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Intersection of the ATF6 and XBP1 ER stress pathways in mouse islet cells

2020; Elsevier BV; Volume: 295; Issue: 41 Linguagem: Inglês

10.1074/jbc.ra120.014173

ISSN

1083-351X

Autores

Rohit B. Sharma, Christine Darko, Laura Alonso,

Tópico(s)

Immune Cell Function and Interaction

Resumo

Success or failure of pancreatic beta cell adaptation to ER stress is a determinant of diabetes susceptibility. The ATF6 and IRE1/XBP1 pathways are separate ER stress-response effectors important to beta cell health and function. ATF6α. and XBP1 direct overlapping transcriptional responses in some cell types. However, the signaling dynamics and interdependence of ATF6α and XBP1 in pancreatic beta cells have not been explored. To assess pathway-specific signal onset, we performed timed exposures of primary mouse islet cells to ER stressors and measured the early transcriptional response. Comparing the time course of induction of ATF6 and XBP1 targets suggested that the two pathways have similar response dynamics. The role of ATF6α in target induction was assessed by acute knockdown using islet cells from Atf6αflox/flox mice transduced with adenovirus expressing Cre recombinase. Surprisingly, given the mild impact of chronic deletion in mice, acute ATF6α knockdown markedly reduced ATF6-pathway target gene expression under both basal and stressed conditions. Intriguingly, although ATF6α knockdown did not alter Xbp1 splicing dynamics or intensity, it did reduce induction of XBP1 targets. Inhibition of Xbp1 splicing did not decrease induction of ATF6α targets. Taken together, these data suggest that the XBP1 and ATF6 pathways are simultaneously activated in islet cells in response to acute stress and that ATF6α is required for full activation of XBP1 targets, but XBP1 is not required for activation of ATF6α targets. These observations improve understanding of the ER stress transcriptional response in pancreatic islets. Success or failure of pancreatic beta cell adaptation to ER stress is a determinant of diabetes susceptibility. The ATF6 and IRE1/XBP1 pathways are separate ER stress-response effectors important to beta cell health and function. ATF6α. and XBP1 direct overlapping transcriptional responses in some cell types. However, the signaling dynamics and interdependence of ATF6α and XBP1 in pancreatic beta cells have not been explored. To assess pathway-specific signal onset, we performed timed exposures of primary mouse islet cells to ER stressors and measured the early transcriptional response. Comparing the time course of induction of ATF6 and XBP1 targets suggested that the two pathways have similar response dynamics. The role of ATF6α in target induction was assessed by acute knockdown using islet cells from Atf6αflox/flox mice transduced with adenovirus expressing Cre recombinase. Surprisingly, given the mild impact of chronic deletion in mice, acute ATF6α knockdown markedly reduced ATF6-pathway target gene expression under both basal and stressed conditions. Intriguingly, although ATF6α knockdown did not alter Xbp1 splicing dynamics or intensity, it did reduce induction of XBP1 targets. Inhibition of Xbp1 splicing did not decrease induction of ATF6α targets. Taken together, these data suggest that the XBP1 and ATF6 pathways are simultaneously activated in islet cells in response to acute stress and that ATF6α is required for full activation of XBP1 targets, but XBP1 is not required for activation of ATF6α targets. These observations improve understanding of the ER stress transcriptional response in pancreatic islets. Pancreatic beta cell dysfunction or loss leads to relative insulin deficiency and diabetes in its many forms. Production and folding of the insulin polypeptide places significant demand on the beta cell endoplasmic reticulum (ER) (1Liu M. Weiss M.A. Arunagiri A. Yong J. Rege N. Sun J. Haataja L. Kaufman R.J. Arvan P. Biosynthesis, structure, and folding of the insulin precursor protein.Diabetes Obes. Metab. 