Correspondence: Chika Yamamoto (E-mail: yamamoto@phar.toho-u.ac.jp) Toshiyuki Kaji (E-mail: t-kaji@rs.tus.ac.jp)*These authors equally contributed to this work.

Induction of metallothionein isoforms in cultured bovine aortic endothelial cells exposed to cadmium

Tomoya Fujie1,*, Yusuke Ozaki2,*, Fukuta Takenaka2,*, Misaki Nishio2, Takato Hara1, Yasuyuki Fujiwara3, Chika Yamamoto1 and Toshiyuki Kaji2

1Department of Environmental Health, Faculty of Pharmaceutical Sciences, Toho University,

2-2-1 Miyama, Funabashi 274-8510, Japan2Department of Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science,

2641 Yamazaki, Noda 278-8510, Japan3Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences,

1432-1 Horinouchi, Hachioji 192-0392, Japan

(Received October 19, 2020; Accepted October 21, 2020)

ABSTRACT — Metallothionein (MT) is an inducible protein with cytoprotective activity against heavy metals such as cadmium, zinc, and copper. MT-1 and MT-2 are the isoforms of MT induced by and bind the heavy metals. Bovine aortic endothelial cells contain three types of MT genes, namely, MT-1A, MT-1E, and MT-2A; however, the associated protein expression of these MT isoforms has not been identified. In the present study, the expression of MT subisoform proteins in cells treated with cadmium chloride was identified using a high-performance liquid chromatography-inductively coupled plasma-mass spectrome-try system. It was revealed that: (1) transcriptional induction of MT-1A by cadmium was markedly more sensitive than that of MT-1E/2A; (2) MT-1A and MT-2A proteins were the predominant MT subisoforms induced by cadmium; and (3) there might be differentiation in the functions of MT-1 and MT-2 against cadmium cytotoxicity, although the actual roles of the MT isoforms in the cells were not distinct. This is the first study to show the differential induction of isoforms of MT proteins in vascular endothelial cells by cadmium. Key words: Cadmium, Metallothionein, Isoform, Subisoform, Vascular endothelial cell

INTRODUCTION

Metallothionein (MT) is a heavy metal-binding pro-tein with cytoprotective effects, such as against heavy metals and reactive oxygen species (Kägi, 1991; Sato and Bremner, 1993). Two isoforms of MT, MT-1 and MT-2, are cytoprotective proteins that are ubiquitous in the organs of mammals and can be induced by cadmium, zinc, and copper (Karin and Richards, 1982; Palmiter, 1994). MT-1 and MT-2 are induced by heavy metals at different levels; in addition, the induction of the tran-script of MT-1 subisoform genes by cadmium, zinc, nick-el, and iron is different among the MT-1 subisoforms in human urothelial cells (McNeill et al., 2019). Atorv-astatin increases mRNA expression of the MT-2 mRNA subisoforms, but not the MT-1 subisoforms, in human osteosarcoma cells (Habel et al., 2013). Cadmium and

copper have a stronger ability to bind MT-1 than MT-2, whereas zinc tends to bind to MT-2 instead of MT-1 (Połeć Pawlak et al., 2002; Artells et al., 2013), sug-gesting that MT isoforms have specific physiological and toxicological roles. However, the difference in func-tions between MT-1 and MT-2 has not been completely described.

Metal response element (MRE)-binding transcription factor-1 (MTF-1) is essential for the induction of MT (Heuchel et al., 1994). MTF-1 requires zinc ions to bind to the MRE in the promoter region of MT genes (Radtke et al., 1993; Zhang et al., 2001). As cadmium does not have the ability to bind to MTF-1 (Koizumi et al., 1992; Heuchel et al., 1994), it is considered that free zinc ions released from zinc-binding proteins by cadmium activate MTF-1 (Otsuka et al., 2007). For these reasons, heavy metals such as cadmium and zinc are typical inducers

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of MT. However, in vascular endothelial cells, cadmium induces MT (Kaji et al., 1992), but zinc does not have the ability to induce MT (Fujie et al., 2016a).

