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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 attenuates HSPA1A expression.(A) Relative luciferase expression of a reporter construct driven by the SRE promoter (downstream target of RhoA/ROCK signaling) in HL-1 cardiomyocytes transfected with empty plasmid (pC, pcDNA3.1+), C3T exoenzyme plasmid (pC3T) or RhoA-WT encoding plasmid (pRhoA). (B) Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in cardiomyocytes transfected with pC, pC3T or pRhoA. (C) Relative HSPA1A-luc expression in cardiomyocytes transfected with pC, pC3T or pRhoA and subjected to a HS (45°C, 10 min), white bars represent control non-HS, whereas black bars represent HS cells. (D) Top panel shows a representative Western blot with HSPA1A levels of cardiomyocytes transfected with pC, pC3T or pRhoA and subjected to a HS. Below, quantified data of HSPA1A/GAPDH levels for conditions as indicated. (E) Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in cardiomyocytes transfected with empty plasmid pC, pC3T, pP190RhoGAP, pRhoA-n19, pRhoA or pRhoA-v14 without (E) and with (F) HS. (G) RhoA activation attenuates HSPA1A expression in human HEK-293 cells. Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in HEK293 cells transfected with empty plasmid pC, pC3T, or pRhoA. *P<0.05, **P<0.01, ***P<0.001 compared to control pC and #P<0.05, ##P<0.01 compared to pC or pC HS. White bar in panel (F) represents control non-HS cells, whereas black bars represent HS cells.
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pone.0133553.g001: RhoA activation attenuates HSPA1A expression.(A) Relative luciferase expression of a reporter construct driven by the SRE promoter (downstream target of RhoA/ROCK signaling) in HL-1 cardiomyocytes transfected with empty plasmid (pC, pcDNA3.1+), C3T exoenzyme plasmid (pC3T) or RhoA-WT encoding plasmid (pRhoA). (B) Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in cardiomyocytes transfected with pC, pC3T or pRhoA. (C) Relative HSPA1A-luc expression in cardiomyocytes transfected with pC, pC3T or pRhoA and subjected to a HS (45°C, 10 min), white bars represent control non-HS, whereas black bars represent HS cells. (D) Top panel shows a representative Western blot with HSPA1A levels of cardiomyocytes transfected with pC, pC3T or pRhoA and subjected to a HS. Below, quantified data of HSPA1A/GAPDH levels for conditions as indicated. (E) Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in cardiomyocytes transfected with empty plasmid pC, pC3T, pP190RhoGAP, pRhoA-n19, pRhoA or pRhoA-v14 without (E) and with (F) HS. (G) RhoA activation attenuates HSPA1A expression in human HEK-293 cells. Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in HEK293 cells transfected with empty plasmid pC, pC3T, or pRhoA. *P<0.05, **P<0.01, ***P<0.001 compared to control pC and #P<0.05, ##P<0.01 compared to pC or pC HS. White bar in panel (F) represents control non-HS cells, whereas black bars represent HS cells.

Mentions: To determine if RhoA signaling affects the HSR, control and heat shocked (HS) HL-1 cardiomyocytes were transfected with a luciferase reporter construct driven by the HSF1 regulated human HSP70 (HSPA1A) promoter (HSPA1A-luc). RhoA activity was inhibited by transfection of the C3-exoenzyme (pC3T) construct, which inhibits the RhoA pathway, or activated by overexpression of RhoA by transfection of a RhoA wild type construct (pRhoA) (S1A and S1B Fig). The effectiveness of these manipulations was validated using co-transfection with a luciferase construct driven by the RhoA-dependent serum response element (SRE) (Fig 1A). pRhoA expression reduced both basal HSPA1A-luc (Fig 1B) and also strongly suppressed the heat-induced increase in HSPA1A-luc expression (Fig 1C). pRhoA also significantly reduced the level of endogenous HSPA1A protein expression in the total fraction of heat-shocked cells (Fig 1D).


