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Riluzole increases the amount of latent HSF1 for an amplified heat shock response and cytoprotection.

Yang J, Bridges K, Chen KY, Liu AY - PLoS ONE (2008)

Bottom Line: The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect.We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection.Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, Rutgers State University of New Jersey, Piscataway, New Jersey, United States of America.

ABSTRACT

Background: Induction of the heat shock response (HSR) and increased expression of the heat shock proteins (HSPs) provide mechanisms to ensure proper protein folding, trafficking, and disposition. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases.

Methodology/principal findings: We used a cell-based hsp70-luciferease reporter gene assay system to identify agents that modulate the HSR and show here that clinically relevant concentrations of the FDA-approved ALS drug riluzole significantly increased the heat shock induction of hsp70-luciferse reporter gene. Immuno-Western and -cytochemical analysis of HSF1 show that riluzole increased the amount of cytosolic HSF1 to afford a greater activation of HSF1 upon heat shock. The increased HSF1 contributed centrally to the cytoprotective activity of riluzole as hsf1 gene knockout negated the synergistic activity of riluzole and conditioning heat shock to confer cell survival under oxidative stress. Evidence of a post-transcriptional mechanism for the increase in HSF1 include: quantitation of mRNA(hsf1) by RT-PCR showed no effect of either heat shock or riluzole treatment; riluzole also increased the expression of HSF1 from a CMV-promoter; analysis of the turnover of HSF1 by pulse chase and immunoprecipitation show that riluzole slowed the decay of [(35)S]labeled-HSF1. The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect.

Conclusion/significance: We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection. Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.

Show MeSH

Related in: MedlinePlus

Dose-response effect of riluzole on the basal and heat shock induced hsp70-luciferase reporter gene expression.HeLa cells were transfected with the hsp 70-firefly luciferase reporter DNA and the internal control Renilla luciferase DNA according to methods described in the text. 6 hr after DNA transfection, cells were plated into 96 Stripwell® plates (Corning/Costar 9102). Riluzole was added to individual wells to final concentrations as indicated and incubated at 37°C for 16 hr. The condition used for heat shock was 2 hr at 42°C followed by recovery at 37°C for 4 hr. Controls were incubated at 37°C for an equivalent time. Luciferase activity was assayed using the Dual-Glo luciferase assay system from Promega (E2920) as described. Result on hsp70-reporter is presented as a ratio of hsp70-firefly luciferase over the internal control Renilla luciferase activity, relative to that of the control (no heat shock and no riluzole; the ratio of firefly/Renilla set at 1). Panels (A) and (B) are the average±SD of the result from four independent experiments each with 4 separate determinations. Panels (C) and (D) are the result from one single experiment with four different sample/determinations. Panels (A) and (C) show results on the reporter gene under both basal (37°C) and heat shock (42°C, 2 hr) condition; panels (B) and (D) show the effect of riluzole on the basal luciferase reporter gene activity on an expanded Y-axis. * and ** denotes, respectively, two-tailed t-test with a probability of difference between 0.01–0.05 (significant) and <0.01 (highly significant).
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pone-0002864-g001: Dose-response effect of riluzole on the basal and heat shock induced hsp70-luciferase reporter gene expression.HeLa cells were transfected with the hsp 70-firefly luciferase reporter DNA and the internal control Renilla luciferase DNA according to methods described in the text. 6 hr after DNA transfection, cells were plated into 96 Stripwell® plates (Corning/Costar 9102). Riluzole was added to individual wells to final concentrations as indicated and incubated at 37°C for 16 hr. The condition used for heat shock was 2 hr at 42°C followed by recovery at 37°C for 4 hr. Controls were incubated at 37°C for an equivalent time. Luciferase activity was assayed using the Dual-Glo luciferase assay system from Promega (E2920) as described. Result on hsp70-reporter is presented as a ratio of hsp70-firefly luciferase over the internal control Renilla luciferase activity, relative to that of the control (no heat shock and no riluzole; the ratio of firefly/Renilla set at 1). Panels (A) and (B) are the average±SD of the result from four independent experiments each with 4 separate determinations. Panels (C) and (D) are the result from one single experiment with four different sample/determinations. Panels (A) and (C) show results on the reporter gene under both basal (37°C) and heat shock (42°C, 2 hr) condition; panels (B) and (D) show the effect of riluzole on the basal luciferase reporter gene activity on an expanded Y-axis. * and ** denotes, respectively, two-tailed t-test with a probability of difference between 0.01–0.05 (significant) and <0.01 (highly significant).

