The No-Go and Nonsense-Mediated RNA Decay

Diabetes Volume 67, October 2018 2019

The No-Go and Nonsense-Mediated RNA Decay Pathways Are Regulated by Inﬂammatory Cytokines in Insulin-Producing Cells and Human Islets and Determine b-Cell Insulin Biosynthesis and Survival

Seyed Mojtaba Ghiasi,1 Nicolai Krogh,2 Björn Tyrberg,3 and Thomas Mandrup-Poulsen1

Diabetes 2018;67:2019–2037 | https://doi.org/10.2337/db18-0073

Stress-related changes in b-cell mRNA levels result from Proinflammatory cytokines have been implied in b-cell a balance between gene transcription and mRNA decay. failure and apoptosis in type 1 and type 2 diabetes (T1D The regulation of RNA decay pathways has not been and T2D) in part by regulation of the b-cell transcriptome investigated in pancreatic b-cells. We found that no-go (1). The levels of ;20% of the .29,000 transcripts in and nonsense-mediated RNA decay pathway compo- human islets are altered by cytokines, including apoptosis- nents (RDPCs) and exoribonuclease complexes were fl and in ammation-related genes (2). Interestingly, 35% of STUDIES ISLET expressed in INS-1 cells and human islets. Pelo, Dcp2, the genes expressed in human islets undergo alternative Dis3L2, Upf2, and Smg1/5/6/7 were upregulated by in- splicing, and cytokines cause substantial changes in the fl ammatory cytokines in INS-1 cells under conditions number of spliced transcripts (2). Most human genes b where central -cell mRNAs were downregulated. These exhibit alternative splicing, but not all alternatively spliced changes in RDPC mRNA or corresponding protein transcripts are translated into functional proteins. This levels were largely confirmed in INS-1 cells and rat/ varies in a cell-specific manner, contributing to tissue human islets. Cytokine-induced upregulation of Pelo, specificity of gene expression (3), and can be modulated Xrn1, Dis3L2, Upf2, and Smg1/6 was reduced by induc- by cellular signals such as those provided by proinflammatory ible nitric oxide synthase inhibition, as were endoplasmic cytokines (4). In pancreatic b-cells, cytokines upregulate reticulum (ER) stress, inhibition of Ins1/2 mRNA, and . accumulated insulin secretion. Reactive oxygen species the expression of 20 genes involved in RNA splicing, and . inhibition or iron chelation did not affect RDPC ex- cytokines induce changes in alternative splicing of 50% fi pression. Pelo or Xrn1 knockdown (KD) aggravated, of the cytokine-modi ed genes as detected by exon array whereas Smg6 KD ameliorated, cytokine-induced INS-1 analysis (4). fi cell death without affecting ER stress; both increased mRNA molecules are prone to damage and delity insulin biosynthesis and medium accumulation but errors, which can cause translation of aberrant mRNA not glucose-stimulated insulin secretion in cytokine- into dysfunctional proteins (5), and so far, an RNA repair exposed INS-1 cells. In conclusion, RDPCs are regu- machinery analogous to that operating to restore damaged lated by inflammatory stress in b-cells. RDPC KD DNA has not been discovered. Rather, cellular RNA sur- improved insulin biosynthesis, likely by preventing veillance mechanisms assure the quality and fidelity of Ins1/2 mRNA clearance. Pelo/Xrn1 KD aggravated, but mRNA molecules (6), resulting in breakdown of damaged Smg6 KD ameliorated, cytokine-mediated b-cell death, RNA. This is generally achieved through targeting aberrant possibly through prevention of proapoptotic and anti- mRNA molecules for degradation by various endogenous apoptotic mRNA degradation, respectively. nucleases.

1Department of Biomedical Sciences, University of Copenhagen, Copenhagen, This article contains Supplementary Data online at http://diabetes Denmark .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0073/-/DC1. 2 Department of Cellular and Molecular Medicine, University of Copenhagen, © 2018 by the American Diabetes Association. Readers may use this article as Copenhagen, Denmark long as the work is properly cited, the use is educational and not for proﬁt, and the 3 Translational Science; Cardiovascular, Renal and Metabolism; and IMED Biotech work is not altered. More information is available at http://www.diabetesjournals Unit, AstraZeneca, Gothenburg, Sweden .org/content/license. Corresponding author: Thomas Mandrup-Poulsen, [email protected]. Received 17 January 2018 and accepted 23 July 2018. 2020 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018

