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, 156 (2), 412-421

Cadmium-Mediated Activation of the HSP90/HSF1 Pathway Regulated by Reactive Persulfides/Polysulfides

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Cadmium-Mediated Activation of the HSP90/HSF1 Pathway Regulated by Reactive Persulfides/Polysulfides

Yasuhiro Shinkai et al. Toxicol Sci.

Abstract

Cadmium is an environmental electrophile that modifies reactive thiols in proteins, indicating that this heavy metal may modulate redox-signal transduction pathways. The current consensus is that reactive persulfides and polysulfides produced by cystathionine γ-lyase (CSE) and cystathionine β-synthase are highly nucleophilic and thus cadmium may be captured by these reactive sulfur species. It has previously been found that electrophile-mediated covalent modifications of the heat shock protein (HSP) are involved in the activation of heat shock factor 1 (HSF1) pathway. The effects of cadmium on the activation of HSP/HSF1 pathway were investigated in this study. Exposure of bovine aortic endothelial cells to cadmium resulted in modification of HSP90 and HSF1 activation, thereby up-regulating the downstream protein HSP70. The siRNA-mediated knockdown of HSF1 enhanced the cytotoxicity induced by cadmium, suggesting that the HSP90/HSF1 pathway contributes to protection against cadmium toxicity. The knockdown of CSE and/or cystathionine β-synthase decreased the levels of reactive sulfur species in the cells and increased the degree of HSP70 induction and cytotoxicity caused by exposure to cadmium. Overexpression of CSE diminished cadmium-mediated up-regulation of HSP70 and cytotoxicity. These results suggest that cadmium activates HSF1 by modifying HSP90 and that reactive sulfur species regulate the redox signal transduction pathway presumably via capture of cadmium, resulting in protection against cadmium toxicity under toxic conditions.

Keywords: cadmium; cystathionine γ-lyase; heat shock factor 1; heat shock protein; persulfides/polysulfides..

Figures

FIG. 1
FIG. 1
Activation of the HSF1/HSE pathway by cadmium in BAECs. A, Translocation of HSF1 into the nucleus by cadmium. Cells were exposed to cadmium chloride (2 or 10 µM) for 1, 3, or 6 h, then the nucleus and cytosol fractions were subjected to Western blotting analysis using the antibodies indicated. G6PD and Lamin B were used as cytosolic maker and nucleus marker, respectively. B, Activation of HSE-driven transcriptional luciferase activity by cadmium. The HSE-driven luciferase activity was measured using cells that had been exposed to cadmium chloride (1, 5, 10, or 15 µM) for 12 h. C, Cadmium-mediated upregulation of heat shock protein (HSP) mRNA expression. Cells were exposed to cadmium chloride (1, 2.5, 5, or 10 µM) for 12 h, then real-time PCR analyses was performed for the HSP70-A1, HSPA6, HSP90α, and HSP90β genes. D, Increase in HSP proteins caused by exposure to cadmium. Cells were exposed to cadmium chloride (1, 2.5, 5, or 10 µM) for 24 h, then the total cell lysates were subjected to Western blotting analysis using the antibodies indicated. Each value is the mean ± SE of 3 independent experiments. * P < .05 and ** P < .01 compared with the controls.
FIG. 2
FIG. 2
Effect of HSF1 knockdown on HSP induction and cytotoxicity caused by the exposure of BAECs to cadmium. A, Cells were transfected with control siRNA, HSF1 siRNA1, or HSF1 siRNA2 for 48 h, then exposed to cadmium chloride (2.5, 5, or 10 µM) for 12 h. The total cell lysates were subjected to Western blotting analysis using the antibodies indicated. B, Cells were transfected with control siRNA, HSF1 siRNA1, or HSF1 siRNA2 for 48 h, then exposed to cadmium chloride (5, 10, 20, or 30 µM) for 24 h. Cell viability was measured using the MTT assay. Each value is the mean ± SE of 3 independent experiments. * P < .05 and ** P < .01 compared with the siControl results.
FIG. 3
FIG. 3
Effect of cadmium on interactions between HSF1 and HSPs, and the modification of HSP90 by cadmium. A, Dissociation of HSF1 from HSP90, caused by cadmium, in BAECs. Cells were exposed to cadmium chloride (2 or 10 µM) for 3 h, then the total cell lysates were subjected to immunoprecipitation assay using the antibodies indicated. B, Chemical modification of HSP90 by cadmium in BAECs. Cells were exposed to cadmium chloride (2 or 10 µM) for 3 h, then the total cell lysates were subjected to the BPM precipitation assay using the antibodies indicated. C, Quantitative band intensity results for the experiments described in part B. D, Chemical modification of recombinant HSP90 by cadmium. Recombinant human HSP90 (1 µM) was incubated with cadmium chloride (1, 5, 25, or 100 µM) in 50 mM Tris–HCl (pH 7.5) at 25 °C for 30 min. The resulting protein was subjected to the BPM-labeling assay. E, Recombinant human HSP90 (2.5 µM) was incubated with cadmium chloride (10 µM) in 50 mM Tris–HCl (pH 7.5) at 25 °C for 30 min. The resulting proteins were digested with trypsin and analyzed by nanoUPLC-MS system as described in the experimental procedures section. The mass spectrometry and MSE data are shown in Tables 3 and 4, respectively. Each value is the mean ± SE of 3 independent experiments. * P < .05 and ** P < .01 compared with the controls.
FIG. 4
FIG. 4
Effect of siRNA-mediated knockdown of CSE and/or CBS on HSP induction and cytotoxicity in BAECs caused by exposure to cadmium. A, Effect of CSE and CBS knockdown on RSS levels. Cells were transfected with control siRNA, CSE siRNA, and/or CBS siRNA for 48 h, then the cellular RSS levels were determined using the fluorescent probe SSP4. The fluorescence images of the cells (left) and the relative intensity (right) were shown. The scale bar indicates 100 µm. B, Effect of CSE/CBS double knockdown on HSP induction caused by exposure to cadmium. Cells were transfected with control siRNA, CSE siRNA, or CBS siRNA for 48 h, then exposed to cadmium chloride (1, 2.5, 5, or 10 µM) for 24 h. The total cell lysates were subjected to Western blotting analysis using the antibodies indicated. C, Effect of CSE and CBS knockdown on cadmium-induced cytotoxicity. Cells were transfected with control siRNA, CSE siRNA, and/or CBS siRNA for 48 h, then exposed to cadmium chloride (5, 10, 20, or 30 µM) for 24 h. Cell viability was measured using the MTT assay. Each value is the mean ± SE of 3 independent experiments. * P < .05 and ** P < .01 compared with the siControl results.
FIG. 5
FIG. 5
Effect of CSE overexpression on HSP induction and cytotoxicity in BAECs caused by exposure to cadmium. A, Effect of CSE overexpression on HSP induction caused by exposure to cadmium. Cells were transfected with pClneo alone or pClneo-HA-CSE for 24 h, then exposed to cadmium chloride (1, 2.5, 5, or 10 µM) for 24 h. The total cell lysates were subjected to Western blotting analysis using the antibodies indicated. B, Effect of CSE overexpression on cadmium-induced cytotoxicity. Cells were transfected with pClneo alone or pClneo-HA-CSE for 24 h, then exposed to cadmium chloride (2.5, 5, 10, 20, or 30 µM) for 24 h. Cell viability was measured using the MTT assay. Each value is the mean ± SE of 3 independent experiments. * P < .05 and ** P < .01 compared with the siControl results.

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