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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.


The MTF-1–MRE pathway mediates Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) MRE-driven transcriptional activity. Vascular endothelial cells transfected with an MRE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and MRE-driven transcriptional activity was determined by performing the MRE-directed reporter assay; (B) siRNA-mediated MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h, and expression of MTF-1 mRNA was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three samples. ** p < 0.01 indicates significantly different from the corresponding siControl; (C) Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
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ijms-17-01381-f003: The MTF-1–MRE pathway mediates Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) MRE-driven transcriptional activity. Vascular endothelial cells transfected with an MRE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and MRE-driven transcriptional activity was determined by performing the MRE-directed reporter assay; (B) siRNA-mediated MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h, and expression of MTF-1 mRNA was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three samples. ** p < 0.01 indicates significantly different from the corresponding siControl; (C) Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.

Mentions: Next, we investigated whether the MTF-1–MRE pathway was involved in Sb35-induced transcription of endothelial MT isoforms. Expression of MTF-1 was detected by real-time RT-PCR because we failed to detect bovine MTF-1 protein by Western blot analysis. Sb35 only slightly increased the MRE-driven transcriptional activity (Figure 3A). In the MTF-1 knockdown (Figure 3B), Sb35-induced expression of MT-1A and MT-2A was suppressed (Figure 3C), suggesting that the constitutive expression and activity of MTF-1 is sufficient for transcriptional induction of endothelial MT-1A and MT-2A. Sb35 did not induce the transcription of MT-1E, although siRNA-mediated knockdown of MTF-1 suppressed this constitutive expression. As reported previously [35], the MTF-1–MRE pathway is involved in Sb35 induction of endothelial MT-1A and MT-2A.


Transcriptional Induction of Metallothionein by Tris(pentafluorophenyl)stibane in Cultured Bovine Aortic Endothelial Cells
The MTF-1–MRE pathway mediates Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) MRE-driven transcriptional activity. Vascular endothelial cells transfected with an MRE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and MRE-driven transcriptional activity was determined by performing the MRE-directed reporter assay; (B) siRNA-mediated MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h, and expression of MTF-1 mRNA was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three samples. ** p < 0.01 indicates significantly different from the corresponding siControl; (C) Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
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ijms-17-01381-f003: The MTF-1–MRE pathway mediates Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A. (A) MRE-driven transcriptional activity. Vascular endothelial cells transfected with an MRE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and MRE-driven transcriptional activity was determined by performing the MRE-directed reporter assay; (B) siRNA-mediated MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h, and expression of MTF-1 mRNA was determined by performing real-time RT-PCR. Data are expressed as the mean ± SE of three samples. ** p < 0.01 indicates significantly different from the corresponding siControl; (C) Sb35-induced transcription of endothelial MT-1A, MT-1E, and MT-2A after MTF-1 knockdown. Vascular endothelial cells transfected with control or MTF-1 siRNA were treated with or without 50 or 100 µM Sb35 for 12 h. Data are expressed as the mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding siControl.
Mentions: Next, we investigated whether the MTF-1–MRE pathway was involved in Sb35-induced transcription of endothelial MT isoforms. Expression of MTF-1 was detected by real-time RT-PCR because we failed to detect bovine MTF-1 protein by Western blot analysis. Sb35 only slightly increased the MRE-driven transcriptional activity (Figure 3A). In the MTF-1 knockdown (Figure 3B), Sb35-induced expression of MT-1A and MT-2A was suppressed (Figure 3C), suggesting that the constitutive expression and activity of MTF-1 is sufficient for transcriptional induction of endothelial MT-1A and MT-2A. Sb35 did not induce the transcription of MT-1E, although siRNA-mediated knockdown of MTF-1 suppressed this constitutive expression. As reported previously [35], the MTF-1–MRE pathway is involved in Sb35 induction of endothelial MT-1A and MT-2A.

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.