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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

HSF1 immuno-fluorescence photomicrographs of control and heat shocked HeLa cells without and with riluzole pre-treatment.Riluzole (2 μM) was added to HeLa cells and incubated at 37°C for 16 hr (panels e–h). Designated plates of cells were heat shocked at 42°C for 2 hr (panels c, d, g, h). Control cells were incubated at 37° for equivalent time periods. Cells were fixed, permeabilized and stained for HSF1 according to methods described in the text (panels a, c, e and g). Nuclei were counter stained with Hoechst 33342 (panels b, d, f and h). Images of representative fields were captured with a SPOT camera system (Diagnostic Instruments, Inc., Sterling Heights, MI).
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pone-0002864-g003: HSF1 immuno-fluorescence photomicrographs of control and heat shocked HeLa cells without and with riluzole pre-treatment.Riluzole (2 μM) was added to HeLa cells and incubated at 37°C for 16 hr (panels e–h). Designated plates of cells were heat shocked at 42°C for 2 hr (panels c, d, g, h). Control cells were incubated at 37° for equivalent time periods. Cells were fixed, permeabilized and stained for HSF1 according to methods described in the text (panels a, c, e and g). Nuclei were counter stained with Hoechst 33342 (panels b, d, f and h). Images of representative fields were captured with a SPOT camera system (Diagnostic Instruments, Inc., Sterling Heights, MI).

Mentions: We also used immunocytochemical staining techniques to evaluate the effects of heat shock and riluzole treatment on the distribution and amount of HSF1. We show in Fig. 3 that HSF1 had a diffuse cytoplasmic and nuclear distribution in unstressed cells (panel a). Heat shock promoted the nuclear localization of HSF1 (panel c). Riluzole increased the diffused HSF1 staining intensity in both the cytosol and nucleus (panel e), and the pretreatment with riluzole followed by heat shock gave a greater increase in the nuclear HSF1 when compared to heat shock alone although there remained some diffuse staining in the cytosol (panel g).


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)

HSF1 immuno-fluorescence photomicrographs of control and heat shocked HeLa cells without and with riluzole pre-treatment.Riluzole (2 μM) was added to HeLa cells and incubated at 37°C for 16 hr (panels e–h). Designated plates of cells were heat shocked at 42°C for 2 hr (panels c, d, g, h). Control cells were incubated at 37° for equivalent time periods. Cells were fixed, permeabilized and stained for HSF1 according to methods described in the text (panels a, c, e and g). Nuclei were counter stained with Hoechst 33342 (panels b, d, f and h). Images of representative fields were captured with a SPOT camera system (Diagnostic Instruments, Inc., Sterling Heights, MI).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481402&req=5

pone-0002864-g003: HSF1 immuno-fluorescence photomicrographs of control and heat shocked HeLa cells without and with riluzole pre-treatment.Riluzole (2 μM) was added to HeLa cells and incubated at 37°C for 16 hr (panels e–h). Designated plates of cells were heat shocked at 42°C for 2 hr (panels c, d, g, h). Control cells were incubated at 37° for equivalent time periods. Cells were fixed, permeabilized and stained for HSF1 according to methods described in the text (panels a, c, e and g). Nuclei were counter stained with Hoechst 33342 (panels b, d, f and h). Images of representative fields were captured with a SPOT camera system (Diagnostic Instruments, Inc., Sterling Heights, MI).
Mentions: We also used immunocytochemical staining techniques to evaluate the effects of heat shock and riluzole treatment on the distribution and amount of HSF1. We show in Fig. 3 that HSF1 had a diffuse cytoplasmic and nuclear distribution in unstressed cells (panel a). Heat shock promoted the nuclear localization of HSF1 (panel c). Riluzole increased the diffused HSF1 staining intensity in both the cytosol and nucleus (panel e), and the pretreatment with riluzole followed by heat shock gave a greater increase in the nuclear HSF1 when compared to heat shock alone although there remained some diffuse staining in the cytosol (panel g).

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