Perk-dependent repression of miR-106b-25 cluster is required for ER stress-induced apoptosis

Abstract

Activation of the unfolded protein response sensor PKR-like endoplasmic reticulum kinase (Perk) attenuates endoplasmic reticulum (ER) stress levels. Conversantly, if the damage is too severe and ER function cannot be restored, this signaling branch triggers apoptosis. Bcl-2 homology 3-only family member Bim is essential for ER stress-induced apoptosis. However, the regulatory mechanisms controlling Bim activation under ER stress conditions are not well understood. Here, we show that downregulation of the miR-106b-25 cluster contributes to ER stress-induced apoptosis and the upregulation of Bim. Hypericin-mediated photo-oxidative ER damage induced Perk-dependent cell death and led to a significant decrease in the levels of miRNAs belonging to miR-106b-25 cluster in wild-type (WT) but not in Perk⁻/⁻ MEFs. Further, we show that expression of miR-106b-25 and Mcm-7 (host gene of miR-106b-25) is co-regulated through the transcription factors Atf4 (activating transcription factor 4) and Nrf2 (nuclear factor-erythroid-2-related factor 2). ER stress increased the activity of WT Bim 3'UTR (untranslated region) construct but not the miR-106b-25 recognition site-mutated Bim 3'UTR construct. Overexpression of miR-106b-25 cluster inhibits ER stress-induced cell death in WT but did not confer any further protection in Bim-knockdown cells. Further, we show downregulation in the levels of miR-106b-25 cluster in the symptomatic SOD1(G86R) transgenic mice. Our results suggest a molecular mechanism whereby repression of miR-106b-25 cluster has an important role in ER stress-mediated increase in Bim and apoptosis.

Figure 1

Downregulation of miRNAs of the miR-106b-25 cluster during conditions of ER stress. (a) H9c2 cardiomyocytes were either untreated (Un) or treated with (1.0 μM) Tg and (1.0 μg/ml) Tm for 24 h. The expression level of indicated miRNAs was quantified by real-time RT-PCR, normalizing against snoRNA. Error bars represent mean±S.D. from three independent experiments performed in triplicate. (b) MCF-7 cells were either untreated (Un) or treated with (2.0 μM) Tg and (2.0 μg/ml) Tm for 24 h and the expression level of indicated miRNAs was quantified by real-time RT-PCR, normalizing against U6 snRNA. Error bars represent mean±S.D. from three independent experiments performed in triplicate. (*P<0.05, **P<0.01, two-tailed unpaired t-test compared with untreated cells)

Figure 2

Perk-dependent increases in the activity of Bim 3′UTR reporter construct during conditions of ER stress. (a) pCDH and miR-106b-25 cells were either untreated (Un) or treated with (0.25 μM) Tg for indicated time points. Total protein was isolated and immunoblotting was performed using antibodies against Bim and β-actin. (b) MCF-7 cells were transfected with Bim 3′UTR reporter plasmid containing binding sites for members of miR-106b-25 (Bim UTR-wt) or Bim 3′UTR reporter plasmid with mutated binding sites for members of miR-106b-25 (Bim UTR-mt). At 24 h after transfection cells were left untreated (Un) or treated with (2.0 μM) Tg and (2.0 μg/ml) Tm, (50 μg/ml) Eto, or (600 μM) H₂O₂. Luciferase activity was measured 48 h after transfection using Dual-Glo assay system and normalized luciferase activity (Renilla/Firefly) is shown. Error bars represent mean±S.D. from three independent experiments performed in duplicate. (c) PC12 cells were transfected with Bim 3′UTR reporter plasmid containing binding sites for members of miR-106b-25 (Bim UTR-wt). At 24 h after transfection cells were left untreated (Un) or treated with (0.25 μM) Tg and (2.0 μg/ml) Tm. Luciferase activity was measured 48 h after transfection using Dual-Glo assay system and normalized luciferase activity (Renilla/Firefly) is shown. Error bars represent mean±S.D. from three independent experiments performed in duplicate. (d) MCF-7 cells were transfected with Bim 3′UTR reporter plasmid containing binding sites for members of miR-106b-25 (Bim UTR-wt) in combination with pcDNA3 (pcDNA), WT Ire1, dominant-negative Ire1 (ΔC Ire1), WT Perk or dominant-negative Perk (K618A Perk) expression plasmids. At 24 h after transfection cells were left untreated (Un) or treated with (2.0 μM) Tg and (2.0 μg/ml) Tm. Luciferase activity was measured 48 h after transfection using Dual-Glo assay system and normalized luciferase activity (Renilla/Firefly) is shown. Error bars represent mean±S.D. from three independent experiments performed in duplicate. (*P<0.05, two-tailed unpaired t-test compared with untreated cells)

