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• W2029175062 abstract "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. 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. When the kidney is rendered ischemic or is obstructed, it is protected against subsequent ischemia for at least 15 days from the time of the initial insult (1Park K.M. Chen A. Bonventre J.V. J. Biol. Chem. 2001; 276: 11870-11876Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 2Park K.M. Kramers C. Vayssier-Taussat M. Chen A. Bonventre J.V. J. Biol. Chem. 2002; 277: 2040-2049Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The mechanisms of the protection afforded by prior ischemic stress are not known; however, many of the downstream signals that mediate ischemia/reperfusion injury have been well characterized. For example, reactive oxygen species (ROS) 1The abbreviations used are: ROS, reactive oxygen species; [Ca2+]i, intracellular free Ca2+ concentration; ER, endoplasmic reticulum; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal kinase; SAPK, stress-activated protein kinase; UPR, unfolded protein response; PKB, protein kinase B; DMEM, Dulbecco's modified Eagle's medium; DTTox, trans-4,5-dihydroxy-1,2-dithiane (oxidized dithiothreitol); LDH, lactate dehydrogenase; EBSS, Earle's balanced salt solution; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ASK1, apoptosis signal-regulated kinase-1; FCS, fetal calf serum. are major mediators of ischemia/reperfusion injury (3Cutrn J.C. Perrelli M.G. Cavalieri B. Peralta C. Rosell Catafau J. Poli G. Free Radic. Biol. Med. 2002; 33: 1200-1208Crossref PubMed Scopus (146) Google Scholar, 4Li C. Jackson R.M. Am. J. Physiol. 2002; 282: C227-C241Crossref PubMed Scopus (898) Google Scholar, 5Droge W. Physiol. Rev. 2002; 82: 47-95Crossref PubMed Scopus (7605) Google Scholar). Although cells contain antioxidant defenses that minimize susceptibility to ROS, ROS generation and oxidative stress often exceed the cell's antioxidant capacity (6Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1942) Google Scholar). Oxidative stress causes a rapid increase in intracellular free Ca2+ concentration ([Ca2+]i) in a number of cell types, including cells derived from the renal tubule (7Ueda N. Shah S.V. Am. J. Physiol. 1992; 263: F214-D221PubMed Google Scholar, 8Wang H. Joseph J.A. Free Radic. Biol. Med. 2000; 28: 1222-1231Crossref PubMed Scopus (83) Google Scholar). Increases in [Ca2+]i can result in enhanced Ca2+ influx into mitochondria, disrupting mitochondrial metabolism and leading to cell death (9Duchen M.R. J. Physiol. (Lond.). 2000; 529: 57-68Crossref Scopus (948) Google Scholar, 10Ermak G. Davies K.J. Mol. Immunol. 2002; 38: 713-721Crossref PubMed Scopus (666) Google Scholar). Changes in [Ca2+]i also modulate gene transcription and proteases and nucleases that control cell apoptosis (9Duchen M.R. J. Physiol. (Lond.). 2000; 529: 57-68Crossref Scopus (948) Google Scholar, 10Ermak G. Davies K.J. Mol. Immunol. 2002; 38: 713-721Crossref PubMed Scopus (666) Google Scholar). Recent investigations using renal epithelial cells indicate that the endoplasmic reticulum (ER) stress response can modulate both oxidative stress and [Ca2+]i after treatment with organic hydroperoxides and alkylating agents (11Liu H. Bowes III, R.C. van de Water B. Sillence C. Nagelkerke J.F. Stevens J.L. J. Biol. Chem. 1997; 272: 21751-21759Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 12Liu H. Miller E. van de Water B. Stevens J.L. J. Biol. Chem. 1998; 273: 12858-12862Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Likewise, increased attention has been paid recently to the possibility that ER stress influences the pathophysiology of acute ischemia in the brain, heart, or kidney (13Bush K.T. Keller S.H. Nigam S.K. J. Clin. Invest. 2000; 106: 621-626Crossref PubMed Scopus (135) Google Scholar, 14Benjamin I.J. McMillan D.R. Circ. Res. 1998; 83: 117-132Crossref PubMed Scopus (790) Google Scholar, 15Paschen W. Cell Calcium. 