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• W1987342012 abstract "We have shown that renal epithelial cell survival depends on the sustained activation of the extracellular signal-regulated protein kinase (ERK) and lack of this activation was associated with death during oxidative stress. ERK is activated via the canonical epidermal growth factor receptor (EGFR)–Ras–MEK pathway, which could be attenuated by oxidants. We now show that the failure to activate ERK in a sustained manner during severe oxidative stress is owing to the activation of the signal transducer and activator of transcription-3 (STAT3) rather than the failure to activate the EGFR. Tyrosine phosphorylation of the EGFR and STAT3 was studied in hydrogen peroxide (H2O2)-treated mouse proximal tubule (TKPTS) cells or in mouse kidney after ischemia/reperfusion (I/R) injury by Western blotting. STAT3 activation was inhibited by either pharmacologically (AG490) through its upstream janus kinase (JAK2) or by a dominant-negative STAT3 adenovirus. EGFR was inhibited by AG1478. Survival was determined by fluorescence-activated cell sorter analysis and trypan blue exclusion. We found that the EGFR was phosphorylated on its major autophosphorylation site (Tyr1173) regardless of the H2O2 dose. On the other hand, both I/R and severe oxidative stress – but not moderate stress – increased tyrosine phosphorylation of STAT3 in an EGFR and JAK2-dependent manner. Inhibition of JAK2 or STAT3 lead to increased ERK activation and survival of TKPTS cells during severe oxidative stress. Our data suggest a role of tyrosine-phosphorylated STAT3 in the suppression of ERK activation. These data suggest that the STAT3 pathway might represent a new target for improved survival of proximal tubule cells exposed to severe oxidant injury. We have shown that renal epithelial cell survival depends on the sustained activation of the extracellular signal-regulated protein kinase (ERK) and lack of this activation was associated with death during oxidative stress. ERK is activated via the canonical epidermal growth factor receptor (EGFR)–Ras–MEK pathway, which could be attenuated by oxidants. We now show that the failure to activate ERK in a sustained manner during severe oxidative stress is owing to the activation of the signal transducer and activator of transcription-3 (STAT3) rather than the failure to activate the EGFR. Tyrosine phosphorylation of the EGFR and STAT3 was studied in hydrogen peroxide (H2O2)-treated mouse proximal tubule (TKPTS) cells or in mouse kidney after ischemia/reperfusion (I/R) injury by Western blotting. STAT3 activation was inhibited by either pharmacologically (AG490) through its upstream janus kinase (JAK2) or by a dominant-negative STAT3 adenovirus. EGFR was inhibited by AG1478. Survival was determined by fluorescence-activated cell sorter analysis and trypan blue exclusion. We found that the EGFR was phosphorylated on its major autophosphorylation site (Tyr1173) regardless of the H2O2 dose. On the other hand, both I/R and severe oxidative stress – but not moderate stress – increased tyrosine phosphorylation of STAT3 in an EGFR and JAK2-dependent manner. Inhibition of JAK2 or STAT3 lead to increased ERK activation and survival of TKPTS cells during severe oxidative stress. Our data suggest a role of tyrosine-phosphorylated STAT3 in the suppression of ERK activation. These data suggest that the STAT3 pathway might represent a new target for improved survival of proximal tubule cells exposed to severe oxidant injury. During ischemia/reperfusion (I/R) injury, the excessive formation of reactive oxygen species and their intermediates, such as hydrogen peroxide (H2O2), contributes to the death of proximal tubules of the kidney.1.Safirstein R.L. Lessons learned from ischemic and cisplatin-induced nephrotoxicity in animals.Ren Fail. 1999; 21: 359-364Crossref PubMed Scopus (12) Google Scholar In cultured mouse proximal tubule cells (TKPTS), we found that a moderate amount of H2O2 (0.5 mmol/l) resulted in sustained activation of extracellular signal-regulated kinase (ERK) and cell survival. By contrast, treatment with excessive H2O2 (1.0 mmol/l) led to a transient activation of ERK and cell death.