12 research outputs found

    Prolyl hydroxylation of ATF4 on aa 60 and 235 by PHD1/3 limits ATF4 availability.

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    <p>(A–B) Specificity of shRNA used to KD PHD1/3. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with scrambled control or PHD1 shRNA and PHD3 shRNA alone or in combination, and treated with TM (1 µg/ml) for 6 h. The relative mRNA levels of PHD1 (A) and PHD3 (B) were determined by qPCR. The results are shown as the mean values ± S.E. of three independent experiments. Three independent shRNA were used to confirm the changes shown. (C) PHD1 and PHD3 cooperation is required and mediate the effect of Siah1a/2 on TM-induced CHOP transcription. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with different PHD1 shRNA or PHD3 shRNA, or their combination, or scrambled shRNA. Cells were treated with TM (1 µg/ml) and collected 6 h later. The relative transcription levels of CHOP were determined by qPCR. (D) PHD1 and PHD3 mediate the effect of Siah1a/2 on hypoxia-induced CHOP transcription. <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were infected with PHD1 shRNA and PHD3 shRNA alone or in combination, or control vector and exposed to 1% O<sub>2</sub> for 6 h. The relative mRNA level of CHOP was determined by qPCR. (E) PHD3 protein is induced in <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells. WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were exposed to 1% O<sub>2</sub> for 24 h prior to the analysis for the expression of HIF-1α, PHD3 and β-actin by Western blotting. The arrow points to the position of the endogenous PHD3 protein. (F) Annotated MS/MS spectra resulting in the identification of proline hydroxylation sites at P60 and P235. Identified fragment ions are shown, as are the detected sites of peptide backbone cleavage; <i>m</i>/<i>z</i>, mass to charge ratio. Note that site determining fragment ions resulted in localization of both sites of proline hydroxylation. (G) Mutations of the two identified proline hydroxylation sites at P235 and P60 to alanine stabilize ATF4 protein. 293T cells were transfected either with Flag-ATF4 or Flag-ATF4 presenting either a mutation at P60, P235, or both. After 24 h from transfection cells were treated overnight with vehicle, DMOG (0.5 mM; upper panel), or MG132 (5 µM; lower panel) followed by cell harvest and immunoblot analysis of ATF4 and β-actin. *** p<0.0005, ** p<0.005, * p<0.05 compared to ad shRNA scr. (A–D) in the same condition (student's t-test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of three independent experiments.</p

    UPR induction of Siah1/2 RNA and protein is ATF4-dependent.

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    <p>(A) TM induces Siah2 protein levels. Litter-matched MEF WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (1 µg/ml) for 10 h. Whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. (B) TM induces Siah1 protein. Litter-matched MEF WT and <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (1 µg/ml) for 10 h. Whole cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. Right panel: ER stress does not induce Siah1b mRNA levels. MEFs from <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> cells were treated with TM (2 µg/ml) for 6 h and the relative expression of Siah1b mRNA was measured by quantitative real time PCR (qPCR). (C) ER stress induces Siah2 mRNA levels. MEFs from WT animals were treated with the indicated concentrations of TM for 6 h and the relative expression of Siah2 mRNA was measured by qPCR. (D) HeLa and Lu1205 cells were treated with the indicated concentrations of TM for 6 h and the relative expression of Siah2 mRNA was measured by qPCR. (E) ER-stress induction of Siah2 mRNA is attenuated in <i>Atf4<sup>−/−</sup></i> MEFs. Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (2 µg/ml), hypoxia (H), or both. RNA prepared 6 h later was used for qPCR to quantify Siah2 transcripts, relative to levels of H3.3A mRNA. (F) CHOP mRNA levels are ATF4 but not hypoxia dependent. MEFs from <i>Atf4</i> WT and <i>Atf4<sup>−/−</sup></i> genotypes were subjected to TM or hypoxia or combined treatment and RNA prepared for qPCR analysis of CHOP transcripts. (G) VEGFA mRNA levels are ATF4 dependent under normoxia. Experiment was performed as indicated in panel E, except that qPCR analysis used the VEGFA primers. (H) <i>Atf4</i> WT and <i>Atf4<sup>−/−</sup></i> MEFs were infected with ATF4-expressing adenovirus. Cell lysates were prepared and the level of ATF4 protein was detected in immunoblots with the respective antibody. β-actin served as the loading control. (I) Ectopic ATF4 expression rescues Siah2 mRNA levels in TM-treated <i>Atf4<sup>−/−</sup></i> MEFs. MEFs of the indicated genotypes were infected with control (β-Gal) or with ATF4 adenoviruses for 24 h, followed by 6 h exposure to TM (2 µg/ml), as indicated. Relative expression of Siah2 mRNA was quantified by qPCR. *** p<0.0005, ** p<0.005, * p<0.05 compared to non treated (C–D) or to ATF4 WT in the same condition (student's t test). The Western blot experiments were repeated three times and the qPCR results are shown as the means ± S.E. of three independent experiments.</p