2001; 29: 1-11Crossref PubMed Scopus (142) Google Scholar). Given the association between ROS, oxidative stress, and ischemic injury in the kidney, we investigated the association between the ER stress response and H2O2 in renal epithelial cells. ROS-induced cell injury has been attributed, in part, to the change in activation of intracellular signaling molecules, including mitogen-activated protein kinases (MAPKs). MAPKs, which include extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK), and p38 subfamilies, are important regulatory proteins that transduce various extracellular signals into intracellular events (16Seger R. Krebs E.G. FASEB J. 1995; 9: 726-735Crossref PubMed Scopus (3218) Google Scholar, 17Mandlekar S. Kong A.N. Apoptosis. 2001; 6: 469-477Crossref PubMed Scopus (341) Google Scholar). The ERK, JNK/SAPK, and p38 subfamilies are all activated in response to oxidative injury. In 1996, Guyton et al. (18Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar) implicated ERK activation as a survival factor following oxidant injury. Subsequent studies from a number of laboratories confirmed these findings in other cell types and with other injurious agents (19Ikeyama S. Kokkonen G. Shack S. Wang X.T. Holbrook N.J. FASEB J. 2002; 16: 114-116Crossref PubMed Scopus (116) Google Scholar, 20Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (691) Google Scholar). The influence of JNK activation on cell survival following oxidative stress remains complex and highly controversial (6Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1942) Google Scholar). Some studies have shown that JNK activation is correlated with cell death or apoptosis induced by agents that act, at least in part, via generation of ROS (21Chen Y.R. Tan T.H. Int. J. Oncol. 2000; 16: 651-662PubMed Google Scholar). Thus, ERK and JNK can have counteracting influences on cell survival during stress, including oxidant injury (18Guyton K.Z. Liu Y. Gorospe M. Xu Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1142) Google Scholar, 20Wang X. Martindale J.L. Liu Y. Holbrook N.J. Biochem. J. 1998; 333: 291-300Crossref PubMed Scopus (691) Google Scholar); and the balance between JNK and ERK activation may determine cell fate after renal ischemia/reperfusion injury (1Park K.M. Chen A. Bonventre J.V. J. Biol. Chem. 2001; 276: 11870-11876Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 22di Mari J.F. Davis R. Safirstein R.L. Am. J. Physiol. 1999; 277: F195-F203PubMed Google Scholar). We investigated the association between ER stress and JNK or ERK phosphorylation. The signals that activate the ER stress response are well characterized, as is the link between ER Ca2+ release and cell injury. The ER is involved in both Ca2+ signaling and posttranslational protein folding and maturation. Release of Ca2+ from the ER may contribute to the ischemia/reperfusion injury of the brain, heart, and kidney (15Paschen W. Cell Calcium. 2001; 29: 1-11Crossref PubMed Scopus (142) Google Scholar, 23Kristian T. Siesjo B.K. Stroke. 1998; 29: 705-718Crossref PubMed Scopus (729) Google Scholar, 24Yamashita J. Itoh M. Kuro T. Kobayashi Y. Ogata M. Takaoka M. Matsumura Y. J. Pharmacol. Exp. Ther. 2001; 296: 412-419PubMed Google Scholar). The response of the ER to unfolded proteins, known as the unfolded protein response (UPR), is currently the best understood model of ER stress signaling. The UPR has been shown to modulate expression of ER chaperones, allowing the cell to tolerate the accumulation of unfolded proteins (15Paschen W. Cell Calcium. 2001; 29: 1-11Crossref PubMed Scopus (142) Google Scholar, 25Sherman M.Y. Goldberg A.L. Neuron. 2001; 29: 15-32Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar, 26Kaufman R.J. Scheuner D. Schroder M. Shen X. Lee K. Liu C.Y. Arnold S.M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 411-421Crossref PubMed Scopus (506) Google Scholar, 27Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar). In mammalian cells, the UPR is activated by agents that prevent protein glycosylation (tunicamycin) and disulfide bond formation (DTTox) and by agents that deplete ER Ca2+ stores such as thapsigargin and the Ca2+ ionophore A23187. The UPR also regulates many genes that affect diverse aspects of cell physiology. In the yeast Saccharomyces cerevisiae, 381 of 6000 genes were found to participate in the UPR, including 208 genes for which some functional information is available (28Travers K.J. Patil C.K. Wodicka L. Lockhart D.J. Weissman J.S. Walter P. Cell. 2000; 101: 249-258Abstract Full Text Full Text PDF PubMed Scopus (1599) Google Scholar). Thus, the current view of the ER stress pathway has broadened from a pathway that simply regulates ER molecular chaperones to one that impacts many aspects of cell physiology (29Spear E. Ng D.T. Traffic. 