Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor, FG4592, Induces Endogenous Metallothionein3 Expression in Human Neuronal Cell Line, ReNcell CX Cells

Mizuki Tsuru; Taisei Ito; Kazuki Komai; Fukuto Kunitomo; Yukie Nakayama; Takanori Murakami; Kazuki Ohuchi; Yasuhiro Shinkai; Tomoki Kimura; Nobuhiko Miura; Yoshito Kumagai; Isao Hozumi; Masatoshi Inden; Hisaka Kurita

doi:10.1248/bpb.b24-00792

Regular Article

Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor, FG4592, Induces Endogenous Metallothionein3 Expression in Human Neuronal Cell Line, ReNcell CX Cells

Mizuki Tsuru ^†, Taisei Ito ^†, Kazuki Komai, Fukuto Kunitomo, Yukie Nakayama, Takanori Murakami, Kazuki Ohuchi, Yasuhiro Shinkai, Tomoki Kimura, Nobuhiko Miura, Yoshito Kumagai, Isao Hozumi, Masatoshi Inden, Hisaka Kurita

Author information

Mizuki Tsuru ^†
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Taisei Ito ^†
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Kazuki Komai
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Fukuto Kunitomo
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Yukie Nakayama
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Takanori Murakami
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Kazuki Ohuchi
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Yasuhiro Shinkai
Environmental Biology Laboratory, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, 1432–1 Horinouchi, Hachioji, Tokyo 192–0392, Japan
Tomoki Kimura
Laboratory of Public Health & Preventive Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45–1 Nagaotoge-cho, Hirakata, Osaka 573–0101, Japan
Nobuhiko Miura
Department of Health Science, Yokohama University of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245–2006, Japan
Yoshito Kumagai
Graduate School of Pharmaceutical Sciences, Kyusyu University, 3–1–1 Maidashi, Higashi-ku, Fukuoka 812–8582, Japan
Isao Hozumi
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Masatoshi Inden
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Hisaka Kurita Corresponding author
Laboratory of Medical Therapeutics and Molecular Therapeutics, Gifu Pharmaceutical University, 1–25–4 Daigaku-nishi, Gifu 501–1196, Japan
Author contributions

†These authors contributed equally: Mizuki Tsuru, Taisei Ito

Keywords: metallothionein3, neuron, hypoxia-inducible factor 1 alpha (HIF1α), HIF-prolyl hydroxylase, FG4592

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Supplementary material

2025 Volume 48 Issue 2 Pages 137-143

DOI https://doi.org/10.1248/bpb.b24-00792

Details

Abstract

Metallothionein (MT) is a small-molecule protein that functions in essential trace element homeostasis. Among MT isoforms, MT3 is involved in neuronal activity, and its expression is reported to be decreased in patients with neurodegenerative conditions such as Alzheimer’s disease; however, only a few effective drugs have been reported to induce MT3 expression. In this study, we evaluated existing drugs for the induction of MT3 expression in the neuronal cell line of ReNcell CX cells. Using recombinant proteins of MT isoforms with the 3× Flag tag, we performed Western blotting (WB) with the primary antibodies against MT3 or Flag tag, and this method of WB for MT3 was confirmed specifically to detect the MT3 protein. We treated ReNcell CX cells with several HIF-PH inhibitors and evaluated MT3 expression via real-time RT-PCR. We found that FG4592 significantly enhanced MT3 expression at both RNA and protein levels. FG4592 treatment increased the amount of hypoxia-inducible factor 1 alpha (HIF1α) binding to the MT3 promoter. These findings indicate that FG4592 induces MT3 expression via increased HIF1α. In conclusion, we found FG4592 to be an endogenous MT3 inducer in the cells of the nervous system in this study. The findings of this study are expected to lead to the development of new MT3-inducing drugs for neurodegenerative diseases based on FG4592.

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