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

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 spectrometry 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.

INTRODUCTION

Metallothionein (MT) is a heavy metal-binding protein 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 transcript of MT-1 subisoform genes by cadmium, zinc, nickel, and iron is different among the MT-1 subisoforms in human urothelial cells (McNeill et al., 2019). Atorvastatin 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), suggesting that MT isoforms have specific physiological and toxicological roles. However, the difference in functions 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 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 identified that copper(II) bis(diethyldithiocarbamate), a copper complex, is an inducer of MT in vascular endothelial cells (Fujie et al., 2016c). Previously, it was shown that the transcription factor nuclear factor-erythroid 2-related 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 functions 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 different MT isoforms in vascular endothelial cells exposed to cadmium.

MATERIALS AND METHODS

Materials

Bovine aortic endothelial cells were purchased from Cell Applications (San Diego, CA, USA). The following materials were purchased from the indicated vendors: Dulbecco’s modified Eagle’s medium and calcium- and magnesium-free phosphate buffered saline from Nissui Pharmaceutical (Tokyo, Japan); fetal bovine serum, Opti-MEM^® Reduced Serum Medium, Lipofectamine^® RNAiMAX Transfection 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 reagent 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 culture

Bovine aortic endothelial cells were cultured at 37°C in 5% CO₂ in Dulbecco’s modified Eagle’s medium supplemented 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 endothelial cells treated with cadmium chloride. Real-time polymerase chain reaction (RT-PCR) analysis was performed in accordance with a method described previously (Fujie et al., 2016c).

Transfection

Bovine 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 sequences of the sense and antisense strands of siRNAs were as follows: bovine MT-1A siRNA, 5’-UCCAUUUGGAGGAAAAGCGAG-3’ (sense) and 5’-CGCUUUUCCUCCAAAUGGACC-3’ (antisense); bovine MT-1E siRNA, 5’-CCACAACAAACUUGCAUUUdTdT-3’ (sense) and 5’-AAAUGCAAGUUUGUUGUGGdTdT-3’ (antisense); bovine MT-2A siRNA, 5’-GUACAAACCUGCAUAUUUUdTdT-3’ (sense) and 5’- AAAAUAUGCAGGUUUGUACdTdT-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-inductively 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 cultures of bovine aortic endothelial cells, with or without the above transfection for 4 hr, were treated with cadmium 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 scraping 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 method (Sunaga et al., 1987). Specifically, the MT isoforms were separated in 25 mM 1,3-diaminopropane-HCl elution 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 connected to the ICP-MS (NexION 300S; PerkinElmer) and were detected by cadmium (m/z = 114) bound to the MT isoforms.

Statistical analysis

The 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 binding 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 induction between the two MT isoforms. However, Richards et al. (1984) reported that the induction of MT-1 was higher than that of MT-2 in human dermal fibroblasts after exposure to cadmium, whereas that of MT-2 was higher than that of MT-1 for zinc. Our previous study suggested a difference in the functions of MT-1 and MT-2 based on the intracellular signaling pathways that mediate the induction of MT isoforms in cultured bovine aortic 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 isoforms/subisoforms has not been identified. In the present study, the induction of MT mRNA and protein by cadmium in bovine aortic endothelial cells was investigated to examine whether there were differences in the function and regulation of induction by cadmium between endothelial MT-1 and MT-2. First, transcriptional induction was determined by the evaluation of MT-1A, MT-1E, and MT-2A mRNA expression in cells exposed to cadmium. 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 expression 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 cadmium in cells.

Fig. 1

mRNA expression of MT-1A, MT-1E, and MT-2A in bovine aortic endothelial cells after exposure to cadmium. 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 panels). Values are presented as the means ± SE of three technical replicates. **Significant difference from the corresponding control, p < 0.01.

We attempted to identify MT-1 and MT-2 proteins in the elution profiles obtained from the separation method using an HPLC-ICP-MS system (Fig. 2). MT proteins 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 markedly reduced the expression of peak II (Fig. 2), indicating that peaks I and II were mainly composed of MT-1A and MT-2A, respectively. Thus, bovine aortic endothelial 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 siRNA-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 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 mechanisms 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 suggested that MT-1A was the predominant MT-1 subisoform induced, as well as the MT-2A subisoform, in bovine aortic endothelial cells after exposure to cadmium. The physiological roles and induction of MT-1E protein remain to be elucidated.

Fig. 2

The HPLC-ICP-MS profiles for cadmium in the cytosol of bovine aortic endothelial cells exposed to cadmium. 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.

