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Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis

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Cell Stress and Chaperones Aims and scope

Abstract

The endoplasmic reticulum (ER) has the ability to signal organelle dysfunction via a complex signaling network known as the unfolded protein response (UPR). In this work, hamster fibroblast cells exhibiting moderate levels of ER stress were compared to those exhibiting severe ER stress. Inhibition of N-linked glycosylation was accomplished via a temperature-sensitive mutation in the Dad1 subunit of the oligosaccharyltransferase (OST) complex or by direct inhibition with tunicamycin (Tm). Temperature shift (TS) treatment generated weak activation of ER stress signaling when compared to doses of Tm that are typically used in ER stress studies (500–1000 nM). A dose-response analysis of key ER stress signaling mediators, inositol-requiring enzyme 1 (IRE1) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), revealed 20–40 nM of Tm to generate activation intensity similar to TS treatment. In parental BHK21 cells, moderate (20–40 nM) and high doses (200–1000 nM) of Tm were compared to identify physiological and signaling-based differences in stress response. Inhibition of ER Ca2+ release via ITPR activity with 2-aminoethoxydiphenyl borate (2-APB) or Xestospongin C (XeC) was sufficient to protect against apoptosis induced by moderate but not higher doses of Tm. Analysis of kinase activation over a range of Tm exposures revealed the p38 stress-activated protein kinase (SAPK) to display increasing activation with Tm dosage. Interestingly, Tm induced the extracellular regulated kinases (Erk1/2) only at moderate doses of Tm. Inhibition of ER transmembrane stress sensors (IRE1, PERK) or cytosolic signaling mediators (p38, Jnk1, Erk1/2) was used to evaluate pathways involved in apoptosis activation during ER stress. Inhibition of either PERK or p38 was sufficient to reduce cell death and apoptosis induced by moderate, but not high, doses of Tm. During ER stress, cells exhibited a rapid decline in anti-apoptotic Mcl-1 and survivin proteins. Inhibition of PERK was sufficient to block this affect. This work reveals moderate doses of ER stress to generate patterns of stress signaling that are distinct from higher doses and that apoptosis activation at moderate levels of stress are dependent upon PERK and p38 signaling. Studies exploring ER stress signaling should recognize that this signaling acts as a rheostat rather than a simple switch, behaving distinctively in a dose-dependent manner.

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Abbreviations

ER:

Endoplasmic reticulum

Tm:

Tunicamycin

TS:

Temperature shift

2-APB:

2-Aminoethoxyphenylborate

OST:

Oligosaccharyltransferase

XeC:

Xestospongin C

IRE1:

Inositol-requiring enzyme 1

PERK:

Protein kinase R (PKR)-like endoplasmic reticulum kinase

Xbp1:

X-box binding protein 1

ITPR:

Inositol 1,4,5-trisphosphate receptor

RT/PCR:

Reverse transcription, polymerase chain reaction

UPR:

Unfolded protein response

References

  • Arai K et al (2004) Involvement of ERK MAP kinase in endoplasmic reticulum stress in SH-SY5Y human neuroblastoma cells. J Neurochem 89(1):232–239

    Article  CAS  PubMed  Google Scholar 

  • Ascher-Landsberg J et al (1999) The effects of 2-aminoethoxydiphenyl borate, a novel inositol 1,4, 5-trisphosphate receptor modulator on myometrial contractions. Biochem Biophys Res Commun 264(3):979–982

    Article  CAS  PubMed  Google Scholar 

  • Ashwell JD (2006) The many paths to p38 mitogen-activated protein kinase activation in the immune system. Nat Rev Immunol 6(7):532–540

    Article  CAS  PubMed  Google Scholar 

  • Blackshaw S et al (2000) Type 3 inositol 1,4,5-trisphosphate receptor modulates cell death. FASEB J 14(10):1375–1379

    Article  CAS  PubMed  Google Scholar 

  • Boehning D et al (2003) Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis. Nat Cell Biol 5(12):1051–1061

    Article  CAS  PubMed  Google Scholar 

  • Boyce M et al (2005) A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress. Science 307(5711):935–939

    Article  CAS  PubMed  Google Scholar 

  • Brewster JL et al (2000) Deletion of Dad1 in mice induces an apoptosis-associated embryonic lethality. Genesis 26:271–278

    Article  CAS  PubMed  Google Scholar 

  • Cnop M et al (2007) Selective inhibition of eukaryotic translation initiation factor 2 alpha dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic beta-cell dysfunction and apoptosis. J Biol Chem 282(6):3989–3997