Based on bio-organometallics research strategies (Fujie et al., 2016b, 2019a, 2019b; Hara et al., 2019), we iden-tified that copper(II) bis(diethyldithiocarbamate), a cop-per complex, is an inducer of MT in vascular endotheli-al cells (Fujie et al., 2016c). Previously, it was shown that the transcription factor nuclear factor-erythroid 2-relat-ed factor 2 (Nrf2) partly contributes to MT induction by cadmium in cells (Shinkai et al., 2016). It was revealed that transcriptional induction of endothelial MT-1 was mediated by both the MTF-1-MRE and Nrf2-antioxidant response element (ARE) pathways, whereas that of MT-2 was mediated only by the MTF-1-MRE pathway (Fujie et al., 2016c, 2016d), suggesting a difference in the func-tions of MT-1 and MT-2.

However, the expression of MT-1 and MT-2 proteins in vascular endothelial cells is unclear. The present study was undertaken to identify the protein expression of dif-ferent MT isoforms in vascular endothelial cells exposed to cadmium.

MATERIALS AND METHODS

MaterialsBovine aortic endothelial cells were purchased

from Cell Applications (San Diego, CA, USA). The following materials were purchased from the indi-cated vendors: Dulbecco’s modified Eagle’s medi-um and calcium- and magnesium-free phosphate buff-ered saline from Nissui Pharmaceutical (Tokyo, Japan); fetal bovine serum, Opti-MEM® Reduced Serum Medium, Lipofectamine® RNAiMAX Trans-fection Reagent, and High-Capacity cDNA Reverse Transcription Kit from Thermo Fisher Scientific (Waltham, MA, USA); cell culture dishes and plates from Corning (Corning, NY, USA); QIAzol lysis rea-gent from QIAGEN (Venlo, The Netherlands); GeneAce SYBR® qPCR Mix α from Nippon Gene (Tokyo, Japan); and 1,3-diaminopropane from Tokyo Chemical Industry (Tokyo, Japan). Other reagents were purchased from Nacalai Tesque (Kyoto, Japan).

Cell cultureBovine aortic endothelial cells were cultured at 37°C in

5% CO2 in Dulbecco’s modified Eagle’s medium supple-mented with 10% fetal bovine serum until they reached confluency. The medium was discarded, and the cells were washed twice with serum-free Dulbecco’s modified Eagle’s medium. The cells were treated with or without

cadmium chloride (0.5, 1, 2, or 5 µM) at 37°C for 3, 6, 12, or 24 hr in serum-free Dulbecco’s modified Eagle’s medium.

Real-time reverse transcription-polymerase chain reaction analysis

Total RNA was extracted from bovine aortic endothe-lial cells treated with cadmium chloride. Real-time polymerase chain reaction (RT-PCR) analysis was per-formed in accordance with a method described previously (Fujie et al., 2016c).

TransfectionBovine aortic endothelial cells were cultured and

small interfering RNAs (siRNAs) (Bioneer, Daejeon, Korea) were transfected using RNAiMAX reagent in accordance with the manufacturer’s instructions, as described previously (Fujie et al., 2016c). The sequenc-es of the sense and antisense strands of siRNAs were as follows: bovine MT-1A siRNA, 5’-UCCAUUUGGAG-GAAAAGCGAG-3’ (sense) and 5’-CGCUUUUCCUC-CAAAUGGACC-3’ (antisense); bovine MT-1E siRNA, 5’-CCACAACAAACUUGCAUUUdTdT-3’ (sense) and 5’-AAAUGCAAGUUUGUUGUGGdTdT-3’ (antisense); bovine MT-2A siRNA, 5’-GUACAAACCUGCAUAUU-UUdTdT-3’ (sense) and 5’- AAAAUAUGCAGGUUU-GUACdTdT-3’ (antisense). A nonspecific sequence was used as the siRNA negative control (QIAGEN).

Separation of MT-1 and MT-2 proteins by high-performance liquid chromatography-inductively coupled plasma-mass spectrometry

The high-performance liquid chromatography-induc-tively coupled plasma-mass spectrometry (HPLC-ICP-MS) method to analyze the MT isoforms separately was constructed based on the method described by Miyayama et al. (2007) and Li et al. (2018). Briefly, confluent cul-tures of bovine aortic endothelial cells, with or without the above transfection for 4 hr, were treated with cadmi-um chloride (0.5, 1, 2, or 5 µM) for 6, 12, 24, or 48 hr. The cells were harvested with Tris-HCl (pH 7.2) by scrap-ing with a rubber policeman and sonicated to prepare cell homogenate. The cell homogenate was centrifuged at 18,000 × g for 10 min at 4°C to obtain the supernatant. Cadmium chloride solution was added to the supernatant at a concentration of 500 µM. The supernatant was left to stand for 10 min and then incubated at 95°C for 5 min. After centrifugation at 105,000 × g for 1 hr at 4°C, the supernatant was used for the determination of MT-1 and MT-2 proteins by the HPLC-ICP-MS analysis meth-od (Sunaga et al., 1987). Specifically, the MT isoforms