2018; 20 (30230185): 28-5010.1111/dom.13378Crossref PubMed Scopus (91) Google Scholar). Adaptation to manageable ER stress can have a positive impact on beta cell mass and function (2Sharma R.B. O'Donnell A.C. Stamateris R.E. Ha B. McCloskey K.M. Reynolds P.R. Arvan P. Alonso L.C. Insulin demand regulates β cell number via the unfolded protein response.J. Clin. Invest. 2015; 125 (26389675): 3831-384610.1172/JCI79264Crossref PubMed Scopus (127) Google Scholar, 3Eizirik D.L. Cnop M. ER stress in pancreatic beta cells: the thin red line between adaptation and failure.Sci. Signal. 2010; 3 (20179270): pe710.1126/scisignal.3110pe7Crossref PubMed Scopus (116) Google Scholar, 4Chan J.Y. Luzuriaga J. Maxwell E.L. West P.K. Bensellam M. Laybutt D.R. The balance between adaptive and apoptotic unfolded protein responses regulates β-cell death under ER stress conditions through XBP1, CHOP and JNK.Mol. Cell Endocrinol. 2015; 413 (26135354): 189-20110.1016/j.mce.2015.06.025Crossref PubMed Scopus (79) Google Scholar, 5Herbert T.P. Laybutt D.R. A reevaluation of the role of the unfolded protein response in islet dysfunction: maladaptation or a failure to adapt?.Diabetes. 2016; 65 (27222391): 1472-148010.2337/db15-1633Crossref PubMed Scopus (49) Google Scholar, 6Sharma R.B. Snyder J.T. Alonso L.C. Atf6α impacts cell number by influencing survival, death and proliferation.Mol Metab. 2019; 27S (31500833): S69-S8010.1016/j.molmet.2019.06.005Crossref PubMed Scopus (16) Google Scholar), but failed beta cell adaptation to ER stress is a well-established contributor to lost beta cell function and survival (3Eizirik D.L. Cnop M. ER stress in pancreatic beta cells: the thin red line between adaptation and failure.Sci. Signal. 2010; 3 (20179270): pe710.1126/scisignal.3110pe7Crossref PubMed Scopus (116) Google Scholar, 4Chan J.Y. Luzuriaga J. Maxwell E.L. West P.K. Bensellam M. Laybutt D.R. The balance between adaptive and apoptotic unfolded protein responses regulates β-cell death under ER stress conditions through XBP1, CHOP and JNK.Mol. Cell Endocrinol. 2015; 413 (26135354): 189-20110.1016/j.mce.2015.06.025Crossref PubMed Scopus (79) Google Scholar, 7Back S.H. Kaufman R.J. Endoplasmic reticulum stress and type 2 diabetes.Annu. Rev. Biochem. 2012; 81 (22443930): 767-79310.1146/annurev-biochem-072909-095555Crossref PubMed Scopus (419) Google Scholar, 8Biden T.J. Boslem E. Chu K.Y. Sue N. Lipotoxic endoplasmic reticulum stress, β cell failure, and type 2 diabetes mellitus.Trends Endocrinol. Metab. 2014; 25 (24656915): 389-39810.1016/j.tem.2014.02.003Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 9Evans-Molina C. Hatanaka M. Mirmira R.G. Lost in translation: endoplasmic reticulum stress and the decline of β-cell health in diabetes mellitus.Diabetes Obes. Metab. 2013; 15: 159-16910.1111/dom.12163Crossref PubMed Scopus (40) Google Scholar, 10Fonseca S.G. Gromada J. Urano F. Endoplasmic reticulum stress and pancreatic beta cell death.Trends Endocrinol. Metab. 2011; 22 (21458293): 266-27410.1016/j.tem.2011.02.008Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 11Cnop M. Toivonen S. Igoillo-Esteve M. Salpea P. Endoplasmic reticulum stress and eIF2α phosphorylation: the Achilles heel of pancreatic β cells.Mol. Metab. 