    Article  CAS  PubMed  Google Scholar 

  • Czabotar PE et al (2014) Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat Rev Mol Cell Biol 15(1):49–63

    Article  CAS  PubMed  Google Scholar 

  • Gardner BM, Walter P (2011) Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response. Science 333(6051):1891–1894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gardner BM et al (2013) Endoplasmic reticulum stress sensing in the unfolded protein response. Cold Spring Harb Perspect Biol 5(3):a013169

    Article  PubMed  PubMed Central  Google Scholar 

  • Ghosh S et al (2015) Cross-talk between endoplasmic reticulum (ER) stress and the MEK/ERK pathway potentiates apoptosis in human triple negative breast carcinoma cells: role of a dihydropyrimidone, nifetepimine. J Biol Chem 290(7):3936–3949

    Article  CAS  PubMed  Google Scholar 

  • Hamanaka RB et al (2009) PERK-dependent regulation of IAP translation during ER stress. Oncogene 28(6):910–920

    Article  CAS  PubMed  Google Scholar 

  • Hassler J, Cao SS, Kaufman RJ (2012) IRE1, a double-edged sword in pre-miRNA slicing and cell death. Dev Cell 23(5):921–923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hetz C (2012) The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat Rev Mol Cell Biol 13(2):89–102

    CAS  PubMed  Google Scholar 

  • Higo T et al (2010) Mechanism of ER stress-induced brain damage by IP(3) receptor. Neuron 68(5):865–878

    Article  CAS  PubMed  Google Scholar 

  • Hiramatsu N et al (2014) Translational and posttranslational regulation of XIAP by eIF2alpha and ATF4 promotes ER stress-induced cell death during the unfolded protein response. Mol Biol Cell 25(9):1411–1420

    Article  PubMed  PubMed Central  Google Scholar 

  • Hiramatsu N et al (2015) Multiple mechanisms of unfolded protein response-induced cell death. Am J Pathol 185:1800–1808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hong NA et al (2000) Mice lacking Dad1, the defender against apoptotic death-1, express abnormal N-linked glycoproteins and undergo increased embryonic apoptosis. Dev Biol 220(1):76–84

    Article  CAS  PubMed  Google Scholar 

  • Hoppins S, Nunnari J (2012) Cell biology. Mitochondrial dynamics and apoptosis—the ER connection. Science 337(6098):1052–1054

    Article  CAS  PubMed  Google Scholar 

  • Jiang CC et al (2007) Inhibition of MEK sensitizes human melanoma cells to endoplasmic reticulum stress-induced apoptosis. Cancer Res 67(20):9750–9761

    Article  CAS  PubMed  Google Scholar 

  • Jiang Q et al (2014) Involvement of p38 in signal switching from autophagy to apoptosis via the PERK/eIF2alpha/ATF4 axis in selenite-treated NB4 cells. Cell Death Dis 5:e1270

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Joseph SK, Hajnoczky G (2007) IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond. Apoptosis 12(5):951–968

    Article  CAS  PubMed  Google Scholar 

  • Kessel D (2006) Protection of Bcl-2 by salubrinal. Biochem Biophys Res Commun 346(4):1320–1323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kim L et al (2005) p38 MAPK autophosphorylation drives macrophage IL-12 production during intracellular infection. J Immunol 174(7):4178–4184

    Article  CAS  PubMed  Google Scholar 

  • Lee AS (2005) The ER chaperone and signaling regulator GRP78/BiP as a monitor of endoplasmic reticulum stress. Methods 35(4):373–381

    Article  CAS  PubMed  Google Scholar 

  • Lim EJ, Heo J, Kim YH (2015) Tunicamycin promotes apoptosis in leukemia cells through ROS generation and downregulation of survivin expression. Apoptosis 20(8):1087–1098

    Article  CAS  PubMed  Google Scholar 

  • Liu Z et al (2015) Protein kinase R-like ER kinase and its role in endoplasmic reticulum stress-decided cell fate. Cell Death Dis 6:e1822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Manni S et al (2012) Protein kinase CK2 protects multiple myeloma cells from ER stress-induced apoptosis and from the cytotoxic effect of HSP90 inhibition through regulation of the unfolded protein response. Clin Cancer Res 18(7):1888–1900

    Article  CAS  PubMed  Google Scholar 

  • Maurel M et al (2014) Getting RIDD of RNA: IRE1 in cell fate regulation. Trends Biochem Sci 39(5):245–254

    Article  CAS  PubMed  Google Scholar 

  • Miyamoto S et al (2000) Xestospongin C, a selective and membrane-permeable inhibitor of IP(3) receptor, attenuates the positive inotropic effect of alpha-adrenergic stimulation in Guinea-pig papillary muscle. Br J Pharmacol 130(3):650–654