At least four forms of cotranslational mRNA quality Denmark). After 48 h of preincubation, cells were exposed control pathways have been described: nonsense-mediated to cytokine mixture at the concentrations indicated in decay (NMD), Staufen1 (STAU1)-mediated mRNA decay figures or figure legends for the indicated time points, (SMD), no-go decay (NGD), and nonstop decay (NSD) glucolipotoxic (GLT) conditions (0.5 mmol/L palmitate con- (7–9). Both NSD and NGD pathways share the ribosomal jugated with 0.1% albumin + 25 mmol/L glucose), or control dissociation factor Pelo, which is necessary for degradation medium (RPMI-glutamax, 10% FBS, 1% penicillin/strepto- of “non-stop codon” mRNAs and damaged mRNAs that mycin, 50 mmol/L 2-mercaptoethanol). cause translational stalling, respectively (10). In SMD, STAU1 recognizes either stem-loop intramolecular base Human and Rat Islet Culture and Cytokine Exposures . pairing of 39-UTR sequences or intermolecular base pairing Islets from six human heart-beating organ donors ( 80% of 39-UTR sequences with long-noncoding RNA via partially purity, donor characteristics listed in Supplementary Table A complementary Alu elements (8). NMD engaging Upf1, 2, 1 ) were isolated by the European Consortium for Islet 3A, and 3B and Smg1, 5, 6, and 7 degrades mRNAs with Transplantation (ECIT) under local approval in Milan, premature termination codons (PTCs). PTCs can arise in Italy, received in fully anonymous form and precultured cellsthroughvariousmechanisms: germline or somatic muta- as described previously (15). There were no apparent differ- tions in DNA, errors in transcription, posttranscriptional ences in results obtained with islets from male or female mRNA damage, or notably errors in mRNA processing, donors, and data were therefore combined. For experiments, such as alternative splicing (9). Failure to recognize and 50 human islets were cultured in RPMI supplemented with eliminate these mRNA transcripts can result in production 2% human serum (Life Technologies, Naerum, Denmark), of truncated and dysfunctional proteins that directly perturb 1% penicillin/streptomycin, and 5.6 mmol/L glucose for 24 h cell function or accumulate in the endoplasmic reticulum exposed with or without cytokine mixture (300 pg/mL (ER) and thus cause ER stress. rrIL-1b +10ng/mLrhIFN-g +10ng/mLrhTNF-a)or PTCs have been implicated in ;30% of all inherited control medium for 4 days. diseases, indicating that the NMD pathway plays a vital Rat islets were isolated from 1-week-old rat pups and role in survival and health. Expressed sequence tag analysis precultured for 72 h before exposure to cytokines for 12, of RNA-sequencing data of the human transcriptome iden- 18, and 24 h as previously described (16). tified 35% of 5,693 alternatively spliced isoforms to be NMD Single-Cell RNA Sequencing of Pancreatic Islets targets, and at least 12% of human genes have a PTC- The expression of genes in islet cell types was determined containing splice isoform (11), thus necessitating regulated by reanalyzing published human islet single-cell sequenc- unproductive splicing and translation (RUST) to link alter- ing data (donor information in Supplementary Table 1B; native splicing and NMD and regulate the abundance of EBI accession number MTAB-5061) (17). mRNA transcripts (12). Accordingly, NMD inhibition leads to an ;15%increaseinPTC-containingmRNAisoforms(13). Apoptosis and Cell Viability Assays Very little is known about the role of RNA decay in Apoptosis assay was performed in duplicate through de- fl stress regulation of the transcriptome. We therefore hy- tection of either caspase-3 activity using the uorimetric pothesized that cytokines regulate RNA decay with con- caspase-3 activity assay kit (Sigma-Aldrich, Copenhagen, sequences for b-cell survival in response to inflammatory Denmark) or DNA/histone complexes released from the stress. We report that RNA decay pathway components nucleus to the cytosol using the Roche cell death assay kit (RDPCs) are regulated by proinflammatory cytokines in (Roche, Mannheim, Germany) according to the manufac- b-cells in a nitric oxide (NO), but not reactive oxygen turers’ protocols. Cell viability was measured by MTT and species (ROS), dependent manner. RDPC knockdown (KD) Alamarblue assays (Life Technologies), as previously de- partially restored insulin transcripts, content and medium scribed (18,19). accumulation, but not glucose-stimulated insulin secretion NO Assay (GSIS), and modulated cytokine-mediated death in cytokine- As a surrogate of NO production, accumulated nitrite was exposed INS-1 cells. measured in supernatants (100 mL) from the wells used for INS-1 cell apoptosis assay by the Griess reagent, as pre- RESEARCH DESIGN AND METHODS viously described (20). Cell Culture and Cytokine Exposures The rat INS-1 cell line provided by Claes Wollheim (Uni- Real-time Reverse Transcriptase Quantitative PCR versity of Geneva, Geneva, Switzerland) tested negative for Total RNA was extracted using the NucleoSpin kit Mycoplasma and was maintained as previously described (Macheray-Nagel, Bethlehem, PA) according to the man- (14). INS-1 cells were seeded in 6-well plates (1 3 106 ufacturer’s instructions. Quality and quantity of the cells/well for RNA isolation), 48-well plates (50,000 cells/ extracted RNA were assessed using a NanoDrop-1000 well for cell death assay), or 96-well plates (30,000 cells/well (Thermo Fisher Scientific). Five-hundred nanograms total for MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- RNA was used for cDNA synthesis with the iScript-cDNA lium-bromide] and Alamarblue assays) (NUNC, Roskilde, kit (Bio-Rad, Copenhagen, Denmark). Real-time reverse diabetes.diabetesjournals.org Ghiasi and Associates 2021