Figure 3

PERK-dependent regulation of miR-106b-25 cluster during conditions of ER stress. (a) WT and Perk knockout (Perk^−/−) MEFs were exposed to phOx stress (200 nM hypericin for 2 h, 2.7 J/cm²). The graph shows the surviving fraction of cells 24 h after treatment. Error bars represent mean±S.D. from three experiments performed in duplicate. (*P<0.05, two-tailed unpaired t-test compared with WT MEFs). (b) WT and Perk knockout (Perk^−/−) MEFs were exposed to phOx stress (200 nM hypericin for 2 h, 2.7J/cm²) and expression levels of Bim was quantified by real-time RT-PCR, normalizing against Gapdh. Error bars represent mean±S.D. from two independent experiments performed in triplicate. (c) WT and Perk knockout (Perk^−/−) MEFs were incubated with 200 nM hypericin for 2 h and irradiated (2.7 J/cm²) (phOx stress). Expression level of miR-106b, miR-25, and miR-93 was quantified by real-time RT-PCR, normalizing against snoRNA. Error bars represent mean±S.D. from two independent experiments performed in triplicate. (*P<0.05, **P<0.01, two-tailed unpaired t-test compared with untreated WT MEFs)

Figure 4

Effect of ectopic Atf4 and Nrf2 on the expression miR-106b-25 cluster and Bim. (a and b) PC12 cells were transfected with pcDNA3 (c), WT Atf4 or Nrf2 expression plasmids and total RNA was isolated at indicated time points. Expression levels of indicated miRNAs were quantified by real-time RT-PCR, normalizing against snoRNA. Error bars represent mean±S.D. from three independent experiments performed in triplicate. (*P<0.05, **P<0.01, two-tailed unpaired t-test as compared with control transfected cells). (c) PC12 cells were transfected with pcDNA3 (c), WT Atf4, or Nrf2 expression plasmids. Total RNA was isolated and expression levels of Chop, Bim, and Mcm-7 were quantified by real-time RT-PCR, normalizing against Gapdh. Error bars represent mean±S.D. from three independent experiments performed in triplicate. (*P<0.05, **P<0.01, two-tailed unpaired t-test as compared with control transfected cells). (d) pTRIPZ-shKeap1-293T cells were treated with (500 ng/ml) of doxycycline for indicated time points and expression levels of miR-106b, miR-93, and miR-25 were quantified by real-time RT-PCR, normalizing against snoRNA. Error bars represent mean±S.D. from four independent experiments performed in triplicate. (*P<0.05, **P<0.01, two-tailed unpaired t-test as compared with uninduced (No Dox) cells)

Figure 5

Downregulation of miRNAs belonging to miR-106b-25 cluster during conditions of ER stress. PC12 cells were either untreated (Un) or treated with (0.25 μM) Tg and (2.0 μg/ml) Tm for 24 h (a), (100 μM) 4-HNE for 6 h (b), and (0.5 mM) tBHQ for 12 h (c). The expression levels of miR-106b, miR-93 and miR-25 were quantified by real-time RT-PCR, normalizing against snoRNA. The expression level of Bim and Mcm-7 was quantified by real-time RT-PCR, normalizing against Gapdh. Error bars represent mean±S.D. from three independent experiments performed in triplicate. (*P<0.05, **P<0.01, two-tailed unpaired t-test as compared with untreated cells)

