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RhoA Activation Sensitizes Cells to Proteotoxic Stimuli by Abrogating the HSF1-Dependent Heat Shock Response.

Meijering RA, Wiersma M, van Marion DM, Zhang D, Hoogstra-Berends F, Dijkhuis AJ, Schmidt M, Wieland T, Kampinga HH, Henning RH, Brundel BJ - PLoS ONE (2015)

Bottom Line: While active RhoA did not preclude HSF1 nuclear accumulation, phosphorylation, acetylation, or sumoylation, it did impair binding of HSF1 to the hsp genes promoter element HSE.Impaired binding results in suppression of HSP expression and sensitized cells to proteotoxic stress.These results reveal that active RhoA negatively regulates the HSR via attenuation of the HSF1-HSE binding and thus may play a role in sensitizing cells to proteotoxic stimuli.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

ABSTRACT

Background: The heat shock response (HSR) is an ancient and highly conserved program of stress-induced gene expression, aimed at reestablishing protein homeostasis to preserve cellular fitness. Cells that fail to activate or maintain this protective response are hypersensitive to proteotoxic stress. The HSR is mediated by the heat shock transcription factor 1 (HSF1), which binds to conserved heat shock elements (HSE) in the promoter region of heat shock genes, resulting in the expression of heat shock proteins (HSP). Recently, we observed that hyperactivation of RhoA conditions cardiomyocytes for the cardiac arrhythmia atrial fibrillation. Also, the HSR is annihilated in atrial fibrillation, and induction of HSR mitigates sensitization of cells to this disease. Therefore, we hypothesized active RhoA to suppress the HSR resulting in sensitization of cells for proteotoxic stimuli.

Methods and results: Stimulation of RhoA activity significantly suppressed the proteotoxic stress-induced HSR in HL-1 atrial cardiomyocytes as determined with a luciferase reporter construct driven by the HSF1 regulated human HSP70 (HSPA1A) promoter and HSP protein expression by Western Blot analysis. Inversely, RhoA inhibition boosted the proteotoxic stress-induced HSR. While active RhoA did not preclude HSF1 nuclear accumulation, phosphorylation, acetylation, or sumoylation, it did impair binding of HSF1 to the hsp genes promoter element HSE. Impaired binding results in suppression of HSP expression and sensitized cells to proteotoxic stress.

Conclusion: These results reveal that active RhoA negatively regulates the HSR via attenuation of the HSF1-HSE binding and thus may play a role in sensitizing cells to proteotoxic stimuli.

No MeSH data available.


Related in: MedlinePlus

RhoA activation inhibits HSF1 transcriptional activity by suppressing HSF1 binding to HSE, which is independent of HSF1 translocation and post-translational modifications.(A) Nuclear accumulation of HSF1 in response to HS with and without RhoA modulation by calpeptin [Calp] in cardiomyocytes. (B) HSF1 and RhoA levels in TX-100 soluble cytosolic fractions and nuclear-containing fractions of cells with and without HS and RhoA modulation by Calp. The nuclear protein poly-ADP ribose polymerase (PARP) is exclusively present in nuclear containing fractions. C is control non treated cells and V is DMSO (solvent) treated cells. (C) Relative HSPA1A-luc expression in cells transfected with pcDNA3.1+, pS230A, pS303/S307A or pS303/S307D with or without calpeptin (Calp) and subjected to a HS (45°C, 10 min). None of the HSF1 mutants were able to rescue the calpeptin-induced HSPA1A suppression. (D) Top panel: Western blot showing sumoylation (SUMO1 and SUMO2-3) and acetylation status (Ac-lys) of immunoprecipitated (IP) HSF1, obtained from TX-100 nuclear containing fractions of cells treated with or without calpeptin (Calp) and subjected to a HS. Lower panel: Quantified data showing no effect of calpeptin on acetylation and sumoylation levels of HSF1. (E) Representative Western blot of HSPA1A expression in heat shocked cells pre- and post-treated with the HSF1 enhancer GGA with and without calpeptin treatment. GGA results in boosting of the HSPA1A expression after heat shock, but was not able to rescue the calpeptin-induced suppression of the HSPA1A expression. (F) Top panel: EMSA for HSF1 binding to the HSE in response to RhoA modulation and HS, with HeLa nuclear cell extract as a positive control and a competition-assay with a non-labeled HSE probe (cold probe) for DMSO (V) and calpeptin (Calp) treated cells, to determine specific binding to the HSE probe. Lower panel: calpeptin significantly attenuated HSF1-HSE binding compared to V.
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pone.0133553.g004: RhoA activation inhibits HSF1 transcriptional activity by suppressing HSF1 binding to HSE, which is independent of HSF1 translocation and post-translational modifications.(A) Nuclear accumulation of HSF1 in response to HS with and without RhoA modulation by calpeptin [Calp] in cardiomyocytes. (B) HSF1 and RhoA levels in TX-100 soluble cytosolic fractions and nuclear-containing fractions of cells with and without HS and RhoA modulation by Calp. The nuclear protein poly-ADP ribose polymerase (PARP) is exclusively present in nuclear containing fractions. C is control non treated cells and V is DMSO (solvent) treated cells. (C) Relative HSPA1A-luc expression in cells transfected with pcDNA3.1+, pS230A, pS303/S307A or pS303/S307D with or without calpeptin (Calp) and subjected to a HS (45°C, 10 min). None of the HSF1 mutants were able to rescue the calpeptin-induced HSPA1A suppression. (D) Top panel: Western blot showing sumoylation (SUMO1 and SUMO2-3) and acetylation status (Ac-lys) of immunoprecipitated (IP) HSF1, obtained from TX-100 nuclear containing fractions of cells treated with or without calpeptin (Calp) and subjected to a HS. Lower panel: Quantified data showing no effect of calpeptin on acetylation and sumoylation levels of HSF1. (E) Representative Western blot of HSPA1A expression in heat shocked cells pre- and post-treated with the HSF1 enhancer GGA with and without calpeptin treatment. GGA results in boosting of the HSPA1A expression after heat shock, but was not able to rescue the calpeptin-induced suppression of the HSPA1A expression. (F) Top panel: EMSA for HSF1 binding to the HSE in response to RhoA modulation and HS, with HeLa nuclear cell extract as a positive control and a competition-assay with a non-labeled HSE probe (cold probe) for DMSO (V) and calpeptin (Calp) treated cells, to determine specific binding to the HSE probe. Lower panel: calpeptin significantly attenuated HSF1-HSE binding compared to V.