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar We also demonstrated that survival after H2O2 treatment requires the activation of the transcription factor cAMP-responsive element-binding protein, a downstream effector of the epidermal growth factor receptor (EGFR)–ERK pathway,3.Arany I. Megyesi J.K. Reusch J.E. et al.CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress.Kidney Int. 2005; 68: 1573-1582Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar and this signaling is interrupted by severe oxidant stress. The activation of ERK has been shown to be executed through the canonical EGFR–Ras–MEK pathway.4.English J. Pearson G. Wilsbacher J. et al.New insights into the control of MAP kinase pathways.Exp Cell Res. 1999; 253: 255-270Crossref PubMed Scopus (367) Google Scholar,5.Rozakis-Adcock M. Fernley R. Wade J. et al.The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.Nature. 1993; 363: 83-85Crossref PubMed Scopus (825) Google Scholar Epidermal growth factor (EGF) and EGF-like ligands bind and activate the intrinsic tyrosine kinase activity of the EGF receptor and initiate autophosphorylation of various tyrosine residues. H2O2 itself can also induce phosphorylation of the EGFR and initiate signaling through the EGFR–Ras–MEK pathway.6.Goldkorn T. Balaban N. Matsukuma K. et al.EGF-receptor phosphorylation and signaling are targeted by H2O2 redox stress.Am J Respir Cell Mol Biol. 1998; 19: 786-798Crossref PubMed Scopus (170) Google Scholar, 7.Gamou S. Shimizu N. Hydrogen peroxide preferentially enhances the tyrosine phosphorylation of epidermal growth factor receptor.FEBS Lett. 1995; 357: 161-164Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 8.Meves A. Stock S.N. Beyerle A. et al.H2O2 mediates oxidative stress-induced epidermal growth factor receptor phosphorylation.Toxicol Lett. 2001; 122: 205-214Crossref PubMed Scopus (46) Google Scholar, 9.Rao G.N. Hydrogen peroxide induces complex formation of SHC-Grb2-SOS with receptor tyrosine kinase and activates Ras and extracellular signal-regulated protein kinases group of mitogen-activated protein kinases.Oncogene. 1996; 13: 713-719PubMed Google Scholar, 10.Ichiki T. Tokunou T. Fukuyama K. et al.Cyclic AMP response element-binding protein mediates reactive oxygen species-induced c-fos expression.Hypertension. 2003; 42: 177-183Crossref PubMed Scopus (41) Google Scholar In vivo studies suggest that H2O2 generated during reperfusion could act as an activator of the EGFR.11.Yano T. Yazima S. Hagiwara K. et al.Activation of epidermal growth factor receptor in the early phase after renal ischemia–reperfusion in rat.Nephron. 1999; 81: 230-233Crossref PubMed Scopus (27) Google Scholar Thus, moderate levels of H2O2 might serve as a second messenger in survival signaling including the EGFR/ERK pathway.12.Peus D. Meves A. Vasa R.A. et al.H2O2 is required for UVB-induced EGF receptor and downstream signaling pathway activation.Free Radical Biol Med. 1999; 27: 1197-1202Crossref PubMed Scopus (78) Google Scholar, 13.Vindis C. Seguelas M.H. Lanier S. et al.Dopamine induces ERK activation in renal epithelial cells through H2O2 produced by monoamine oxidase.Kidney Int. 2001; 59: 76-86Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 14.Frank G.D. Eguchi S. Inagami T. et al.N-acetylcysteine inhibits angiotensin ii-mediated activation of extracellular signal-regulated kinase and epidermal growth factor receptor.Biochem Biophys Res Commun. 2001; 280: 1116-1119Crossref PubMed Scopus (53) Google Scholar Proximal tubules of the kidney, which undergo necrotic cell death, express high levels of EGFR15.Okada T. Iwamoto A. Kusakabe K. et al.Perinatal development of the rat kidney: proliferative activity and epidermal growth factor.Biol Neonate. 