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. 2017 Aug 1;158(2):275-285.
doi: 10.1093/toxsci/kfx096.

Endoplasmic Reticulum Stress-Induced CHOP Inhibits PGC-1α and Causes Mitochondrial Dysfunction in Diabetic Embryopathy

Xi Chen  1   2 , Jianxiang Zhong  2 , Daoyin Dong  2 , Gentao Liu  1 , Peixin Yang  1   3
Affiliations

Endoplasmic Reticulum Stress-Induced CHOP Inhibits PGC-1α and Causes Mitochondrial Dysfunction in Diabetic Embryopathy

Xi Chen et al. Toxicol Sci. .

Abstract

Endoplasmic reticulum (ER) stress has been implicated in the development of maternal diabetes-induced neural tube defects (NTDs). ER stress-induced C/EBP homologous protein (CHOP) plays an important role in the pro-apoptotic execution pathways. However, the molecular mechanism underlying ER stress- and CHOP-induced neuroepithelium cell apoptosis in diabetic embryopathy is still unclear. Deletion of the Chop gene significantly reduced maternal diabetes-induced NTDs. CHOP deficiency abrogated maternal diabetes-induced mitochondrial dysfunction and neuroepithelium cell apoptosis. Further analysis demonstrated that CHOP repressed the expression of peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α), an essential regulator for mitochondrial biogenesis and function. Both CHOP deficiency in vivo and knockdown in vitro restore high glucose-suppressed PGC-1α expression. In contrast, CHOP overexpression mimicked inhibition of PGC-1α by high glucose. In response to the ER stress inducer tunicamycin, PGC-1α expression was decreased, whereas the ER stress inhibitor 4-phenylbutyric acid blocked high glucose-suppressed PGC-1α expression. Moreover, maternal diabetes in vivo and high glucose in vitro promoted the interaction between CHOP and the PGC-1α transcriptional regulator CCAAT/enhancer binding protein-β (C/EBPβ), and reduced C/EBPβ binding to the PGC-1α promoter leading to markedly decrease in PGC-1α expression. Together, our findings support the hypothesis that maternal diabetes-induced ER stress increases CHOP expression which represses PGC-1α through suppressing the C/EBPβ transcriptional activity, subsequently induces mitochondrial dysfunction and ultimately results in NTDs.

Keywords: C/EBPβ; CHOP; ER stress; PGC-1α; diabetic embryopathy; mitochondrial dysfunction; neural tube defects.

