title: Moderate endoplasmic reticulum stress activates a PERK and p38-dependent apoptosis.
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.
authors.affiliation: Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA.
authors.forename: Emily C
authors.initials: EC
authors.lastname: Lumley
authors.affiliation: Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA.
authors.forename: Acadia R
authors.initials: AR
authors.lastname: Osborn
authors.affiliation: Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA.
authors.forename: Jessica E
authors.initials: JE
authors.lastname: Scott
authors.affiliation: Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA.
authors.forename: Amanda G
authors.initials: AG
authors.lastname: Scholl
authors.affiliation: Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA.
authors.forename: Vicki
authors.initials: V
authors.lastname: Mercado
authors.affiliation: Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA.
authors.forename: Young T
authors.initials: YT
authors.lastname: McMahan
authors.affiliation: Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA.
authors.forename: Zachary G
authors.initials: ZG
authors.lastname: Coffman
authors.affiliation: Natural Science Division, Pepperdine University, 24255 Pacific Coast Highway, Malibu,, CA, 90263, USA. jay.brewster@pepperdine.edu.
authors.forename: Jay L
authors.initials: JL
authors.lastname: Brewster
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001896:Boron Compounds / Q000494:pharmacology; D002460:Cell Line; D002934:Cinnamates / Q000633:toxicity; D006224:Cricetinae; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D006031:Glycosylation / Q000187:drug effects; D006602:Hexosyltransferases / Q000235:genetics / Q000378:metabolism; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D047028:Macrocyclic Compounds / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology; D012326:RNA Splicing / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013696:Temperature; D013890:Thiourea / Q000031:analogs & derivatives / Q000633:toxicity; D014415:Tunicamycin / Q000633:toxicity; D000071717:X-Box Binding Protein 1 / Q000235:genetics / Q000378:metabolism; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D001896:Boron Compounds; D002934:Cinnamates; D051034:Inhibitor of Apoptosis Proteins; D047028:Macrocyclic Compounds; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010080:Oxazoles; D012333:RNA, Messenger; D000071717:X-Box Binding Protein 1; C496827:salubrinal; C511704:xestospongin C; D014415:Tunicamycin; C109986:2-aminoethoxydiphenyl borate; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase; D019892:eIF-2 Kinase; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D013890:Thiourea
keywords: Apoptosis; Endoplasmic reticulum; PERK; Protein folding; Protein kinase R (PKR)-like endoplasmic reticulum kinase; Unfolded protein response; p38 kinase