2017; 6 (28951826): 1024-103910.1016/j.molmet.2017.06.001Crossref PubMed Scopus (144) Google Scholar). Understanding the normal beta cell response to stress is critical to learning more about how beta cells fail. The unfolded protein response (UPR) is the primary mechanism of cellular adaptation to ER stress. The UPR is an elegant multipathway stress-response initiated when unfolded peptides recruit chaperone GRP78 away from ER transmembrane proteins PERK, IRE1, and ATF6, allowing them to activate downstream signals (7Back S.H. Kaufman R.J. Endoplasmic reticulum stress and type 2 diabetes.Annu. Rev. Biochem. 2012; 81 (22443930): 767-79310.1146/annurev-biochem-072909-095555Crossref PubMed Scopus (419) Google Scholar). PERK activation results in phosphorylation of eIF2α, which slows translation of certain proteins to decrease new peptide translocation into the ER (7Back S.H. Kaufman R.J. Endoplasmic reticulum stress and type 2 diabetes.Annu. Rev. Biochem. 2012; 81 (22443930): 767-79310.1146/annurev-biochem-072909-095555Crossref PubMed Scopus (419) Google Scholar). PERK and eIF2α have been extensively studied in pancreatic beta cells (11Cnop M. Toivonen S. Igoillo-Esteve M. Salpea P. Endoplasmic reticulum stress and eIF2α phosphorylation: the Achilles heel of pancreatic β cells.Mol. Metab. 2017; 6 (28951826): 1024-103910.1016/j.molmet.2017.06.001Crossref PubMed Scopus (144) Google Scholar, 12Gao Y. Sartori D.J. Li C. Yu Q.-C. Kushner J.A. Simon M.C. Diehl J.A. PERK is required in the adult pancreas and is essential for maintenance of glucose homeostasis.Mol. Cell Biol. 2012; 32 (23071091): 5129-513910.1128/MCB.01009-12Crossref PubMed Scopus (77) Google Scholar, 13Gupta S. McGrath B. Cavener D.R. PERK (EIF2AK3) regulates proinsulin trafficking and quality control in the secretory pathway.Diabetes. 2010; 59 (20530744): 1937-194710.2337/db09-1064Crossref PubMed Scopus (105) Google Scholar, 14Wang R. McGrath B.C. Kopp R.F. Roe M.W. Tang X. Chen G. Cavener D.R. Insulin secretion and Ca2+ dynamics in β-cells are regulated by PERK (EIF2AK3) in concert with calcineurin.J. Biol. Chem. 2013; 288 (24114838): 33824-3383610.1074/jbc.M113.503664Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 15Wang R. Munoz E.E. Zhu S. McGrath B.C. Cavener D.R. Perk gene dosage regulates glucose homeostasis by modulating pancreatic β-cell functions.PLoS One. 2014; 9 (24915520): e9968410.1371/journal.pone.0099684Crossref PubMed Scopus (19) Google Scholar, 16Back S.H. Scheuner D. Han J. Song B. Ribick M. Wang J. Gildersleeve R.D. Pennathur S. Kaufman R.J. Translation attenuation through eIF2α phosphorylation prevents oxidative stress and maintains the differentiated state in β cells.Cell Metab. 2009; 10 (19583950): 13-2610.1016/j.cmet.2009.06.002Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 17Scheuner D. Song B. McEwen E. Liu C. Laybutt R. Gillespie P. Saunders T. Bonner-Weir S. Kaufman R.J. Translational control is required for the unfolded protein response and in vivo glucose homeostasis.Mol. Cell. 2001; 7 (11430820): 1165-117610.1016/S1097-2765(01)00265-9Abstract Full Text Full Text PDF PubMed Scopus (1085) Google Scholar, 18Scheuner D. Vander Mierde D. Song B. Flamez D. Creemers J.W.M. Tsukamoto K. Ribick M. Schuit F.C. Kaufman R.J. Control of mRNA translation preserves endoplasmic reticulum function in beta cells and maintains glucose homeostasis.Nat. Med. 2005; 11 (15980866): 757-76410.1038/nm1259Crossref PubMed Scopus (320) Google Scholar). The other two arms of the UPR, ATF6α and IRE1/XBP1, are less well-studied in beta cells. IRE1 activation initiates an atypical cytoplasmic RNA splicing event, converting unspliced uXbp1 to spliced sXbp1, which when translated generates a transcription factor, XBP1 (7Back S.H. Kaufman R.J. Endoplasmic reticulum stress and type 2 diabetes.Annu. Rev. Biochem. 