30 research outputs found

    Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

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    <div><p>Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate <i>NLRP1</i> gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to <i>NLRP1</i> promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for <i>NLRP1</i> induction. This study provides further evidence that links ER stress with innate</p></div

    The IRE1α/XBP1s Pathway Is Essential for the Glucose Response and Protection of β Cells

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    <div><p>Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α) to initiate X-box-binding protein 1 (<i>Xbp1</i>) mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional <i>Xbp1</i> mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated <i>Ire1α</i> deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, <i>Ire1α</i> deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon <i>Ire1α</i> deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, <i>Ire1α</i> deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific <i>Ire1α</i> deletion. The results demonstrate that glucose activates IRE1α-mediated <i>Xbp1</i> splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure.</p></div

    Novel Bioinformatics Method for Identification of Genome-Wide Non-Canonical Spliced Regions Using RNA-Seq Data

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    <div><p>Setting</p><p>During endoplasmic reticulum (ER) stress, the endoribonuclease (RNase) <i>Ire1</i>α initiates removal of a 26 nt region from the mRNA encoding the transcription factor <i>Xbp1</i> via an unconventional mechanism (atypically within the cytosol). This causes an open reading frame-shift that leads to altered transcriptional regulation of numerous downstream genes in response to ER stress as part of the unfolded protein response (UPR). Strikingly, other examples of targeted, unconventional splicing of short mRNA regions have yet to be reported.</p><p>Objective</p><p>Our goal was to develop an approach to identify non-canonical, possibly very short, splicing regions using RNA-Seq data and apply it to ER stress-induced <i>Ire1</i>α heterozygous and knockout mouse embryonic fibroblast (MEF) cell lines to identify additional <i>Ire1</i>α targets.</p><p>Results</p><p>We developed a bioinformatics approach called the Read-Split-Walk (RSW) pipeline, and evaluated it using two <i>Ire1</i>α heterozygous and two <i>Ire1</i>α-null samples. The 26 nt non-canonical splice site in <i>Xbp1</i> was detected as the top hit by our RSW pipeline in heterozygous samples but not in the negative control <i>Ire1</i>α knockout samples. We compared the <i>Xbp1</i> results from our approach with results using the alignment program BWA, Bowtie2, STAR, Exonerate and the Unix “<i>grep</i>” command. We then applied our RSW pipeline to RNA-Seq data from the SKBR3 human breast cancer cell line. RSW reported a large number of non-canonical spliced regions for 108 genes in chromosome 17, which were identified by an independent study.</p><p>Conclusions</p><p>We conclude that our RSW pipeline is a practical approach for identifying non-canonical splice junction sites on a genome-wide level. We demonstrate that our pipeline can detect novel splice sites in RNA-Seq data generated under similar conditions for multiple species, in our case mouse and human.</p></div

    Discovery of Sulfonamidebenzamides as Selective Apoptotic CHOP Pathway Activators of the Unfolded Protein Response

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    Cellular proteins that fail to fold properly result in inactive or disfunctional proteins that can have toxic functions. The unfolded protein response (UPR) is a two-tiered cellular mechanism initiated by eukaryotic cells that have accumulated misfolded proteins within the endoplasmic reticulum (ER). An adaptive pathway facilitates the clearance of the undesired proteins; however, if overwhelmed, cells trigger apoptosis by upregulating transcription factors such as C/EBP-homologous protein (CHOP). A high throughput screen was performed directed at identifying compounds that selectively upregulate the apoptotic CHOP pathway while avoiding adaptive signaling cascades, resulting in a sulfonamidebenzamide chemotype that was optimized. These efforts produced a potent and selective CHOP inducer (AC<sub>50</sub> = 0.8 μM; XBP1 > 80 μM), which was efficacious in both mouse embryonic fibroblast cells and a human oral squamous cell cancer cell line, and demonstrated antiproliferative effects for multiple cancer cell lines in the NCI-60 panel

    GADD34 CA-AAV effectively inhibits eIF2α phosphorylation in 5XFAD brain but does not block amyloid-associated BACE1 elevation.