2001; 2: 515-523Crossref PubMed Scopus (47) Google Scholar). It is not entirely clear, however, how ER stress protects cells. In particular, it is not known whether the cellular MAPK and Akt/protein kinase B (PKB) pathways are effectors of the ER stress response (30Urano F. Bertolotti A. Ron D. J. Cell Sci. 2000; 113: 3697-3702Crossref PubMed Google Scholar, 31Ron D. J. Clin. Invest. 2002; 110: 1383-1388Crossref PubMed Scopus (745) Google Scholar). Although the ER stress response has been implicated in the pathophysiology of ischemic injury (13Bush K.T. Keller S.H. Nigam S.K. J. Clin. Invest. 2000; 106: 621-626Crossref PubMed Scopus (135) Google Scholar, 14Benjamin I.J. McMillan D.R. Circ. Res. 1998; 83: 117-132Crossref PubMed Scopus (790) Google Scholar, 15Paschen W. Cell Calcium. 2001; 29: 1-11Crossref PubMed Scopus (142) Google Scholar, 32Treiman M. Trends Cardiovasc. Med. 2002; 12: 57-62Crossref PubMed Scopus (36) Google Scholar), the role of individual ER stress proteins has not been addressed. Glucose-regulated proteins are the prototypical ER chaperones induced by ER stress (33Lee A.S. Curr. Opin. Cell Biol. 1992; 4: 267-273Crossref PubMed Scopus (396) Google Scholar). Induction of glucose-regulated proteins has been associated with protection against an increase in [Ca2+]i (11Liu H. Bowes III, R.C. van de Water B. Sillence C. Nagelkerke J.F. Stevens J.L. J. Biol. Chem. 1997; 272: 21751-21759Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 12Liu H. Miller E. van de Water B. Stevens J.L. J. Biol. Chem. 1998; 273: 12858-12862Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 15Paschen W. Cell Calcium. 2001; 29: 1-11Crossref PubMed Scopus (142) Google Scholar) and facilitation of protein folding (25Sherman M.Y. Goldberg A.L. Neuron. 2001; 29: 15-32Abstract Full Text Full Text PDF PubMed Scopus (889) Google Scholar, 27Lee A.S. Trends Biochem. Sci. 2001; 26: 504-510Abstract Full Text Full Text PDF PubMed Scopus (925) Google Scholar, 34Kaufman R.J. Genes Dev. 1999; 13: 1211-1233Crossref PubMed Scopus (1944) Google Scholar). Overexpression, antisense, and ribozyme approaches in tissue culture systems have led to the conclusion that GRP78, GRP94, and Adapt78 protect cells against cell death (11Liu H. Bowes III, R.C. van de Water B. Sillence C. Nagelkerke J.F. Stevens J.L. J. Biol. Chem. 1997; 272: 21751-21759Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 35Jamora C. Dennert G. Lee A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7690-7694Crossref PubMed Scopus (247) Google Scholar, 36Miyake H. Hara I. Arakawa S. Kamidono S. J. Cell. Biochem. 2000; 77: 396-408Crossref PubMed Scopus (106) Google Scholar, 37Morris J.A. Dorner A.J. Edwards C.A. Hendershot L.M. Kaufman R.J. J. Biol. Chem. 1997; 272: 4327-4334Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 38Reddy R.K. Lu J. Lee A.S. J. Biol. Chem. 1999; 274: 28476-28483Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Overexpression of the ER molecular chaperones also correlates with increased survival of renal epithelial cells subjected to ATP depletion (39Bush K.T. George S.K. Zhang P.L. Nigam S.K. Am. J. Physiol. 1999; 277: F211-F218PubMed Google Scholar). Therefore, glucose-regulated proteins might also be involved in ER protection from ischemic injury. In this study, we investigated the effect of preconditioning the renal epithelial cell line LLC-PK1 with different inducers of ER stress to determine whether ER stress can protect against injury caused by H2O2, the prototypical ROS thought to contribute to ischemia/reperfusion injury. Our results demonstrate that ER stress and GRP78 protect epithelial cells against oxidative stress by preventing the increase in [Ca2+]i that normally follows H2O2 exposure. In naïve renal epithelial cells, H2O2 treatment activated both ERK1/2 and JNK phosphorylation. With prior ER stress, cells survived H2O2 challenge and had increased ERK1/2 relative to JNK phosphorylation. The results indicate that ERK1/2 activation is downstream of the ER stress response