Abstract
Abstract Metallothionein(MT) is a high metal content, cysteine-rich low molecular weight protein ubiquitous in animal cells. Its expression can be induced by metals、chemicals or under various stresses. To understand the factors involved in the induction of MT protein expression by metals, in this study we have selected and characterized several potential inhibitors of protein kinase and protein phosphatase that may involve in signaling pathway of MT gene expression. We have also investigated whether reactive oxygen species (ROS) is involved in the pathway for activation of MT gene or not by using various chemicals influenced the redox-oxidation reaction in cells. We found that rottlerin, a PKCd specific inhibitor, can effectively activate the MT gene expression, and the mechanism of MT gene induced by rottlerin was also investigated in this study. Result from Northern blot analysis showed that most of the protein kinase inhibitors exhibited no or little effect on the induction of MT gene expression by metals. Inhibitors have been tested in this study including classical PKC inhibitors (HBDDE, Gö6976), phospholipase inhibitors (neomycin, RHC-80267, U-73122), calmodulin inhibitor (W7), protein tyrosine kinase inhibitor (herbimycine A) and MEK1 inhibitor (PD98059). H7, a broad range PKC inhibitor, is the only inhibitor conferring inhibition activity on MT gene expression induced by metals. Manoalide, a phospholipase-A2 inhibitor, and okadaic acid, a protein phosphatase-2A and protein phosphatase-1 inhibitor, partially inhibited MT gene expression. However, rottlerin, a PKCd inhibitor, and vanadate, a tyrosine phosphatase, increase MT gene expression. There were no effect detected on MT gene expression induced by metals when cells were pre-treated with hydrogen peroxide to increase ROS, DL-buthionine-[S, R]-sulfoximine (BSO) to inhibit synthesis of glutathione (GSH), or ascorbic acid or a-tocopherol to scavenge ROS. However, MT gene expression can be effectively inhibited by either N-acetyl-L-cysteine (NAC), a chemical elevates cellular GSH synthesis, or 3-AT, a catalase inhibitor. Although mannitol can act as a hydroxy radical scavenger, it just partial inhibits MT gene expression induced by cadmium, but not zinc ion. Whether the signaling pathway of metal induced MT gene expression in CdR cells involves in phosphorylation cascade or ROS synthesis is remain to be determined. Rottlerin activated MT gene expression can be detected by Northern blot and RT-PCR. Although cells treated with rottlerin inhibited metal uptake into cells and did not increase cellular MRE-binding transcription factor-1 (MTF-1) content, metal responsive element (MRE) binding activity of MTF-1 was elevated. These results indicate that intracellular zinc ion is possibly re-distributed after cells were treated by rottlerin. However, no effect aws observed when GH3 cells were treated with rottlerin at the same condition. The results showed that rottlerin did not increase MTF-1 binding activity to MRE, whereas induced MT gene expression and inhibited Cd uptake. These results indicate that induction mechanism of MT gene expression by rottlerin is cell type specific.