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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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Consequences of genetic and pharmacological blockade of protein degradation pathways on the regulation of HSF1.(A) Effects of riluzole on HSF1 in NIH-3T3 versus Lamp2A RNAi-knockdown cells. Cells in 60 mm plates were treated with 2 μM riluzole at 37°C for 16 hr. For heat shock, cells were placed in a 42°C incubator for 2 hrs. Cells were harvested and aliquots of the RIPA cell extracts containing 10 μg protein were used for immuno-Western blot analysis of HSF1 according to methods described in the text. The position on the gel of the HSF1 and of the heat induced hyperphosphorylated HSF1 is indicated by an *. The relative abundance of the HSF1 protein is indicated at the bottom of the figure. (B) Effects of chloroquine on HSF1. HeLa cells were treated with 0.2 mM chloroquine at 37°C for time periods as indicated. Aliquots of the RIPA cell extract were used for immuno-Western blot detection of HSF1. Extracts from control and heat-shocked cells were included as controls. The position on the gel of the HSF1 and of the heat induced hyperphosphorylated HSF1 is indicated by an *.
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pone-0002864-g007: Consequences of genetic and pharmacological blockade of protein degradation pathways on the regulation of HSF1.(A) Effects of riluzole on HSF1 in NIH-3T3 versus Lamp2A RNAi-knockdown cells. Cells in 60 mm plates were treated with 2 μM riluzole at 37°C for 16 hr. For heat shock, cells were placed in a 42°C incubator for 2 hrs. Cells were harvested and aliquots of the RIPA cell extracts containing 10 μg protein were used for immuno-Western blot analysis of HSF1 according to methods described in the text. The position on the gel of the HSF1 and of the heat induced hyperphosphorylated HSF1 is indicated by an *. The relative abundance of the HSF1 protein is indicated at the bottom of the figure. (B) Effects of chloroquine on HSF1. HeLa cells were treated with 0.2 mM chloroquine at 37°C for time periods as indicated. Aliquots of the RIPA cell extract were used for immuno-Western blot detection of HSF1. Extracts from control and heat-shocked cells were included as controls. The position on the gel of the HSF1 and of the heat induced hyperphosphorylated HSF1 is indicated by an *.

Mentions: There are at least three different lysosomal mechanisms of protein degradation: autophagy (aka, macroautophagy, MA), chaperone-mediated autophagy (CMA), and micro-autophagy pathways. Macro-autophagy is considered a non-selective process of “self-eating” involving the formation of intracellular biomembrane that sequester a portion of the cytosol and whole organelle to form the autophagosome. Chaperone-mediated autophagy, on the other hand, requires the binding of specific substrate protein to the constitutively expressed HSC70 cognate protein and binding of the complex to the lysosomal receptor Lamp2A for the importation and intra-lysosomal degradation of the substrate. We showed in Fig. 7 that whereas riluzole up-regulated the expression of HSF1 of both the control and heat-shocked NIH-3T3 cells, riluzole had little effect in the Lamp2A(-) RNAi knock-down cells [32], [33]. The effect of riluzole is distinct from that of lysozomotropic agents such as chloroquine or ammonium chloride. We show in Fig. 7B that chloroquine (0.2 mM) gave a time-dependent increase in the hyperphosphorylation (supershift in gel) of HSF1. Further, in despite the activation of HSF1, chloroquine treatment had dire consequence in cell viability–most of the cells were dying/dead after 8 hr incubation in the presence of 0.2 mM chloroquine. These effects of chloroquine are qualitatively different from that of riluzole.


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)

Consequences of genetic and pharmacological blockade of protein degradation pathways on the regulation of HSF1.(A) Effects of riluzole on HSF1 in NIH-3T3 versus Lamp2A RNAi-knockdown cells. Cells in 60 mm plates were treated with 2 μM riluzole at 37°C for 16 hr. For heat shock, cells were placed in a 42°C incubator for 2 hrs. Cells were harvested and aliquots of the RIPA cell extracts containing 10 μg protein were used for immuno-Western blot analysis of HSF1 according to methods described in the text. The position on the gel of the HSF1 and of the heat induced hyperphosphorylated HSF1 is indicated by an *. The relative abundance of the HSF1 protein is indicated at the bottom of the figure. (B) Effects of chloroquine on HSF1. HeLa cells were treated with 0.2 mM chloroquine at 37°C for time periods as indicated. Aliquots of the RIPA cell extract were used for immuno-Western blot detection of HSF1. Extracts from control and heat-shocked cells were included as controls. The position on the gel of the HSF1 and of the heat induced hyperphosphorylated HSF1 is indicated by an *.
© Copyright Policy
Related In: Results  -  Collection

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pone-0002864-g007: Consequences of genetic and pharmacological blockade of protein degradation pathways on the regulation of HSF1.(A) Effects of riluzole on HSF1 in NIH-3T3 versus Lamp2A RNAi-knockdown cells. Cells in 60 mm plates were treated with 2 μM riluzole at 37°C for 16 hr. For heat shock, cells were placed in a 42°C incubator for 2 hrs. Cells were harvested and aliquots of the RIPA cell extracts containing 10 μg protein were used for immuno-Western blot analysis of HSF1 according to methods described in the text. The position on the gel of the HSF1 and of the heat induced hyperphosphorylated HSF1 is indicated by an *. The relative abundance of the HSF1 protein is indicated at the bottom of the figure. (B) Effects of chloroquine on HSF1. HeLa cells were treated with 0.2 mM chloroquine at 37°C for time periods as indicated. Aliquots of the RIPA cell extract were used for immuno-Western blot detection of HSF1. Extracts from control and heat-shocked cells were included as controls. The position on the gel of the HSF1 and of the heat induced hyperphosphorylated HSF1 is indicated by an *.
Mentions: There are at least three different lysosomal mechanisms of protein degradation: autophagy (aka, macroautophagy, MA), chaperone-mediated autophagy (CMA), and micro-autophagy pathways. Macro-autophagy is considered a non-selective process of “self-eating” involving the formation of intracellular biomembrane that sequester a portion of the cytosol and whole organelle to form the autophagosome. Chaperone-mediated autophagy, on the other hand, requires the binding of specific substrate protein to the constitutively expressed HSC70 cognate protein and binding of the complex to the lysosomal receptor Lamp2A for the importation and intra-lysosomal degradation of the substrate. We showed in Fig. 7 that whereas riluzole up-regulated the expression of HSF1 of both the control and heat-shocked NIH-3T3 cells, riluzole had little effect in the Lamp2A(-) RNAi knock-down cells [32], [33]. The effect of riluzole is distinct from that of lysozomotropic agents such as chloroquine or ammonium chloride. We show in Fig. 7B that chloroquine (0.2 mM) gave a time-dependent increase in the hyperphosphorylation (supershift in gel) of HSF1. Further, in despite the activation of HSF1, chloroquine treatment had dire consequence in cell viability–most of the cells were dying/dead after 8 hr incubation in the presence of 0.2 mM chloroquine. These effects of chloroquine are qualitatively different from that of riluzole.

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