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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.

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Related in: MedlinePlus

Synergistic effects of riluzle and conditioning heat shock in conferring cell survival under oxidative stress requires a functional HSF1.(A) Dose response effect of riluzole and conditioning heat shock on cell viability in the absence and presence of oxidative stress challenge. HeLa cells in 96 Strip-well plate were used. The conditions for riluzole treatment and conditioning heat shock were as described in the text. To test for cell survival under conditions of oxidative stress, 20 μM sodium arsenite was added and incubated at 37°C for 24 hr. Viability of the cells was determined using the CellTiter-Glo luminescent reagent from Promega Inc. Cell viability signal, relative to that of the untreated control, is plotted as a function of the concentration of riluzole added. Result represents the average of four independent determinations±standard deviation. * and ** denotes, respectively, two-tailed t-test with a probability of difference between 0.01–0.05 (significant) and <0.01 (highly significant) of the riluzole-treated samples from that of the minus riluzole control. (B) The cytoprotective activity of riluzole and conditioning heat shock requires a functional HSF1 protein. Murine embryo fibroblasts derived from hsf1−/− knockout mice [25] and its hsf1+/+ normal littermate were plated in 96 a Stripwell plate. The conditions used from the treatment of cells with riluzole, conditioning heat shock at 42°C for 2 hr, and assessment of the “cell-kill” effects of arsenite were as described in the text. The figure presents data on viability of the arsenite-challenged cells pretreated with various concentrations of riluzole, without and with conditioning heat shock. Data on viability of the control cells (i.e. without arsenic challenge) are not included in Fig. 4B as they were qualitatively similar to that of the HeLa cells shown in Fig. 4A.
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pone-0002864-g004: Synergistic effects of riluzle and conditioning heat shock in conferring cell survival under oxidative stress requires a functional HSF1.(A) Dose response effect of riluzole and conditioning heat shock on cell viability in the absence and presence of oxidative stress challenge. HeLa cells in 96 Strip-well plate were used. The conditions for riluzole treatment and conditioning heat shock were as described in the text. To test for cell survival under conditions of oxidative stress, 20 μM sodium arsenite was added and incubated at 37°C for 24 hr. Viability of the cells was determined using the CellTiter-Glo luminescent reagent from Promega Inc. Cell viability signal, relative to that of the untreated control, is plotted as a function of the concentration of riluzole added. Result represents the average of four independent determinations±standard deviation. * and ** denotes, respectively, two-tailed t-test with a probability of difference between 0.01–0.05 (significant) and <0.01 (highly significant) of the riluzole-treated samples from that of the minus riluzole control. (B) The cytoprotective activity of riluzole and conditioning heat shock requires a functional HSF1 protein. Murine embryo fibroblasts derived from hsf1−/− knockout mice [25] and its hsf1+/+ normal littermate were plated in 96 a Stripwell plate. The conditions used from the treatment of cells with riluzole, conditioning heat shock at 42°C for 2 hr, and assessment of the “cell-kill” effects of arsenite were as described in the text. The figure presents data on viability of the arsenite-challenged cells pretreated with various concentrations of riluzole, without and with conditioning heat shock. Data on viability of the control cells (i.e. without arsenic challenge) are not included in Fig. 4B as they were qualitatively similar to that of the HeLa cells shown in Fig. 4A.

Mentions: Induction of the HSPs provides an important cytoprotective mechanism for survival under stress. In this context, our observation that riluzole increased HSF1 reserve to allow for a more robust mobilization of HSF1 and induction of the HSR would suggest that riluzole and conditioning heat shock could have synergistic effect in protecting cells against stress-induced injury and death. In Fig. 4A, we show the dose-dependent effects of riluzole and conditioning heat shock on cell viability in the absence and presence of sodium arsenite-induced oxidative stress challenge [23], [24]. Cell viability was expressed as a % of that of the control (no riluzole, no heat shock, no arsenite). We showed that: (1) arsenite (20 μM, 24 hr) by itself decreased cell viability by 75%; this cytotoxic effect was countered somewhat by the pretreatment of cells with riluzole with an optimal protection observed at 1 μM (viability 25 & 45% for 0 and 1 μM riluzole, respectively; solid circle, solid line). (2) Conditioning heat shock (pre-HS) increase cell survival from 25 to 34% in the arsenite-challenged cells. Further, the treatment of cells with riluzole followed by conditioning heat shock had a synergistic effect in promoting cell survival when challenged with arsenite (solid triangle, solid line); at 1 μM riluzole, the percentage of viable cells was 75 versus 45%, with and without conditioning heat shock, respectively. (3) In the un-challenged cells (no arsenite): riluzole by itself had a small but reproducible effect in promoting cell growth/viability (open circle), whereas conditioning heat shock reduced cell growth/viability by ∼10% (open triangle).


