Metallothionein mediates the level and activity of nuclear factor κB 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.