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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 effects depend on a posttranscriptional mechanism. (A) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. Western blot analysis showed that H2O2 treatment decreased pAKT (Ser-473) and pFOXO3 (Thr-32) levels and increased pJNK (Thr-183/Tyr-185) levels both in pNEO-Hsp27 neurons and in control neurons. β-Actin served as loading control. (B) CGNs were cotransfected with the luciferase reporter containing the bim promoter sequence and either pNEO-Hsp27 or a control construct. Bim promoter activation was significantly increased after H2O2 (37.5 μM) addition both in pNEO-Hsp27 neurons and in control neurons (*p < 0.05; n = 3). (C) CGNs were pretreated with the proteasome inhibitor epoxomicin (50 nM) or vehicle 45 min before H2O2 (37.5/50 μM) addition. Western blot analysis revealed that H2O2 treatment down-regulated Hsp25 protein level (*p < 0.05; n = 3) and up-regulated Bim levels. Epoxomicin pretreatment prevented Hsp25 down-regulation and increased Bim protein levels induced by H2O2. β-Actin served as loading control. (D) CGNs were transfected with pNEO-Hsp27 or a control construct and pretreated with epoxomicin (50 nM) or vehicle 45 min before H2O2 (37.5 μM) addition. pNEO-Hsp27 neurons presented lower Bim protein levels after H2O2 addition than control neurons in the presence of epoxomicin or vehicle (*p < 0.05; n = 3). β-Actin served as loading control. (E) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. qPCR analysis showed that H2O2 treatment up-regulated bim mRNA levels (*p < 0.05; n = 3). pNEO-Hsp27 neurons treated with H2O2 showed significant higher levels of bim mRNA than H2O2-treated control neurons (*p < 0.05; n = 3). (F) ATF4 mRNA levels were also quantified by qPCR. H2O2 (37.5 μM) addition increased ATF4 mRNA levels (*p < 0.05; n = 3); however pNEO-Hsp27 transfection did not have any significant effect on these levels.
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Figure 3: Hsp27 effects depend on a posttranscriptional mechanism. (A) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. Western blot analysis showed that H2O2 treatment decreased pAKT (Ser-473) and pFOXO3 (Thr-32) levels and increased pJNK (Thr-183/Tyr-185) levels both in pNEO-Hsp27 neurons and in control neurons. β-Actin served as loading control. (B) CGNs were cotransfected with the luciferase reporter containing the bim promoter sequence and either pNEO-Hsp27 or a control construct. Bim promoter activation was significantly increased after H2O2 (37.5 μM) addition both in pNEO-Hsp27 neurons and in control neurons (*p < 0.05; n = 3). (C) CGNs were pretreated with the proteasome inhibitor epoxomicin (50 nM) or vehicle 45 min before H2O2 (37.5/50 μM) addition. Western blot analysis revealed that H2O2 treatment down-regulated Hsp25 protein level (*p < 0.05; n = 3) and up-regulated Bim levels. Epoxomicin pretreatment prevented Hsp25 down-regulation and increased Bim protein levels induced by H2O2. β-Actin served as loading control. (D) CGNs were transfected with pNEO-Hsp27 or a control construct and pretreated with epoxomicin (50 nM) or vehicle 45 min before H2O2 (37.5 μM) addition. pNEO-Hsp27 neurons presented lower Bim protein levels after H2O2 addition than control neurons in the presence of epoxomicin or vehicle (*p < 0.05; n = 3). β-Actin served as loading control. (E) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. qPCR analysis showed that H2O2 treatment up-regulated bim mRNA levels (*p < 0.05; n = 3). pNEO-Hsp27 neurons treated with H2O2 showed significant higher levels of bim mRNA than H2O2-treated control neurons (*p < 0.05; n = 3). (F) ATF4 mRNA levels were also quantified by qPCR. H2O2 (37.5 μM) addition increased ATF4 mRNA levels (*p < 0.05; n = 3); however pNEO-Hsp27 transfection did not have any significant effect on these levels.

Mentions: We next sought to determine the mechanism used by Hsp27 to prevent Bim induction during oxidative stress–induced cell death. First, we analyzed a possible effect of Hsp27 on bim promoter activation by the AKT/FOXO3 pathway. Overexpression of the pNEO-Hsp27 construct in CGNs did not prevent the down-regulation of pAKT (Ser-473) and pFOXO3 (Thr-32) levels induced by H2O2 (37.5 μM) treatment (Figure 3A). We also examined the JNK/AP1 signaling pathway, which is activated by oxidative stress and involved in bim promoter activation (Torres and Forman, 2003; Biswas et al., 2007). Treatment of CGNs with H2O2 (37.5 μM) increased JNK activation, as detected by the up-regulation of pJNK (Thr-183/Tyr-185) levels (Figure 3A). However, overexpression of the pNEO-Hsp27 did not prevent this effect. Finally, we cotransfected CGNs with the luciferase reporter plasmid bearing the bim promoter sequence and the pNEO-Hsp27 construct or alternatively its control vector. Hsp27 overexpression did not prevent the up-regulation of the bim promoter after H2O2 addition (Figure 3B). These results indicate that the effect of Hsp27 on Bim expression does not depend on the regulation of its promoter.


