miR-216b regulation of c-Jun mediates GADD153/CHOP-dependent apoptosis

Abstract

The ability of the unfolded protein response, UPR, to regulate cell homeostasis through both gene expression and protein synthesis has been well documented. One primary pro-apoptotic protein that responds to both PERK and Ire1 signalling is the CHOP/GADD153 transcription factor. Although CHOP deficiency delays onset of cell death, questions remain regarding how CHOP regulates apoptosis. Here, we provide evidence demonstrating that CHOP/GADD153-dependent apoptosis reflects expression of micro-RNA, miR-216b. MiR-216b accumulation requires PERK-dependent induction of CHOP/GADD153, which then directly regulates miR-216b expression. As maximal expression of miR-216b is antagonized by Ire1, miR-216b accumulation reflects the convergence of PERK and Ire1 activities. Functionally, miR-216b directly targets c-Jun, thereby reducing AP-1-dependent transcription and sensitizing cells to ER stress-dependent apoptosis. These results provide direct insight into the molecular mechanisms of CHOP/GADD153-dependent cell death.

Figure 1. ER stress induced miR-216b expression.

(a) Heat map depicting differentially expressed microRNAs (at least two-fold) following exposure to tunicamycin (Tm) as indicated. (b,c) qPCR analysis of CHOP mRNA levels and miR-216b expression in NIH3T3 cells treated with Tm (1 μg ml⁻¹) or thapsigargin (TG, 500 nM) for indicated times. Averages are calculated from three independent experiments. (d) qPCR analysis of miR-216b expression and CHOP mRNA levels in NIH3T3 cells cultured in low-glucose medium for indicated times. (e) qPCR analysis of miR-216b expression and CHOP mRNA levels in U2OS cells treated with paclitaxel (100 nM) for indicated times. Data are mean±s.d. of three independent experiments. Statistical analysis was analysed by Student's t-test. (*P<0.05, treatment versus non-treatment).

Figure 2. PERK-dependent miR-216b induction.

(a) PERK^+/+ and PERK^−/− MEFs were treated with 500 nM TG for indicated times. MiR-216b was assessed by qPCR (left graph); PERK and CHOP were assessed by immunoblot (right). (b) MiR-216b levels were quantified by qPCR following exposure of cells to thapsigargin and a small-molecule PERK inhibitor (left). PERK, eIF2α-p and CHOP induction were assessed by immunoblot (right). (c–e) MEFs of the indicated genotype were treated with TG (500 nM) for indicated intervals. Protein extracts from these cells were immunoblotted for the proteins as indicated (lower panels) and miR-216b levels were quantified by qPCR (upper panels; n=3). (f) CHOP^−/− MEFs were transfected with vector or CHOP and 2 days later treated with TG (500 nM) for indicated intervals. Protein extracts from these cells were immunoblotted for CHOP and miR-216b levels quantified by qPCR (n=3). (g) MiR-216b expression and (h) c-Jun mRNA levels were analysed in MMTV-Neu tumours from either PERK^+/+ or a PERK^−/− background. Data represent mean±s.d. of three independent observations. Statistical significance was analysed analysed by Student's t-test. (*P<0.05, WT versus −/−).

Figure 3. CHOP binds to miR-216b promoter region.

(a) Occupancy of CHOP on the promoter of mouse miR-216b. NIH3T3 cells (left panel) and U2OS (right panel) were treated with/without TG for 5 h, followed by ChIP assay using IgG or an anti-CHOP antibody. Precipitated DNA was subjected to qPCR analysis. All ChIP data represent the mean and standard error of the mean from three independent experiments. (b) Schematic representation of miR-216b promoter reporter (216b-pro-pGL4; left panel). (c) 293T cells were co-transfected with 216b-pro-pGL4, 216b-mut-pGL4 and Renilla-Luc as an internal control. 48 h post transfection, cells were treated with PERK inhibitor followed by TG treatment for intervals as indicated. Values are the ratios of firefly to Renilla luminescence (mean±s.d. of triplicate experiments). (d) NIH3T3 cells were treated with/without actinomycin D (2 μg ml⁻¹) for 1 h and challenged with 500 nM TG as indicated. MiR-216b levels were measured with qPCR (mean±s.d., n=3). (e) Dicer^flox/flox, Dicer^+/− and Dicer^−/− MEFs were treated with 500 nM TG, qPCR assessment of miR-216b (left) and chop mRNA (right). Data represent mean±s.d. of three independent experiments and statistical analysis was analysed by Student's t-test (*P<0.05).

Figure 4. Ire1α suppresses miR-216b expression.