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nakashima T et al (1993) Molecular cloning of a human cDNA encoding a novel protein, DAD1, whose defect causes apoptotic cell death in hamster BHK21 cells. Mol Cell Biol 13(10):6367–6374

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Niederer KE et al (2005) Cypermethrin blocks a mitochondria-dependent apoptotic signal initiated by deficient N-linked glycosylation within the endoplasmic reticulum. Cell Signal 17:177–186

    Article  CAS  PubMed  Google Scholar 

  • Nikonov AV et al (2002) Active translocon complexes labeled with GFP-Dad1 diffuse slowly as large polysome arrays in the endoplasmic reticulum. J Cell Biol 158(3):497–506

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Opferman JT (2015) Attacking cancer’s Achilles heel: antagonism of anti-apoptotic BCL-2 family members. FEBS J. doi:10.1111/febs.13472

    PubMed  Google Scholar 

  • Ota A et al (2010) Specific regulation of noncanonical p38alpha activation by Hsp90-Cdc37 chaperone complex in cardiomyocyte. Circ Res 106(8):1404–1412

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ruggiano A, Foresti O, Carvalho P (2014) Quality control: ER-associated degradation: protein quality control and beyond. J Cell Biol 204(6):869–879

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rutkowski DT et al (2006) Adaptation to ER stress is mediated by differential stabilities of pro-survival and pro-apoptotic mRNAs and proteins. PLoS Biol 4(11):e374

    Article  PubMed  PubMed Central  Google Scholar 

  • Saleem H et al (2014) Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor. Br J Pharmacol 171(13):3298–3312

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sanjay A, Fu J, Kreibich G (1998) DAD1 is required for the function and the structural integrity of the oligosaccharyltransferase complex. J Biol Chem 273(40):26094–26099

    Article  CAS  PubMed  Google Scholar 

  • Schroder M (2008) Endoplasmic reticulum stress responses. Cell Mol Life Sci 65(6):862–894

    Article  CAS  PubMed  Google Scholar 

  • Sohn J et al (2003) The effect of ursodeoxycholic acid on the survivin in thapsigargin-induced apoptosis. Cancer Lett 191(1):83–92

    Article  CAS  PubMed  Google Scholar 

  • Upton JP et al (2012) IRE1alpha cleaves select microRNAs during ER stress to derepress translation of proapoptotic caspase-2. Science 338(6108):818–822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walter P, Ron D (2011) The unfolded protein response: from stress pathway to homeostatic regulation. Science 334(6059):1081–1086

    Article  CAS  PubMed  Google Scholar 

  • Wang X et al (2011) Bcl-2 proteins regulate ER membrane permeability to luminal proteins during ER stress-induced apoptosis. Cell Death Differ 18(1):38–47

    Article  PubMed  Google Scholar 

  • Wek RC, Jiang HY, Anthony TG (2006) Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans 34(Pt 1):7–11

    Article  CAS  PubMed  Google Scholar 

  • Wu L et al (2014) Salubrinal protects against rotenone-induced SH-SY5Y cell death via ATF4-parkin pathway. Brain Res 1549:52–62

    Article  CAS  PubMed  Google Scholar 

  • Zhu X et al (2014) Ubiquitination of inositol-requiring enzyme 1 (IRE1) by the E3 ligase CHIP mediates the IRE1/TRAF2/JNK pathway. J Biol Chem 289(44):30567–30577

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zong WX et al (2003) Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis. J Cell Biol 162(1):59–69

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

The authors wish to recognize the helpful contributions of research students Celina Jones, Emily Blair, and Tina Johnson. This work was supported by grants from the National Institutes of Health (R15GM065139), the National Science Foundation (DBI-1062721), and research funding from Pepperdine University.

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Authors and Affiliations

  1. Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA

    Emily C. Lumley, Acadia R. Osborn, Jessica E. Scott, Amanda G. Scholl, Vicki Mercado, Young T. McMahan, Zachary G. Coffman & Jay L. Brewster

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  1. Emily C. Lumley
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  2. Acadia R. Osborn
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  3. Jessica E. Scott
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  4. Amanda G. Scholl
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  6. Young T. McMahan
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  7. Zachary G. Coffman
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  8. Jay L. Brewster
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Correspondence to Jay L. Brewster.

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Lumley, E.C., Osborn, A.R., Scott, J.E. et al. Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis. Cell Stress and Chaperones 22, 43–54 (2017). https://doi.org/10.1007/s12192-016-0740-2

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  • DOI: https://doi.org/10.1007/s12192-016-0740-2

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