2001; 79: 46-53Crossref PubMed Scopus (9) Google Scholar and activate the EGFR during I/R injury;11.Yano T. Yazima S. Hagiwara K. et al.Activation of epidermal growth factor receptor in the early phase after renal ischemia–reperfusion in rat.Nephron. 1999; 81: 230-233Crossref PubMed Scopus (27) Google Scholar,16.Safirstein R. Price P.M. Saggi S.J. et al.Changes in gene expression after temporary renal ischemia.Kidney Int. 1990; 37: 1515-1521Abstract Full Text PDF PubMed Scopus (154) Google Scholar yet, we and others have failed to demonstrate activation of ERK or other downstream elements of the canonical pathway in proximal tubule segments following I/R injury.17.di Mari J.F. Davis R. Safirstein R.L. MAPK activation determines renal epithelial cell survival during oxidative injury.Am J Physiol Renal Physiol. 1999; 277: F195-F203PubMed Google Scholar,18.Andreucci M. Michael A. Kramers C. et al.Renal ischemia/reperfusion and ATP depletion/repletion in LLC-PK1 cells result in phosphorylation of FKHR and FKHRL1.Kidney Int. 2003; 64: 1189-1198Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar In seeking an explanation for this observation we considered at least two possibilities. Severe stress might fail to activate the EGFR, usually accomplished by phosphorylation at a key activation site on the receptor,6.Goldkorn T. Balaban N. Matsukuma K. et al.EGF-receptor phosphorylation and signaling are targeted by H2O2 redox stress.Am J Respir Cell Mol Biol. 1998; 19: 786-798Crossref PubMed Scopus (170) Google Scholar, 7.Gamou S. Shimizu N. Hydrogen peroxide preferentially enhances the tyrosine phosphorylation of epidermal growth factor receptor.FEBS Lett. 1995; 357: 161-164Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 8.Meves A. Stock S.N. Beyerle A. et al.H2O2 mediates oxidative stress-induced epidermal growth factor receptor phosphorylation.Toxicol Lett. 2001; 122: 205-214Crossref PubMed Scopus (46) Google Scholar, 9.Rao G.N. Hydrogen peroxide induces complex formation of SHC-Grb2-SOS with receptor tyrosine kinase and activates Ras and extracellular signal-regulated protein kinases group of mitogen-activated protein kinases.Oncogene. 1996; 13: 713-719PubMed Google Scholar, 19.Chen K. Vita J.A. Berk B.C. et al.c-Jun N-terminal kinase activation by hydrogen peroxide in endothelial cells involves SRC-dependent epidermal growth factor receptor transactivation.J Biol Chem. 2001; 276: 16045-16050Crossref PubMed Scopus (182) Google Scholar or by a post-receptor mechanism involving the activation of transduction signaling arising from an alternate pathway that represses EGFR downstream signaling. Such an EGFR-repressing mechanism has been observed during reactive oxygen species-activated janus kinase (JAK)/STAT signaling both in vitro as well as during I/R-induced injury,20.Megyesi J. Andrade L. Vieira Jr, J.M. et al.Positive effect of the induction of p21WAF1/CIP1 on the course of ischemic acute renal failure.Kidney Int. 2001; 60: 2164-2172Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,21.Park J.I. Strock C.J. Ball D.W. et al.The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine–paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway.Mol Cell Biol. 2003; 23: 543-554Crossref PubMed Scopus (104) Google Scholar whereby the activation of the signal transducer and activator of transcription-3 (STAT3) has been observed to downregulate the ERK1/2 pathway. It would appear that these alternate signaling pathways induced by severe stress result in the inhibition of the EGFR-mediated survival pathway. We therefore postulated that the ERK survival signaling pathway is interrupted by severe oxidant stress in TKPTS. Accordingly, we tested the hypotheses that severe oxidant stress fails to activate ERK either by failure to