(a) Ire1α^−/− and Ire1wt MEFs were treated with 500 nM TG and miR-216b (right) was measured by qPCR. N=3. (b) Ire1α was knocked down using three independent shRNAs 2, 3, 5 followed by treatment of cells with 500 nM TG. Ire1α knockdown was confirmed by immunoblot, and miR-216b levels were quantified by qPCR. (c) Illustration of the potential Ire1α cleavage sites within pre-miR-216b. (d) γ-³²PATP-labelled pre-miR-216b or Xbp-1 oligonucleotides were incubated with recombinant protein Ire1α (aa 465–977) for 2, 4 and 6 h, and cleavage products were resolved by a urea-polyacrylamide gel electrophoresis and visualized by autoradiography. (e) Ire1α^−/− and Ire1wt MEFs were treated with TG (500 nM) for indicated intervals. Ire1α, Bip and CHOP were assessed by immunoblot. (f) Xbp1s expression and (g) miR-216b levels in U2OS cells transfected with vector or Xbp1s were quantified by qPCR following exposure of cells to thapsigargin. All panels provide values that are the average of three independent experiments and error bars indicate standard deviation among all the replicates. Statistical analysis was analysed by Student's t-test (*P<0.05).

Figure 5. MiR-216b targets the c-Jun 3′UTR.

(a) miR-216b reporter construct (left) was expressed in mimic control (ctrl), miR-216b or A-miR-216b-expressing U2OS cells. Luciferase activity was measured and normalized to internal transfection control Renilla. Error bars represent standard deviation for three independent experiments. (b) MiR-216b seed sequence matches in the c-Jun 3′UTR. (c) NIH3T3 cells were treated with PERK inhibitor for 1 h and followed by 500 nM thapsigargin (TG); c-Jun and CHOP protein levels assessed by immunoblot. (d) U2OS stable cell lines expressing control, miR-216b or A-miR-216b were treated with 500 nM TG as indicated. Levels of the indicated proteins were measured by immunoblot. (e) An AP-1 luciferase reporter was transiently introduced into NIH3T3 cells along with a Renilla control. Cells were co-transfected as indicated with control, miR-216b or A-miR-216b. 48 h post transfection, cells were treated with TG before quantifying luciferase relative to Renilla control. (f) CHOP^+/+ and CHOP^−/− MEFs were treated with 500 nM TG for indicated intervals. CHOP and c-Jun were immunoblotted. (g–j) qPCR analysis of CHOP mRNA levels downstream target genes of c-Jun in U2OS treated as in d. Data present the mean of three independent experiments, error bars indicate the standard deviation. Statistical significance was analysed analysed by Student's t-test (*P<0.05).

Figure 6. MiR-216b expression sensitizes cells to ER stress.

(a) Representative FACS analysis histograms of U2OS stable cells expressing control, miR-216b or A-miR-216b after treatment with 500 nm TG. (b) U2OS cells stably expressing control, miR-216b or A-miR-216b were plated and treated with 400 nM TG for 1 or 3 h, and then returned to normal growth medium for 12 days (d). Plates were assessed for colony formation by staining with Giemsa staining (left). Quantification of colonies (right). (c) Cell doubling was quantified over 6 days. Values are means±s.d. (n=3) and statistical significance was analysed by Student's t-test. (*P<0.05; ^#P>0.05).

Figure 7. MiR-216b sensitizes cells to ER stress-dependent apoptosis via regulation of cJun.

(a) c-Jun knockdown with three independent shRNA (top panel). Total and cleaved PARP and cJun were assessed by immunoblot (middle panel). (b) U2OS cells were infected with virus encoding either miR-216b or sh-c-Jun # 2 (from panel a) at varying viral concentrations (top). C-Jun and cleaved caspase 3 were quantified by immunoblot 48 h post infection. (c) U2OS cells stably expressing control, miR-216b or A-miR-216b were infected with retrovirus coding MIGR1 or c-Jun for 3 days, cells were plated at 2 × 10³ cells per 60 mm dish, treated with 400 nM thapsigargin (TG) for 1–3 h, and then returned to growth medium for 12 days. (d) Quantification of Giemsa-stained colonies from c. Average cell survival fractions (TG treatment/untreated). Values are means±s.d. (n=3). (e) miR-216b was introduced into CHOP^−/− cells, treated as indicated with TG and assessed for accumulation of cleaved caspase 3. (f) Model depicting CHOP-dependent regulation of miR-216b and CHOP-dependent apoptosis. Data are mean±s.d. of three independent experiments. Statistical analysis was analysed by Student's t-test (*P<0.05).