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    <p>5XFAD or non-Tg pups were injected on postnatal day 0 into lateral ventricles with 2 µl per hemisphere of 6.6×10<sup>10</sup> viral genomes of GADD34 CA-AAV or GADD 34 cont–AAV plus 6.9×10<sup>10</sup> viral genomes of CaMKII tTA-AAV. Mice were aged to 6 months and brains harvested for immunoblot and immunofluorescence microscopy analysis. (A) 20 µg/lane of cortex or hippocampus homogenate from 6 month-old 5XFAD (+) and non-Tg (–) mice either GADD34 CA-AAV injected (CA) or GADD 34 cont–AAV injected (cont) were subjected to immunoblot analysis for BACE1, total eIF2α, phosphorylated (p)-eIF2α, and βIII-tubulin as a loading control. All samples were transferred onto a single piece of PVDF membrane, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101643#s2" target="_blank">Methods</a>, and representative blots are shown here. (B) BACE1 immunosignal intensities were normalized to those of βIII-tubulin. Phosphorylated and total eIF2α immunosignal intensities were measured and phosphorylated:total eIF2α (phospho/total eIF2α) ratio calculated. All measures are displayed as percentage of GADD 34 cont–AAV injected non-Tg control. BACE1 levels were elevated in GADD 34 cont–AAV transduced 5XFAD cortex and hippocampus compared to non-Tg, as expected. Importantly, GADD34 CA-AAV transduction reduced p-eIF2α levels by ∼85–90% compared to GADD34 cont-AAV transduction in both 5XFAD and non-Tg cortex and hippocampus. Despite this dramatic inhibition of eIF2α phosphorylation, BACE1 levels were elevated in GADD34 CA-AAV transduced 5XFAD cortex and hippocampus to the same extent as in GADD34 cont-AAV transduced 5XFAD cortex and hippocampus. n = 6–15 mice per group, bars represent SEM, asterisks (*) indicate significant changes compared to non-Tg GADD34 cont-AAV control, NS  =  not significant, p<0.05*, p<0.01**, p<0.001***, (#) represents significant difference between 5XFAD GADD34 cont-AAV and 5XFAD GADD34 CA-AAV p<0.001 ###. (C) Cortical homogenates from 5XFAD mice injected with either GADD 34 cont–AAV or GADD34 CA-AAV were prepared for measurement of total (soluble plus insoluble) Aβ42 levels (ng/mg total protein) by ELISA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101643#s2" target="_blank">Methods</a>). No significant difference in total Aβ42 level between GADD34 CA-AAV and GADD34 cont-AAV brain transduction was observed. Bars represent SEM (D) Coronal brain sections of representative GADD34 CA-AAV or GADD34 cont-AAV transduced 5XFAD mice co-stained with anti-BACE1 antibody (green) and thiazine red (ThR) for fibrillar amyloid, then imaged by fluorescence microscopy. Both the intensities of BACE1 immunostaining and fibrillar plaque load signal appear unaffected by reduction of eIF2α phosphorylation via GADD34 CA-AAV transduction, thus corroborating our immunoblot analysis that phosphorylated eIF2α does not mediate amyloid-associated BACE1 elevation. Each image is taken at 10x objective magnification, at the same exposure, from the cortex just above hippocampal region CA3. Scale bar = 100 µm.</p

    <i>KO</i> islets exhibit ER stress.