Mentions: As active RhoA suppresses the transcription of all HSF1-regulated HSPs examined, we investigated its action on the main steps of HSF1 transcriptional activation, i.e. nuclear accumulation, post-translational modifications (phosphorylation, acetylation and/or sumoylation), and binding to the HSE. In control cells, HSF1 was mainly located in the cytosol (Fig 4A), but after heat shock, HSF1 accumulates in cell nuclei (Fig 4A) and the TX-100 nuclear-containing fraction (Fig 4B). Activation of RhoA, by calpeptin, did not affect the heat shock-induced HSF1 nuclear translocation (Fig 4A and 4B).


RhoA Activation Sensitizes Cells to Proteotoxic Stimuli by Abrogating the HSF1-Dependent Heat Shock Response.

Meijering RA, Wiersma M, van Marion DM, Zhang D, Hoogstra-Berends F, Dijkhuis AJ, Schmidt M, Wieland T, Kampinga HH, Henning RH, Brundel BJ - PLoS ONE (2015)

RhoA activation inhibits HSF1 transcriptional activity by suppressing HSF1 binding to HSE, which is independent of HSF1 translocation and post-translational modifications.(A) Nuclear accumulation of HSF1 in response to HS with and without RhoA modulation by calpeptin [Calp] in cardiomyocytes. (B) HSF1 and RhoA levels in TX-100 soluble cytosolic fractions and nuclear-containing fractions of cells with and without HS and RhoA modulation by Calp. The nuclear protein poly-ADP ribose polymerase (PARP) is exclusively present in nuclear containing fractions. C is control non treated cells and V is DMSO (solvent) treated cells. (C) Relative HSPA1A-luc expression in cells transfected with pcDNA3.1+, pS230A, pS303/S307A or pS303/S307D with or without calpeptin (Calp) and subjected to a HS (45°C, 10 min). None of the HSF1 mutants were able to rescue the calpeptin-induced HSPA1A suppression. (D) Top panel: Western blot showing sumoylation (SUMO1 and SUMO2-3) and acetylation status (Ac-lys) of immunoprecipitated (IP) HSF1, obtained from TX-100 nuclear containing fractions of cells treated with or without calpeptin (Calp) and subjected to a HS. Lower panel: Quantified data showing no effect of calpeptin on acetylation and sumoylation levels of HSF1. (E) Representative Western blot of HSPA1A expression in heat shocked cells pre- and post-treated with the HSF1 enhancer GGA with and without calpeptin treatment. GGA results in boosting of the HSPA1A expression after heat shock, but was not able to rescue the calpeptin-induced suppression of the HSPA1A expression. (F) Top panel: EMSA for HSF1 binding to the HSE in response to RhoA modulation and HS, with HeLa nuclear cell extract as a positive control and a competition-assay with a non-labeled HSE probe (cold probe) for DMSO (V) and calpeptin (Calp) treated cells, to determine specific binding to the HSE probe. Lower panel: calpeptin significantly attenuated HSF1-HSE binding compared to V.
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Related In: Results  -  Collection

