Luteolin protects DYT-PRKRA cells from apoptosis by suppressing PKR activation

Figures

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  FIGURE 1. (A) PKR is hyperactive in DYT-PRKRA patient lymphoblasts. PKR activity assay and western blot analysis for p-PKR and total PKR. PKR kinase activity assay was performed using PKR immunoprecipitated from wt and DYT-PRKRA lymphoblast extracts using a monoclonal PKR antibody (R&D Systems) and protein A-sepharose beads. PKR activity was assessed without any externally added activator and the bands represent endogenous activity levels of PKR. The PKR band intensities were quantified using Imagequant TL (Cytiva), and the bar graph shows data from 3 independent experiments and the p values are as indicated. Blue bar: wt and orange bar: DYT-PRKRA. Whole cell extracts from normal (wt) and DYT-PRKRA patient derived lymphoblasts were analyzed. Blots were probed for p-PKR, total PKR, p-PERK, and total PERK. Best of three representative blots are shown. (B) Western blot analysis for p-PKR, p-eIF2α and GADD34. Normal (wt) and DYT-PRKRA patient derived lymphoblasts treated with 5 μg/ml of tunicamycin (TM) and cell extracts were prepared at various time points as indicated above the lanes after treatment and from untreated cells. Western blot analysis was performed with the indicated antibodies.…

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  FIGURE 2. (A) PKR inhibition by C16. Kinase activity assay was performed using PKR immunoprecipitated from HeLa cell extracts using a monoclonal PKR antibody (R&D Systems) and protein A-sepharose beads. Either 1 μg/ml polyI:polyC (lanes 2, 4, 5) or 4 ng recombinant wt PACT (lanes 3, 6, 7) were used as PKR activators. C16 was added either at 0.05 μM (lanes 4 and 6) or 0.1 μM (lanes 5 and 7) as indicated on the top of the lanes. Lanes 1: no activator added. (B) Inhibition of PKR activation and eIF2α phosphorylation in lymphoblasts by C16. The normal (wt) and DYT-PRKRA patient derived lymphoblasts were treated with either 5 μg/ml of tunicamycin (TM), TM + 0.1 μM C16 or TM + 0.5 μM C16 for 2 h. For the C16 treated samples, the cells were pretreated with C16 for 24 h before tunicamycin treatment. Whole cell extracts were prepared at 2 h after the tunicamycin treatment and were analyzed by western blot analysis. Blots were probed for p-PKR, total PKR, p-eIFα, and total eIF2α. Best of four representative blots are shown. (C) Western blot analysis for cleaved PARP1. The normal (wt) and DYT-PRKRA patient derived lymphoblasts were treated with either 5 μg/ml of tunicamycin (TM), TM + 0.1 μM C16 or…

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  FIGURE 3. (A) Luteolin disrupts the interaction between PKR and PACT. (A) Co-IP of endogenous PKR and PACT proteins. Lymphoblasts from unaffected family member (wt) or DYT-PRKRA patient (patient) were treated with 50 μM luteolin. The cell extracts were prepared 24 h after the treatment, and endogenous PKR protein was immunoprecipitated using anti-PKR mAb and protein A-sepharose, which immunoprecipitates total PKR. The immunoprecipitates were analyzed by western blot analysis with anti-PACT monoclonal antibody (Co-IP panel). The blot was stripped and re-probed with anti-PKR mAb to ascertain an equal amount of PKR was immunoprecipitated in each lane (IP panel). Input blot: Western blot analysis of total proteins in the extract with anti-PACT mAb showing equal amount of PACT in all samples. (B) Mammalian two-hybrid analysis. HeLa cells were transfected with 250 ng of each of the two test plasmids encoding proteins to be tested for interaction, 50 ng of the reporter plasmid pG5Luc, and 1 ng of plasmid pRL-Null to normalize transfection efficiency. 2 h after transfection, one set of samples were left untreated, and one set was treated with 50 μg/ml luteolin. Cells were harvested 24 h after…

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  FIGURE 4. Luteolin protects DYT-PRKRA cells from apoptosis in response to ER stress. (A) Western blot analysis for cleaved PARP1. The normal (wt) and DYT-PRKRA patient derived lymphoblasts were treated with 5 μg/ml of tunicamycin (TM) either without pretreatment with luteolin or with 50 µM luteolin pretreatment for 24 h. Whole cell extracts prepared at the indicated time points after TM treatment were analyzed using anti-cleaved PARP1 and anti-β-actin antibodies. (B) Caspase-Glo 3/7 activity. The normal (wt) and DYT-PRKRA patient derived lymphoblasts were treated with 5 μg/ml of tunicamycin (TM) either without pretreatment with luteolin or with 50 µM luteolin pretreatment for 24 h. The caspase 3/7 activities were measured at 0 h and 24 h. Blue bars: wt cells, and orange bars: DYT-PRKRA cells, filled bars: tunicamycin treated and unfilled bars: luteolin and tunicamycin treated. The data is an average of three independent experiments and the p values are as indicated.