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 effects depend on a posttranscriptional mechanism. (A) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. Western blot analysis showed that H2O2 treatment decreased pAKT (Ser-473) and pFOXO3 (Thr-32) levels and increased pJNK (Thr-183/Tyr-185) levels both in pNEO-Hsp27 neurons and in control neurons. β-Actin served as loading control. (B) CGNs were cotransfected with the luciferase reporter containing the bim promoter sequence and either pNEO-Hsp27 or a control construct. Bim promoter activation was significantly increased after H2O2 (37.5 μM) addition both in pNEO-Hsp27 neurons and in control neurons (*p < 0.05; n = 3). (C) CGNs were pretreated with the proteasome inhibitor epoxomicin (50 nM) or vehicle 45 min before H2O2 (37.5/50 μM) addition. Western blot analysis revealed that H2O2 treatment down-regulated Hsp25 protein level (*p < 0.05; n = 3) and up-regulated Bim levels. Epoxomicin pretreatment prevented Hsp25 down-regulation and increased Bim protein levels induced by H2O2. β-Actin served as loading control. (D) CGNs were transfected with pNEO-Hsp27 or a control construct and pretreated with epoxomicin (50 nM) or vehicle 45 min before H2O2 (37.5 μM) addition. pNEO-Hsp27 neurons presented lower Bim protein levels after H2O2 addition than control neurons in the presence of epoxomicin or vehicle (*p < 0.05; n = 3). β-Actin served as loading control. (E) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. qPCR analysis showed that H2O2 treatment up-regulated bim mRNA levels (*p < 0.05; n = 3). pNEO-Hsp27 neurons treated with H2O2 showed significant higher levels of bim mRNA than H2O2-treated control neurons (*p < 0.05; n = 3). (F) ATF4 mRNA levels were also quantified by qPCR. H2O2 (37.5 μM) addition increased ATF4 mRNA levels (*p < 0.05; n = 3); however pNEO-Hsp27 transfection did not have any significant effect on these levels.
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Figure 3: Hsp27 effects depend on a posttranscriptional mechanism. (A) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. Western blot analysis showed that H2O2 treatment decreased pAKT (Ser-473) and pFOXO3 (Thr-32) levels and increased pJNK (Thr-183/Tyr-185) levels both in pNEO-Hsp27 neurons and in control neurons. β-Actin served as loading control. (B) CGNs were cotransfected with the luciferase reporter containing the bim promoter sequence and either pNEO-Hsp27 or a control construct. Bim promoter activation was significantly increased after H2O2 (37.5 μM) addition both in pNEO-Hsp27 neurons and in control neurons (*p < 0.05; n = 3). (C) CGNs were pretreated with the proteasome inhibitor epoxomicin (50 nM) or vehicle 45 min before H2O2 (37.5/50 μM) addition. Western blot analysis revealed that H2O2 treatment down-regulated Hsp25 protein level (*p < 0.05; n = 3) and up-regulated Bim levels. Epoxomicin pretreatment prevented Hsp25 down-regulation and increased Bim protein levels induced by H2O2. β-Actin served as loading control. (D) CGNs were transfected with pNEO-Hsp27 or a control construct and pretreated with epoxomicin (50 nM) or vehicle 45 min before H2O2 (37.5 μM) addition. pNEO-Hsp27 neurons presented lower Bim protein levels after H2O2 addition than control neurons in the presence of epoxomicin or vehicle (*p < 0.05; n = 3). β-Actin served as loading control. (E) CGNs were transfected with pNEO-Hsp27 or a control construct before H2O2 (37.5 μM) addition. qPCR analysis showed that H2O2 treatment up-regulated bim mRNA levels (*p < 0.05; n = 3). pNEO-Hsp27 neurons treated with H2O2 showed significant higher levels of bim mRNA than H2O2-treated control neurons (*p < 0.05; n = 3). (F) ATF4 mRNA levels were also quantified by qPCR. H2O2 (37.5 μM) addition increased ATF4 mRNA levels (*p < 0.05; n = 3); however pNEO-Hsp27 transfection did not have any significant effect on these levels.
Mentions: We next sought to determine the mechanism used by Hsp27 to prevent Bim induction during oxidative stress–induced cell death. First, we analyzed a possible effect of Hsp27 on bim promoter activation by the AKT/FOXO3 pathway. Overexpression of the pNEO-Hsp27 construct in CGNs did not prevent the down-regulation of pAKT (Ser-473) and pFOXO3 (Thr-32) levels induced by H2O2 (37.5 μM) treatment (Figure 3A). We also examined the JNK/AP1 signaling pathway, which is activated by oxidative stress and involved in bim promoter activation (Torres and Forman, 2003; Biswas et al., 2007). Treatment of CGNs with H2O2 (37.5 μM) increased JNK activation, as detected by the up-regulation of pJNK (Thr-183/Tyr-185) levels (Figure 3A). However, overexpression of the pNEO-Hsp27 did not prevent this effect. Finally, we cotransfected CGNs with the luciferase reporter plasmid bearing the bim promoter sequence and the pNEO-Hsp27 construct or alternatively its control vector. Hsp27 overexpression did not prevent the up-regulation of the bim promoter after H2O2 addition (Figure 3B). These results indicate that the effect of Hsp27 on Bim expression does not depend on the regulation of its promoter.

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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Related in: MedlinePlus