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Hsp27 binding to the 3'UTR of bim mRNA prevents neuronal death during oxidative stress-induced injury: a novel cytoprotective mechanism.

Dávila D, Jiménez-Mateos EM, Mooney CM, Velasco G, Henshall DC, Prehn JH - Mol. Biol. Cell (2014)

Bottom Line: This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27.Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3'UTR mRNA sequence was cloned downstream of a luciferase gene.These results suggest that repression of bim mRNA translation through binding to the 3'UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics and RCSI Centre for the Study of Neurological Disorders, Royal College of Surgeons in Ireland, Dublin 2, Ireland Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain daviddav@ucm.es prehn@rcsi.ie.

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Hsp27 regulates Bim protein levels and oxidative stress–induced cell death. (A) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. pNEO-Hsp27 neurons displayed significantly lower Bim protein levels than did control neurons after H2O2 treatment (*p < 0.05; n = 3). β-Actin served as loading control. (B) At 4 h after H2O2 (37.5 μM) treatment, pNEO-Hsp27 neurons displayed lower cytosolic cytochrome C protein levels than in control neurons. Mitochondrial membrane levels of cytochrome C were also higher in pNEO-Hsp27 neurons than in control neurons. IκB-α protein served as marker of cytosolic fraction and LAMP1 as marker of the membrane fraction, including mitochondrial membranes. (C) CGNs transfected with pNEO-Hsp27 or a control construct were treated with H2O2 (37.5 μM) or sham conditions and live-stained with Hoechst 4–6 h later. pNEO-Hsp27 neurons showed a significantly lower percentage of cells with pyknotic nuclei than control neurons after H2O2 addition (*p < 0.05; n = 3). Bar, 2.5 μm. (D) CGNs were electroporated with Hsp25 siRNA (rodent homologue of Hsp27; 100/200 pmol) or control siRNA (200 pmol), and Bim and Hsp25 protein levels were analyzed after 72 h by Western blot. Hsp25 levels were depleted in Hsp25 siRNA (200 pmol) neurons, which also displayed significantly higher levels of Bim than control siRNA neurons (*p < 0.05; n = 3). β-Actin served as loading control. (E) CGNs electroporated with Hsp25 siRNA or control siRNA were live-stained with Hoechst. Hsp25 siRNA neurons presented a significantly higher percentage of cells with pyknotic nuclei than control neurons (*p < 0.05; n = 3). Bar, 2.5 μm.
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Figure 2: Hsp27 regulates Bim protein levels and oxidative stress–induced cell death. (A) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. pNEO-Hsp27 neurons displayed significantly lower Bim protein levels than did control neurons after H2O2 treatment (*p < 0.05; n = 3). β-Actin served as loading control. (B) At 4 h after H2O2 (37.5 μM) treatment, pNEO-Hsp27 neurons displayed lower cytosolic cytochrome C protein levels than in control neurons. Mitochondrial membrane levels of cytochrome C were also higher in pNEO-Hsp27 neurons than in control neurons. IκB-α protein served as marker of cytosolic fraction and LAMP1 as marker of the membrane fraction, including mitochondrial membranes. (C) CGNs transfected with pNEO-Hsp27 or a control construct were treated with H2O2 (37.5 μM) or sham conditions and live-stained with Hoechst 4–6 h later. pNEO-Hsp27 neurons showed a significantly lower percentage of cells with pyknotic nuclei than control neurons after H2O2 addition (*p < 0.05; n = 3). Bar, 2.5 μm. (D) CGNs were electroporated with Hsp25 siRNA (rodent homologue of Hsp27; 100/200 pmol) or control siRNA (200 pmol), and Bim and Hsp25 protein levels were analyzed after 72 h by Western blot. Hsp25 levels were depleted in Hsp25 siRNA (200 pmol) neurons, which also displayed significantly higher levels of Bim than control siRNA neurons (*p < 0.05; n = 3). β-Actin served as loading control. (E) CGNs electroporated with Hsp25 siRNA or control siRNA were live-stained with Hoechst. Hsp25 siRNA neurons presented a significantly higher percentage of cells with pyknotic nuclei than control neurons (*p < 0.05; n = 3). Bar, 2.5 μm.

Mentions: Having observed the down-regulation of Hsp25 levels induced by H2O2 treatment, we analyzed its possible relation with the expression of Bim. For this purpose, we transfected CGNs with a construct expressing the wild-type form of the human Hsp27 (pNEO-Hsp27). Despite the fact that we also observed a decrease in levels of Hsp27 in H2O2-treated CGNs, transfection of Hsp27 prevented the up-regulation of Bim protein levels (Figure 2A) and subsequent apoptotic events such as cytosolic accumulation of cytochrome C (Figure 2B) or accumulation of the active caspase 3 subunit (Supplemental Figure S1A). Hsp27 transfection in CGNs also prevented the nuclear pyknosis induced by H2O2 treatment (Figure 2C). On the other hand, depletion of the endogenous Hsp25 levels in CGNs by expression of a specific small interfering RNA (siRNA) targeting Hsp25 (200 pmol) was able to mimic the effect of H2O2 treatment, up-regulating significantly Bim protein levels and inducing nuclear pyknosis (Figure 2, D and E).


Hsp27 binding to the 3'UTR of bim mRNA prevents neuronal death during oxidative stress-induced injury: a novel cytoprotective mechanism.