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were separated in 25 mM 1,3-diaminopropane-HCl elu-tion buffer (pH 9.0) at a flow rate of 0.5 mL/min using an HPLC system (Flexar FX-20; PerkinElmer, Waltham, MA, USA) equipped with a DEAE column (ES-502N; Asahipak, Tokyo, Japan, 7.5 × 100 mm) directly connect-ed to the ICP-MS (NexION 300S; PerkinElmer) and were detected by cadmium (m/z = 114) bound to the MT iso-forms.

Statistical analysisThe data were analyzed for statistical significance

using analysis of variance and Bonfferoni-type multiple t-test for the multiple comparison with Statcel3 (OMS, Tokyo, Japan) when possible. P values of less than 0.05 were considered statistically significant.

RESULTS AND DISCUSSION

MT-1 and MT-2 are generally induced by bind-ing heavy metals such as cadmium, zinc, and copper (Piotrowski and Szymańska, 1976; Eaton et al., 1980). In addition, they were found to be induced to similar levels (Yagle and Palmiter, 1985), suggesting that there is little or no difference in the function and regulation of induc-tion between the two MT isoforms. However, Richards et al. (1984) reported that the induction of MT-1 was high-er than that of MT-2 in human dermal fibroblasts after exposure to cadmium, whereas that of MT-2 was high-er than that of MT-1 for zinc. Our previous study sug-gested a difference in the functions of MT-1 and MT-2 based on the intracellular signaling pathways that medi-ate the induction of MT isoforms in cultured bovine aor-tic endothelial cells (Fujie et al., 2016c, 2016d). These cells have three subisoform genes of MT: MT-1A, MT-1E, and MT-2A; however, the protein expression of MT iso-forms/subisoforms has not been identified. In the present study, the induction of MT mRNA and protein by cad-mium in bovine aortic endothelial cells was investigat-ed to examine whether there were differences in the func-tion and regulation of induction by cadmium between endothelial MT-1 and MT-2. First, transcriptional induc-tion was determined by the evaluation of MT-1A, MT-1E, and MT-2A mRNA expression in cells exposed to cadmi-um. As shown in Fig. 1, cadmium significantly elevated the mRNA expression of MT-1A, MT-1E, and MT-2A in a concentration- and time-dependent manner. However, the degree of elevation differed between MT-1A and MT-1E/MT-2A mRNAs. Specifically, MT-1A mRNA expres-sion was significantly increased several hundred-fold by cadmium, whereas MT-1E and MT-2A mRNA expression was increased several ten-fold, suggesting that MT-1A

gene was the most sensitive to cadmium in bovine aortic endothelial cells. This also suggests that MT-1A may be one of the dominant MT-1 subisoforms induced by cad-mium in cells.

We attempted to identify MT-1 and MT-2 proteins in the elution profiles obtained from the separation meth-od using an HPLC-ICP-MS system (Fig. 2). MT pro-teins were eluted as two major peaks at 12-13 min and 17-20 min, termed peak I and peak II, respectively. MT-1A siRNA selectively reduced the expression of peak I, MT-1E siRNA did not affect the expression of either peak I or peak II, and MT-2A siRNA selectively and mark-edly reduced the expression of peak II (Fig. 2), indicat-ing that peaks I and II were mainly composed of MT-1A and MT-2A, respectively. Thus, bovine aortic endotheli-al cells predominantly expressed MT-1A and MT-2A as MT isoforms after exposure to cadmium. The expression of MT-1A, MT-1E, and MT-2A mRNAs were decreased to approximately 10, 20, and 6%, respectively, in siR-NA-treated cells after exposure to cadmium. A partial reduction of peak I by MT-1A siRNA was postulated to be due to a phenomenon that MT-1 can be induced by a

Fig. 1. mRNA expression of MT-1A, MT-1E, and MT-2A in bovine aortic endothelial cells after exposure to cad-mium. Confluent cultures of the cells were treated with cadmium chloride at 0.5, 1, 2, and 5 µM for 12 hr (left panels) or at 2 µM for 3, 6, 12, and 24 hr (right pan-els). Values are presented as the means ± SE of three technical replicates. **Significant difference from the corresponding control, p < 0.01.

