Pifithrin-µ Induces Stress Granule Formation, Regulates Cell Survival, and Rewires Cellular Signaling

Abstract

(1) Background: Stress granules (SGs) are cytoplasmic protein-RNA condensates that assemble in response to various insults. SG production is driven by signaling pathways that are relevant to human disease. Compounds that modulate SG characteristics are therefore of clinical interest. Pifithrin-µ is a candidate anti-tumor agent that inhibits members of the hsp70 chaperone family. While hsp70s are required for granulostasis, the impact of pifithrin-µ on SG formation is unknown. (2) Methods: Using HeLa cells as model system, cell-based assays evaluated the effects of pifithrin-µ on cell viability. Quantitative Western blotting assessed cell signaling events and SG proteins. Confocal microscopy combined with quantitative image analyses examined multiple SG parameters. (3) Results: Pifithrin-µ induced bona fide SGs in the absence of exogenous stress. These SGs were dynamic; their properties were determined by the duration of pifithrin-µ treatment. The phosphorylation of eIF2α was mandatory to generate SGs upon pifithrin-µ exposure. Moreover, the formation of pifithrin-µ SGs was accompanied by profound changes in cell signaling. Pifithrin-µ reduced the activation of 5'-AMP-activated protein kinase, whereas the pro-survival protein kinase Akt was activated. Long-term pifithrin-µ treatment caused a marked loss of cell viability. (4) Conclusions: Our study identified stress-related changes in cellular homeostasis that are elicited by pifithrin-µ. These insights are important knowledge for the appropriate therapeutic use of pifithrin-µ and related compounds.

Keywords: 5′-AMP-activated protein kinase (AMPK); eIF2α phosphorylation; pifithrin-µ; protein kinase Akt; stress granules.

Figure 1

PFT-µ triggers the assembly of bona fide cytoplasmic stress granules. (A) HeLa cells were incubated with vehicle or PFT-µ (50 µM, 1 h), and the proteins indicated were detected by immunolocalization. Cytoplasmic condensates induced by PFT-µ accumulated the SG markers G3BP1 and HuR. (B–D) HeLa cells were treated with vehicle or PFT-µ (50 µM, 2 h). (B) The treatment with vehicle or PFT-µ was conducted in the absence or presence of 10 µg/mL cycloheximide (CHX). G3BP1 provided the SG marker. (C) The translation initiation factor eIF4G and G3BP1 were visualized by immunofluorescence in cells incubated with vehicle or PFT-µ. (D) PolyA⁺-containing RNA and G3BP1 were detected with a combination of in situ hybridization with oligo-dT50 and immunostaining. All scale bars are 20 µm.

Figure 2

The formation of PFT-µ SGs relies on eIF2α phosphorylation. (A) HeLa cells were incubated with vehicle, PFT-µ, or arsenite. Crude extracts were evaluated for the phosphorylation of eIF2α on S51 (p-eIF2α) and total eIF2α. Actin provided a reference for loading. The molecular mass of marker proteins is indicated in kD at the right margin. The relative phosphorylation of eIF2α (p/total eIF2α) and the abundance of total eIF2α were quantified for at least three independent experiments. Results normalized to vehicle controls are depicted as average + standard error of the mean (SEM). One-way ANOVA combined with Bonferroni post hoc analysis identified significant differences between groups. Comparison to vehicle: **, p < 0.01. Comparison between PFT-µ and arsenite, # p < 0.05. (B) Wildtype and mutant mouse embryonic fibroblasts (MEFs) were incubated with PFT-µ. The formation of SGs was assessed with the SG marker proteins G3BP1 and HuR. Cells that produce mutant eIF2α (S51A) also synthesize GFP (Materials and Methods). All images were acquired with identical settings; scale bar is 20 µm. Several SGs are marked with arrowheads. Note that MEFs with mutant eIF2α do not generate SGs when treated with PFT-µ.

Figure 3

Kinetics of SG formation in response to PFT-µ treatment. HeLa cells were incubated with the vehicle DMSO (V) or PFT-µ (P) for the times indicated [h]. Scale bar is 20 µm. The arrowhead points to a pyknotic nucleus, which indicates cell death. For the measurements of SG properties, all images were acquired with identical settings. SG parameters were quantified for two independent experiments, each set with at least 112 cells per condition for each experiment. Results were normalized to the 1 h PFT-µ datapoint. Bar graphs depict data as average + SEM. Statistical evaluation was performed with one-way ANOVA and Bonferroni post hoc analysis. The 1 h vehicle control was used as reference for pairwise comparisons; *, p < 0.05; **, p < 0.01; ***, p < 0.001. AU, arbitrary units.