in the cell protective pathway. Accordingly, modulating the balance between JNK and ERK1/2 phosphorylation with inhibitors or genetic manipulations that alter upstream MAPK signaling shows that cell survival by ER stress preconditioning is linked to an increase in ERK1/2 and a decrease in JNK activation. These results place ERK as a distal mediator of the ER stress response in protection against oxidative injury and may be important for understanding how renal preconditioning affects mechanisms of ischemia/reperfusion injury in vivo. Materials—Fetal calf serum and Dulbecco's modified Eagle's medium (DMEM) were obtained from Invitrogen. The acetoxymethyl ester of Fura-2 (Fura-2/AM) and Pluronic F-127 were purchased from Molecular Probes, Inc. (Eugene, OR). H2O2, tunicamycin, thapsigargin, and trans-4,5-dihydroxy-1,2-dithiane (DTTox) were obtained from Sigma. LY294002, U0126, and SP600125 (Calbiochem) were dissolved in Me2SO and stored as 10 mm (LY294002 and U0126) and 40 mm (SP600125) stock solutions. The anti-phospho-Akt antibody was obtained from Cell Signaling (Beverly, MA). The anti-total Akt and anti-total ERK antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-GRP94 and anti-calreticulin antibodies were obtained from Stressgen Biotech Corp. (Victoria, British Columbia, Canada). All other antibodies were purchased from New England Biolabs Inc. (Beverly, MA). Cell Cultures and Experimental Treatments—LLC-PK1 cells (a porcine renal epithelial cell line with proximal tubule epithelial characteristics) were obtained from American Type Culture Collection (Manassas, VA). LLC-PK1 cells were maintained in DMEM supplemented with 10% fetal calf serum. ER stress preconditioning was produced as described previously (11Liu H. Bowes III, R.C. van de Water B. Sillence C. Nagelkerke J.F. Stevens J.L. J. Biol. Chem. 1997; 272: 21751-21759Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 12Liu H. Miller E. van de Water B. Stevens J.L. J. Biol. Chem. 1998; 273: 12858-12862Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Briefly, confluent cells were treated with DTTox (10 mm) for 3 h and returned to complete medium for 12–16 h. Alternatively, cells pretreated for 12–16 h in complete medium containing tunicamycin (1.5 μg/ml), thapsigargin (0.3 μg/ml), or A23187 (7 μm) were washed with phosphate-buffered saline and returned to complete medium. To prevent the bias of pre-selection, cell injury was measured both immediately and 24 h after returning to complete medium. There was no significant difference in lactate dehydrogenase (LDH) release in cells with and without ER stress preconditioning at either time point (data not shown). Preconditioned and control cells were treated with 1 mm H2O2 for 15 min in Earle's balanced salt solution (EBSS) and allowed to recover in complete medium. Alternatively, preconditioned cells were serum-deprived for 3–4 h and then treated with 250 μm H2O2 by directly adding H2O2 to the medium without changing the medium to prevent an effect of medium change on signaling pathways during oxidative stress. The later protocol was used when activation of members of the MAPK family was determined to avoid serum stimulation upon refeeding. Specific inhibitors were added 1 h before H2O2 treatment. Cell injury was determined by LDH release as a percent of total LDH as described previously (11Liu H. Bowes III, R.C. van de Water B. Sillence C. Nagelkerke J.F. Stevens J.L. J. Biol. Chem. 1997; 272: 21751-21759Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). In some experiments, the effect of altering expression of specific ER stress proteins was examined. LLC-PK1 cells expressing an antisense RNA targeted to GRP78 (pkASgrp78 cells) or overexpressing human calreticulin (pkCRT cells) as well as controls transfected with the same pcDNA3 plasmid (used to construct both cell lines) containing no insert (pkNEO cells) were established as described previously (11Liu H. Bowes III, R.C. van de Water B. Sillence C. Nagelkerke J.F. Stevens J.L. J. Biol. Chem. 1997; 272: 21751-21759Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 12Liu H. Miller E. van de Water B. Stevens J.L. J. Biol. Chem. 1998; 273: 12858-12862Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). When the effects of ER stress on H2O2-induced cell injury and MAPK activation were tested using pkASgrp78 or pkCRT cells, three independent clones of both lines as well as three pkNEO lines were compared to avoid bias that might have occurred randomly through selection of individual clones. Measurement of Intracellular Free Calcium Concentration—[Ca2+]i was determined with the Ca2+-sensitive fluorescent dye Fura-2/AM according to Chen et al. (40Chen Q. Jones T.W. Stevens J.L. J. Cell. Physiol. 