Figure 1—Cytokines or GLT increase the expression of genes regulating RNA decay components in INS-1 cells and human islets. mRNA levels of RDPCs in INS-1 cells exposed to cytokine combination (Cyt;150 pg/mL IL-1b + 0.1 ng/mL IFN-g)(A), GLT (0.5 mmol/L palmitate conjugated with 0.1% albumin + 25 mmol/L glucose) (B), or control (Ctl) medium for 12, 18, and 24 h were quantified by RT-qPCR and normalized to 18S rRNA (n = 6). The basal relative mRNA level is shown in Supplementary Fig. 1F. C: Expression level of key components of RNA decay pathways in dispersed donor human islet b-cells from healthy subjects (n = 6) and patients with T2D (n = 4) was determined by single-cell RNA sequencing and quantified by reads per kilobase per million mapped reads (RPKM) per cell. The symbol t indicates ANOVA with Bonferroni-corrected post hoc Student t test P value of T2D vs. healthy donors. D: The protein levels of key candidates of RDPCs in INS-1 cells exposed to the cytokine combination for the given times were determined by WB and quantified normalized to tubulin (n = 3, but n = 2 for Smg6 in INS-1). Blots are shown in Supplementary Fig. 1G. E: mRNA levels of RDPCs and UPR markers in human islets exposed to Cyt (300 pg/mL IL-1b + 10 ng/mL IFN-g + 10 ng/mL TNF-a) for 12, 18, and 24 h were quantified by RT-qPCR and normalized to Hprt1 (n = 6). The basal relative mRNA level is shown in Supplementary Fig. 1H. F: The protein levels of key candidates of RDPCs in human islets exposed to the cytokine combination for the given times were determined by WB and quantified normalized to tubulin (n = 3). Blots are shown in Supplementary Fig. 1I. G–J: INS-1 cells or human islets were exposed to Cyt (for INS-1 cells: 150 pg/mL IL-1b + 0.1 ng/mL IFN-g; for human islets: 300 pg/mL rrIL-1b + 10 ng/mL rhIFN-g + 10 ng/mL rhTNF-a) with or without coincubation with 10 or 100 mg/mL PTC124, respectively. Apoptosis was measured in human islets (n =3)(G) and INS-1 cells (n =3)(H). NO was measured in the collected supernatant from INS-1 cells (n =3)(I), and mRNA levels of UPR markers were measured in INS-1 cells by RT-qPCR and normalized to 18S rRNA (n =6)(J). Cyt 15, 15 pg/mL IL-1b + 0.1 ng/mL IFN-g; Cyt 37, 37 pg/mL IL-1b + 0.1 ng/mL IFN-g; Cyt 150, 150 pg/mL IL-1b + 0.1 ng/mL IFN-g. Data are means 6 SEM. The symbols t and * indicate the Bonferroni-corrected paired Student t test P values of treatments vs. Ctl and Cyt conditions, respectively, unless otherwise stated. * or t, #0.05; ** or tt, #0.01; *** or ttt, #0.001; **** or tttt, #0.0001. 2022 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018

Figure 1—Continued.

transcriptase quantitative PCR (RT-qPCR) was performed 2ΔCt analysis, and for simplicity some figures were pre- on 12 ng cDNA with SYBR Green PCR Master Mix (Life sented as fold change versus control, which was calculated Technologies) and specific primers (Supplementary Table by the 2ΔΔCt method. 2) and run in a real-time PCR machine (Applied Biosys- tems, Naerum, Denmark). NormFinder software (21) was Northern Blotting Analysis used to select the most stable reference gene among Seven micrograms total RNA was blotted to a positive hypoxanthine-guanine phosphor-ribosyl-transferase 1 charged nylon membrane (Ambion, Naerum, Denmark) (Hprt1), actin, and 18S rRNA. Statistical analysis was and probed against insulin-2 (Ins2) mRNA and 18S rRNA performed on the gene expression levels normalized to using radiolabeled-specific probes as described in detail in the chosen reference gene (see figure legends) through the Supplementary Data. diabetes.diabetesjournals.org Ghiasi and Associates 2023

Figure 1—Continued.

Western Blotting Analysis Lentiviral Short Hairpin RNA Gene KD Western blotting (WB) was performed as described in the GIPZ lentiviral short hairpin RNA (shRNA) particles Supplementary Data (22) using antibodies against Pelo (Dharmacon, Søborg, Denmark) against Pelo, Xrn1, (Novus Biologicals, Littleton, CO), Xrn1, Dis3L1, Dis3L2, Smg6, and Upf1 genes along with a nonsilencing shRNA a-tubulin (Sigma-Aldrich), Smg1, Smg5, Smg6, Smg7, (NS) vector as negative control were produced using the Upf1, or Upf2 (Santa Cruz Biotechnology, Heidelberg, Trans-Lentiviral shRNA Packaging System (Dharmacon) Germany). in HEK293 cells according to the manufacturer’s instructions and as described in detail in the Supplementary GSIS Data. INS-1 cells (300,000) were cultured in 12-well plates (Nunc) and incubated for 2 days before being exposed Ins2 mRNA Decay Kinetics in CHO Cells to cytokine mixture (150 pg/mL IL-1b + 0.1 ng/mL IFN-g) The Ins2 gene was cloned into CHO cells (CHO-Ins), and or control medium. GSIS was performed using Krebs- its mRNA half-life and decay kinetics were determined Ringer buffer containing 2 or 17 mmol/L glucose as pre- using the Lenti-X Tet-One Inducible Expression System viously described (23). as fully described in the Supplementary Data.