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small amount of MT-1A mRNA (Miyayama et al., 2007). Nine genes, MT-1A, MT-1B, MT-1E, MT-1F, MT-1G, MT-1H, MT-1L, MT-1M, and MT-1X, have been identified as MT-1 subisoforms in humans; the only subisoform of MT-2 is MT-2A (Kimura and Kambe, 2016). However, little is known about the functions and induction mech-anisms of MT subisoforms, as separation of the protein subisoforms has not yet been achieved because of their very high homology. In the present study, it was suggest-ed that MT-1A was the predominant MT-1 subisoform induced, as well as the MT-2A subisoform, in bovine aor-tic endothelial cells after exposure to cadmium. The phys-

iological roles and induction of MT-1E protein remain to be elucidated.

The induction of endothelial MT isoform after expo-sure to cadmium was investigated, as shown in Fig. 3. The MT isoforms were induced in a concentration- and time-dependent manner (left and right panels, respec-tively). The peak area for MT-2 was greater than that for MT-1, by approximately 1.6-, 2.5-, 1.9-, and 1.8-fold after exposure to cadmium at 0.5, 1, 2, and 5 µM, respective-ly, for 24 hr (Fig. 3, left panels), suggesting that both MT isoforms contributed to the protective effects against cad-mium cytotoxicity. Transcriptional induction of endothe-

Fig. 2. The HPLC-ICP-MS profiles for cadmium in the cyto-sol of bovine aortic endothelial cells exposed to cad-mium. Confluent cultures of the cells were transfected with the siRNA of Control (siControl), MT-1A (siMT-1A), MT-1E (siMT-1E), or MT-2A (siMT-2A) and then treated with cadmium chloride at 2 µM for 24 hr. The profiles of 114cadmium (114Cd) were determined using an ES-502N column and HPLC-ICP-MS.

Fig. 3. The HPLC-ICP-MS profiles for cadmium in the cy-tosol of bovine aortic endothelial cells exposed to cadmium. Confluent cultures of the cells were treated with cadmium chloride at 0.5, 1, 2, and 5 µM for 24 hr (left panels) or at 2 µM for 6, 12, 24, and 48 hr (right panels). The profiles of 114cadmium (114Cd) were deter-mined using an ES-502N column and HPLC-ICP-MS.

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lial MT-1 is mediated by both the MTF-1-MRE and Nrf2-ARE pathways, whereas that of MT-2 is mediated only by the MTF-1-MRE pathway (Fujie et al., 2016c, 2016d), suggesting that cadmium could cause sufficient activa-tion of these two pathways to induce MT-1 and MT-2 at the same, or very similar, concentrations. A time-course study showed that the MT isoform peaks appeared after exposure to 2 µM cadmium for 24 hr or longer. Present-ly, the peak area of MT-2 was greater than that of MT-1, by approximately 1.9- and 2.4-fold after 24 and 48 hr, respectively (Fig. 3, right panels), suggesting that MT-1 was induced at a faster rate than MT-2, but the maximum protein expression of MT-1 was lower than that of MT-2 in vascular endothelial cells after exposure to cadmium. This result partly supports the hypothesis that MT-1 par-ticipates in the biological defense system, whereas MT-2 mainly regulates intracellular zinc metabolism (Fujie et al., 2016c), although this does not mean that MT iso-forms cannot assume a share of the other role.

The present study revealed that: (1) transcription-al induction of MT-1A by cadmium was markedly more sensitive than that of MT-1E/MT-2A; (2) MT-1A and MT-2A proteins were the predominant MT subisoforms induced by cadmium; and (3) there might be differenti-ation in the functions of MT-1 and MT-2 against cadmi-um cytotoxicity, although the actual roles of the MT iso-forms in the cells were not distinct. Further studies should be performed to clarify whether there are differences in the functions and the induction of MT-1 and MT-2 in vas-cular endothelial cells after exposure to agents other than cadmium.

ACKNOWLEDGMENTS

This work was supported by JSPS KAKENHI Grant Numbers 19K16361 (to T.F.) and 19K07089 (to T.K.).

Conflict of interest---- The authors declare that there is no conflict of interest.

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