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Figures

Figure 1
Figure 1
CHOP knockout ameliorates maternal diabetes-induced NTD formation and neuroepithelial cell apoptosis. A, The average blood glucose level in non-diabetic and diabetic groups. The mRNA abundance (B) and protein abundance of CHOP (C) in E8.5 embryos. D, Morphology and H&E staining images of E10.5 embryos. Arrow shows the NTD. Scale bars: 300 µm. E, NTD incidences of E10.5 embryos in each group. F, Representative images of the TUNEL assays in E8.5 embryos. Apoptotic cells were labeled and nuclei were labeled by DAPI. The dense blue V shape areas are the neural tubes. The bar graph showed the quantification of apoptotic cell number. Experiments were repeated using 3 embryos (N=3) from different dams and 3 images were obtained from each embryo. Scale bars: 70 µm. The protein abundance of cleaved caspase 3 (G) and caspase 8 (H) in E8.5 embryos. Bar graphs for protein abundance were quantitative data from 3 independent experiments. ND: non-diabetic dams; DM: diabetic dams; NTD: neural tube defect; KO: CHOP knockout. * indicate significant difference (p < .05) compared with other groups.
Figure 2
Figure 2
CHOP knockout restores maternal diabetes-disturbed mitochondrial function. A, Defective mitochondria (mito) rates (%). Defective mitochondria rate = the number of defective mitochondria divided by total number of mitochondria per image area (image size: 9.43 μm2) in neuroepithelial cells. Neuroepithelia from 3 embryos (N = 3) derived from different dams were used. Three serial sections per embryo were analyzed. Scale bars: 200 nm. B, JC-1 dye-stained isolated neuroepithelial cells were analyzed for the mitochondrial potential by a flow cytometer. Bar graphs for rate of fluorescence were quantitative data from 3 independent experiments. R1 indicates the membrane intact mitochondria. R2 indicates the membrane detective mitochondrial. C, The protein abundance of Puma, Bak, Bax, and Bim in isolated mitochondria from E8.5 embryos. D, The protein abundance of tBid (cleaved), pBad (phosphorylated) in E8.5 embryos. Bar graphs for protein abundance were quantitative data from 3 independent experiments. *indicate significant difference compared with other groups. ND: non-diabetic dams; DM: diabetic dams; KO: CHOP knockout. * indicate significant difference (p < .05) compared with other groups.
Figure 3
Figure 3
Maternal diabetes or high glucose-increased CHOP inhibits PGC-1α expression. A, The mRNA abundance of PGC-1α in E8.5 embryos. B, The mRNA abundance of CHOP in C17.2 cells transfected with or without CHOP siRNA under normal or high glucose conditions. C, The mRNA abundance of PGC-1α in C17.2 cells transfected with or without CHOP siRNA under normal or high glucose conditions. D, The protein abundance of PGC-1α and CHOP in C17.2 cells transfected with or without CHOP siRNA under normal or high glucose conditions. Bar graphs for protein abundance were quantitative data from 3 independent experiments. E, The mRNA abundance of PGC-1α and CHOP in C17.2 cells transfected with or without CHOP expression vector under normal or high glucose conditions. F, The protein abundance of PGC-1α and CHOP in C17.2 cells transfected with or without CHOP expression vector under normal or high glucose conditions. Bar graphs for protein abundance were quantitative data from 3 independent experiments. G, PGC-1α promoter activity analyzed by relative luciferase reporter activities in C17.2 cells. Experiments were repeated 3 times. ND: non-diabetic dams; DM: diabetic dams; KO: CHOP knockout; NG: normal glucose; HG: high glucose. Control: blank pcDNA3 vector. *indicate significant difference (p < .05) compared with other groups.
Figure 4
Figure 4
ER stress inducer Tunicamycin-increased CHOP inhibits PGC-1α expression. A, The mRNA abundance of CHOP and PGC-1α in C17.2 cells treated with tunicamycin under different time. B, The protein abundance of CHOP and PGC-1α in C17.2 cells treated with tunicamycin or high glucose combined with 4-PBA. Bar graphs for protein abundance were quantitative data from 3 independent experiments. C, PGC-1α promoter activity analyzed by relative luciferase reporter activities in C17.2 cells treated with tunicamycin or high glucose combined with 4-PBA. Experiments were repeated 3 times. NG: normal glucose; HG: high glucose. *indicate significant difference (p < .05) compared with 0 h group in A and NG-Vehicle group in B and C. # indicate significant difference (p < .05) compared with HG-vehicle group in B and C.
Figure 5
Figure 5
C/EBPβ overexpression restores high glucose/tunicamycin-suppressed PGC-1α expression. A, The protein abundance of PGC-1α, CHOP, and C/EBPβ in C17.2 cells transfected with C/EBPβ expression vector under normal or high glucose conditions. B, The protein abundance of PGC-1α, CHOP, and C/EBPβ in C17.2 cells transfected with C/EBPβ expression vector and treated with tunicamycin (1 μg/ml). Bar graphs for protein abundance were quantitative data from 3 independent experiments. NG: normal glucose; HG: high glucose; Tuni: tunicamycin. Different letter indicates significant difference (p < .05).
Figure 6
Figure 6
CHOP inhibits C/EBPβ DNA-binding activity by forming CHOP-C/EBPβ heterodimers. A, Representative images of Co-immunoprecipitation using an anti-CHOP antibody in C17.2 cells transfected with CHOP siRNA or scramble control siRNA under normal or high glucose conditions. B, Representative images of Co-immunoprecipitation using an anti-CHOP antibody in E8.5 embryos. In the bar graphs, the protein level of C/EBPβ was assessed in CHOP immunoprecipitates and was normalized by 10% input. Normal rabbit IgG was used as control. C, Potential binding sites for C/EBPβ in the PGC-1α promoter. D, Immunoprecipitated chromatin was analyzed by PCR for the C/EBPβ site and quantification of C/EBPβ occupancy on the C/EBPβ site in C17.2 cells transfected with CHOP siRNA or scramble control siRNA under normal or high glucose conditions. E, Immunoprecipitated chromatin was analyzed by PCR for the C/EBPβ site and quantification of C/EBPβ occupancy on the C/EBPβ site in E8.5 embryos. ND: non-diabetic dams; DM: diabetic dams; NG: normal glucose; HG: high glucose; KO: CHOP knockout. *indicate significant difference (p < .05) compared with other groups.
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