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 attenuates HSPA1A expression.(A) Relative luciferase expression of a reporter construct driven by the SRE promoter (downstream target of RhoA/ROCK signaling) in HL-1 cardiomyocytes transfected with empty plasmid (pC, pcDNA3.1+), C3T exoenzyme plasmid (pC3T) or RhoA-WT encoding plasmid (pRhoA). (B) Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in cardiomyocytes transfected with pC, pC3T or pRhoA. (C) Relative HSPA1A-luc expression in cardiomyocytes transfected with pC, pC3T or pRhoA and subjected to a HS (45°C, 10 min), white bars represent control non-HS, whereas black bars represent HS cells. (D) Top panel shows a representative Western blot with HSPA1A levels of cardiomyocytes transfected with pC, pC3T or pRhoA and subjected to a HS. Below, quantified data of HSPA1A/GAPDH levels for conditions as indicated. (E) Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in cardiomyocytes transfected with empty plasmid pC, pC3T, pP190RhoGAP, pRhoA-n19, pRhoA or pRhoA-v14 without (E) and with (F) HS. (G) RhoA activation attenuates HSPA1A expression in human HEK-293 cells. Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in HEK293 cells transfected with empty plasmid pC, pC3T, or pRhoA. *P<0.05, **P<0.01, ***P<0.001 compared to control pC and #P<0.05, ##P<0.01 compared to pC or pC HS. White bar in panel (F) represents control non-HS cells, whereas black bars represent HS cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4508109&req=5

pone.0133553.g001: RhoA activation attenuates HSPA1A expression.(A) Relative luciferase expression of a reporter construct driven by the SRE promoter (downstream target of RhoA/ROCK signaling) in HL-1 cardiomyocytes transfected with empty plasmid (pC, pcDNA3.1+), C3T exoenzyme plasmid (pC3T) or RhoA-WT encoding plasmid (pRhoA). (B) Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in cardiomyocytes transfected with pC, pC3T or pRhoA. (C) Relative HSPA1A-luc expression in cardiomyocytes transfected with pC, pC3T or pRhoA and subjected to a HS (45°C, 10 min), white bars represent control non-HS, whereas black bars represent HS cells. (D) Top panel shows a representative Western blot with HSPA1A levels of cardiomyocytes transfected with pC, pC3T or pRhoA and subjected to a HS. Below, quantified data of HSPA1A/GAPDH levels for conditions as indicated. (E) Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in cardiomyocytes transfected with empty plasmid pC, pC3T, pP190RhoGAP, pRhoA-n19, pRhoA or pRhoA-v14 without (E) and with (F) HS. (G) RhoA activation attenuates HSPA1A expression in human HEK-293 cells. Relative luciferase expression of a reporter construct driven by the HSPA1A promoter in HEK293 cells transfected with empty plasmid pC, pC3T, or pRhoA. *P<0.05, **P<0.01, ***P<0.001 compared to control pC and #P<0.05, ##P<0.01 compared to pC or pC HS. White bar in panel (F) represents control non-HS cells, whereas black bars represent HS cells.
Mentions: To determine if RhoA signaling affects the HSR, control and heat shocked (HS) HL-1 cardiomyocytes were transfected with a luciferase reporter construct driven by the HSF1 regulated human HSP70 (HSPA1A) promoter (HSPA1A-luc). RhoA activity was inhibited by transfection of the C3-exoenzyme (pC3T) construct, which inhibits the RhoA pathway, or activated by overexpression of RhoA by transfection of a RhoA wild type construct (pRhoA) (S1A and S1B Fig). The effectiveness of these manipulations was validated using co-transfection with a luciferase construct driven by the RhoA-dependent serum response element (SRE) (Fig 1A). pRhoA expression reduced both basal HSPA1A-luc (Fig 1B) and also strongly suppressed the heat-induced increase in HSPA1A-luc expression (Fig 1C). pRhoA also significantly reduced the level of endogenous HSPA1A protein expression in the total fraction of heat-shocked cells (Fig 1D).

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