Insulin Assay Statistical Analysis Insulin concentration (pmol/L) was measured using in- Data are presented as means 6 SEM. Statistical analysis sulin-competitive ELISA assay as previously described (24), was performed on raw data even in cases where normalized with the modiﬁcation that the enzyme substrate 1-Step data are shown. Group comparisons were conducted as Ultra TMB (3,39,5,5-tetramethylbenzidine) (Life Technol- two- or one-way ANOVA as appropriate. Two-way ANOVA ogies) was used. P values are shown in Supplementary Tables 3 and 4. 2024 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018

Figure 1—Continued.

One-way ANOVA P values are given next to the related ﬁg- paired Student t test was chosen to normalize for in- ures. Signiﬁcant ANOVAs were followed by post hoc paired terpassage variability in outcome parameters. Since the Student t test with Bonferroni correction using GraphPad experimental conditions did not allow sequential sam- Prism version 6 (GraphPad Software, La Jolla, CA). The pling from the same cell culture, parallel control and diabetes.diabetesjournals.org Ghiasi and Associates 2025

Figure 1—Continued.

interventional plate wells were considered to be paired RESULTS observations and analyzed accordingly statistically. If Cytokines and GLT Differentially Regulate the the post hoc paired Student t test did not reveal the Expression of RNA Decay Components in INS-1 Cells comparison carrying the ANOVA statistical difference, and Human Islets individual paired Student t tests were performed and Since cytokines and, to a lesser extent, GLT downregulated corrected for multiple comparisons. Bonferroni-corrected mRNA levels of insulin and b-cell–speciﬁc transcription P values #0.05 were considered signiﬁcant and #0.10 factors (TFs) (Supplementary Fig. 1A and B) and inhibited a trend. accumulated insulin release (Supplementary Fig. 1C), we 2026 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018

Figure 1—Continued.

examined whether cytokines or GLT regulate proteins Since NMD may lead to synthesis of mutated proteins belonging to either NGD or NMD pathways in INS-1 cells that elicit an unfolded protein response (UPR) (9,26), we or rat/human islets, respectively. RT-qPCR analysis showed examined UPR mRNA levels in cytokine-exposed human that cytokines at one or more time points significantly islets by RT-qPCR. The Bip mRNA level was significantly upregulated the mRNA level of the ribosomal dissociation upregulated at 18 and 24 h (Fig. 1E). However, likely due to factor of the NGD pathway (Pelo), NMD components (Upf2, high baseline levels of Chop and ATF4 due to handling stress Smg1, Smg5, Smg6, and Smg7), and the exosomal system ofthehumanislets,wewereunabletodetectsignificant (mRNA decapping [Dcp2] and exoribonuclease [Dis3L2]) cytokine-induced changes in the expression of these ER but did not change Hbsl1, Abce1, Upf1, DcpS, Xrn1, stress markers. Because cytokines induce alternatively Dis3L1, or Dis3 mRNA levels in INS-1 cells (Fig. 1A). The spliced b-cell mRNA isoforms potentially containing PTCs, NMD component Upf3B was downregulated. the translation of which is expected to trigger apoptosis Since glucotoxicity at least in part involves activation of through ER stress, we next asked if forcing ribosomal read- b-cell–derived IL-1b production and secretion (25), we through of such PTC-containing mRNAs with the drug PTC124 investigated if exposure of INS-1 cells to GLT conditions would aggravate cytokine-induced apoptosis in human induced alteration in the RDPC expression similar to those islets and INS-1 cells, which was the case (Fig. 1G and H). increased by cytokines. The mRNA levels of Pelo/Smg1/ PTC124 also potentiated INS-1 cell NO production (Fig. 1I) Smg5 were significantly increased in GLT-exposed INS-1 and expression of the ER stress markers Bip (P = 0.0005) and cells at one or more time points (Fig. 1B). Hbsl1 and Abce1 Chop (P = 0.06) (Fig. 1J). Taken together, these results show were unaffected. Single-cell RNA sequencing of b-cells (Fig. that cytokines and GLT conditions differentially regulate the 1C), but not of the other endocrine cell subtypes (Supple- expression of RDPCs in INS-1 cells and human islets. mentary Fig. 1D), derived from four deceased patients with T2D and six control subjects (Supplementary Table 1B) Pelo and Xrn1 KD Aggravates, Whereas Smg6 KD confirmed the significant upregulation of Pelo observed Reduces, Cytokine-Induced INS-1 Cell Viability in the GLT-exposed INS-1 cells (Fig. 1B). We next investigated the impact of Pelo, Upf1, Smg6, and WB analysis confirmed significant upregulation (or trends Xrn1 stable KD (Supplementary Fig. 2) on cytokine-induced for upregulation) of Pelo, Xrn1, Dis3L2, Upf1 (P = 0.08), cell death. Pelo (Fig. 2A)orXrn1KD(Fig.2B) modestly, but Upf2, Smg5, Smg6 (no P value, n = 2), and Smg7 protein significantly, aggravated, whereas Smg6 (Fig. 2C) mod- (P = 0.059) but downregulation of Smg1 protein in cyto- estly, but significantly, ameliorated cytokine-induced cell kine-exposed INS-1 cells (Fig. 1D). WB analysis of two toxicity measured either by the caspase-3 activity assay kit independent experiments also confirmed the increase in or by MTT and Alamarblue assays. In addition, the effect of protein levels of the key RNA decay proteins (Pelo, Xrn1, the KD of RDPCs on cytokine-induced toxicity was con- Dis3L2, Upf1, and Smg6) in rat islets at one or more time firmed by measuring cell death using the Roche apoptosis points (Supplementary Fig. 1E). detection kit (Supplementary Fig. 3). Smg6 KD also im- In human islets exposed to cytokines, mRNA levels of Xrn1, proved viability measured by MTT in the absence of cyto- Upf2, Upf3B, Smg1, and Smg7, but not Pelo, Dis3L1/2, Upf3A, kines (Fig. 2C). or Smg5, were significantly upregulated at one or more time These data indicate that in cytokine-induced b-cell points with a trend for upregulation of Smg6 (Fig. 1E). apoptosis, the NGD and exoribonuclease pathways serve The protein levels of Xrn1, Upf1, and Smg6 largely reflected protective functions, whereas the NMD pathway poten- changes observed for the corresponding mRNA levels (Fig. 1F). tiates toxicity. diabetes.diabetesjournals.org Ghiasi and Associates 2027

