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, 7 (6), e39586

CHOP Potentially Co-Operates With FOXO3a in Neuronal Cells to Regulate PUMA and BIM Expression in Response to ER Stress

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CHOP Potentially Co-Operates With FOXO3a in Neuronal Cells to Regulate PUMA and BIM Expression in Response to ER Stress

Arindam P Ghosh et al. PLoS One.

Abstract

Endoplasmic reticulum (ER) stress-induced apoptosis has been implicated in various neurodegenerative diseases including Parkinson Disease, Alzheimer Disease and Huntington Disease. PUMA (p53 upregulated modulator of apoptosis) and BIM (BCL2 interacting mediator of cell death), pro-apoptotic BH3 domain-only, BCL2 family members, have previously been shown to regulate ER stress-induced cell death, but the upstream signaling pathways that regulate this response in neuronal cells are incompletely defined. Consistent with previous studies, we show that both PUMA and BIM are induced in response to ER stress in neuronal cells and that transcriptional induction of PUMA regulates ER stress-induced cell death, independent of p53. CHOP (C/EBP homologous protein also known as GADD153; gene name Ddit3), a critical initiator of ER stress-induced apoptosis, was found to regulate both PUMA and BIM expression in response to ER stress. We further show that CHOP knockdown prevents perturbations in the AKT (protein kinase B)/FOXO3a (forkhead box, class O, 3a) pathway in response to ER stress. CHOP co-immunoprecipitated with FOXO3a in tunicamycin treated cells, suggesting that CHOP may also regulate other pro-apoptotic signaling cascades culminating in PUMA and BIM activation and cell death. In summary, CHOP regulates the expression of multiple pro-apoptotic BH3-only molecules through multiple mechanisms, making CHOP an important therapeutic target relevant to a number of neurodegenerative conditions.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Tunicamycin produces a concentration- and time-dependent decrease in cell viability in SH-SY5Y cells concomitant with caspase-3 activation.
(A) Treatment of SH-SY5Y cells with tunicamycin (0.1–5µM) for 24 h caused a progressive decrease in cell viability as measured by Calcein AM cleavage assay. (B) Treatment with tunicamycin (1µM) for 6–48 h caused a time-dependent decrease in cell viability and an increase in caspase-3 like enzymatic activity (C), as measured by the DEVD-AMC cleavage assay. (D) Treatment with tunicamycin (1µM) for 24h also produced an increase in cleaved caspase-3 immunoreactivity. (E) Treatment with tunicamycin (1µM) also produced an increase in levels of cleaved caspase-3 as measured by immunoblotting. (F) Tunicamycin (1µM) treatment of SH-SY5Y cells upregulated markers of the UPR: BiP, phospho eIF2α and CHOP on immunoblotting. Data points represent mean ± SEM, with n = 6.*p<0.05 by one-way ANOVA with Bonferroni post hoc test versus UT controls.
Figure 2
Figure 2. ER stress in neuronal cells induces the expression of pro-apoptotic proteins PUMA and BIM.
(A) Tunicamycin (1µM) induces a time-dependent increase in PUMA and BIM protein levels. Though AraC caused an increase in PUMA and phospho p53 protein levels, tunicamycin-induced increase in PUMA protein levels was not associated with an increase in phospho p53 levels. (B) Tunicamycin treatment also caused a robust increase in Puma mRNA levels as measured by quantitative PCR. Data points represent mean ± SEM, with n = 3.*p<0.01 by one-way ANOVA/Bonferroni post hoc test versus UT controls.
Figure 3
Figure 3. Loss of Puma but not p53, protects against tunicamycin-induced cell death and caspase-3 activation.
(A) PUMA-deficient telencephalic neurons exhibited significantly less death after exposure to tunicamycin in comparison to wild-type telencephalic neurons from litter mate controls. Similarly, PUMA-deficient telencephalic neurons were protected against AraC-induced cell death (B) PUMA-deficient telencephalic neurons exhibited a significant attenuation in caspase-3 activation after exposure to tunicamycin or AraC in comparison to wild-type neurons. (C) p53-deficient telencephalic neurons did not exhibit significant protection against tunicamycin-induced cell death although they were protected from AraC-induced cell death in comparison to wild-type telencephalic neurons. (D) p53-deficient telencephalic neurons exhibited a significant attenuation in caspase-3 activation after exposure to AraC in comparison to wild-type litter control neurons but not after treatment with tunicamycin. (E) BIM-deficient telencephalic neurons demonstrated no significant protection against tunicamycin-induced cell death in comparison to wild-type littermates. The data represent mean ± SEM, with n = 5. *p<0.01 by two-way ANOVA/Bonferroni post hoc test compared to both the wild-type and the knock-out treated group.
Figure 4
Figure 4. ER stress causes a change in the AKT/FOXO3a axis.
Tunicamycin (1µM) induced a progressive dephosphorylation of AKT (Ser 473) accompanied by a progressive dephosphorylation of FOXO3a (Thr 32). In comparison to ER stress, genotoxic stress does not induce changes in phospho AKT or phospho FOXO3a levels.
Figure 5
Figure 5. CHOP knockdown inhibits the induction of PUMA and BIM in neuronal cells in response to ER stress.
(A) Knockdown of CHOP in SH-SY5Y cells using shRNA ameliorated the induction of PUMA with tunicamycin in comparison to control cells. (B) Knockdown of CHOP also attenuates the transcriptional induction of Puma mRNA in comparison to controls. (C) Knockdown of CHOP also causes a decrease in the activation of BIM in comparison to control cells after treatment with tunicamycin. Data points represent mean ± SEM, with n = 3. *p<0.05 by 2-way ANOVA/Bonferroni post hoc test vs. treated controls and **p<0.05 by 2-way ANOVA/Bonferroni post hoc test vs. treated CHOP knockdowns.
Figure 6
Figure 6. CHOP knockdown prevents dephosphorylation of AKT and FOXO3a.
(A) CHOP knockdown prevents dephosphorylation of AKT (Ser 473) in comparison to control cells treated with tunicamycin. (B) CHOP knockdown prevents dephosphorylation of FOXO3a (Thr 32) in comparison to control cells treated with tunicamycin.
Figure 7
Figure 7. CHOP directly interacts with FOXO3a in response to ER stress.
(A) Nuclear and cytoplasmic fractions from untreated SH-SY5Y cells and those treated with tunicamycin (1µM) for 24h were examined for levels of total and phosphorylated FOXO3a and CHOP. GAPDH was used as a loading control for whole cell lysates or cytoplasmic fractions, while HDAC was used as a loading control for nuclear fractions (B) Cell lysates of SH-SY5Y cells treated with tunicamycin or controls were subjected to immunoprecipitation using FOXO3a polyclonal antibody or control IgG. CHOP protein was detected from immunoprecipitates by western blotting in comparison to input controls.
Figure 8
Figure 8. Proposed model for direct and indirect activation of PUMA and BIM by CHOP.

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