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)

Synergistic effects of riluzle and conditioning heat shock in conferring cell survival under oxidative stress requires a functional HSF1.(A) Dose response effect of riluzole and conditioning heat shock on cell viability in the absence and presence of oxidative stress challenge. HeLa cells in 96 Strip-well plate were used. The conditions for riluzole treatment and conditioning heat shock were as described in the text. To test for cell survival under conditions of oxidative stress, 20 μM sodium arsenite was added and incubated at 37°C for 24 hr. Viability of the cells was determined using the CellTiter-Glo luminescent reagent from Promega Inc. Cell viability signal, relative to that of the untreated control, is plotted as a function of the concentration of riluzole added. Result represents the average of four independent determinations±standard deviation. * and ** denotes, respectively, two-tailed t-test with a probability of difference between 0.01–0.05 (significant) and <0.01 (highly significant) of the riluzole-treated samples from that of the minus riluzole control. (B) The cytoprotective activity of riluzole and conditioning heat shock requires a functional HSF1 protein. Murine embryo fibroblasts derived from hsf1−/− knockout mice [25] and its hsf1+/+ normal littermate were plated in 96 a Stripwell plate. The conditions used from the treatment of cells with riluzole, conditioning heat shock at 42°C for 2 hr, and assessment of the “cell-kill” effects of arsenite were as described in the text. The figure presents data on viability of the arsenite-challenged cells pretreated with various concentrations of riluzole, without and with conditioning heat shock. Data on viability of the control cells (i.e. without arsenic challenge) are not included in Fig. 4B as they were qualitatively similar to that of the HeLa cells shown in Fig. 4A.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481402&req=5

pone-0002864-g004: Synergistic effects of riluzle and conditioning heat shock in conferring cell survival under oxidative stress requires a functional HSF1.(A) Dose response effect of riluzole and conditioning heat shock on cell viability in the absence and presence of oxidative stress challenge. HeLa cells in 96 Strip-well plate were used. The conditions for riluzole treatment and conditioning heat shock were as described in the text. To test for cell survival under conditions of oxidative stress, 20 μM sodium arsenite was added and incubated at 37°C for 24 hr. Viability of the cells was determined using the CellTiter-Glo luminescent reagent from Promega Inc. Cell viability signal, relative to that of the untreated control, is plotted as a function of the concentration of riluzole added. Result represents the average of four independent determinations±standard deviation. * and ** denotes, respectively, two-tailed t-test with a probability of difference between 0.01–0.05 (significant) and <0.01 (highly significant) of the riluzole-treated samples from that of the minus riluzole control. (B) The cytoprotective activity of riluzole and conditioning heat shock requires a functional HSF1 protein. Murine embryo fibroblasts derived from hsf1−/− knockout mice [25] and its hsf1+/+ normal littermate were plated in 96 a Stripwell plate. The conditions used from the treatment of cells with riluzole, conditioning heat shock at 42°C for 2 hr, and assessment of the “cell-kill” effects of arsenite were as described in the text. The figure presents data on viability of the arsenite-challenged cells pretreated with various concentrations of riluzole, without and with conditioning heat shock. Data on viability of the control cells (i.e. without arsenic challenge) are not included in Fig. 4B as they were qualitatively similar to that of the HeLa cells shown in Fig. 4A.
Mentions: Induction of the HSPs provides an important cytoprotective mechanism for survival under stress. In this context, our observation that riluzole increased HSF1 reserve to allow for a more robust mobilization of HSF1 and induction of the HSR would suggest that riluzole and conditioning heat shock could have synergistic effect in protecting cells against stress-induced injury and death. In Fig. 4A, we show the dose-dependent effects of riluzole and conditioning heat shock on cell viability in the absence and presence of sodium arsenite-induced oxidative stress challenge [23], [24]. Cell viability was expressed as a % of that of the control (no riluzole, no heat shock, no arsenite). We showed that: (1) arsenite (20 μM, 24 hr) by itself decreased cell viability by 75%; this cytotoxic effect was countered somewhat by the pretreatment of cells with riluzole with an optimal protection observed at 1 μM (viability 25 & 45% for 0 and 1 μM riluzole, respectively; solid circle, solid line). (2) Conditioning heat shock (pre-HS) increase cell survival from 25 to 34% in the arsenite-challenged cells. Further, the treatment of cells with riluzole followed by conditioning heat shock had a synergistic effect in promoting cell survival when challenged with arsenite (solid triangle, solid line); at 1 μM riluzole, the percentage of viable cells was 75 versus 45%, with and without conditioning heat shock, respectively. (3) In the un-challenged cells (no arsenite): riluzole by itself had a small but reproducible effect in promoting cell growth/viability (open circle), whereas conditioning heat shock reduced cell growth/viability by ∼10% (open triangle).

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