1994; 161: 293-302Crossref PubMed Scopus (26) Google Scholar) with modifications. Cells grown on coverslips coated with bovine collagen type I were rinsed with phosphate-buffered saline and loaded with 3 μm Fura-2/AM in EBSS. Pluronic F-127 (20%) at a 1:1000 (v/v) dilution was added to Fura-2/AM to facilitate cell loading. In addition, 2 mm probenecid was added to prevent intracellular compartment transport or extrusion of Fura-2-free acid. After incubation with Fura-2/AM for 1 h at 37 °C, cells were washed two to three times with EBSS in the presence of probenecid. The coverslips were positioned in a quartz cuvette, containing 2.5 ml of EBSS with probenecid, for fluorescence analysis using a Shimadzu RF-5000 spectrofluorophotometer. [Ca2+]i was calculated as equal to K d (224 nm) × (R – R min)/(R max – R) according to Grynkiewicz et al. (41Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Fluorescence emission was monitored at 505 nm. R is the ratio of the fluorescence at 340 nm excitation to that at 380 nm excitation. Construction of Recombinant Adenoviral Vectors—MEK1-DD, a constitutively active mutant of MAPK/ERK kinase-1 (MEK1), the upstream activator of ERK1/2, was created by PCR using primers to substitute serines 218 and 222 with aspartic acid residues as previously described (42Huang W. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (133) Google Scholar). This mutant has been shown to activate ERK1/2 when expressed in COS-7 cells as well as in NIH3T3 cells (42Huang W. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8960-8963Crossref PubMed Scopus (133) Google Scholar, 43Welch D.R. Sakamaki T. Pioquinto R. Leonard T.O. Goldberg S.F. Hon Q. Erikson R.L. Rieber M. Rieber M.S. Hicks D.J. Bonventre J.V. Alessandrini A. Cancer Res. 2000; 60: 1552-1556PubMed Google Scholar). A recombinant adenoviral vector carrying the MEK1-DD cDNA (AdMEK1-DD) was constructed as previously described (44Choukroun G. Hajjar R. Kyriakis J.M. Bonventre J.V. Rosenzweig A. Force T. J. Clin. Invest. 1998; 102: 1311-1320Crossref PubMed Scopus (179) Google Scholar). Protein expression was confirmed by immunoblotting and by assay of ERK activity in mouse mesangial cells. 2G. Choukroun and J. V. Bonventre, manuscript in preparation. The recombinant adenovirus carrying the Escherichia coli LacZ gene (AdLacZ) encoding β-galactosidase was kindly provided by Dr. Roger Hajjar (Cardiovascular Research Center, Massachusetts General Hospital, Boston, MA). Western Blot Analysis—Cells were lysed in a solution containing 70 mm β-glycerophosphate (pH 7.2), 0.1 mm sodium orthovanadate, 2 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 0.5% Triton X-100, 10% glycerol, and protease inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, and 100 μg/ml phenylmethylsulfonyl fluoride) for 30 min. Lysates were centrifuged at 14,000 rpm for 10 min at 4 °C. Supernatants (30–50 μg) were boiled in 1× sample buffer (500 mm Tris-HCl (pH 6.8), 10% SDS, 20% glycerol, 0.05% bromphenol blue, and 1% 2-mercaptoethanol) for 5 min and separated on 10% polyacrylamide gels. Proteins were electrotransferred to Immobilon-P membranes (Millipore Corp., Bedford, MA) and blotted with the indicated antibodies at 4 °C overnight in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk. Membranes were then incubated with horseradish peroxidase-conjugated antibodies at room temperature for 45 min, and the signal was detected using chemiluminescence (ECL, Amersham Biosciences), followed by exposure to X-Omat AR film (Eastman Kodak Co.). Blots were stripped and reblotted with the indicated antibodies to determine equal loading of samples. Stripping of the initial antibody probe was accomplished by submerging the membrane in buffer containing 100 mm 2-mercaptoethanol, 20% SDS, and 62.5 mm Tris-HCl (pH 6.8) at 