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

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–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.


Expression of Nrf2 and its downstream proteins, Sb35-induced ARE-driven transcriptional activity, and Sb35-induced proteasome inhibition in vascular endothelial cells. (A) Expression of Nrf2, HO-1, and GCLM. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (left panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (right panels), and expression of Nrf2, HO-1, and GCLM was determined by performing Western blotting; (B) ARE-driven transcriptional activity. Vascular endothelial cells transfected with an ARE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and ARE-driven transcriptional activity was determined by performing ARE-directed reporter assay. Data are represented as mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding control; (C) Sb35-induced proteasome inhibition. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 24 h, and ubiquitinated proteins were determined by performing Western blotting.
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ijms-17-01381-f004: Expression of Nrf2 and its downstream proteins, Sb35-induced ARE-driven transcriptional activity, and Sb35-induced proteasome inhibition in vascular endothelial cells. (A) Expression of Nrf2, HO-1, and GCLM. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (left panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (right panels), and expression of Nrf2, HO-1, and GCLM was determined by performing Western blotting; (B) ARE-driven transcriptional activity. Vascular endothelial cells transfected with an ARE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and ARE-driven transcriptional activity was determined by performing ARE-directed reporter assay. Data are represented as mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding control; (C) Sb35-induced proteasome inhibition. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 24 h, and ubiquitinated proteins were determined by performing Western blotting.

Mentions: Sb35 increased the intracellular accumulation of Nrf2 in a concentration- and time-dependent manner and upregulated Nrf2 target proteins such as HO-1 and GCLM (Figure 4A). Sb35 significantly increased the ARE-driven transcriptional activity in a concentration-dependent manner (Figure 4B), confirming that it activates the Nrf2–ARE pathway in vascular endothelial cells. It was shown that Sb35 exhibited proteasome inhibitory activity in a concentration-dependent manner (Figure 4C), suggesting that activation of Nrf2 by Sb35 is at least partly due to the proteasome inhibition that stabilizes Nrf2. Since Sb35 did not generate reactive oxygen species (Figure S2), it is unlikely that activation of Nrf2 by Sb35 is mediated by reactive oxygen species.


Transcriptional Induction of Metallothionein by Tris(pentafluorophenyl)stibane in Cultured Bovine Aortic Endothelial Cells
Expression of Nrf2 and its downstream proteins, Sb35-induced ARE-driven transcriptional activity, and Sb35-induced proteasome inhibition in vascular endothelial cells. (A) Expression of Nrf2, HO-1, and GCLM. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (left panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (right panels), and expression of Nrf2, HO-1, and GCLM was determined by performing Western blotting; (B) ARE-driven transcriptional activity. Vascular endothelial cells transfected with an ARE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and ARE-driven transcriptional activity was determined by performing ARE-directed reporter assay. Data are represented as mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding control; (C) Sb35-induced proteasome inhibition. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 24 h, and ubiquitinated proteins were determined by performing Western blotting.
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Related In: Results  -  Collection

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ijms-17-01381-f004: Expression of Nrf2 and its downstream proteins, Sb35-induced ARE-driven transcriptional activity, and Sb35-induced proteasome inhibition in vascular endothelial cells. (A) Expression of Nrf2, HO-1, and GCLM. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 12 h (left panels) or 100 µM Sb35 for 3, 6, 12, 24, or 48 h (right panels), and expression of Nrf2, HO-1, and GCLM was determined by performing Western blotting; (B) ARE-driven transcriptional activity. Vascular endothelial cells transfected with an ARE reporter vector were treated with or without 10, 50, 100, 150, or 200 µM Sb35 or 5 µM cadmium for 12 h, and ARE-driven transcriptional activity was determined by performing ARE-directed reporter assay. Data are represented as mean ± SE of three representative samples, with each sample obtained from three independent experiments. ** p < 0.01 indicates significantly different from the corresponding control; (C) Sb35-induced proteasome inhibition. Vascular endothelial cells were treated with or without 10, 50, 100, 150, or 200 µM Sb35 for 24 h, and ubiquitinated proteins were determined by performing Western blotting.
Mentions: Sb35 increased the intracellular accumulation of Nrf2 in a concentration- and time-dependent manner and upregulated Nrf2 target proteins such as HO-1 and GCLM (Figure 4A). Sb35 significantly increased the ARE-driven transcriptional activity in a concentration-dependent manner (Figure 4B), confirming that it activates the Nrf2–ARE pathway in vascular endothelial cells. It was shown that Sb35 exhibited proteasome inhibitory activity in a concentration-dependent manner (Figure 4C), suggesting that activation of Nrf2 by Sb35 is at least partly due to the proteasome inhibition that stabilizes Nrf2. Since Sb35 did not generate reactive oxygen species (Figure S2), it is unlikely that activation of Nrf2 by Sb35 is mediated by reactive oxygen species.

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.