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    <p>(A) qRT-PCR of UPR genes in islets isolated 6 wk post-Tam and incubated in 11 mM glucose 16 h ([<i>n</i> = 5], [<i>p</i> ≤ 0.05]). (B) Immunofluorescence microscopy of pancreas sections stained for KDEL (BIP and GRP94) (green), the plasma membrane protein GLUT2 (red), and nuclei DAPI (blue). Overlap of red/green channels represents defective compartmentalization that was found to be increased in the <i>KO</i><sup><i>Fe/-; Cre</i></sup> as shown in yellow. Scale bars, 400x = 50 μm, 1,000x = 10 μm, 5,180x = 2 μm and 10,500x = 1 μM. Additional examples are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.s007" target="_blank">S3B Fig</a>. (C) EM of adult mouse (16 wk old) islets and their β cells from mice 2 wk post-Tam. Scale bars, both panels, 1 μm. Distended mitochondria are outlined with yellow dashes. (D) Conventional PCR flanking the 26 nt intron in <i>Xbp1</i> mRNA spliced by IRE1α from the islet complementary DNAs (cDNAs) used for mRNA-Seq analysis, 6 mM versus 18 mM glucose. Results representative of <i>n</i> = 5 per genotype. (E) Global heatmap for the ~22,000 mRNAs detected by mRNA-Seq for 18 mM <i>KO</i><sup><i>Fe/-; Cre</i></sup> & <i>WT</i><sup><i>Fe/+</i></sup> samples; green and red indicate increased and decreased expression. The blue box indicates genes with inverse expression dependent on IRE1α and high glucose.</p

    mRNA sequencing identifies IRE1α- and glucose-dependent mRNAs in islets.

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    <p>(A) mRNA-Seq data on β cell-specific mRNAs. The results show no significant change to INS1 or INS2 in the <i>KO</i><sup><i>Fe/-; Cre</i></sup> samples, while MAFA, GCG, and PC5 are increased by deletion ([<i>n</i> = 5], [18 mM <i>KO</i><sup><i>Fe/-; Cre</i></sup>, <i>p</i>-values ≤ 0.05]). mRNA-Seq expression fold changes were normalized relative to the 6 mM <i>WT</i><sup><i>Fe/+</i></sup> islet context. (B) Four-way Venn diagrams of <i>WT</i><sup><i>Fe/+</i></sup> versus <i>KO</i><sup><i>Fe/-; Cre</i></sup> islets during 6 mM versus 1 8mM glucose exposur<i>e</i> for 72 h. <i>Ire1α</i>-dependent mRNAs are in bold italics, while those also dependent on high glucose are in bold, italicized, and underlined font. At the center, bar graphs representing the <i>Ire1α</i>- and glucose-dependent trends of interest are labeled “Induction” and “Repression.” (C) Combined <b>DAVID</b> (the Database for Annotation, Visualization and Integrated Discovery) and “ConceptGen” GO analysis of <i>Ire1α-</i> and glucose-dependent mRNAs. Categories shown are specifically found in the genotype, while the shared categories have been omitted for simplicity, although no single mRNA was common between the groups. (D) Mass spectrometry of murine islets infected with <i>Ad-IREα-K907A (Ad-ΔR)</i> versus <i>Ad-β-Galactosidase</i> (<i>β-Gal</i>). Proteins with ≥5 unique peptides detected per protein increased or decreased upon infection in triplicate were analyzed for GO using ConceptGen and DAVID web resources (<i>n</i> = 3). The proteins shown (Fig 3D) exhibit the same expression dependence for IRE1α as measured by mRNA-Seq (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002277#pbio.1002277.s002" target="_blank">S2 Data</a>).</p

    Genetic reduction of eIF2α phosphorylation via eIF2α S51A knockin mutation does not block amyloid-associated BACE1 elevation in 5XFAD brain.

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    <p>5XFAD mice were crossed with mice harboring the eIF2α S51A targeted replacement mutation to generate 5XFAD (+) or non-Tg (–) offspring that were either heterozygous for the eIF2α S51A knockin mutation (S/A) or wild-type (S/S). Mice were aged to 12 months, brains harvested, and homogenates prepared. 20 µg/lane of brain homogenate were subjected to immunoblot analysis for transgenic human (h) APP, BACE1, total eIF2α, and phosphorylated (p)-eIF2α. All samples were transferred onto a single piece of PVDF membrane and stained with ponceau S as a protein loading control, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101643#s2" target="_blank">Methods</a>. For quantification, BACE1 immunosignal intensity was normalized to ponceau S staining intensity for a given lane. Phosphorylated and total eIF2α immunosignal intensities were measured and phosphorylated:total eIF2α (phospho/total eIF2α) ratio calculated for a given lane. The means of each group were calculated and means displayed as percentage of the mean non-Tg S/S control. Both non-Tg and 5XFAD mice that were also heterozygous for the eIF2α S51A knockin mutation had a ∼40% reduction in phospho/total eIF2α ratio compared to their S/S counterparts; presumably, the phospho/total eIF2α ratios did not reach the expected 50% reduction because of a high non-specific background on the p-eIF2α immunoblot or partial compensatory increased phosphorylation of the wild type allele. Importantly, BACE1 level in 5XFAD; S/A brain showed an amyloid-associated elevation that was equivalent to that of 5XFAD; S/S brain, despite the 40% reduction in phospho/total eIF2α ratio. n = 19–30 mice per group. Bars represent SEM, asterisks (*) indicate significant changes compared to non-Tg S/S control, p<0.05*, p<0.01**, p<0.001***, NS  =  not significant, (#) indicates significant difference between 5XFAD S/S and 5XFAD S/A p<0.001 ###.</p

