Metallothionein mediates the level and activity of nuclear factor kappa B in murine fibroblasts.
The zinc-binding protein metallothionein (MT) is associated with resistance to apoptosis. We examined whether MT regulates the zinc-dependent antiapoptotic transcription factor nuclear factor KappaB (NF-KappaB), which is up-regulated under many conditions that lead to elevated MT expression. NF-KappaB protein levels and NF-KappaB-dependent reporter gene activity were examined in clonal MT(+) (MT-WT) and MT(-) (MT-KO) fibroblastic cell lines. The amount of cellular NF-KappaB p65 protein in MT-KO was less than 20% of the amount in MT-WT cells, in accord with increased sensitivity of MT-KO cells to apoptosis. NF-KappaB p65 mRNA levels, and NF-KappaB p50 subunit and IKappaBalpha protein levels, were unchanged. NF-KappaB activity assessed by expression of a transfected NF-KappaB reporter construct was less than half that observed in MT-KO cells. Decreased nuclear localization of NF-KappaB p65 in MT-KO clones was not responsible for differences in activity. In fact, MT-KO cells had higher nuclear levels of NF-KappaB p65 than did MT-WT cells, despite a lower cellular NF-KappaB level and function, suggesting that metallothionein mediated the specific activity of NF-KappaB. Reconstitution of MT by stable incorporation of an MT-1 expression vector in MT-KO cells resulted in increased NF-KappaB p65 (but not IKappaBalpha or NF-KappaB p50), increased NF-KappaB-dependent reporter activity, and increased resistance to apoptosis. These data support the hypothesis that metallothionein positively regulates the cellular level and activity of NF-KappaB.
Butcher
Heather L
HL
Department of Physiology and Pharmacology, University of Western Ontario, London, Ontario, Canada.
Kennette
Wendy A
WA
Collins
Olga
O
Zalups
Rudolfs K
RK
Koropatnick
James
J
D000818:Animals; D045744:Cell Line, Tumor; D005347:Fibroblasts / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D008668:Metallothionein / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D051996:Transcription Factor RelA; D014162:Transfection
D016328:NF-kappa B; D051996:Transcription Factor RelA; D008668:Metallothionein