metallothionein and chelerythrine

Recently, we discovered that beta-thujaplicin (BT) induces metallothionein (MT) expression in mouse keratinocytes, both in vivo and in vitro. However, the molecular mechanisms by which BT exerts its biological effects have not been elucidated. The purpose of this study is to explore the signal transduction pathway involved in the MT mRNA induction by BT. Using a HaCaT keratinocyte cell line, Northern blotting was performed for analyzing the human MT-IIA mRNA expression levels in combination with BT and a number of protein kinase (PK) inhibitors including H7, HA1004 and a PKC-specific inhibitor chelerythrin. CAT assays with the MT-IIA gene promorter-CAT construct were conducted for examining the transcriptional regulation by BT of MT. A free radical scavenger N-acetylcysteine (NAC) was used for analyzing a role of oxidative stress for the MT gene induction by BT. BT increased MT-IIA gene transcript levels and CAT activity in a dose-dependent fashion in HaCaT cells. The increase in MT-IIA mRNA levels and CAT activity were completely suppressed by H7 but not by HA1004. In addition, chelerythrin prevented BT-inducible MT-IIA promoter activation. Furthermore, NAC suppressed BT-inducible MT-IIA promoter activation. These results demonstrate that BT is a potent activator of the MT-IIA gene promoter and that PKC activation and reactive oxygen species are implicated in BT-inducible MT-IIA gene expression. BT may be a useful tool for dissecting the signal transduction pathway mediating MT-IIA promoter activation.

Topics: Acetylcysteine; Alkaloids; Benzophenanthridines; Blotting, Western; Enzyme Inhibitors; Epidermal Cells; Free Radical Scavengers; Genes, Reporter; Humans; Keratinocytes; Metallothionein; Monoterpenes; Phenanthridines; Plasmids; Protein Kinase C; Reactive Oxygen Species; Signal Transduction; Tropolone; Up-Regulation

The effects of protein kinase C (PKC) inhibitors on the metallothionein (MT) gene expression induced by metals were investigated. When PKC inhibitor (H7 or chelerythrine) was administered to Cd resistant, MT gene-amplified Chinese hamster ovary (CdR) cells, the induction of MT mRNA by Cd or Zn was blocked. Treating the CdR cells with a PKA-specific inhibitor, HA1004, did not cause an inhibition of metal-induced MT gene transcription. The inhibitory effect was effectuated by adding inhibitors within 40 min of exposing the cells to Cd. Apparently, AP1 was not involved in this down-regulatory effect of PKC inhibitor on MT gene expression since the inducibility of MT promoter was blocked by H7 even in the absence of the AP1-binding sequence. For Cd-treated cells, Cd accumulation in the cell was similar with or without H7 treatment. However, H7 markedly reduced cellular Zn accumulation when the cells were treated with Zn. Cycloheximide treatment increased the level of MT mRNA. This elevation can also be blocked by treating the cell with PKC inhibitor. Results in this study suggest that PKC participates in the process of metal-induced MT gene expression.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adaptor Protein Complex 1; Adaptor Protein Complex alpha Subunits; Adaptor Proteins, Vesicular Transport; Alkaloids; Animals; Benzophenanthridines; Cadmium; CHO Cells; Cricetinae; Cycloheximide; DNA-Binding Proteins; Drug Resistance; Enzyme Inhibitors; Gene Expression Regulation; Membrane Proteins; Metallothionein; Phenanthridines; Promoter Regions, Genetic; Protein Kinase C; Protein Synthesis Inhibitors; Recombinant Fusion Proteins; RNA, Messenger; Signal Transduction; Transcription Factor MTF-1; Transcription Factors; Zinc