Mentions: The effects of riluzole on the basal (control; 37°C) and heat shock-induced (42°) hsp70-luciferase reporter gene activity in the human HeLa cell line is shown in Fig. 1. Fig. 1A and 1B represent the average±standard deviation of four separate experiments each with 4 separate determinations, and Fig. 1C and D is the average±standard deviation of four separate determinations from one experiment. We show in fig. 1A that expression of the hsp70-reporter was induced 36 fold on average by heat shock. Pre-incubation of the cells with riluzole for 16 hr followed by heat shock gave a riluzole dose-dependent amplification of the heat shock induction of hsp70-firefly luciferase reporter gene expression (solid symbol, Fig. 1A); at the optimal riluzole concentration of 1–2 μM, the heat shock induced hsp70-reporter gene activity was ∼2.7×higher than that of heat shock control (without riluzole). Analysis of the effects of riluzole on the basal (i.e. 37°C) expression of hsp70-luciferase as shown in Fig. 1B revealed a qualitatively similar effect: both in terms of the optimal concentration of riluzole (1–2 μM) and the fold of enhancement (2.5–2.8 fold without/with riluzole). The large standard deviation of the heat induced reporter gene activity shown in Fig. 1A (solid symbol) is due to experiment-to-experiment variation in the basal (37°C) reporter gene activity and this translates to a wide range in the fold of induction by heat shock (e.g. in the absence of riluzole, the range of heat shock induction was 15 to 71 fold over that of the 37°C control for the four different experiments in Fig. 1A). Similar variation in the basal HSF1 activity under normal conditions has previously been noted [16]. Within a given experiment using the same pool of transfected cell, however, the sample-to-sample variation was <10% (Fig. 1C and 1D). This pattern was consistently observed throughout the many experiments done over a two-year period. The effect of riluzole in amplifying the hsp70-reporter gene expression required a pre-incubation period: the addition of riluzole at or within 1–2 hr of heat shock (before or after) had little effect (data not shown).


Riluzole increases the amount of latent HSF1 for an amplified heat shock response and cytoprotection.

Yang J, Bridges K, Chen KY, Liu AY - PLoS ONE (2008)

Dose-response effect of riluzole on the basal and heat shock induced hsp70-luciferase reporter gene expression.HeLa cells were transfected with the hsp 70-firefly luciferase reporter DNA and the internal control Renilla luciferase DNA according to methods described in the text. 6 hr after DNA transfection, cells were plated into 96 Stripwell® plates (Corning/Costar 9102). Riluzole was added to individual wells to final concentrations as indicated and incubated at 37°C for 16 hr. The condition used for heat shock was 2 hr at 42°C followed by recovery at 37°C for 4 hr. Controls were incubated at 37°C for an equivalent time. Luciferase activity was assayed using the Dual-Glo luciferase assay system from Promega (E2920) as described. Result on hsp70-reporter is presented as a ratio of hsp70-firefly luciferase over the internal control Renilla luciferase activity, relative to that of the control (no heat shock and no riluzole; the ratio of firefly/Renilla set at 1). Panels (A) and (B) are the average±SD of the result from four independent experiments each with 4 separate determinations. Panels (C) and (D) are the result from one single experiment with four different sample/determinations. Panels (A) and (C) show results on the reporter gene under both basal (37°C) and heat shock (42°C, 2 hr) condition; panels (B) and (D) show the effect of riluzole on the basal luciferase reporter gene activity on an expanded Y-axis. * and ** denotes, respectively, two-tailed t-test with a probability of difference between 0.01–0.05 (significant) and <0.01 (highly significant).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2481402&req=5