2012; 81 (22443930): 767-79310.1146/annurev-biochem-072909-095555Crossref PubMed Scopus (419) Google Scholar). XBP1 is an important part of the beta cell ER stress response (4Chan J.Y. Luzuriaga J. Maxwell E.L. West P.K. Bensellam M. Laybutt D.R. The balance between adaptive and apoptotic unfolded protein responses regulates β-cell death under ER stress conditions through XBP1, CHOP and JNK.Mol. Cell Endocrinol. 2015; 413 (26135354): 189-20110.1016/j.mce.2015.06.025Crossref PubMed Scopus (79) Google Scholar, 19Cunha D.A. Gurzov E.N. Naamane N. Ortis F. Cardozo A.K. Bugliani M. Marchetti P. Eizirik D.L. Cnop M. JunB protects β-cells from lipotoxicity via the XBP1-AKT pathway.Cell Death Differ. 2014; 21 (24786832): 1313-132410.1038/cdd.2014.53Crossref PubMed Scopus (32) Google Scholar, 20Lee A.-H. Heidtman K. Hotamisligil G.S. Glimcher L.H. Dual and opposing roles of the unfolded protein response regulated by IRE1alpha and XBP1 in proinsulin processing and insulin secretion.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21555585): 8885-889010.1073/pnas.1105564108Crossref PubMed Scopus (192) Google Scholar, 21Kirkpatrick C.L. Wiederkehr A. Baquié M. Akhmedov D. Wang H. Gauthier B.R. Akerman I. Ishihara H. Ferrer J. Wollheim C.B. Hepatic nuclear factor 1alpha (HNF1alpha) dysfunction down-regulates X-box–binding protein 1 (XBP1) and sensitizes beta-cells to endoplasmic reticulum stress.J. Biol. Chem. 2011; 286 (21784843): 32300-3231210.1074/jbc.M111.247866Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 22Moore B.D. Jin R.U. Lo H. Jung M. Wang H. Battle M.A. Wollheim C.B. Urano F. Mills J.C. Transcriptional regulation of X-box–binding protein one (XBP1) by hepatocyte nuclear factor 4α (HNF4Α) is vital to beta-cell function.J. Biol. Chem. 2016; 291 (26792861): 6146-615710.1074/jbc.M115.685750Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 23Tsuchiya Y. Saito M. Kadokura H. Miyazaki J.-I. Tashiro F. Imagawa Y. Iwawaki T. Kohno K. IRE1-XBP1 pathway regulates oxidative proinsulin folding in pancreatic β cells.J. Cell Biol. 2018; 217 (29507125): 1287-130110.1083/jcb.201707143Crossref PubMed Scopus (67) Google Scholar) but can have negative effects if unregulated (24Allagnat F. Christulia F. Ortis F. Pirot P. Lortz S. Lenzen S. Eizirik D.L. Cardozo A.K. Sustained production of spliced X-box binding protein 1 (XBP1) induces pancreatic beta cell dysfunction and apoptosis.Diabetologia. 2010; 53 (20349222): 1120-113010.1007/s00125-010-1699-7Crossref PubMed Scopus (93) Google Scholar). ATF6 pathway members are bZip transcription factors, the most studied of which is ATF6α. When released from GRP78, ATF6α, a single-pass ER transmembrane protein, translocates to the Golgi where a cleavage event releases a cytoplasmic domain which is a potent but short-lived transcription factor (6Sharma R.B. Snyder J.T. Alonso L.C. Atf6α impacts cell number by influencing survival, death and proliferation.Mol Metab. 2019; 27S (31500833): S69-S8010.1016/j.molmet.2019.06.005Crossref PubMed Scopus (16) Google Scholar). ATF6α, similar to XBP1, may have both positive (2Sharma R.B. O'Donnell A.C. Stamateris R.E. Ha B. McCloskey K.M. Reynolds P.R. Arvan P. Alonso L.C. Insulin demand regulates β cell number via the unfolded protein response.J. Clin. Invest. 2015; 125 (26389675): 3831-384610.1172/JCI79264Crossref PubMed Scopus (127) Google Scholar, 25Usui M. Yamaguchi S. Tanji Y. Tominaga R. Ishigaki Y. Fukumoto M. Katagiri H. Mori K. Oka Y. Ishihara H. Atf6α-null mice are glucose intolerant due to pancreatic β-cell failure on a high-fat diet but partially resistant to diet-induced insulin resistance.Metabolism. 