activate the EGFR or by activation of the JAK2/STAT3 pathway. Furthermore, we sought to re-establish ERK signaling by manipulation of these pathways in order to restore cell survival during severe oxidant stress. TKPTS cells were treated with 0.5 or 1 mmol/l H2O2 or 10 ng/ml EGF for 30 min. Phosphorylation of the EGFR at one of the major autophosphorylation sites (tyrosine(Tyr) 1173) and the unphosphorylated EGFR was determined by Western blotting (Figure 1a). The results show that the EGFR was significantly phosphorylated at Tyr 1173 both at 0.5 and 1.0 mmol/l H2O2 similar to treatment by its cognate ligand EGF (Figure 1b). Pretreatment of TKPTS cells with 50 μM/l AG1478 (an EGFR inhibitor) 1 h before treatment with 0.5 mmol/l H2O2 resulted in extensive necrotic cell death similar to that seen after treatment with 1 mmol/l H2O2 (Figure 1c), suggesting that EGFR activation is critical for cell survival. For comparison control (untreated), 0.5 mmol/l- and 1 mmol/l H2O2-treated cells are also included. Note the G2/M arrest induced by 0.5 mmol/l H2O2, which we have previously shown to accompany cell survival under these conditions.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar These results suggest that attenuation of ERK phosphorylation by 1 mmol/l H2O22.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar may be downstream from the activated EGFR. We next explored the role of STAT3 in the interruption of EGFR-induced ERK1/2 activation.22.Kritikou E.A. Sharkey A. Abell K. et al.A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland.Development. 2003; 130: 3459-3468Crossref PubMed Scopus (146) Google Scholar TKPTS cells were treated with 0.5 or 1 mmol/l H2O2 for 30 min and Tyr-705 phosphorylated STAT3 was determined by Western blotting (Figure 2a). The results demonstrate that only 1 mmol/l H2O2 phosphorylates STAT3 significantly at Tyr-705 (Figure 2b). Tyrosine phosphorylation of STAT3 is also detectable in lysates from mouse kidneys undergoing I/R injury (Figure 2c). As is shown in Figure 2, 30 min as well as 24 h reperfusion significantly increased tyrosine phosphorylation of STAT3 in the mouse kidney. The relative decrease in STAT3 tyrosine phosphorylation 24 h after reperfusion is owing to the fact that ischemic injury also increased total STAT3 protein at that time point (Figure 2d). Regardless, the levels of tyrosine-phosphorylated STAT3 are still significantly higher compared to the normal kidney. These results suggest that tyrosine phosphorylation of STAT3 might be a key negative regulator of ERK activation. Furthermore, tyrosine-phosphorylated STAT3 might be an important element in death signaling. We next asked if the activation of STAT3 is mediated by the EGFR or JAK2.23.Burova E.B. Grudinkin P.S. Bardin A.A. et al.H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2.Tsitologiia. 2001; 43: 1153-1161PubMed Google Scholar, 24.Simon A.R. Rai U. Fanburg B.L. et al.Activation of the JAK–STAT pathway by reactive oxygen species.Am J Physiol Cell Physiol. 1998; 275: C1640-C1652PubMed Google Scholar, 25.Gorina R. Petegnief V. Chamorro A. et al.AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: involvement of the Jak2/STAT pathway.J Neurochem. 2005; 92: 505-518Crossref PubMed Scopus (56) Google Scholar, 26.Andl C.D. Mizushima T. Oyama K. et al.EGFR-induced cell migration is mediated predominantly by the JAK-STAT pathway in primary esophageal keratinocytes.