Rottlerin Stimulates Metallothionein Gene Expression but Inhibits Metal Transport in Chinese Hamster Ovary Cells

Abstract

Metallothionein (MT) can be induced by various metals. We have shown previously that H7, a protein kinase C (PKC) inhibitor, inactivates metal-induced MT gene expression. To investigate whether a specific PKC isoform is involved in the induction process, inhibitors for various PKC isoforms were administered to cadmium-resistant Chinese hamster ovary (CdR) cells. None of the inhibitors used can reduce metal-induced MT gene expression. However, a PKCδ inhibitor, rottlerin, induced MT mRNA expression in CdR cells in the presence or absence of Cd. Notably, the induction occurs through the activation of the MT transcriptional factor (MTF-1) and is not related to an increase of metal influx. Furthermore, metal accumulation is reduced in the presence of rottlerin. Pulse-labeling analysis indicated that MT protein synthesis increased in CdR cells upon rottlerin treatment. These results suggest that rottlerin blocks metal transport but stimulates MT synthesis in CdR cells. Since rottlerin is capable of reducing the cellular accumulation of Cd, it was expected that the cytotoxic effect of Cd would decrease in the presence of rottlerin. Treating the parental cell of CdR with Cd and rottlerin together indeed showed a decline of cytotoxicity compared to cells treated with Cd alone. We further examined how MTF-1 was activated by rottlerin. Rottlerin-induced MTF-1 activity was not affected in CdR cells by the addition of EDTA. It was, however, diminished by administering an intracellular Zn chelator, TPEN. The result implies a mobilization of intracellular Zn ions after rottlerin treatment in CdR cells. To investigate whether the described results occur in all types of cells, another cell line (GH3) was used to study the effect of rottlerin on MT gene expression. The result revealed that rottlerin did not increase the amount of MT mRNA in GH3 cells. This differential effect between cell lines may be useful for investigating the regulatory mechanism of MT gene expression.