2012; 61 (22386934): 1118-112810.1016/j.metabol.2012.01.004Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 26Engin F. Yermalovich A. Nguyen T. Ngyuen T. Hummasti S. Fu W. Eizirik D.L. Mathis D. Hotamisligil G.S. Restoration of the unfolded protein response in pancreatic β cells protects mice against type 1 diabetes.Sci. Transl. Med. 2013; 5 (24225943): 211ra15610.1126/scitranslmed.3006534Crossref PubMed Scopus (203) Google Scholar) and negative (10Fonseca S.G. Gromada J. Urano F. Endoplasmic reticulum stress and pancreatic beta cell death.Trends Endocrinol. Metab. 2011; 22 (21458293): 266-27410.1016/j.tem.2011.02.008Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 27Cnop M. Abdulkarim B. Bottu G. Cunha D.A. Igoillo-Esteve M. Masini M. Turatsinze J.-V. Griebel T. Villate O. Santin I. Bugliani M. Ladriere L. Marselli L. McCarthy M.I. Marchetti P. et al.RNA sequencing identifies dysregulation of the human pancreatic islet transcriptome by the saturated fatty acid palmitate.Diabetes. 2014; 63 (24379348): 1978-199310.2337/db13-1383Crossref PubMed Scopus (188) Google Scholar) effects in beta cells. The relative timing of activation of UPR pathways has been reported to influence cellular outcome (28Walter F. Schmid J. Düssmann H. Concannon C.G. Prehn J.H.M. Imaging of single cell responses to ER stress indicates that the relative dynamics of IRE1/XBP1 and PERK/ATF4 signalling rather than a switch between signalling branches determine cell survival.Cell Death Differ. 2015; 22 (25633195): 1502-151610.1038/cdd.2014.241Crossref PubMed Scopus (82) Google Scholar). In other cell types, ATF6α and XBP1 are known to co-regulate some genes (29Yamamoto K. Yoshida H. Kokame K. Kaufman R.J. Mori K. Differential contributions of ATF6 and XBP1 to the activation of endoplasmic reticulum stress-responsive cis-acting elements ERSE, UPRE and ERSE-II.J. Biochem. 2004; 136 (15598891): 343-35010.1093/jb/mvh122Crossref PubMed Scopus (328) Google Scholar, 30Shoulders M.D. Ryno L.M. Genereux J.C. Moresco J.J. Tu P.G. Wu C. Yates J.R. Su A.I. Kelly J.W. Wiseman R.L. Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments.Cell Rep. 2013; 3 (23583182): 1279-129210.1016/j.celrep.2013.03.024Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 31Yamamoto K. Sato T. Matsui T. Sato M. Okada T. Yoshida H. Harada A. Mori K. Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1.Dev. Cell. 2007; 13 (17765680): 365-37610.1016/j.devcel.2007.07.018Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar). The dynamics of ATF6 and XBP1 pathway activation in beta cells following stress are currently unknown; similarly, whether these pathways cooperatively regulate genes in beta cells has not been tested. The goals of this study were to define how pancreatic islet cells activate known ATF6α and XBP1 pathway transcriptional targets in response to ER stress, focusing on the time course of induction and any interdependence of the pathways. Primary mouse islet cells were tested using two parallel chemical stressor paradigms with subtly different onset of action to sensitively probe ATF6 and XBP1 stress-response dynamics. The results suggest that the transcriptional response to the ATF6 and XBP1 pathways occurs simultaneously in islet cells. XBP1 target induction depended upon ATF6α, whereas the converse was not true. Taken together, these results clarify the molecular dynamics and interdependence of the ATF6 and XBP1 pathways in mouse islet cells. To assess the time frame of activation of the ATF6 and XBP1 pathways, we designed experiments to capture the early response after acute stress induction. Building on our prior work (2Sharma R.B. O'Donnell A.C. Stamateris R.E. Ha B. McCloskey K.M. Reynolds P.R. Arvan P. Alonso L.C. Insulin demand regulates β cell number via the unfolded protein response.J. Clin. Invest. 