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G1227-G1237Crossref PubMed Scopus (88) Google Scholar Pretreatment of TKPTS cells with either the JAK2 inhibitor AG490 or the EGFR inhibitor AG1478 partially but significantly attenuated 1 mmol/l H2O2-induced STAT3 tyrosine phosphorylation (Figure 3b), suggesting that both JAK2 and EGFR participate in STAT3 phosphorylation. To determine whether ERK activation and survival could be restored by inhibition of the JAK2/STAT3 pathway, TKPTS cells were pretreated with either the JAK2 inhibitor AG490 or infected with a dominant-negative STAT3 adenovirus (Ad-dnSTAT3) before treatment with 1 mmol/l H2O2. After 24 h, cell numbers were counted. As shown in Figure 4a, pretreatment with AG490 significantly increased survival of cells after treatment with 1 mmol/l H2O2. Similarly, inhibition of STAT3 also reduced 1 mmol/l H2O2-induced cell death (Figure 4b). We next explored whether JAK2/STAT3 inhibition restores ERK activation. Accordingly, TKPTS cells were either pretreated with AG490 (Figure 5) or infected with an adeno-dnSTAT3 (Figure 6) before treatment with 1 mmol/l H2O2. The phosphorylation status of ERK was determined 2 and 4 h after treatment with H2O2 by Western blotting. Figures 5 and 6 show that 1 mmol/l H2O2 significantly inhibited the phosphorylation of ERK at those time points. However, pretreatment with AG490 (Figure 5) or by direct inhibition of STAT3 via a dominant-negative mutant (Figure 6) restored ERK activation (phosphorylation).Figure 6Effects of STAT3 inhibition on ERK phosphorylation in TKPTS cells treated with 1 mmol/l H2O2. (a) TKPTS cells were treated with 1 mmol/l H2O2 for 2 and 4 h in the presence or absence of a dnSTAT3 adenovirus. ERK phosphorylation was determined by Western blotting. Data shown are representatives of three independent experiments. (b) Densitometric analysis of Western blots as shown in (a). The extent of ERK phosphorylation was determined by normalizing phospho-ERK1 levels to the total ERK1. Values are given as mean±s.d. (n=3). *P<0.05 compared to the untreated control. #P<0.05 compared to the 1 mmol/l H2O2-treated cells, @P<0.05 compared to the 1 mmol/l H2O2-treated cells.View Large Image Figure ViewerDownload (PPT) We have shown that survival of renal epithelial cells during oxidant injury depends on the activation of the ERK–cAMP-responsive element-binding protein pathway.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar,3.Arany I. Megyesi J.K. Reusch J.E. et al.CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress.Kidney Int. 2005; 68: 1573-1582Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar We now show that an activated EGFR is indispensable for survival as AG1478, an EGFR inhibitor, attenuates survival of TKPTS cells during moderate oxidant stress (Figure 1c). This is the first demonstration of EGFR-dependent survival signaling in renal proximal tubules. Although proximal tubules activate the EGFR after renal I/R injury,27.Breyer M.D. Redha R. Breyer J.A. Segmental distribution of epidermal growth factor binding sites in rabbit nephron.Am J Physiol. 1990; 259: F553-F558PubMed Google Scholar they fail to activate ERK or other downstream elements of the proximal tubule survival pathway17.di Mari J.F. Davis R. Safirstein R.L. MAPK activation determines renal epithelial cell survival during oxidative injury.Am J Physiol Renal Physiol. 1999; 277: F195-F203PubMed Google Scholar,18.Andreucci M. Michael A. Kramers C. et al.Renal ischemia/reperfusion and ATP depletion/repletion in LLC-PK1 cells result in phosphorylation of FKHR and FKHRL1.Kidney Int. 2003; 64: 1189-1198Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and undergo necrotic death. We considered at least two possibilities to explain this observation: failure to phosphorylate the EGFR or activation of alternative pathways that interrupt EGFR–ERK signaling. We found that EGFR is phosphorylated on one of its major autophosphorylation site (Tyr 1173) regardless of the dose of oxidant in proximal tubule cells in vitro (Figure 1a). We then focused our attention on activation of alternate pathways that may interrupt EGFR-mediated survival signaling. We next explored the effects of severe oxidant stress signaling at post-receptor levels. Aware that reactive oxygen species activates the JAK/STAT pathway,24.Simon A.R. Rai U. Fanburg B.L. et al.Activation of the JAK–STAT pathway by reactive oxygen species.Am J Physiol Cell Physiol. 