The induction of endothelial MT isoform after exposure 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, respectively). 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, respectively, for 24 hr (Fig. 3, left panels), suggesting that both MT isoforms contributed to the protective effects against cadmium cytotoxicity. Transcriptional induction of endothelial 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 activation 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. Presently, 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 participates in the biological defense system, whereas MT-2 mainly regulates intracellular zinc metabolism (Fujie et al., 2016c), although this does not mean that MT isoforms cannot assume a share of the other role.

Fig. 3

The HPLC-ICP-MS profiles for cadmium in the cytosol 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 determined using an ES-502N column and HPLC-ICP-MS.

The present study revealed that: (1) transcriptional 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 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. Further studies should be performed to clarify whether there are differences in the functions and the induction of MT-1 and MT-2 in vascular 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.

REFERENCES

• Artells, E., Palacios, Ò., Capdevila, M. and Atrian, S. (2013): Mammalian MT1 and MT2 metallothioneins differ in their metal binding abilities. Metallomics, 5, 1397-1410.
• Eaton, D.L., Stacey, N.H., Wong, K.L. and Klaassen, C.D. (1980): Dose-response effects of various metal ions on rat liver metallothionein, glutathione, heme oxygenase, and cytochrome P-450. Toxicol. Appl. Pharmacol., 55, 393-402.
• Fujie, T., Segawa, Y., Uehara, A., Nakamura, T., Kimura, T., Yoshida, E., Yamamoto, C., Uchiyama, M., Naka, H. and Kaji, T. (2016a): Zinc diethyldithiocarbamate as an inducer of metallothionein in cultured vascular endothelial cells. J. Toxicol. Sci., 41, 217-224.
• Fujie, T., Hara, T. and Kaji, T. (2016b): Toxicology of organic-inorganic hybrid molecules: bio-organometallics and its toxicology. J. Toxicol. Sci., 41 (Special), SP81-SP88.
• Fujie, T., Segawa, Y., Yoshida, E., Kimura, T., Fujiwara, Y., Yamamoto, C., Satoh, M., Naka, H. and Kaji, T. (2016c): Induction of metallothionein isoforms by copper diethyldithiocarbamate in cultured vascular endothelial cells. J. Toxicol. Sci., 41, 225-232.
• Fujie, T., Murakami, M., Yoshida, E., Yasuike, S., Kimura, T., Fujiwara, Y., Yamamoto, C. and Kaji, T. (2016d): Transcriptional induction of metallothionein by tris(pentafluorophenyl)stibane in cultured bovine aortic endothelial cells. Int. J. Mol. Sci., 17, 1381.
• Fujie, T., Takenaka, F., Yoshida, E., Yasuike, S., Fujiwara, Y., Shinkai, Y., Kumagai, Y., Yamamoto, C. and Kaji, T. (2019a): Possible mechanisms underlying transcriptional induction of metallothionein isoforms by tris(pentafluorophenyl)stibane, tris(pentafluorophenyl)arsane, and tris(pentafluorophenyl)phosphane in cultured bovine aortic endothelial cells. J. Toxicol. Sci., 44, 327-333.
• Fujie, T., Yamamoto, T., Yamamoto, C. and Kaji, T. (2019b): Bis(1,4-dihydro-2-methyl-1-phenyl-4-thioxo-3-pyridiolato)zinc(II) exhibits strong cytotoxicity and a high intracellular accumulation in cultured vascular endothelial cells. J. Toxicol. Sci., 44, 113-120.
• Habel, N., Hamidouche, Z., Girault, I., Patiño-García, A., Lecanda, F., Marie, P.J. and Fromigué, O. (2013): Zinc chelation: a metallothionein 2A’s mechanism of action involved in osteosarcoma cell death and chemotherapy resistance. Cell Death Dis., 4, e874.
• Hara, T., Nakano, S., Kitamura, Y., Yamamoto, C., Yasuike, S. and Kaji, T. (2019): Intracellular accumulation-independent cytotoxicity of pentavalent organoantimony compounds in cultured vascular endothelial cells. J. Toxicol. Sci., 44, 845-848.
• Heuchel, R., Radtke, F., Georgiev, O., Stark, G., Aguet, M. and Schaffner, W. (1994): The transcription factor MTF-1 is essential for basal and heavy metal-induced metallothionein gene expression. EMBO J., 13, 2870-2875.