Figure 1—Continued. 2028 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018

Figure 2—KD of Pelo and Xrn1 reduces b-cell viability, whereas KD of Smg6 rescues INS-1 cell viability when exposed to cytokines. The INS-1 cell lines with the most efﬁcient stable KD of Pelo (three shRNAs) (A), Xrn1 (three shRNAs) (B), Smg6 (two shRNAs) (C), and NS were exposed to cytokine combination (Cyt; 150 pg/mL IL-1b + 0.1 ng/mL IFN-g) or control (Ctl) medium for 24 h. Cell viability was measured by ﬂuorometric caspase-3 activity assay kit or Alamarblue and MTT assays. The data are means 6 SEM of n = 6. The symbols t and * indicate the Bonferroni-corrected paired Student t test P values of treatments vs. Ctl and Cyt conditions, respectively. * or t, #0.05; ** or tt, #0.01; *** or ttt, #0.001; tttt, #0.0001.

KD of the RNA Decay Effectors Pelo, Xrn1, and Smg6 increased accumulated insulin secretion (Fig. 3G). Xrn1 Restores Insulin mRNA, Content and Accumulated, but and Smg6 KD also increased GSIS (Fig. 3E and F). Because Not GSIS in Cytokine-Exposed INS-1 Cells only one shRNA against Pelo had the same effect (Fig. 3D), In non–cytokine-exposed cells, KD of Pelo and Xrn1, but we cannot exclude an off-target effect of this particular not Smg6, increased Ins1/2 mRNAs (Fig. 3A–C) and insulin shRNA. As expected, cytokines dramatically reduced accu- content (Fig. 3D–F). KD of all three RDPC effectors mulated insulin secretion (Fig. 3G) and GSIS (Fig. 3D–F) diabetes.diabetesjournals.org Ghiasi and Associates 2029

Figure 3—KD of the RNA decay effectors Pelo, Xrn1, and Smg6 restores insulin mRNA, content and accumulated, but not GSIS in cytokine- exposed INS-1 cells. A–G: The INS-1 cell lines with the most efficient KD of Pelo and Xrn1 (three shRNAs of each), Smg6 (two shRNAs), and NS were exposed to cytokine combination (Cyt; 150 pg/mL IL-1b + 0.1 ng/mL IFN-g) or without as control (Ctl) for 18 h. The mRNA levels of Ins1/2, central b-cell–specific TFs, UPR markers, Ica512, and Gck genes were determined in Pelo (A), Xrn1 (B), and Smg6 (C) KD INS-1 cell lines by RT-qPCR and normalized to 18S rRNA. GSIS and insulin contents were investigated in Pelo (D), Xrn1 (E), and Smg6 (F) KD INS-1 cell lines. Insulin concentrations (pmol/L) were measured by ELISA (n = 6). Accumulated insulin (pmol/L) over 18 h was measured by insulin ELISA in the supernatants of Pelo, Xrn1, and Smg6 (G) KD INS-1 cell lines. H: Pelo or Xrn1 was transiently knocked down in CHO-Ins cells using NS as Ctl. The Pelo or Xrn1 KD or NS CHO-Ins cell lines were treated with 100 ng/mL Dox for 24 h, and the Ins2 mRNA decay was studied using qPCR at 128, 256, 512, and 1,024 min after Dox removal. The RT-qPCR data were normalized to 18S rRNA. The area under the curve of Ins2 mRNA decay was calculated for Pelo, Xrn1, and NS KD cell lines (n = 6). Data are means 6 SEM. The symbols t and * indicate the Bonferroni- corrected paired Student t test P values of treatments vs. Ctl and Cyt conditions, respectively. * or t, #0.05; ** or tt, #0.01; *** or ttt, #0.001; **** or tttt, #0.0001. ns, not significant.