1998; 275: C1640-C1652PubMed Google Scholar,28.Stephanou A. Role of STAT-1 and STAT-3 in ischaemia/reperfusion injury.J Cell Mol Med. 2004; 8: 519-525Crossref PubMed Scopus (87) Google Scholar we found that 1 mmol/l but not 0.5 mmol/l H2O2 phosphorylated STAT3 at Tyr 705 in TKPTS cells (Figure 2a, b). Similarly, I/R injury increased tyrosine phosphorylation of STAT3 in the kidney (Figure 2c, and d). We also found that tyrosine phosphorylation of STAT3 was mediated by both the EGFR and JAK2, as inhibition of either the EGFR or JAK2 diminished tyrosine phosphorylation of STAT3 in vitro (Figure 3). Thus, activation of STAT3 by 1 mmol/l H2O2 requires both the EGFR and JAK2. Inhibition of JAK2 by AG490 or inhibition of STAT3 function by Ad-dnSTAT3 reduced cell death caused by 1 mmol/l H2O2 treatment (Figure 4a, and b), thus revealing a pro-death role of this pathway in oxidant injury in these cells. STAT3 is capable of suppressing the ERK pathway,22.Kritikou E.A. Sharkey A. Abell K. et al.A dual, non-redundant, role for LIF as a regulator of development and STAT3-mediated cell death in mammary gland.Development. 2003; 130: 3459-3468Crossref PubMed Scopus (146) Google Scholar,29.Winston L.A. Hunter T. Intracellular signalling: putting JAKs on the kinase MAP.Curr Biol. 1996; 6: 668-671Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar although the mechanism of this inhibition is not clear. A direct role of STAT3 in the inhibition of ERK activation was revealed in experiments in which inhibition of either JAK2 or STAT3 increased ERK phosphorylation after treatment with 1 mmol/l H2O2 (Figures 5 and 6). One possibility is that the tyrosine-phosphorylated STAT3 competes with growth factor receptor-bound protein 2 for the binding site on the activated EGFR30.Zhang T. Ma J. Cao X. Grb2 regulates Stat3 activation negatively in epidermal growth factor signalling.Biochem J. 2003; 376: 457-464Crossref PubMed Scopus (37) Google Scholar and thus terminates ERK activation. Oxidative stress can also trigger STAT3-mediated activation of SOCS3,31.Carballo M. Conde M. El Bekay R. et al.Oxidative stress triggers STAT3 tyrosine phosphorylation and nuclear translocation in human lymphocytes.J Biol Chem. 1999; 274: 17580-17586Crossref PubMed Scopus (221) Google Scholar which can inhibit EGFR signaling.32.Xia L. Wang L. Chung A.S. et al.Identification of both positive and negative domains within the epidermal growth factor receptor COOH-terminal region for signal transducer and activator of transcription (STAT) activation.J Biol Chem. 2002; 277: 30716-30723Crossref PubMed Scopus (90) Google Scholar To our knowledge, this is also the first determination of the pro-death role of the JAK/STAT pathway in the kidney. The precise mechanism by which the activated (tyrosine phosphorylated) STAT3 suppresses ERK activation remains to be determined. Activation of the JAK/STAT pathway following I/R injury has broad implications. STAT3 can mediate inflammatory responses as well33.Inagaki-Ohara K. Hanada T. Yoshimura A. Negative regulation of cytokine signaling and inflammatory diseases.Curr Opin Pharmacol. 2003; 3: 435-442Crossref PubMed Scopus (31) Google Scholar by increasing the expression of tumor necrosis factor α34.Chappell V.L. Le L.X. La Grone L. et al.Stat proteins play a role in tumor necrosis factor alpha gene expression.Shock. 2000; 14 (discussion 2–3): 400-402Crossref PubMed Scopus (30) Google Scholar or interleukin-8,35.Yeh M. Gharavi N.M. Choi J. et al.Oxidized phospholipids increase interleukin 8 (IL-8) synthesis by activation of the c-src/signal transducers and activators of transcription (STAT)3 pathway.J Biol Chem. 2004; 279: 30175-30181Crossref PubMed Scopus (108) Google Scholar both of which are induced during renal I/R36.Bonventre J.V. Zuk A. Ischemic acute renal failure: an inflammatory disease?.Kidney Int. 2004; 66: 480-485Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar,37.Safirstein R. Megyesi J. Saggi S.J. et al.Expression of cytokine-like genes JE and KC is increased during renal ischemia.Am J Physiol Renal Physiol. 