2015; 125 (26389675): 3831-384610.1172/JCI79264Crossref PubMed Scopus (127) Google Scholar), we isolated pancreatic islets from normal healthy adult male and female C57BL/6J mice, allowed them to recover from the stress of isolation, and then dispersed and plated the islet cells on plastic. Because we consistently observe an unexplained integrated stress response suggesting nutrient depletion when we plate dispersed islet cells in 5 mm glucose (Ref. 2Sharma R.B. O'Donnell A.C. Stamateris R.E. Ha B. McCloskey K.M. Reynolds P.R. Arvan P. Alonso L.C. Insulin demand regulates β cell number via the unfolded protein response.J. Clin. Invest. 2015; 125 (26389675): 3831-384610.1172/JCI79264Crossref PubMed Scopus (127) Google Scholar and data not shown), these experiments were performed in nutrient-replete conditions with 15 mm glucose for 72 h (Fig. 1a). In separate experiments, we determined that culture media glucose concentration does not meaningfully decline during 72 h of islet cell culture (Fig. S1). To assess whether islet dispersion impacts the stress response, we performed parallel experiments in whole islets. Two different stress paradigms were studied: thapsigargin, which causes ER calcium depletion stress, and tunicamycin, which causes ER glycosylation stress. Both chemicals were applied in relatively high doses to achieve robust acute activation. To confirm ER stress, we quantified splicing of the Xbp1 transcript, an early event in activation of the XBP1 pathway. Following thapsigargin exposure, spliced Xbp1 (sXbp1) levels increased rapidly within 1 h, accompanied by an equally rapid decrease in unspliced Xbp1 (uXbp1) (Fig. 1, b and c). However, following tunicamycin exposure, the increase in sXbp1 and decrease in uXbp1 levels were slower, not reaching maximal activation until 4–6 h (Fig. 1, d and e). Quantification of band intensity confirmed that thapsigargin exposure activated Xbp1 splicing earlier than tunicamycin (Fig. 1, f–i). The relative ratio of spliced/unspliced Xbp1 (s/uXbp1) also demonstrated this pattern (Fig. 1, j and k). Perhaps surprisingly, the time course of Xbp1 splicing was quite similar in whole islets and dispersed islet cells. The abundance of Grp78 transcript, a sensitive readout of ER stress response that is induced by both ATF6 and XBP1 pathways (30Shoulders M.D. Ryno L.M. Genereux J.C. Moresco J.J. Tu P.G. Wu C. Yates J.R. Su A.I. Kelly J.W. Wiseman R.L. Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments.Cell Rep. 2013; 3 (23583182): 1279-129210.1016/j.celrep.2013.03.024Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar), confirmed activation of a general ER stress response and a slightly accelerated time course with thapsigargin compared with tunicamycin in dispersed but not whole islets (Fig. 1, l and m). In sum, these conditions allowed a sensitive mapping of the early response to ER stress in mouse islet cells using two different stresses with onset ranging from 1 to 4 h.Figure 1A sensitive system to detect the time course and intensity of islet cell response to ER stress. a, mouse islet dispersed cells (left panels) or whole islets (right panels) were plated, allowed to recover for 1 day, and then cultured in 15 mm glucose medium for 2 days before addition of 1000 nm Tg (black lines in f–m) or 2000 ng/ml Tm (green lines in f–m) for 1–24 h before RNA isolation. b–e, a gel-based assay was used to quantify the time course of Xbp1 splicing over 24 h of stress induced by Tg (b and c) or Tm (d and e). f–m, unspliced Xbp1 (uXbp1; f and g) or