Figure 4

PFT-µ elevates the phosphorylation of eIF2α in a time-dependent fashion. HeLa cells were incubated with vehicle (V) or PFT-µ (P) for the hours [h] indicated. Crude extracts were assessed for eIF2α phosphorylation on S51 (p-eIF2α) and total eIF2α. Actin provided a loading reference. The molecular mass of marker proteins is shown in kD at the right margin. ECL signals were measured and normalized to the vehicle control for each time point. Two independent experiments were evaluated for p-eIF2α, and at least three independent experiments were performed for total eIF2α. Results are shown as average + SEM. Statistical evaluation was performed with one-way ANOVA, followed by Bonferroni post hoc analysis. Significant differences were identified relative to the vehicle control (*, p < 0.05; ***, p < 0.001). Pairwise comparisons showed significant differences between PFT-µ-treated samples (#, p < 0.05; ##, p < 0.01; ###, p < 0.001). AU, arbitrary units.

Figure 5

Impact of PFT-µ treatment on the abundance of SG nucleators, HuR, and hsp70. The levels of different SG components and hsp70 were determined by Western blotting as described for Figure 4. The molecular mass of marker proteins is depicted in kD at the right margin. Three to four independent experiments were conducted for each protein analyzed. Graphs show averages + SEM. Statistical evaluation was conducted with one-way ANOVA and Bonferroni post hoc analysis. Significant differences are indicated relative to the vehicle control (*, p < 0.05; **, p < 0.01, ***, p < 0.001). Pairwise comparisons identified significant differences relative to the 21 h PFT-µ treatment (#, p < 0.05; ##, p < 0.01; ###, p < 0.001). V, vehicle; P, PFT-µ; AU, arbitrary units.

Figure 6

PFT-µ induces cell death in a time-dependent fashion. HeLa cells were incubated with vehicle (V), PFT-µ (P), or staurosporine (STS) for the hours [h] indicated. (A) PFT-µ reduced the metabolic activity of HeLa cells. Results for each condition were normalized to the vehicle control. Data are depicted as average + SEM for three independent experiments. One-way ANOVA combined with Bonferroni post hoc analysis was used for statistical evaluation. The vehicle control served as reference. *, p < 0.05; ***, p < 0.01. Pairwise comparison demonstrated significant differences between 4 h and 21 h PFT-µ treatments; ###, p < 0.001. AU, arbitrary units. (B) Crude extracts were prepared for HeLa cells that were treated as described for part A. Western blotting evaluated the cleavage of lamin A and PARP1. Actin served as loading reference. The molecular mass of marker proteins is shown in kD at the right margin.

Figure 7

PFT-µ diminishes the phosphorylation of AMPK on T172. Cells were incubated with vehicle (V) or PFT-µ for the hours [h] depicted. Crude extracts were evaluated for the phosphorylation of AMPK on T172 (p-AMPK). The same samples were also probed with antibodies against total AMPK (t-AMPK) and actin. The molecular mass of marker proteins in kD is indicated at the right margin. ECL signals were quantified for three independent experiments. Data were normalized to vehicle controls for each time point. Bars show average + SEM for each datapoint. Statistical evaluation was performed with one-way ANOVA combined with Bonferroni post hoc analysis. Changes were assessed relative to the vehicle control; ***, p < 0.001. AU, arbitrary units.

Figure 8

PFT-µ modulates Akt activation. HeLa cells were incubated with vehicle (V) or PFT-µ (P) for the hours [h] specified. Crude extracts were evaluated for Akt phosphorylation on T308 (p-T308) or S473 (p-S473) and for total Akt. The bands quantified for p-T308, p-S473, and total Akt are marked with square brackets. Actin was used as loading reference. The molecular mass of marker proteins is indicated at the right margin in kD. ECL signals were quantified and normalized to the vehicle control for each time point. Results for four to seven independent experiments are depicted as average + SEM. Statistical evaluation was performed with one-way ANOVA followed by Bonferroni post hoc analysis. Significant differences were identified relative to the vehicle control (**, p < 0.01). Pairwise comparisons between PFT-µ-treated samples showed a significant difference for p-S473 (#, p < 0.05). AU, arbitrary units.

Figure 9

Simplified model of the cellular responses triggered by PFT-µ. Stress granules are depicted as green spherical compartments in the cytoplasm. Short-term refers to the incubation period up to 4 h; chronic represents a 21 h treatment. ↑, upregulation; ↓, downregulation. See text for details.