1991; 261: F1095-F1101PubMed Google Scholar and may contribute to the pathogenesis of tubular injury. Tumor necrosis factor α is a known mediator of tubular cell death and plasma or urinary interleukin-8 levels predict high mortality in patients with acute renal failure.38.Ahlstrom A. Hynninen M. Tallgren M. et al.Predictive value of interleukins 6, 8 and 10, and low HLA-DR expression in acute renal failure.Clin Nephrol. 2004; 61: 103-110Crossref PubMed Scopus (18) Google Scholar,39.Simmons E.M. Himmelfarb J. Sezer M.T. et al.Plasma cytokine levels predict mortality in patients with acute renal failure.Kidney Int. 2004; 65: 1357-1365Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar Tumor necrosis factor α34.Chappell V.L. Le L.X. La Grone L. et al.Stat proteins play a role in tumor necrosis factor alpha gene expression.Shock. 2000; 14 (discussion 2–3): 400-402Crossref PubMed Scopus (30) Google Scholar and interleukin-835.Yeh M. Gharavi N.M. Choi J. et al.Oxidized phospholipids increase interleukin 8 (IL-8) synthesis by activation of the c-src/signal transducers and activators of transcription (STAT)3 pathway.J Biol Chem. 2004; 279: 30175-30181Crossref PubMed Scopus (108) Google Scholar possess a STAT3 binding site in their promoter proximal region. Thus, activation of STAT3 in addition to its demonstrated role in suppressing EGFR–ERK signaling might regulate transcription and secretion of inflammatory cytokines that could contribute to tubular epithelial cell death. In summary, we found that severe oxidant stress leads to the tyrosine phosphorylation of STAT3. This event depends on the activation of the EGFR and JAK2 kinase in renal tubular epithelial cells. The pathway is directly linked to cell death as inhibition of STAT3 function enables cells to survive severe oxidant stress. Part of the mechanism entails interruption of ERK activation via an activated EGFR. Thus, the STAT3 pathway might represent an important new target to protect proximal tubule cells from oxidant injury. These results also suggest a dual function for the EGFR (pro-survival or pro-death) and might explain the death of proximal tubules in the kidney in the presence of activated EGFR during I/R injury. The immortalized TKPTS cells are gift from Dr Elsa Bello-Reuss.40.Ernest S. Bello-Reuss E. Expression and function of P-glycoprotein in a mouse kidney cell line.Am J Physiol. 1995; 269: C323-C333PubMed Google Scholar They were maintained in Dulbecco's modified Eagle's medium: Ham's F12 media supplemented with 8% fetal calf serum at 37°C and 5% CO2 atmosphere. Experiments were performed on logarithmically growing cells (approximately 50–60% confluency). Cell viability was determined by trypan blue exclusion. Male 129Sv mice (6–8 weeks old) were anesthetized with 50 mg/kg body weight of sodium pentobarbital and a 50 min period of ischemia was induced by bilateral renal hilum clamping, as described earlier.20.Megyesi J. Andrade L. Vieira Jr, J.M. et al.Positive effect of the induction of p21WAF1/CIP1 on the course of ischemic acute renal failure.Kidney Int. 2001; 60: 2164-2172Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Sham operation was also performed without induction of ischemia. The clamps were removed and kidneys were harvested for Western blotting at various time points after re-establishing perfusion. Monolayers of TKPTS cells were lysed in a radioimmunoprecipitation assay buffer that contained 100 μg/ml phenylmethylsulfonyl fluoride (Sigma, St Louis, MO, USA), 100 mmol/l sodium orthovanadate (Sigma, St Louis, MO, USA), and 50 μl/ml of proteinase inhibitor cocktail (Sigma, St Louis, MO, USA) as described earlier.