spliced Xbp1 (sXbp1; h and i) were quantified relative to Actin abundance or as the ratio of s/uXbp1 (j and k). Transcriptional evidence for global UPR activation was determined by qPCR for Grp78 (l and m). For f–m the black (Tg) and green (Tm) arrows represent the earliest time points with p < 0.05 relative to time 0. Statistical analysis was by one-way ANOVA for each drug, comparing each point to time 0. For readability, significance is labeled only for the earliest time point achieving p < 0.05 (arrows) and the 24-h time point. Sample size was n = 3–4 for all panels. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Using this system, we first assessed the time course of activation of the ATF6 and XBP1 pathways, as determined by timing of increase of pathway-specific transcriptional targets, in both dispersed islet cells and whole islets. Pathway-selective targets were chosen based on a carefully performed unbiased analysis of the stress-independent transcriptional impact of ATF6 or XBP1 activation in HEK293 cells (30Shoulders M.D. Ryno L.M. Genereux J.C. Moresco J.J. Tu P.G. Wu C. Yates J.R. Su A.I. Kelly J.W. Wiseman R.L. Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments.Cell Rep. 2013; 3 (23583182): 1279-129210.1016/j.celrep.2013.03.024Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar) and have been confirmed in other studies (20Lee A.-H. Heidtman K. Hotamisligil G.S. Glimcher L.H. Dual and opposing roles of the unfolded protein response regulated by IRE1alpha and XBP1 in proinsulin processing and insulin secretion.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21555585): 8885-889010.1073/pnas.1105564108Crossref PubMed Scopus (192) Google Scholar, 35Dadey D.Y.A. Kapoor V. Khudanyan A. Urano F. Kim A.H. Thotala D. Hallahan D.E. The ATF6 pathway of the ER stress response contributes to enhanced viability in glioblastoma.Oncotarget. 2016; 7 (26716508): 2080-209210.18632/oncotarget.6712Crossref PubMed Scopus (68) Google Scholar, 36Shaffer A.L. Shapiro-Shelef M. Iwakoshi N.N. Lee A.-H. Qian S.-B. Zhao H. Yu X. Yang L. Tan B.K. Rosenwald A. Hurt E.M. Petroulakis E. Sonenberg N. Yewdell J.W. Calame K. et al.XBP1, downstream of Blimp-1, expands the secretory apparatus and other organelles, and increases protein synthesis in plasma cell differentiation.Immunity. 2004; 21 (15345222): 81-9310.1016/j.immuni.2004.06.010Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar, 37Correll R.N. Grimes K.M. Prasad V. Lynch J.M. Khalil H. Molkentin J.D. Overlapping and differential functions of ATF6α versus ATF6β in the mouse heart.Sci. Rep. 2019; 9 (30765833): e205910.1038/s41598-019-39515-5Crossref PubMed Scopus (20) Google Scholar). All ATF6 targets tested increased under both stress conditions (Fig. 2, a–h). Similar to Grp78, induction of ATF6 targets by tunicamycin was delayed relative to induction by thapsigargin. This difference was more evident in dispersed islet cells than in whole islets, but the results were statistically similar between the two, possibly related to subtle differences in drug responses in whole islets in the early time points (Fig. S2). One target, Herpud1, consistently rose earlier than the others, suggesting a different mechanism of activation (Fig. 2, c and d). All three XBP1 targets tested were also increased in islet cells and whole islets by ER stressors; induction of all three were slightly earlier for thapsigargin than tunicamycin (Fig. 2, i–n). Intriguingly, although some ATF6 target induction was slower (Fig. 2, a and b) or faster (Fig. 2, c and d), on the whole the timing of XBP1 target increase was very similar to the timing of ATF6 target increase. Taken together, the ATF6 and XBP1 