from nonsense (NS) cells. Pelo, Xrn1, or Smg6 KD partially critical for b-cell secretory function. Indeed, Pelo KD restored cytokine-inhibited insulin content (Fig. 3D–F) markedly increased Ins1/2 mRNA levels in the absence and accumulated insulin secretion (Fig. 3G)butnotGSIS of cytokines, restored cytokine-induced reductions in (Fig. 3D–F). Taken together, these results suggest that KD Ins1/2, and partially restored cytokine-induced reductions of all three RDPC effectors improves insulin biosynthesis, in Pdx-1, Isl-1, glucokinase (Gck), and islet cell autoantigen but not GSIS, in cytokine-exposed INS-1 cells. 512 (Ica512) (Fig. 3A). Xrn1 KD increased Ins1/2 mRNA We reasoned that the increase in insulin secretion and levels in the absence of cytokines and partially restored content could be related to a reduced decay of mRNAs cytokine-induced reductions in Ins1/2 and Ica512 mRNAs 2030 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018

Figure 3—Continued.

(Fig. 3B). In contrast, Smg6 KD did not affect Ins1/2 We then asked if RNA decay effector KD caused ER stress. mRNA levels in the absence of cytokines but completely In the absence of cytokines, Pelo KD modestly increased restored cytokine-induced reductions in Ins1 (P = 0.052), Atf4 (P =0.06)andBip(P = 0.031), and Xrn1 KD increased Ins2 (P = 0.008), and Ica512 (P = 0.0006) mRNAs and Bip (P = 0.029). Pelo, Xrn1, and Smg6 KD did not signif- partially restored Pdx-1 and Isl-1 mRNAs (Fig. 3C). The icantly alter cytokine-induced ER stress markers, although relative changes in insulin mRNA levels observed in Pelo, there was a trend for Pelo KD to increase (P = 0.059) and for Xrn1, and Smg6 KD were confirmed by Northern blotting Smg6 KD to reduce sXbp1 (P = 0.08) (Fig. 3A–C). analysis (Supplementary Fig. 4). These results suggest that the NGD and NMD, but not We next investigated if the increase in insulin RNA levels the exoribonuclease, pathways mediate cytokine-induced was associated with the prolonged half-life of insulin mRNA reductions in mRNAs encoding proteins regulating glucose due to impaired RNA decay. We created CHO cells trans- sensing and insulin gene transcription and exocytosis. fected with Ins2 mRNA under doxycycline (Dox) induction Cytokine-Induced Regulation of RNA Decay Effectors using a tetracycline-on/off expression system, termed CHO- and ER Stress Marker Expression Is NO, but Not ROS, Ins cells. Transient KD of Pelo, but not Xrn1, in CHO-Ins Dependent cells caused a significant increase in the Ins2 mRNA area Inflammatory cytokines increase the production of ROS in under the curve level compared with NS control (Fig. 3H). b-cells through iron catalysis of the Fenton reaction (27) diabetes.diabetesjournals.org Ghiasi and Associates 2031

Figure 3—Continued.

and via activation of NF-kB–mediated inducible nitric oxide and B that NMA and the combination of NMA with synthase (iNOS) gene expression and NO production (28). NAC and DFO markedly reduced NO synthesis while Further, cytokine-mediated gene regulation is in many cases not affecting iNOS mRNA expression. NAC and DFO NO dependent (29). We therefore investigated whether modestly but signiﬁcantly reduced NO synthesis but inhibition of nitroxidative or oxidative stress by the use not iNOS mRNA expression, in agreement with previ- of the iNOS inhibitor NG-methyl-L-arginine acetate (NMA), ous reports demonstrating that ROS facilitates iNOS the ROS scavenger N-acetyl cysteine (NAC), and the iron activity (30). chelator deferoxamine (DFO) alone or in combination The counterregulation of the RNA decay pathways by alleviated cytokine-induced regulation of RNA decay iNOS inhibition, but not by ROS inhibition, alone was components and ensuing ER stress. Several RNA decay associated with partial normalization of Ins1/2 mRNA levels components were regulated in cytokine-exposed INS-1 and ER stress markers (Fig. 4B). In summary, these data cells (as also found in Fig. 1A), and these changes were show that cytokine-induced regulation of RDPCs and ER largely NO dependent in that they were reversed or stress marker expression is NO, but not ROS, dependent. partially reversed by NMA alone or in combination with NAC and DFO, but not by NAC or DFO alone (Fig. 4A). To DISCUSSION verify that the effect of NMA was mediated via iNOS In this study, we found that a decrease of steady-state inhibition, we demonstrated in Supplementary Fig. 5A mRNA levels of Ins1/2 and central b-cell–speciﬁc TFs in 2032 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018

Figure 3—Continued.