• Karin, M. and Richards, R.I. (1982): Human metallothionein genes--primary structure of the metallothionein-II gene and a related processed gene. Nature, 299, 797-802.
• Kägi, J.H. (1991): Overview of metallothionein. Methods Enzymol., 205, 613-626.
• Kaji, T., Mishima, A., Koyanagi, E., Yamamoto, C., Sakamoto, M. and Kozuka, H. (1992): Possible mechanism for zinc protection against cadmium cytotoxicity in cultured vascular endothelial cells. Toxicology, 76, 257-270.
• Kimura, T. and Kambe, T. (2016): The functions of metallothionein and ZIP and ZnT transporters: an overview and perspective. Int. J. Mol. Sci., 17, 336.
• Koizumi, S., Yamada, H., Suzuki, K. and Otsuka, F. (1992): Zinc-specific activation of a HeLa cell nuclear protein which interacts with a metal responsive element of the human metallothionein-IIA gene. Eur. J. Biochem., 210, 555-560.
• Li, S., Yang, Z., Cao, J., Qiu, B. and Li, H. (2018): Determination of metallothionein isoforms in fish by cadmium saturation combined with anion exchange HPLC-ICP-MS. Chromatographia, 81, 881-889.
• McNeill, R.V., Mason, A.S., Hodson, M.E., Catto, J.W. and Southgate, J. (2019): Specificity of the metallothionein-1 response by cadmium-exposed normal human urothelial cells. Int. J. Mol. Sci., 20, 1344.
• Miyayama, T., Ogra, Y. and Suzuki, K.T. (2007): Separation of metallothionein isoforms extracted from isoform-specific knockdown cells on two-dimensional micro high-performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry. J. Anal. At. Spectrom., 22, 179-182.
• Otsuka, F., Ohno, S., Suzuki, K., Takahashi, K., Ohsawa, M. and Koizumi, S. (2007): [Mechanism of metallothionein gene activation mediated by heavy-metal dependent transcription factor MTF-1]. Yakugaku Zasshi, 127, 675-684.
• Palmiter, R.D. (1994): Regulation of metallothionein genes by heavy metals appears to be mediated by a zinc-sensitive inhibitor that interacts with a constitutively active transcription factor, MTF-1. Proc. Natl. Acad. Sci. USA, 91, 1219-1223.
• Piotrowski, J.K. and Szymańska, J.A. (1976): Influence of certain metals on the level of metallothionein-like proteins in the liver and kidneys of rats. J. Toxicol. Environ. Health, 1, 991-1002.
• Połeć Pawlak, K., Palacios, O., Capdevila, M., González-Duarte, P. and Lobiński, R. (2002): Monitoring of the metal displacement from the recombinant mouse liver metallothionein Zn₇-complex by capillary zone electrophoresis with electrospray MS detection. Talanta, 57, 1011-1017.
• Radtke, F., Heuchel, R., Georgiev, O., Hergersberg, M., Gariglio, M., Dembic, Z. and Schaffner, W. (1993): Cloned transcription factor MTF-1 activates the mouse metallothionein I promoter. EMBO J., 12, 1355-1362.
• Richards, R.I., Heguy, A. and Karin, M. (1984): Structural and functional analysis of the human metallothionein-IA gene: differential induction by metal ions and glucocorticoids. Cell, 37, 263-272.
• Sato, M. and Bremner, I. (1993): Oxygen free radicals and metallothionein. Free Radic. Biol. Med., 14, 325-337.
• Shinkai, Y., Kimura, T., Itagaki, A., Yamamoto, C., Taguchi, K., Yamamoto, M., Kumagai, Y. and Kaji, T. (2016): Partial contribution of the Keap1-Nrf2 system to cadmium-mediated metallothionein expression in vascular endothelial cells. Toxicol. Appl. Pharmacol., 295, 37-46.
• Sunaga, H., Kobayashi, E., Shimojo, N. and Suzuki, K.T. (1987): Detection of sulfur-containing compounds in control and cadmium-exposed rat organs by high-performance liquid chromatography--vacuum-ultraviolet inductively coupled plasma-atomic emission spectrometry (HPLC-ICP). Anal. Biochem., 160, 160-168.
• Yagle, M.K. and Palmiter, R.D. (1985): Coordinate regulation of mouse metallothionein I and II genes by heavy metals and glucocorticoids. Mol. Cell. Biol., 5, 291-294.
• Zhang, B., Egli, D., Georgiev, O. and Schaffner, W. (2001): The Drosophila homolog of mammalian zinc finger factor MTF-1 activates transcription in response to heavy metals. Mol. Cell. Biol., 21, 4505-4514.