Metallothionein Mediates the Level and Activity of Nuclear Factor 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 B (NFB), which is up-regulated under many conditions that lead to elevated MT expression. NFB protein levels and NFB-dependent reporter gene activity were examined in clonal MT( ) (MT-WT) and MT( ) (MT-KO) fibroblastic cell lines. The amount of cellular NFB 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. NFB p65 mRNA levels, and NFB p50 subunit and I B protein levels, were unchanged. NFB activity assessed by expression of a transfected NFB reporter construct was less than half that observed in MT-KO cells. Decreased nuclear localization of NFB p65 in MT-KO clones was not responsible for differences in activity. In fact, MT-KO cells had higher nuclear levels of NFB p65 than did MT-WT cells, despite a lower cellular NFB level and function, suggesting that metallothionein mediated the specific activity of NFB. Reconstitution of MT by stable incorporation of an MT-1 expression vector in MT-KO cells resulted in increased NFB p65 (but not I B or NFB p50), increased NFB-dependent reporter activity, and increased resistance to apoptosis. These data support the hypothesis that metallothionein positively regulates the cellular level and activity of NFB. Metallothioneins (MTs) are small ( 10 kDa) metal-binding proteins found in all eukaryotes. MTs bind a variety of metal ions (Ag, Hg, Cu, Cd, and others) but are associated primarily with zinc in most mammalian cells (reviewed in DeMoor et al., 2001). MTs are highly homologous and of similar structure, implying common biological function. However, an essential function for MTs has yet to be identified. Among the four MT isoforms identified, MT-1 and MT-2 are expressed in virtually all mammalian cells. MT-3 is limited to brain, pancreas, and intestine (Ebadi et al., 1995) and MT-4 to squamous epithelial cells in skin and tongue (Quaife et al., 1994). MT-3 and MT-4 are constitutively expressed, whereas MT-1 and MT-2 are both constitutive and highly inducible. MTs are associated with proliferation in human tumor cells in culture (Koropatnick et al., 1995) and in situ (Kontozoglou et al., 1989), compensatory renal growth (Zalups et al., 1995), and monocyte activation (Leibbrandt and Koropatnick, 1994; Leibbrandt et al., 1994). MT expression and intracellular localization is altered in some cells and tissues undergoing differentiation (Koropatnick and Duerksen, 1987; Quaife et al., 1994) Thus, MTs are associated with a broad range of physiological events. Roles in response to cellular and tissue stress have been attributed to MT, including detoxification of heavy metals, protection against oxidative injury (including apoptosis), and homeostatic regulation of essential metals (Koropatnick and Zalups, 2000). This has led to the hypothesis that MT mediates gene expression by donating zinc, directly or indirectly, to transcription factors and other proteins that contribute to chemoand radio-resistance by inhibiting apoptosis. 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). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.104.066126. ABBREVIATIONS: MT, metallothionein; NFB, nuclear factor of the -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. 0022-3565/04/3102-589–598$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 310, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 66126/1156274 JPET 310:589–598, 2004 Printed in U.S.A.