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Kidneys were homogenized in the same buffer as described earlier.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 3.Arany I. Megyesi J.K. Reusch J.E. et al.CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress.Kidney Int. 2005; 68: 1573-1582Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 41.Arany I. Megyesi J.K. Kaneto H. et al.Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells.Am J Physiol Renal Physiol. 2004; 287: F543-F549Crossref PubMed Scopus (192) Google Scholar Protein content was determined by using a BioRad Protein Determination assay (BioRad Hercules, CA, USA) as described earlier.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar Proteins (50–100 μg) from cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidine difluoride membrane (BioRad, Hercules, CA, USA). The filters were hybridized with the appropriate primary antibodies followed by a horseradish peroxidase-conjugated secondary antibody. The bands were visualized by an enhanced chemiluminescence method (Amersham) and quantified by densitometry (UnScan-It, Silk Scientific, Ore, UT, USA). TKPTS cells were grown in six-well-plates and incubated in the presence of 25 multiplicity of infection/ml adenovirus vector that contained a dominant-negative STAT3 construct21.Park J.I. Strock C.J. Ball D.W. et al.The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine–paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway.Mol Cell Biol. 2003; 23: 543-554Crossref PubMed Scopus (104) Google Scholar for 24 h at 37°C as described earlier.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar,3.Arany I. Megyesi J.K. Reusch J.E. et al.CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress.Kidney Int. 2005; 68: 1573-1582Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Total cell lysates from these infected cells were prepared for later analysis. For survival studies the infected cells were treated with H2O2 for additional 24 h and cell counts were determined by trypan blue exclusion. Earlier we determined that the efficiency of infection was around 80% using a control (Ad-green fluorescent protein) adenovirus (unpublished data). We also determined that infection with a control adenovirus (Ad-green fluorescent protein) did not influence cell survival or activation of ERK.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar,3.Arany I. Megyesi J.K. Reusch J.E. et al.CREB mediates ERK-induced survival of mouse renal tubular cells after oxidant stress.Kidney Int. 2005; 68: 1573-1582Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar Cell cycle analysis was performed by propidium iodide staining as described earlier.2.Arany I. Megyesi J.K. Kaneto H. et al.Activation of ERK or inhibition of JNK ameliorates H2O2 cytotoxicity in mouse renal proximal tubule cells.Kidney Int. 2004; 65: 1231-1239Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar,41.Arany I. Megyesi J.K. Kaneto H. et al.Cisplatin-induced cell death is EGFR/src/ERK signaling dependent in mouse proximal tubule cells.Am J Physiol Renal Physiol. 2004; 287: F543-F549Crossref PubMed Scopus (192) Google Scholar Briefly, TKPTS cells were collected after trypsinization and fixed in 70% ethanol overnight. After RNAse treatment, cells were incubated with 5 μg/ml propidium iodide and analyzed with a Becton Dickinson FACSCalibur analyzer. The cell cycle profile was analyzed using the CellQuest software. This work was financially supported by an NIH/NIDDK Grant (PO1 DK58324-01A1 to RLS). This material is the result of work supported in part with resources and the use of facilities at the Central Arkansas Veteran Healthcare System, Little Rock, AR, USA." @default.
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• W1987342012 title "STAT3 attenuates EGFR-mediated ERK activation and cell survival during oxidant stress in mouse proximal tubular cells" @default.
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