Protection of renal epithelial cells against oxidative injury by endoplasmic reticulum stress preconditioning is mediated by ERK1/2 activation.
We investigated the role of the endoplasmic reticulum (ER) stress response in intracellular Ca2+ regulation, MAPK activation, and cytoprotection in LLC-PK1 renal epithelial cells in an attempt to identify the mechanisms of protection afforded by ER stress. Cells preconditioned with trans-4,5-dihydroxy-1,2-dithiane, tunicamycin, thapsigargin, or A23187 expressed ER stress proteins and were resistant to subsequent H2O2-induced cell injury. In addition, ER stress preconditioning prevented the increase in intracellular Ca2+ concentration that normally follows H2O2 exposure. Stable transfection of cells with antisense RNA targeted against GRP78 (pkASgrp78 cells) prevented GRP78 induction, disabled the ER stress response, sensitized cells to H2O2-induced injury, and prevented the development of tolerance to H2O2 that normally occurs with preconditioning. ERK and JNK were transiently (30-60 min) phosphorylated in response to H2O2. ER stress-preconditioned cells had more ERK and less JNK phosphorylation than control cells in response to H2O2 exposure. Preincubation with a specific inhibitor of JNK activation or adenoviral infection with a construct that encodes constitutively active MEK1, the upstream activator of ERKs, also protected cells against H2O2 toxicity. In contrast, the pkASgrp78 cells had less ERK and more JNK phosphorylation upon H2O2 exposure. Expression of constitutively active ERK also conferred protection on native as well as pkAS-grp78 cells. These results indicate that GRP78 plays an important role in the ER stress response and cytoprotection. ER stress preconditioning attenuates H2O2-induced cell injury in LLC-PK1 cells by preventing an increase in intracellular Ca2+ concentration, potentiating ERK activation, and decreasing JNK activation. Thus, the ER stress response modulates the balance between ERK and JNK signaling pathways to prevent cell death after oxidative injury. Furthermore, ERK activation is an important downstream effector mechanism for cellular protection by ER stress.
Medical Services, Massachusetts General Hospital and Brigham and Women's Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115, USA.
Cheng-Chieh
CC
Hung
Takaharu
T
Ichimura
James L
JL
Stevens
Joseph V
JV
Bonventre
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000032:analysis; D037282:Calreticulin / Q000235:genetics; D002460:Cell Line; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000201:enzymology / Q000648:ultrastructure; D015870:Gene Expression; D006861:Hydrogen Peroxide / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure*; D048369:MAP Kinase Kinase 1; D048670:MAP Kinase Kinase 4; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D016296:Mutagenesis; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011994:Recombinant Proteins; D015398:Signal Transduction; D013552:Swine; D019284:Thapsigargin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D037282:Calreticulin; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014415:Tunicamycin; D000001:Calcimycin; D019284:Thapsigargin; D006861:Hydrogen Peroxide; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D002118:Calcium; D004229:Dithiothreitol