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pone.0133553.g004: RhoA activation inhibits HSF1 transcriptional activity by suppressing HSF1 binding to HSE, which is independent of HSF1 translocation and post-translational modifications.(A) Nuclear accumulation of HSF1 in response to HS with and without RhoA modulation by calpeptin [Calp] in cardiomyocytes. (B) HSF1 and RhoA levels in TX-100 soluble cytosolic fractions and nuclear-containing fractions of cells with and without HS and RhoA modulation by Calp. The nuclear protein poly-ADP ribose polymerase (PARP) is exclusively present in nuclear containing fractions. C is control non treated cells and V is DMSO (solvent) treated cells. (C) Relative HSPA1A-luc expression in cells transfected with pcDNA3.1+, pS230A, pS303/S307A or pS303/S307D with or without calpeptin (Calp) and subjected to a HS (45°C, 10 min). None of the HSF1 mutants were able to rescue the calpeptin-induced HSPA1A suppression. (D) Top panel: Western blot showing sumoylation (SUMO1 and SUMO2-3) and acetylation status (Ac-lys) of immunoprecipitated (IP) HSF1, obtained from TX-100 nuclear containing fractions of cells treated with or without calpeptin (Calp) and subjected to a HS. Lower panel: Quantified data showing no effect of calpeptin on acetylation and sumoylation levels of HSF1. (E) Representative Western blot of HSPA1A expression in heat shocked cells pre- and post-treated with the HSF1 enhancer GGA with and without calpeptin treatment. GGA results in boosting of the HSPA1A expression after heat shock, but was not able to rescue the calpeptin-induced suppression of the HSPA1A expression. (F) Top panel: EMSA for HSF1 binding to the HSE in response to RhoA modulation and HS, with HeLa nuclear cell extract as a positive control and a competition-assay with a non-labeled HSE probe (cold probe) for DMSO (V) and calpeptin (Calp) treated cells, to determine specific binding to the HSE probe. Lower panel: calpeptin significantly attenuated HSF1-HSE binding compared to V.
Mentions: As active RhoA suppresses the transcription of all HSF1-regulated HSPs examined, we investigated its action on the main steps of HSF1 transcriptional activation, i.e. nuclear accumulation, post-translational modifications (phosphorylation, acetylation and/or sumoylation), and binding to the HSE. In control cells, HSF1 was mainly located in the cytosol (Fig 4A), but after heat shock, HSF1 accumulates in cell nuclei (Fig 4A) and the TX-100 nuclear-containing fraction (Fig 4B). Activation of RhoA, by calpeptin, did not affect the heat shock-induced HSF1 nuclear translocation (Fig 4A and 4B).

Bottom Line: While active RhoA did not preclude HSF1 nuclear accumulation, phosphorylation, acetylation, or sumoylation, it did impair binding of HSF1 to the hsp genes promoter element HSE.Impaired binding results in suppression of HSP expression and sensitized cells to proteotoxic stress.These results reveal that active RhoA negatively regulates the HSR via attenuation of the HSF1-HSE binding and thus may play a role in sensitizing cells to proteotoxic stimuli.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacy and Pharmacology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

ABSTRACT

Background: The heat shock response (HSR) is an ancient and highly conserved program of stress-induced gene expression, aimed at reestablishing protein homeostasis to preserve cellular fitness. Cells that fail to activate or maintain this protective response are hypersensitive to proteotoxic stress. The HSR is mediated by the heat shock transcription factor 1 (HSF1), which binds to conserved heat shock elements (HSE) in the promoter region of heat shock genes, resulting in the expression of heat shock proteins (HSP). Recently, we observed that hyperactivation of RhoA conditions cardiomyocytes for the cardiac arrhythmia atrial fibrillation. Also, the HSR is annihilated in atrial fibrillation, and induction of HSR mitigates sensitization of cells to this disease. Therefore, we hypothesized active RhoA to suppress the HSR resulting in sensitization of cells for proteotoxic stimuli.

Methods and results: Stimulation of RhoA activity significantly suppressed the proteotoxic stress-induced HSR in HL-1 atrial cardiomyocytes as determined with a luciferase reporter construct driven by the HSF1 regulated human HSP70 (HSPA1A) promoter and HSP protein expression by Western Blot analysis. Inversely, RhoA inhibition boosted the proteotoxic stress-induced HSR. While active RhoA did not preclude HSF1 nuclear accumulation, phosphorylation, acetylation, or sumoylation, it did impair binding of HSF1 to the hsp genes promoter element HSE. Impaired binding results in suppression of HSP expression and sensitized cells to proteotoxic stress.

Conclusion: These results reveal that active RhoA negatively regulates the HSR via attenuation of the HSF1-HSE binding and thus may play a role in sensitizing cells to proteotoxic stimuli.

No MeSH data available.


Related in: MedlinePlus