Persistent activation of nuclear factor-kappaB in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IkappaB 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-kappaB (NF-kappaB) dimers. Subsequent culturing was associated with prolonged expression of active p50:p65 and persistent induction of a high-mobility NF-kappaB 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 alpha (TNF-alpha), and was maintained at basal levels of expression by a soluble HSC-derived factor. An NF-kappaB-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 IkappaB-alpha. Physiological significance of the lower persistent NF-kappaB activities was also demonstrated by the ability of long-term cultured HSCs to support the activity of the NF-kappaB-dependent human intercellular adhesion molecule-1 (ICAM-1) promoter. Freshly isolated HSCs expressed high levels of IkappaB-alpha and IkappaB-beta. Culture activation was accompanied by a long-term reduction in levels of IkappaB-alpha with no detectable expression in the nuclear fraction of cells, under these conditions p50:p65 was detected in the nucleus. IkappaB-beta expression was transiently reduced and, upon replenishment, was associated with appearance of a lower-mobility IkappaB-beta 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-kappaB DNA binding activity by gliotoxin was associated with increased numbers of apoptotic cells. We suggest that activation of NF-kappaB 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.
Elsharkawy
A M
AM
University of Medicine, Southampton General Hospital, Southampton, England.
Wright
M C
MC
Hay
R T
RT
Arthur
M J
MJ
Hughes
T
T
Bahr
M J
MJ
Degitz
K
K
Mann
D A
DA
D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D005912:Gliotoxin / Q000494:pharmacology; D020672:I-kappa B Proteins; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D051841:Transcription Factor RelB; D014157:Transcription Factors / Q000096:biosynthesis*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; D011518:Proto-Oncogene Proteins; C497595:Relb protein, rat; D014157:Transcription Factors; D018799:Intercellular Adhesion Molecule-1; D000072000:NF-KappaB Inhibitor alpha; D051841:Transcription Factor RelB; D005912:Gliotoxin; D004247:DNA