Figure 6

Inhibition of ER stress-induced apoptosis by miR-106b-25 cluster is mediated by repression of Bim. (a) The control (Neo) and Bim-shRNA expressing (Bim-shRNA) PC12 cells were either untreated (Un) or treated with (0.25 μM) Tg, (2.0 μg/ml) Tm, (0.5 mM) tBHQ, and (100 μM) 4-4-HNE for 48 h. Apoptosis was determined with annexin-V/PI staining followed by FACS analysis. Percentages of cells positive for both annexin-V and annexin-V/PI are shown. Average and error bars represent mean±S.D. from three independent experiments. (*P<0.005, two-tailed unpaired t-test as compared with control (Neo) cells). (b) Upper panel, The indicated cells were either untreated (Un) or treated with (0.25 μM) Tg for indicated 24 h. Total protein was isolated and immunoblotting was performed using antibodies against Bim, Chop and β-actin. Lower panel, in the experiment described in (a), induction of Bim and Chop was calculated after densitometric analysis of the autoradiographs with Image J. Average and error bars represent mean±S.D. from three independent experiments.(**P<0.05, two-tailed unpaired t-test as compared with miR-106b-25 expressing (Neo/miR-106b-25) cells). (c) The indicated cells were either untreated or treated with (0.25 μM) Tg, (2.0 μg/ml) Tm, (0.5 mM) tBHQ, and (100 μM) 4-4-HNE for 48 h. Apoptosis was determined with annexin-V/PI staining followed by FACS analysis. Percentages of cells positive for both annexin-V and annexin-V/PI are shown. Average and error bars represent mean±S.D. from three independent experiments. (*P<0.005, two-tailed unpaired t-test as compared with control (Neo/pCDH) cells). (d) The indicated cells were either untreated or treated with (0.25 μM) Tg for 24 h or (2.0 μg/ml) Tm for 24 h, (0.5 mM) tBHQ for 12 h, and (100 μM) 4-4-HNE for 12 h, and DEVDase activity was measured as described in Materials and Methods. Average and error bars represent mean±S.D. from three independent experiments. (*P<0.005, two-tailed unpaired t-test as compared with control (Neo/pCDH) cells)

Figure 7

Downregulation of miRNAs belonging to miR-106b-25 cluster in the SOD1 mutant mice. (a) The total protein was isolated from the cortex of WT and symptomatic SOD1^G86R mice. A total of 40 μg protein per sample used for immunoblotting was analyzed using antibodies against Bim and HSP90. Upper panel, western blot analysis of Bim expression in cortex of WT and symptomatic SOD1^G86R mice. Lower panel, average of Bim expression in the cortex of WT (n=2) and symptomatic SOD1^G86R (n=7) mice are shown and error bars represent mean±S.D. (**P<0.05, two-tailed unpaired t-test as compared with WT mice). (b–d) The total RNA was isolated from the cortex of WT and symptomatic SOD1^G86R mice and expression levels of miR-106b, miR-93, and miR-25 were quantified by real-time RT-PCR, normalizing against U6 snRNA. Box plots showing median and distribution of miR-106b (b), miR-25, (c) and miR-93 (d) in the cortex of WT (n=6) and symptomatic SOD1^G86R (n=6) mice are shown. (*P<0.05, Wilcoxon signed rank test as compared with WT mice)

Figure 8

Effect of miR-106b-25 expression on sensitivity to ER stress-induced cell death. (a) The expression of miR-106b-25 cluster members was measured by qRT-PCR from total RNA isolated from lysates of the indicated cell lines. Results are presented as relative expression using Z30 as an internal control and employing the delta-delta C_t method. Error bars represent mean±S.E. from three independent experiments performed in triplicate. Statistical significance was determined by ANOVA with Bonferroni post hoc correction; *P<0.05, **P<0.01, ***P<0.001, compared with H69 cells. (b) Indicated cell lines were treated with (2.0 μM) Tg and (2.0 μg/ml) Tm for 24 h. After treatment the apoptotic nuclei were determined as described in Materials and Methods section. Error bars represent mean±S.E. from three independent experiments performed in triplicate. Statistical significance was determined by ANOVA with Bonferroni post hoc correction; *P<0.05, **P<0.01, ***P<0.001, compared with H69 cells

Figure 9

Schematic summary detailing the main conclusions of this manuscript. During ER stress the transcription factors Atf4 and Nrf2 are activated and downregulate the Mcm7, host gene of miR-106b-25 cluster in a Perk dependent manner, thus contributing to induction of Bim and ER stress-induced apoptosis (see text for details)