    BACE1-YFP expressed from a transgene with a truncated BACE1 mRNA 5′ UTR is also elevated and accumulates around amyloid plaques in 5XFAD brain.

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    <p>(A) 5′ UTR of BACE1-YFP transgene. The BACE1-YFP coding region (green) was subcloned into the tetO promoter vector PMM400 (black) via an NheI site (gray) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101643#pone.0101643-BuggiaPrevot1" target="_blank">[52]</a>, leaving a severely truncated BACE1 mRNA 5′ UTR (orange) consisting of only eleven nucleotides that lack the uORFs required for de-repression of translation by phosphorylated eIF2α. (B) 5XFAD mice were crossed with BACE1-YFP transgenic mice to generate 5XFAD (+) and non-Tg (–) offspring that also expressed the BACE1-YFP transgene. 5XFAD and non-Tg offspring that lacked the BACE1-YFP transgene were also generated. At 6–8 months of age, cortices of 5XFAD; BACE1-YFP, non-Tg; BACE1-YFP, 5XFAD, and non-Tg littermates (n = 5 for each group) were harvested, homogenized, and 20 µg/lane of homogenates were subjected to immunoblot analysis for BACE1 using the 3D5 anti-BACE1 antibody. The immunoblot was stained with ponceau S as a protein loading control. Representative BACE1-YFP immunoblot signals are shown. BACE1-YFP runs at ∼90 kDa on SDS-PAGE, compared to ∼65 kDa for endogenous (endog.) BACE1. For quantification, BACE1 and BACE1-YFP immunosignal intensities were normalized to ponceau S staining intensity for a given lane. The means of each group were calculated and means displayed as percentage of the mean BACE1 level in non-Tg control. The BACE1-YFP transgene is expressed at levels that are ∼4-fold higher than that of endogenous BACE1. As expected, endogenous BACE1 level is significantly elevated in 5XFAD brain compared to non-Tg brain. Most importantly, BACE1-YFP levels also exhibit a significant amyloid-associated elevation with the 5XFAD genotype compared to the non-Tg genotype, despite the complete absence of uORFs necessary for regulation by eIF2α phosphorylation. Bars represent SEM, asterisks (*) indicate significant changes compared to respective non-Tg control, p<0.05*. (C) Sagittal section of representative 5XFAD; BACE1-YFP cortex stained with anti-BACE1 antibody and imaged for BACE1 immunofluorescence (red) and YFP fluorescence (green) by confocal microscopy. Upper row shows lower magnification of several amyloid plaques (stars) each surrounded by an annulus of punctate accumulations of BACE1 and BACE1-YFP. Lower row shows higher magnification image of boxed inset in upper row. Our previous work has identified these BACE1 accumulations as swollen dystrophic axons and presynaptic terminals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101643#pone.0101643-Kandalepas1" target="_blank">[24]</a>. Note the extensive co-localization of BACE1 and BACE1-YFP, although their relative levels appear to vary somewhat in different dystrophies. These results demonstrate that BACE1-YFP accumulates around plaques in the same pattern as endogenous BACE1. Blue in the center of the annulus represents the amyloid plaque core, marked with (*). Blue outside of the annulus indicates DAPI-stained nuclei.</p
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