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  FIGURE 5. (A) Effect of luteolin on PKR activation and ISR in response to tunicamycin in normal and DYT-PRKRA patient lymphoblasts: western blot analysis for p-PKR and p-eIF2α. Whole cell extracts from normal (wt) and DYT-PRKRA patient derived lymphoblasts treated with 5 μg/ml of tunicamycin (TM) without any luteolin pretreatment or after 24 h pretreatment with 50 µM luteolin were analyzed at indicated time points. Blots were probed for p-eIF2α, total eIF2α, p-PKR, and total PKR. Best of four representative blots are shown. (B) The signal intensities of p-eIF2α and total eIF2α bands were quantified using Imagequant TL (Cytiva) and the ratio p-eIF2α/eIF2α was calculated for each time point using four separate experiments. The p values for differences between − lut and + lut for both wt and DYT-PRKRA cells were all below 0.001. Blue lines: wt and orange lines: DYT-PRKRA. Solid lines: without luteolin and dotted lines: with luteolin.

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  FIGURE 6. (A) Effect of luteolin on PKR activation and ISR in response to tunicamycin in normal and DYT-PRKRA patient lymphoblasts: western blot analysis for ATF4 and CHOP. Whole cell extracts from normal (wt) and DYT-PRKRA patient derived lymphoblasts treated with 5 μg/ml of tunicamycin (TM) without any luteolin pretreatment or after 24 h pretreatment with 50 µM luteolin were analyzed at indicated time points. Blots were probed for ATF4, and CHOP. Best of four representative blots are shown. β-actin was used as a loading control to ensure equal amounts of protein was loaded in each lane. (B) DYT-PRKRA patient lymphoblasts express higher levels of ATF4 and CHOP mRNAs in response to tunicamycin and luteolin treatment downregulates ATF4 and CHOP induction. Quantitative RT-PCR of ATF4 and CHOP in wt (blue bars) and DYT-PRKRA (orange bars) lymphoblasts. The hatched bars indicate untreated control values, solid filled bars indicate tunicamycin treated values, and unfilled bars indicate luteolin and tunicamycin treated values. The RQ values indicate that ATF4 and CHOP expression was upregulated in response to tunicamycin and this upregulation was suppressed by luteolin pre-treatment in both wt…

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  FIGURE 7. Luteolin allows for a transient PACT-PKR interaction after ER stress. (A) Co-IP of endogenous PKR and PACT proteins. The DYT-PRKRA lymphoblasts were treated with 5 μg/ml tunicamycin (TM) either with or without luteolin pre-treatment for 24 h. The cell extracts were prepared at indicated time points after the tunicamycin treatment, and endogenous PKR protein was immunoprecipitated using anti-PKR mAb and protein A-sepharose, which immunoprecipitates total PKR. The immunoprecipitates were analyzed by western blot analysis with anti-PACT monoclonal antibody (Co-IP panel). The blot was stripped and re-probed with anti-PKR mAb to ascertain an equal amount of PKR was immunoprecipitated in each lane (IP panel). Input blot: Western blot analysis of total proteins in the extract with anti-PACT mAb showing equal amount of PACT in all samples. (B) Mammalian two-hybrid analysis. HeLa cells were transfected with 250 ng of each of the two test plasmids encoding proteins to be tested for interaction, 50 ng of the reporter plasmid pG5Luc, and 1 ng of plasmid pRL-Null to normalize transfection efficiency. 2 h after transfection, one set of samples were left untreated and one set was treated with…

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  FIGURE 8. A schematic model for ER stress response in wt and DYT-PRKRA cells. (A) ER stress response in wt cells. In the absence of stress, PACT is not phosphorylated and PKR is not activated. After ER stress, PACT is phosphorylated and PACT-PACT and PACT-PKR interactions are enhanced thereby causing a transient PKR activation and eIF2α phosphorylation. This response leads to restoration of homeostasis promoting survival. (B) ER stress response in DYT-PRKRA cells. In the absence of stress, mutant PACT is not phosphorylated but forms strong PACT-PACT as well as PACT-PKR interactions and PKR is activated. After ER stress, PACT is phosphorylated and PACT-PACT and PACT-PKR interactions are further enhanced thereby causing a persistent PKR activation and eIF2α phosphorylation promoting apoptosis. (C) ER stress response in DYT-PRKRA cells in the presence of luteolin. In the absence of stress, mutant PACT is not phosphorylated and the PACT-PKR interactions are disrupted by luteolin and PKR is not activated. After ER stress, PACT is phosphorylated and PACT-PACT and PACT-PKR interactions are enhanced thereby causing a transient PKR activation and eIF2α phosphorylation. This transient response leads…