    Siah1/2-dependent gene expression analysis confirms an ER stress signature.

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    <p>(A) Heat map showing genes involved in diabetes, metabolism and ROS functions (light blue bar), hypoxia and related signaling (brown bar), and pathogen infection and related signaling (blue bar). Each bar represents a block of significant genes, which is involved in the same functional group. The selected genes have p-values less than or equal to 0.05 and fold-changes are greater than or equal to 2 (both directions). In the heatmap, the normalized expression signals are shown from green to red (lower signal to higher signal). The bars on the left of the heatmap indicate the functional groups. Each bar represents a block of significant genes, which involved in the same functional group. (B–C) Venn diagram representing the overlaps of significantly down- (green), and up- (red) expressed genes involved in the functional groups as indicated in the heatmap. Panel B shows genes from pairwise comparisons: knock out glucose deprivation (koGD) versus wtGD, ko oxygen deprivation (koOD) versus wtOD and ko oxygen and glucose deprivation (koOGD) versus wtOGD. Panel c shows genes from pairwise comparisons: ko versus wt, koTG versus wtTG and koTM versus wtTM. (D) Principal component analysis (PCA) 3D plot for the microarray data set. Each spot represents an individual array, and is colored based on treatment group. (E–H) Validation of representative genes from each of the main pathways identified to be Siah1/2-dependent. Siah WT and <i>Siah1a<sup>+/−</sup>::Siah2<sup>−/−</sup></i> (double knock out; DKO) MEF cells were subjected to OGD for 12 h. The relative transcription level was determined by qPCR. Shown are representative genes of the hypoxia (panel E and F), and diabetes/metabolism (panel G and H) pathways found to exhibit increase or decrease expression upon KO of Siah1a/2. The results are shown as the mean values ± S.E. of three independent experiments. (I–K) RNA prepared from Siah1a/2 WT and Siah DKO MEF cells was used for qPCR analysis. The expression of the transcripts from HSPA5 (I), Rab6 (J), GOLT1b (K) were validated by real time qPCR analysis. The results are shown as the mean values ± S.E. of three independent experiments.</p

    Siah1/2 mutant animals are protected from neuronal ischemia-induced cell death.

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    <p>(A) The mortality rate was calculated as the number of dead mice at 24 h after induction of MCAO in <i>Siah1a<sup>+/+</sup>::Siah2<sup>+/+</sup></i> and <i>Siah1a<sup>+/−</sup>::Siah2<sup>−/−</sup></i> mice. HT = heterozygous genotype of the Siah1a animals. (B) Brain sections from <i>Siah1a<sup>+/+</sup>::Siah2<sup>+/+</sup></i> and <i>Siah1a<sup>+/−</sup>::Siah2<sup>−/−</sup></i> mice were stained with Triphenyltetrazolium chloride (TTC) at 24 h after direct MCAO. The infarct volumes of brain sections were calculated as described in the Methods section. Five representative sections from two different mice of each group are shown. (C) At 24 h after MCAO, the brain sections were subjected to fixation, sectioned and further stained with H&E and ApopTag (TUNEL). Magnified TUNEL signal (red) is shown in the insets. The adjacent sections (#1, #2, #5 and #6) were stained with TTC solution. The scale bars indicate 50 µm. (D) Tissue sections adjacent to the ischemic core areas were analyzed by immunohistochemistry using the indicated antibodies. The square inset in the cartoon marks the area from which images were taken. The ischemic core was localized below the yellow line.</p

    Siah1/2 transcription is induced by ATF4 and sXBP1 upon UPR.

