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Transcriptional Induction of Metallothionein by Tris(pentafluorophenyl)stibane in Cultured Bovine Aortic Endothelial Cells

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ABSTRACT

Vascular endothelial cells cover the luminal surface of blood vessels and contribute to the prevention of vascular disorders such as atherosclerosis. Metallothionein (MT) is a low molecular weight, cysteine-rich, metal-binding, inducible protein, which protects cells from the toxicity of heavy metals and active oxygen species. Endothelial MT is not induced by inorganic zinc. Adequate tools are required to investigate the mechanisms underlying endothelial MT induction. In the present study, we found that an organoantimony compound, tris(pentafluorophenyl)stibane, induces gene expression of MT-1A and MT-2A, which are subisoforms of MT in bovine aortic endothelial cells. The data reveal that MT-1A is induced by activation of both the MTF-1–MRE and Nrf2–ARE pathways, whereas MT-2A expression requires only activation of the MTF-1–MRE pathway. The present data suggest that the original role of MT-1 is to protect cells from heavy metal toxicity and oxidative stress in the biological defense system, while that of MT-2 is to regulate intracellular zinc metabolism.

No MeSH data available.


Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Structure of Sb35; (B) Sb35-induced transcription of MT. Vascular endothelial cells were incubated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (upper panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (lower panels). MT-1A, MT-1E, and MT-2A mRNA expression was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. * p < 0.05 and ** p < 0.01 indicate significantly different from the corresponding control.
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ijms-17-01381-f002: Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Structure of Sb35; (B) Sb35-induced transcription of MT. Vascular endothelial cells were incubated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (upper panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (lower panels). MT-1A, MT-1E, and MT-2A mRNA expression was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. * p < 0.05 and ** p < 0.01 indicate significantly different from the corresponding control.

Mentions: Figure 1B depicts the locations of MREs and AREs in the upstream regions of MT genes of the bovine cells. Bovine cells express three MT subisoforms—MT-1A, MT-1E, and MT-2A—and each of their genes has MRE and ARE consensus sequences in the promoter region. The number of MT-1 subisoforms is eight in human and two in bovine cells. To investigate the difference in the intracellular signaling between MT-1 and MT-2 isoforms, the subsequent experiments were performed using bovine endothelial cells having only two subisoforms of MT-1. In bovine aortic endothelial cells, Sb35 (Figure 2a) induced MT-1A and MT-2A gene expression in a concentration-dependent manner when treated for 12 h (Figure 2b, upper panels). We observed maximum induction of MT-1A and MT-2A by 100 µM Sb35 at 12 and 24 h, respectively (Figure 2b, lower panels), indicating that Sb35 stimulates the transcriptional induction of MT-1 and MT-2 isoform genes in the cells; decrease in the induction at 48 h is possibly due to the metabolism of Sb35 and/or downregulation of the intracellular signaling activated by Sb35, although the details are unclear. However, induction of MT protein by Sb35 was not observed in Western blot analysis (Figure S1), suggesting that Sb35 compound is a tool to analyze the transcriptional induction of endothelial MT but not an agent for MT induction to protect cells from the toxicity of heavy metals and generated reactive oxygen species. In addition, Sb35 did not generate reactive oxygen species (Figure S2), suggesting that the transcriptional induction of MT by Sb35 is not mediated by reactive oxygen species. In addition, MT-1A, MT-1E, and MT-2A mRNA levels increased by approximately 12, three, and six fold, respectively, after Sb35 treatment. On the other hand, cadmium, a typical MT inducer, increased MT-1A, MT-1E, and MT-2A mRNA levels by approximately 1000-, 15-, and 15-fold, respectively, suggesting that Sb35 induced MT more weakly than cadmium. This appears to be the reason why Sb35 cannot induce MT at the protein level.


Transcriptional Induction of Metallothionein by Tris(pentafluorophenyl)stibane in Cultured Bovine Aortic Endothelial Cells
Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Structure of Sb35; (B) Sb35-induced transcription of MT. Vascular endothelial cells were incubated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (upper panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (lower panels). MT-1A, MT-1E, and MT-2A mRNA expression was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. * p < 0.05 and ** p < 0.01 indicate significantly different from the corresponding control.
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Related In: Results  -  Collection

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ijms-17-01381-f002: Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) Structure of Sb35; (B) Sb35-induced transcription of MT. Vascular endothelial cells were incubated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (upper panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (lower panels). MT-1A, MT-1E, and MT-2A mRNA expression was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. * p < 0.05 and ** p < 0.01 indicate significantly different from the corresponding control.
Mentions: Figure 1B depicts the locations of MREs and AREs in the upstream regions of MT genes of the bovine cells. Bovine cells express three MT subisoforms—MT-1A, MT-1E, and MT-2A—and each of their genes has MRE and ARE consensus sequences in the promoter region. The number of MT-1 subisoforms is eight in human and two in bovine cells. To investigate the difference in the intracellular signaling between MT-1 and MT-2 isoforms, the subsequent experiments were performed using bovine endothelial cells having only two subisoforms of MT-1. In bovine aortic endothelial cells, Sb35 (Figure 2a) induced MT-1A and MT-2A gene expression in a concentration-dependent manner when treated for 12 h (Figure 2b, upper panels). We observed maximum induction of MT-1A and MT-2A by 100 µM Sb35 at 12 and 24 h, respectively (Figure 2b, lower panels), indicating that Sb35 stimulates the transcriptional induction of MT-1 and MT-2 isoform genes in the cells; decrease in the induction at 48 h is possibly due to the metabolism of Sb35 and/or downregulation of the intracellular signaling activated by Sb35, although the details are unclear. However, induction of MT protein by Sb35 was not observed in Western blot analysis (Figure S1), suggesting that Sb35 compound is a tool to analyze the transcriptional induction of endothelial MT but not an agent for MT induction to protect cells from the toxicity of heavy metals and generated reactive oxygen species. In addition, Sb35 did not generate reactive oxygen species (Figure S2), suggesting that the transcriptional induction of MT by Sb35 is not mediated by reactive oxygen species. In addition, MT-1A, MT-1E, and MT-2A mRNA levels increased by approximately 12, three, and six fold, respectively, after Sb35 treatment. On the other hand, cadmium, a typical MT inducer, increased MT-1A, MT-1E, and MT-2A mRNA levels by approximately 1000-, 15-, and 15-fold, respectively, suggesting that Sb35 induced MT more weakly than cadmium. This appears to be the reason why Sb35 cannot induce MT at the protein level.

View Article: PubMed Central - PubMed

ABSTRACT

Vascular endothelial cells cover the luminal surface of blood vessels and contribute to the prevention of vascular disorders such as atherosclerosis. Metallothionein (MT) is a low molecular weight, cysteine-rich, metal-binding, inducible protein, which protects cells from the toxicity of heavy metals and active oxygen species. Endothelial MT is not induced by inorganic zinc. Adequate tools are required to investigate the mechanisms underlying endothelial MT induction. In the present study, we found that an organoantimony compound, tris(pentafluorophenyl)stibane, induces gene expression of MT-1A and MT-2A, which are subisoforms of MT in bovine aortic endothelial cells. The data reveal that MT-1A is induced by activation of both the MTF-1&ndash;MRE and Nrf2&ndash;ARE pathways, whereas MT-2A expression requires only activation of the MTF-1&ndash;MRE pathway. The present data suggest that the original role of MT-1 is to protect cells from heavy metal toxicity and oxidative stress in the biological defense system, while that of MT-2 is to regulate intracellular zinc metabolism.

No MeSH data available.