pathways seem to be simultaneously activated during acute ER stress in mouse islet cells. ATF6 pathway members include a family of ER transmembrane proteins that are activated in SREBP1-like fashion by cleavage in the Golgi, leading to release of a transcription factor (38Kondo S. Saito A. Asada R. Kanemoto S. Imaizumi K. Physiological unfolded protein response regulated by OASIS family members, transmembrane bZIP transcription factors.IUBMB Life. 2011; 63 (21438114): 233-23910.1002/iub.433Crossref PubMed Scopus (60) Google Scholar). Deletion of ATF6α itself is relatively well-tolerated in mice (31Yamamoto K. Sato T. Matsui T. Sato M. Okada T. Yoshida H. Harada A. Mori K. Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1.Dev. Cell. 2007; 13 (17765680): 365-37610.1016/j.devcel.2007.07.018Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar, 39Wu J. Rutkowski D.T. Dubois M. Swathirajan J. Saunders T. Wang J. Song B. Yau G.D.-Y. Kaufman R.J. ATF6α optimizes long-term endoplasmic reticulum function to protect cells from chronic stress.Dev. Cell. 2007; 13 (17765679): 351-36410.1016/j.devcel.2007.07.005Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar), but deletion of both ATF6α and ATF6β results in early lethality (31Yamamoto K. Sato T. Matsui T. Sato M. Okada T. Yoshida H. Harada A. Mori K. Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6alpha and XBP1.Dev. Cell. 2007; 13 (17765680): 365-37610.1016/j.devcel.2007.07.018Abstract Full Text Full Text PDF PubMed Scopus (781) Google Scholar). To assess the specific role of ATF6α in the acute response to ER stress, we isolated islets from mice bearing genomic loxP sites flanking exons 8 and 9 such that expressing bacterial Cre recombinase results in a null allele (26Engin F. Yermalovich A. Nguyen T. Ngyuen T. Hummasti S. Fu W. Eizirik D.L. Mathis D. Hotamisligil G.S. Restoration of the unfolded protein response in pancreatic β cells protects mice against type 1 diabetes.Sci. Transl. Med. 2013; 5 (24225943): 211ra15610.1126/scitranslmed.3006534Crossref PubMed Scopus (203) Google Scholar). Transducing Atf6αflox/flox islet cells with an adenovirus expressing Cre (Fig. 3a) markedly reduced Atf6α mRNA levels relative to control LacZ-transduced cultures (Fig. 3b), suggesting successful knockdown. ATF6α knockdown modestly reduced mRNA abundance of ATF6 pathway targets Grp78, Hyou1, Herpud1, and Pdia4 under basal conditions (Fig. 3c), suggesting tonic ATF6 pathway activation, mediated by ATF6α, in glucose-replete culture conditions. Abundance of mRNA of PERK pathway members Atf4 and Chop were unchanged by Atf6α knockdown (Fig. 3d), confirming selectivity of the response. Intriguingly, contrary to our expectation that knockdown of Atf6α might induce a compensatory increase in expression of Atf6β, levels actually decreased (Fig. 3e). To assess the role of ATF6α in the islet cell response to acute stress, the early induction of ATF6 pathway targets was quantified after ATF6α knockdown. Although Atf6α itself was strongly induced by thapsigargin in control islet cells, it remained low in Cre-transduced cultures, confirming ongoing effective knockdown (Fig. 4a). Induction of ATF6 pathway targets over the first 6 h of ER stress was markedly blunted by ATF6α knockdown (Fig. 4, b–e). When the duration of stress was extended to 24 h, the impact of ATF6α knockdown was still evident, although most genes showed some continued increase for both thapsigargin and tunicamycin (Fig. 4, f–m). This was despite lack of meaningful recovery of Atf6α transcript abundance (Fig. 4, n and o). Atf6β levels were found to increase after ER stress, in a strong

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