Induction of Metallothionein by Manganese Is Completely Dependent on Interleukin-6 Production Kazuo Kobayashi, Junji Kuroda, Nobuo Shibata, Tatsuya Hasegawa, Yoshiyuki Seko, Masahiko Satoh, Chiharu Tohyama, Hirohisa Takano, Nobumasa Imura, Kou Sakabe, Hitomi Fujishiro, and Seiichiro Himeno Toxicology Research Laboratory, R&D, Kissei Pharmaceutical Co., Ltd., Nagano, Japan (K.K., J.K., N.S.); Department of Environmental Biochemistry, Yamanashi Institute of Environmental Sciences, Yamanashi, Japan (T.H., Y.S.); Environmental Health Sciences Division, National Institute for Environmental Studies, Tsukuba, Japan (M.S., C.T., H.T.); Department of Public Health and Molecular Toxicology, School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan (N.I., K.S.); and Laboratory of Molecular Nutrition and Toxicology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan (H.F., S.H.) Received August 23, 2006; accepted October 19, 2006 ABSTRACT Metallothionein (MT) is a cysteine-rich protein that binds to and is inducible by heavy metals such as cadmium and zinc. How- ever, the precise mechanism of MT induction by other metals remains unclear. In the present study, we investigated the mechanism of MT induction by manganese, focusing on the involvement of cytokine production. Administration of MnCl 2 to mice resulted in the induction of MT dose-dependently in the liver with little accumulation of manganese. Speciation analysis of metals in the liver cytosol showed that the major metal bound to the induced MT was zinc. Administration of MnCl 2 caused an increase in mRNA levels of interleukin-6 (IL-6) in the liver as well as an increase in serum levels of IL-6 but not those of other inflammatory cytokines. Subsequently, serum levels of serum amyloid A (SAA), an acute-phase protein induced by IL-6, in- creased with a peak at 24 h. However, no increase in serum alanine aminotransferase activity was observed, suggesting that manganese enhanced the production of IL-6 and SAA without causing liver injury. In response to IL-6, the expression of a zinc transporter, ZIP14, was enhanced in the liver, possibly contributing to the synthesis of hepatic zinc-MT. In IL-6-null mice, the induction of hepatic MT by treatment with MnCl 2 was completely suppressed to the control level. These results sug- gest that manganese is a unique metal that induces the syn- thesis of hepatic MT completely depending on the production of IL-6 without accompanying liver injury. Metallothionein (MT) is a cysteine-rich low-molecular- weight protein with a high affinity for heavy metals. Physi- ological roles of MT encompass a broad spectrum, including detoxification of heavy metals, scavenging of free radicals, regulation of cell growth, and maintenance of homeostasis of trace metals such as zinc and copper (Suzuki et al., 1993). The most prominent characteristic of MT is its inducibility not only by metals but also by various factors such as hor- mones, cytokines, organic chemicals, starvation, and physical stress (Ka ¨ gi, 1993). It is generally perceived that the expres- sion of the MT gene in response to a metal load is regulated by metal-responsive transcription factor 1 (MTF-1) that binds to the metal-response elements of the promoter region of MT genes (Heuchel et al., 1994). The mechanisms under- lying the activation of the MT gene through the interaction of MTF-1 and metal-response elements have been investigated extensively (Andrews, 2001; Otsuka, 2004), but the precise mechanism of MT induction by nonzinc metals, including cadmium has been poorly understood (Daniels et al., 2002; Wang et al., 2004). In addition to potent MT-inducing metals such as zinc, cadmium, copper, mercury, silver, and bismuth, other metals such as chromium, iron, cobalt, nickel, arsenic, and manga- nese can also induce MT but to the lesser levels (Fleet et al., 1990; Albores et al., 1992). These weak MT-inducing metals This research was supported by Grant-in-Aid for Scientific Research C 15590112 from the Japan Society for the Promotion of Science. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.112912. ABBREVIATIONS: MT, metallothionein; MTF-1, metal-responsive transcription factor 1; TNF, tumor necrosis factor; IL, interleukin; IFN, interferon; STAT, signal transducer and activator of transcription; ALT, alanine aminotransferase; BUN, blood urea nitrogen; SAA, serum amyloid A; ELISA, enzyme-linked immunosorbent assay; HPLC, high-performance liquid chromatography; ICP-MS, inductively coupled argon plasma-mass spec- trometry; RT, reverse transcription; PCR, polymerase chain reaction; ME3738, 22-methoxyolean-12-ene-3,24(4)-diol. 0022-3565/07/3202-721–727$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 320, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 112912/3166906 JPET 320:721–727, 2007 Printed in U.S.A. 721 at ASPET Journals on January 28, 2016 jpet.aspetjournals.org Downloaded from