cytokine-exposed INS-1 and rat or human islets was implication that aberrant mRNA surveillance by NMD associated with upregulation of RDPCs, exoribonucleases, exacerbates inflammation (31); thus, Smg1 depletion and key UPR components in an NO, but not ROS, de- results in chronic inflammation in the mouse model (32). pendent manner. When considering the low sensitivity of The cytokine-induced changes in RDPCs may seem single-cell RNA sequencing of human islets ex vivo, it is modest but are similar to the compensatory effect sizes remarkable that increased Pelo expression was detectable caused by mutations in UPF3B associated with mental in T2D islets, compatible with GLT-mediated upregulation retardation in humans (33). of Pelo in INS-1 cells. Interestingly, this response was iNOS inhibition reversed cytokine-mediated ER stress b-cell specific. KD of Pelo or Xrn1 aggravated, whereas as expected, but also upregulation of RNA decay ef- Smg6 KD ameliorated, cytokine-induced INS-1 cell death fector mRNAs and downregulation of Ins1/2 mRNA. without affecting ER stress; both increased insulin bio- NO-mediated nitration activates the NF-kB (34,35), p53 synthesis and medium accumulation but not GSIS in (36), AP-1 (37), and other signaling pathways (35). Alter- cytokine-exposed INS-1 cells. natively, nucleotide nitration leading to 8-nitroguanosine This study represents, to the best of our knowledge, the modifications of cellular RNAs may cause ribosomal stall- first comprehensive transcriptional and translational pro- ing (7,38) and mediate RDPC expressional upregulation as filing of the b-cell mRNA surveillance and decay system a protective response. Measurement of 8-nitroguanosine and the first description in any cell of how this system is modifications in cytokine-exposed INS-1 cells failed due to regulated by inflammatory and metabolic stress. Studies a lack of specific and sensitive methods (data not shown). have so far focused on how the mRNA surveillance sys- Finally, the possibility that induction of NMD effector tem regulates inflammatory cytokine transcripts with the expression is secondary to NO-mediated alternative diabetes.diabetesjournals.org Ghiasi and Associates 2033

Figure 3—Continued.

splicing (2,29) and generation of PTC-containing tran- islets with PTC124 (39) in an inﬂammatory setting scripts remains to be investigated. expected to enhance generation of alternatively spliced To mimic the cellular outcome of a mismatch between b-cell mRNA isoforms potentially containing PTCs. We ribosomal load with PTC-containing RNAs and RNA decay, anticipated, as shown, that the forced translation would we forced ribosomal readthrough in INS-1 cells and human trigger apoptosis associated with ER stress through the 2034 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018

Figure 4—Cytokine-induced expressional regulations of RNA decay effectors, Ins1/2, and ER stress markers are NO, but not ROS, dependent. INS-1 cells were exposed to cytokine combination (Cyt; 150 pg/mL IL-1b + 0.1 ng/mL IFN-g) and cotreated with 1 mmol/L NAC, 1 mmol/L NMA, 0.1 mmol/L DFO, or a mixture (Mix) of the three for 12 h. The levels of mRNA encoding for proteins belonging to the RNA decay pathway (A) and UPR markers and Ins1/2 (B) were quantiﬁed by RT-qPCR and normalized to 18S rRNA (n = 6). Data are means 6 SEM. The symbols t and * indicate the Bonferroni-corrected paired Student t test P values of treatments vs. control (Ctl) and Cyt conditions, respectively. * or t, #0.05; ** or tt, #0.01; *** or ttt, #0.001; ****, #0.0001.

ATF6 and PERK pathways. We did not observe ER stress in Similarly, Smg6 KD ameliorated INS-1 cell sensitivity to response to RDPC KD likely due to qualitative differences cytokine-induced cell death without affecting ER stress, in the ER load of unfolded protein, expected to be higher which would have been expected due to cytokine-induced after forced readthrough, even though NO induction was alternative splicing with the generation of PTC-containing comparable. mRNAs, leading to the formation of “mutated” proteins in Pelo and Xrn1 KD increased INS-1 cell sensitivity to turn unfolded or misfolded in the ER (2). This expectation cytokine-induced cell death without affecting ER stress, was supported by our observation of Bip and a trend for and to the best of our knowledge, there is no evidence that Chop induction in INS-1 cells exposed to cytokines in the Pelo or Xrn1 deletion induces ER stress in any cell. Possibly presence of PTC124, forcing readthrough of PTC-containing cytokine-induced nitration of mRNA causing ribosomal mRNAs. Either the incomplete (;50%) KD or redundancy stalling or NO-induced eIF2a phosphorylation limiting provided by the several Smg sub- and isoforms may explain translation initiation may limit protein influx into the the absence of ER stress in Smg6-deficient cytokine-exposed ER, thereby not impeding ER folding/chaperoning capac- INS-1 cells. ity. Accordingly, deletion of Dom34, the yeast homolog of The balance of pro- and antiapoptotic transcripts is Pelo, causes ribosomal stalling, blocking ER protein influx regulated by alternative splicing and thereby defines (40,41). apoptosis (42). Thus, Pelo or Xrn1 KD may prevent diabetes.diabetesjournals.org Ghiasi and Associates 2035

Figure 4—Continued.