55 °C for 50 min, followed by washing twice with Tris-buffered saline and 0.1% Tween 20 for 10 min each. Statistical Analyses—Student's t test was used to determine whether there was a significant difference between two groups (p < 0.05). When multiple means were compared, significance (p < 0.05) was determined by analysis of variance, followed by Fisher's protected least significant difference test. For analysis of variance, letter designations are used to indicate significant differences. Means with a common letter designation are not different, and those with a different letter designation are significantly different from all other means with different letter designations. StatView software (SAS Institute, Cary, NC) was used as a statistical tool in this study. H 2 O 2 Increases [Ca2 + ] i prior to Injury to LLC-PK 1 Cells—To establish the temporal relationship between an increase in [Ca2+]i and cell injury in LLC-PK1 cells under oxidative stress, we examined [Ca2+]i at various times after H2O2 treatment. Cells were treated with 1 mm H2O2 for 15 min in EBSS and then returned to EBSS and loaded for 1 h with Fura-2/AM. An increase in [Ca2+]i was detected as early as 1 h after H2O2 treatment and reached a maximum at 2 h. In contrast, a significant amount of H2O2-induced cell death, as measured by LDH release, was not observed until 2 h and increased steadily through 4 h (Fig. 1). Thus, the increase in [Ca2+]i preceded significant cell injury in LLC-PK1 cells treated with H2O2. ER Stress Preconditioning Prevents H 2 O 2 -induced Cell Injury and Ca2 + Accumulation—We next examined the effect of prior ER stress on H2O2-induced cell injury. LLC-PK1 cells preconditioned with different ER stress inducers were protected against subsequent H2O2-induced cell injury at 4 h (Fig. 2A). Prior ER stress induced by DTTox, tunicamycin, thapsigargin, or A23187 prevented the rise in [Ca2+]i normally occurring 2 h after H2O2 treatment (Fig. 2B). To confirm the biological significance of the protection afforded by ER stress preconditioning, we assessed cell injury at later time points under two different conditions of H2O2 exposure. Under conditions of transient (Fig. 3, A and B) or continuous (C and D) H2O2 exposure, the protective effect afforded by previous ER stress persisted up to 24 h. Thus, ER stress preconditioning modified the magnitude of oxidant-induced cell injury and not just the kinetics of the process.Fig. 3Effect of ER stress preconditioning on the time course of H2O2-induced cell injury. LLC-PK1 cells pretreated with vehicle, tunicamycin (TUNIC; 1.5 μg/ml), or thapsigargin (THAPS; 0.3 μg/ml), for 16 h to induce ER stress, were treated with 0.5 or 1 mm H2O2 in EBSS for 15 min and then washed and returned to DMEM plus 10% FCS at time 0 (A and B). Alternatively, cells were washed and serum-deprived for 3 h before exposure to 125 or 250 μm H2O2 at time 0, with no subsequent medium change (C and D). At the various time points, cell injury was determined by measurement of LDH release. The data are the means ± S.E. of three independent measurements. Significant differences (*, p < 0.05) between tunicamycin or thapsigargin pretreatment versus vehicle pretreatment in each group (Gr) at various time points are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Blocking Expression of GRP78 Disrupts the Effect of ER Stress Preconditioning—Because prior ER stress prevented both the increase in [Ca2+]i and cell death after H2O2, we determined whether disruption of the ER stress response alters the sensitivity to oxidant injury and prevents adaptation. We tested the H2O2 sensitivity of pkASgrp78 cells, which" @default.
• W2029175062 created "2016-06-24" @default.
• W2029175062 creator A5000500792 @default.
• W2029175062 creator A5015505628 @default.
• W2029175062 creator A5080273574 @default.
• W2029175062 creator A5089781525 @default.
• W2029175062 date "2003-08-01" @default.
• W2029175062 modified "2023-10-16" @default.
• W2029175062 title "Protection of Renal Epithelial Cells against Oxidative Injury by Endoplasmic Reticulum Stress Preconditioning Is Mediated by ERK1/2 Activation" @default.
• W2029175062 cites W1510592624 @default.
• W2029175062 cites W1556531268 @default.
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