Dávila D, Jiménez-Mateos EM, Mooney CM, Velasco G, Henshall DC, Prehn JH - Mol. Biol. Cell (2014)

Hsp27 regulates Bim protein levels and oxidative stress–induced cell death. (A) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. pNEO-Hsp27 neurons displayed significantly lower Bim protein levels than did control neurons after H2O2 treatment (*p < 0.05; n = 3). β-Actin served as loading control. (B) At 4 h after H2O2 (37.5 μM) treatment, pNEO-Hsp27 neurons displayed lower cytosolic cytochrome C protein levels than in control neurons. Mitochondrial membrane levels of cytochrome C were also higher in pNEO-Hsp27 neurons than in control neurons. IκB-α protein served as marker of cytosolic fraction and LAMP1 as marker of the membrane fraction, including mitochondrial membranes. (C) CGNs transfected with pNEO-Hsp27 or a control construct were treated with H2O2 (37.5 μM) or sham conditions and live-stained with Hoechst 4–6 h later. pNEO-Hsp27 neurons showed a significantly lower percentage of cells with pyknotic nuclei than control neurons after H2O2 addition (*p < 0.05; n = 3). Bar, 2.5 μm. (D) CGNs were electroporated with Hsp25 siRNA (rodent homologue of Hsp27; 100/200 pmol) or control siRNA (200 pmol), and Bim and Hsp25 protein levels were analyzed after 72 h by Western blot. Hsp25 levels were depleted in Hsp25 siRNA (200 pmol) neurons, which also displayed significantly higher levels of Bim than control siRNA neurons (*p < 0.05; n = 3). β-Actin served as loading control. (E) CGNs electroporated with Hsp25 siRNA or control siRNA were live-stained with Hoechst. Hsp25 siRNA neurons presented a significantly higher percentage of cells with pyknotic nuclei than control neurons (*p < 0.05; n = 3). Bar, 2.5 μm.
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Related In: Results  -  Collection

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Figure 2: Hsp27 regulates Bim protein levels and oxidative stress–induced cell death. (A) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. pNEO-Hsp27 neurons displayed significantly lower Bim protein levels than did control neurons after H2O2 treatment (*p < 0.05; n = 3). β-Actin served as loading control. (B) At 4 h after H2O2 (37.5 μM) treatment, pNEO-Hsp27 neurons displayed lower cytosolic cytochrome C protein levels than in control neurons. Mitochondrial membrane levels of cytochrome C were also higher in pNEO-Hsp27 neurons than in control neurons. IκB-α protein served as marker of cytosolic fraction and LAMP1 as marker of the membrane fraction, including mitochondrial membranes. (C) CGNs transfected with pNEO-Hsp27 or a control construct were treated with H2O2 (37.5 μM) or sham conditions and live-stained with Hoechst 4–6 h later. pNEO-Hsp27 neurons showed a significantly lower percentage of cells with pyknotic nuclei than control neurons after H2O2 addition (*p < 0.05; n = 3). Bar, 2.5 μm. (D) CGNs were electroporated with Hsp25 siRNA (rodent homologue of Hsp27; 100/200 pmol) or control siRNA (200 pmol), and Bim and Hsp25 protein levels were analyzed after 72 h by Western blot. Hsp25 levels were depleted in Hsp25 siRNA (200 pmol) neurons, which also displayed significantly higher levels of Bim than control siRNA neurons (*p < 0.05; n = 3). β-Actin served as loading control. (E) CGNs electroporated with Hsp25 siRNA or control siRNA were live-stained with Hoechst. Hsp25 siRNA neurons presented a significantly higher percentage of cells with pyknotic nuclei than control neurons (*p < 0.05; n = 3). Bar, 2.5 μm.
Mentions: Having observed the down-regulation of Hsp25 levels induced by H2O2 treatment, we analyzed its possible relation with the expression of Bim. For this purpose, we transfected CGNs with a construct expressing the wild-type form of the human Hsp27 (pNEO-Hsp27). Despite the fact that we also observed a decrease in levels of Hsp27 in H2O2-treated CGNs, transfection of Hsp27 prevented the up-regulation of Bim protein levels (Figure 2A) and subsequent apoptotic events such as cytosolic accumulation of cytochrome C (Figure 2B) or accumulation of the active caspase 3 subunit (Supplemental Figure S1A). Hsp27 transfection in CGNs also prevented the nuclear pyknosis induced by H2O2 treatment (Figure 2C). On the other hand, depletion of the endogenous Hsp25 levels in CGNs by expression of a specific small interfering RNA (siRNA) targeting Hsp25 (200 pmol) was able to mimic the effect of H2O2 treatment, up-regulating significantly Bim protein levels and inducing nuclear pyknosis (Figure 2, D and E).

Bottom Line: This effect could not be explained by proteasomal degradation of Bim or bim promoter inhibition; however, it was associated with a specific increase in the levels of bim mRNA and with its binding to Hsp27.Finally, we determined that enhanced Hsp27 expression in neurons exposed to H2O2 or glutamate prevented the translation of a reporter plasmid where bim-3'UTR mRNA sequence was cloned downstream of a luciferase gene.These results suggest that repression of bim mRNA translation through binding to the 3'UTR constitutes a novel cytoprotective mechanism of Hsp27 during stress in neurons.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics and RCSI Centre for the Study of Neurological Disorders, Royal College of Surgeons in Ireland, Dublin 2, Ireland Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain daviddav@ucm.es prehn@rcsi.ie.

Show MeSH
Related in: MedlinePlus