clearance of proapoptotic mRNAs, and Smg6 KD may why cytokine-mediated reductions in accumulated insulin prevent clearance of antiapoptotic mRNAs. To support secretion are modestly improved by RDPC KD. Using this speculation, overexpression of the apoptotic inhib- a tetracycline-on/off inducible expression system of the itor growth arrest and DNA damage–inducible 45b Ins2 gene in CHO cells to avoid confounding by endoge- (Gadd45b) in INS-1E cells decreases IL-1b–induced apo- nous Ins gene expression in INS-1 cells, we confirm ptosis (43), and shRNA-mediated KD of Gadd45b aggra- restoration of Ins2 mRNA in Pelo but not in Xrn1 KD vates neuron cell apoptosis (44). Gadd45b mRNA is an CHO cells. There are several Xrn1 isomers but only one NMD target (45) and would therefore be expected to be Pelo gene, and the lack of effect of Xrn1 KD on Ins2 mRNA more stable in Smg6 KD cells. We found that Upf1 KD did levels in Ins-CHO cells may be explained by species differ- not affect cytokine-induced apoptosis, in line with the ences in Xrn1 isomer redundancy (47) or failure of Xrn1 observation that Upf1 or Upf2 depletion does not sig- capacity to deal with forced overexpression of Ins2. nificantly influence sensitivity to staurosporine-induced In conclusion, RDPCs are regulated by inflammatory apoptosis (46). stress in b-cells in an NO, but not ROS, dependent We demonstrate that KD of all three RDPC effectors manner. RDPC KD partially restored insulin transcript partially restores insulin mRNA levels and contents but levels, content and medium accumulation, but not GSIS not GSIS in cytokine-exposed INS-1 cells. These observa- in cytokine-exposed INS-1 cells, likely explained by the tions indicate that the RDPC effectors regulate insulin effect of RDPC KD on insulin mRNA stability leading to biosynthesis but not directly the stimulus-secretion cou- increased insulin biosynthesis but not directly affecting pling, leading to insulin retention, which drives insulin stimulus-secretion coupling. Pelo/Xrn1 KD aggravated, but release via the constitutive pathway, thereby explaining Smg6 KD ameliorated, cytokine-mediated b-cell death, 2036 Cytokine Regulation of RNA Decay in b-Cells Diabetes Volume 67, October 2018 possibly through preventing proapoptotic and antiapop- 12. Lareau LF, Brooks AN, Soergel DA, Meng Q, Brenner SE. The coupling of totic mRNA degradation, respectively. alternative splicing and nonsense-mediated mRNA decay. Adv Exp Med Biol 2007; 623:190–211 13. Pan Q, Saltzman AL, Kim YK, et al. Quantitative microarray profiling provides Acknowledgments. The authors thank Claes Wollheim for generously evidence against widespread coupling of alternative splicing with nonsense- providing INS-1 cells, Dr. J.B. Hansen and Dr. Z. Gerhart-Hines (both from Section mediated mRNA decay to control gene expression. Genes Dev 2006;20:153–158 for Metabolic Receptology, Novo Nordisk Foundation Center for Basic Metabolic 14. Sekine N, Fasolato C, Pralong WF, Theler JM, Wollheim CB. Glucose-induced Research, University of Copenhagen, Copenhagen, Denmark) for providing DMT-1 insulin secretion in INS-1 cells depends on factors present in fetal calf serum and KO mice, Henrik E. Poulsen (Clinical Pharmacology, State University Hospital, rat islet-conditioned medium. Diabetes 1997;46:1424–1433 Copenhagen, Denmark) and Peter E. Nielsen (Department of Cellular and Molecular 15. Bugliani M, Syed F, Masini M, et al. Direct effects of rosuvastatin on Medicine, University of Copenhagen, Copenhagen, Denmark) for helpful discus- pancreatic human beta cells. Acta Diabetol 2013;50:983–985 sions and comments, N. Billestrup (Department of Biomedical Sciences, University 16. Nielsen JH, Svensson C, Galsgaard ED, Møldrup A, Billestrup N. Beta cell of Copenhagen, Copenhagen, Denmark) for providing neonatal rat islets, Henrik proliferation and growth factors. J Mol Med (Berl) 1999;77:62–66 Nielsen (Department of Cellular and Molecular Medicine, University of Copenhagen, 17. Segerstolpe Å, Palasantza A, Eliasson P, et al. Single-cell transcriptome Copenhagen, Denmark) for providing materials and laboratory facilities for profiling of human pancreatic islets in health and type 2 diabetes. Cell Metab 2016; Northern blotting analyses, and the ECIT (Milan, Italy) for providing human islets. 24:593–607 Funding. This project was funded by the Danish Diabetes Academy (DDA), the 18. Plumb JA. Cell sensitivity assays: the MTT assay. Methods Mol Med 2004;88: Department of Biomedical Sciences (BMI) at the University of Copenhagen, the 165–169 Augustinus Foundation, and the Bjarne Jensen Foundation. 19. Kover K, Yan Y, Tong PY, et al. Osteocalcin protects pancreatic beta cell Duality of Interest. This project was funded by Zealand Pharma A/S. B.T. is function and survival under high glucose conditions. Biochem Biophys Res employed by AstraZeneca. No other potential conflicts of interest relevant to this Commun 2015;462:21–26 article were reported. 20. Størling J, Binzer J, Andersson AK, et al. Nitric oxide contributes to cytokine- Author Contributions. S.M.G. initiated the study, developed the protocols induced apoptosis in pancreatic beta cells via potentiation of JNK activity and for the experiments, conducted experiments, performed statistical analysis, inhibition of Akt. Diabetologia 2005;48:2039–2050 constructed figures and tables, and wrote the first draft of the manuscript. N.K. 21. 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