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    <p>(A,B) ER-stress induction of both Siah1 and Siah2 mRNA is attenuated in <i>Atf4<sup>−/−</sup></i> MEFs (A) and <i>Ire1α<sup>−/−</sup></i> MEFs (B). Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (2 µg/ml) or TG (1 µM) for 6 h and the relative expression of Siah1 and Siah2 mRNA was measured by qPCR. (C) IRE1α is required for ER-induced Siah2 mRNA levels. Ectopic expression of sXBP1 restores TM-induction of Siah2 mRNA in <i>Ire1α<sup>−/−</sup></i> MEFs. <i>Ire1α<sup>−/−</sup></i> MEFs were infected either with adenovirus encoding either β-gal or sXBP1. After 24 h, RNA was prepared and quantified using qPCR for the relative levels of Siah2 mRNA. (D) ER-stress induction of Siah2 mRNA levels but not of Siah1 mRNA are attenuated in <i>Atf6α<sup>−/−</sup></i> MEFs. Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (2 µg/ml) TG (1 µM) for 6 h and the relative expression of Siah1 and Siah2 mRNAs were measured by qPCR. (E) ER Stress induction of Siah1/2 transcripts is not HIF1-dependent. WT and HIF1α KO MEFs were subjected to TM (2 µg/ml) or TG (1 µM) treatment and RNA prepared 6 h later was subjected to analysis of Siah1 or Siah2 transcripts. *** p<0.0005, ** p<0.005, * p<0.07 compared to Ire1 KO (C) or to WT under the same condition (student's t test). The results are shown as the mean values ± S.E. of three independent experiments.</p

    Severe ER stress conditions are required to induce Siah1/2 transcription by UPR.

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    <p>(A) Various forms of stress increase Siah2 mRNA levels. MEFs were treated with H<sub>2</sub>O<sub>2</sub> (50 µM), TM (1 µg/ml), TG (1 µM) or histidinol (2 µM) for 8 h and Siah2 mRNA was analyzed by qPCR. Relative expression shown represents Siah2 transcript level. (B–E) CHO cells were treated with doxycycline (100 ng/ml) (83) and cells were collected after 0, 6, 24, and 96 h. The relative mRNA level of Factor VII (B), CHOP (C), Siah1 (D) and Siah2 (E) were determined by qPCR. The results are shown as the means ± S.E. of three independent experiments. (F) MEF cells were treated with increasing amount of TM and after 3 h the relative mRNA levels of Siah1 and Siah2 were determined by qPCR. (G) Siah2 is induced upon oxygen and glucose deprivation. WT or <i>Siah1a<sup>−/−</sup>::Siah2<sup>−/−</sup></i> MEFs were subjected to glucose derivation (GD), oxygen deprivation (1%O<sub>2</sub>; OD) or their combination (OGD) for 12 h followed by cell harvest and RNA analysis using qPCR for levels of Siah2 transcripts. (H–I) ER-stress induction of Siah2 protein and mRNA levels is attenuated in eIF2α AA MEFs. Littermate-matched MEFs of the indicated genotypes were subjected to treatment with TM (1 µg/ml) or TG (1 µM). Cell lysates were prepared 8 h later and levels of ATF4, Siah2 and CHOP proteins were detected by immunoblots. β-actin served as the loading control (H). RNA was prepared 6 h after treatment with TM or TG and was used for qPCR to quantify the levels of Siah2 transcript, relative to levels of H3.3A mRNA (I). *** p<0.0005, ** p<0.005, * p<0.05 compared to control (panels A–G) * p<0.08 compared to WT (panels H–I) (student's t-test). The Western blot experiments were repeated three times and the qPCR results are shown as the mean values ± S.E. of at least three independent experiments.</p

    List of common significant differential expression genes among KO versus WT, koOD versus wtOD, and koOGD versus wtOGD (Figure 4c Venn diagram).

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    <p>List of common significant differential expression genes among KO versus WT, koOD versus wtOD, and koOGD versus wtOGD (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004348#pgen-1004348-g004" target="_blank">Figure 4c</a> Venn diagram).</p

    List of common significant differential expression genes among KO versus WT, koTG versus wtTG, and koTM versus wtTM (Figure 4b Venn diagram).

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    <p>List of common significant differential expression genes among KO versus WT, koTG versus wtTG, and koTM versus wtTM (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004348#pgen-1004348-g004" target="_blank">Figure 4b</a> Venn diagram).</p
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