pone-0002864-g001: Dose-response effect of riluzole on the basal and heat shock induced hsp70-luciferase reporter gene expression.HeLa cells were transfected with the hsp 70-firefly luciferase reporter DNA and the internal control Renilla luciferase DNA according to methods described in the text. 6 hr after DNA transfection, cells were plated into 96 Stripwell® plates (Corning/Costar 9102). Riluzole was added to individual wells to final concentrations as indicated and incubated at 37°C for 16 hr. The condition used for heat shock was 2 hr at 42°C followed by recovery at 37°C for 4 hr. Controls were incubated at 37°C for an equivalent time. Luciferase activity was assayed using the Dual-Glo luciferase assay system from Promega (E2920) as described. Result on hsp70-reporter is presented as a ratio of hsp70-firefly luciferase over the internal control Renilla luciferase activity, relative to that of the control (no heat shock and no riluzole; the ratio of firefly/Renilla set at 1). Panels (A) and (B) are the average±SD of the result from four independent experiments each with 4 separate determinations. Panels (C) and (D) are the result from one single experiment with four different sample/determinations. Panels (A) and (C) show results on the reporter gene under both basal (37°C) and heat shock (42°C, 2 hr) condition; panels (B) and (D) show the effect of riluzole on the basal luciferase reporter gene activity on an expanded Y-axis. * and ** denotes, respectively, two-tailed t-test with a probability of difference between 0.01–0.05 (significant) and <0.01 (highly significant).
Mentions: The effects of riluzole on the basal (control; 37°C) and heat shock-induced (42°) hsp70-luciferase reporter gene activity in the human HeLa cell line is shown in Fig. 1. Fig. 1A and 1B represent the average±standard deviation of four separate experiments each with 4 separate determinations, and Fig. 1C and D is the average±standard deviation of four separate determinations from one experiment. We show in fig. 1A that expression of the hsp70-reporter was induced 36 fold on average by heat shock. Pre-incubation of the cells with riluzole for 16 hr followed by heat shock gave a riluzole dose-dependent amplification of the heat shock induction of hsp70-firefly luciferase reporter gene expression (solid symbol, Fig. 1A); at the optimal riluzole concentration of 1–2 μM, the heat shock induced hsp70-reporter gene activity was ∼2.7×higher than that of heat shock control (without riluzole). Analysis of the effects of riluzole on the basal (i.e. 37°C) expression of hsp70-luciferase as shown in Fig. 1B revealed a qualitatively similar effect: both in terms of the optimal concentration of riluzole (1–2 μM) and the fold of enhancement (2.5–2.8 fold without/with riluzole). The large standard deviation of the heat induced reporter gene activity shown in Fig. 1A (solid symbol) is due to experiment-to-experiment variation in the basal (37°C) reporter gene activity and this translates to a wide range in the fold of induction by heat shock (e.g. in the absence of riluzole, the range of heat shock induction was 15 to 71 fold over that of the 37°C control for the four different experiments in Fig. 1A). Similar variation in the basal HSF1 activity under normal conditions has previously been noted [16]. Within a given experiment using the same pool of transfected cell, however, the sample-to-sample variation was <10% (Fig. 1C and 1D). This pattern was consistently observed throughout the many experiments done over a two-year period. The effect of riluzole in amplifying the hsp70-reporter gene expression required a pre-incubation period: the addition of riluzole at or within 1–2 hr of heat shock (before or after) had little effect (data not shown).

Bottom Line: The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect.We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection.Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Neuroscience, Rutgers State University of New Jersey, Piscataway, New Jersey, United States of America.

ABSTRACT

Background: Induction of the heat shock response (HSR) and increased expression of the heat shock proteins (HSPs) provide mechanisms to ensure proper protein folding, trafficking, and disposition. The importance of HSPs is underscored by the understanding that protein mis-folding and aggregation contribute centrally to the pathogenesis of neurodegenerative diseases.

Methodology/principal findings: We used a cell-based hsp70-luciferease reporter gene assay system to identify agents that modulate the HSR and show here that clinically relevant concentrations of the FDA-approved ALS drug riluzole significantly increased the heat shock induction of hsp70-luciferse reporter gene. Immuno-Western and -cytochemical analysis of HSF1 show that riluzole increased the amount of cytosolic HSF1 to afford a greater activation of HSF1 upon heat shock. The increased HSF1 contributed centrally to the cytoprotective activity of riluzole as hsf1 gene knockout negated the synergistic activity of riluzole and conditioning heat shock to confer cell survival under oxidative stress. Evidence of a post-transcriptional mechanism for the increase in HSF1 include: quantitation of mRNA(hsf1) by RT-PCR showed no effect of either heat shock or riluzole treatment; riluzole also increased the expression of HSF1 from a CMV-promoter; analysis of the turnover of HSF1 by pulse chase and immunoprecipitation show that riluzole slowed the decay of [(35)S]labeled-HSF1. The effect of riluzole on HSF1 was qualitatively different from that of MG132 and chloroquine, inhibitors of the proteasome and lysosome, respectively, and appeared to involve the chaperone-mediated autophagy pathway as RNAi-mediated knockdown of CMA negated its effect.

Conclusion/significance: We show that riluzole increased the amount of HSF1 to amplify the HSR for cytoprotection. Our study provides novel insight into the mechanism that regulates HSF1 turnover, and identifies the degradation of HSF1 as a target for therapeutics intervention.

Show MeSH
Related in: MedlinePlus