‐κB (NF‐κB) dimers. Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high‐mobility NF‐κB DNA binding complex consisting of potentially novel Rel‐like protein(s). Formation of the latter complex was competed for by specific double‐stranded oligonucleotides, was up‐regulated by treatment of HSCs with tumor necrosis factor α (TNF‐α), and was maintained at basal levels of expression by a soluble HSC‐derived factor. An NF‐κB–responsive CAT reporter gene was highly active in early cultured HSCs but was also trans‐activated at a lower but significant level in longer‐term cultured cells and could be completely suppressed by expression of dominant negative IκB‐α. Physiological significance of the lower persistent NF‐κB activities was also demonstrated by the ability of long‐term cultured HSCs to support the activity of the NF‐κB–dependent human intercellular adhesion molecule‐1 (ICAM‐1) promoter. Freshly isolated HSCs expressed high levels of IκB‐α and IκB‐β. Culture activation was accompanied by a long‐term reduction in levels of IκB‐α with no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. IκB‐β expression was transiently reduced and, upon replenishment, was associated with appearance of a lower‐mobility IκB‐β antibody‐reactive species. Bcl3 expression was absent in freshly isolated HSC but was induced during culturing and became a persistent feature of the activated HSC. Inhibition of NF‐κB DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF‐κB in cultured HSC is required for expression of specific genes associated with the activated phenotype such as ICAM‐1 and may be antiapoptotic for rat HSCs.