Abstract

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 ΚB (NF-ΚB), which is up-regulated under many conditions that lead to elevated MT expression. NF-ΚB protein levels and NF-ΚB-dependent reporter gene activity were examined in clonal MT(+) (MT-WT) and MT(–) (MT-KO) fibroblastic cell lines. The amount of cellular NF-ΚB 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-ΚB p65 mRNA levels, and NF-ΚB p50 subunit and IΚBα protein levels, were unchanged. NF-ΚB activity assessed by expression of a transfected NF-ΚB reporter construct was less than half that observed in MT-KO cells. Decreased nuclear localization of NF-ΚB p65 in MT-KO clones was not responsible for differences in activity. In fact, MT-KO cells had higher nuclear levels of NF-ΚB p65 than did MT-WT cells, despite a lower cellular NF-ΚB level and function, suggesting that metallothionein mediated the specific activity of NF-ΚB. Reconstitution of MT by stable incorporation of an MT-1 expression vector in MT-KO cells resulted in increased NF-ΚB p65 (but not IΚBα or NF-ΚB p50), increased NF-ΚB-dependent reporter activity, and increased resistance to apoptosis. These data support the hypothesis that metallothionein positively regulates the cellular level and activity of NF-ΚB.