Human metallothionein gene expression is upregulated by beta-thujaplicin: possible involvement of protein kinase C and reactive oxygen species.
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.
Nakano
Hajime
H
Department of Dermatology, Hirosaki University School of Medicine, Hirosaki, Japan. hnakano@cc.hirosaki-u.ac.jp
Ikenaga
Satsuki
S
Aizu
Takayuki
T
Kaneko
Takahide
T
Matsuzaki
Yasushi
Y
Tsuchida
Shigeki
S
Hanada
Katsumi
K
Arima
Yaeno
Y
D000111:Acetylcysteine / Q000494:pharmacology; D000470:Alkaloids; D053119:Benzophenanthridines; D015153:Blotting, Western; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000078404:Epidermal Cells; D016166:Free Radical Scavengers / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D008668:Metallothionein / Q000235:genetics*; D039821:Monoterpenes / Q000494:pharmacology*; D010617:Phenanthridines / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014334:Tropolone / Q000031:analogs & derivatives* / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects*
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D039821:Monoterpenes; D010617:Phenanthridines; D017382:Reactive Oxygen Species; D014334:Tropolone; D008668:Metallothionein; C016299:chelerythrine; D011493:Protein Kinase C; C009479:beta-thujaplicin; D000111:Acetylcysteine