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.
Footnotes
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This work was supported by grants from the Canadian Institutes of Health Research to J.K. and from the National Institute of Environmental Health Research to R.K.Z. (Grants ES05157 and ES05980) and to R.K.Z. and J.K. (Grant ES11288).
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DOI: 10.1124/jpet.104.066126.
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ABBREVIATIONS: MT, metallothionein; NF-ΚB, nuclear factor of the Κ-enhancer in B cells; TNF, tumor necrosis factor; KO, knockout; WT, wild-type; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; bp, base pair(s); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; DTT, dithiothreitol; DELFIA, dissociation-enhanced lanthanide fluoroimmunoassay; TBH, tert-butylhydroperoxide; ANOVA, analysis of variance.
- Received January 27, 2004.
- Accepted March 23, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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