Tyrosine Phosphorylation of Ikappa Balpha Activates NFkappa B through a Redox-regulated and c-Src-dependent Mechanism Following Hypoxia/Reoxygenation*

Chenguang FanDagger §, Qiang Li§, Dan Ross§, and John F. Engelhardt§||

From the Dagger  Molecular Biology Graduate Program, the § Department of Anatomy and Cell Biology, and the  Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242

Received for publication, July 7, 2002, and in revised form, November 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

NFkappa B is a critical transcription factor involved in modulating cellular responses to environmental injuries. Tyrosine 42 phosphorylation of Ikappa Balpha has been shown to mediate NFkappa B activation following hypoxia/reoxygenation (H/R) or pervanadate treatment. This pathway differs from the canonical proinflammatory pathways, which mediate NFkappa B activation through serine phosphorylation of Ikappa Balpha by the IKK complex. In the present study, we investigated the involvement of c-Src in the redox activation of NFkappa B following H/R or pervanadate treatment. Our results demonstrate that pervanadate or H/R treatment leads to tyrosine phosphorylation of Ikappa Balpha and NFkappa B transcriptional activation independent of the IKK pathway. In contrast, inhibition of c-Src by pp2 treatment or in c-Src (-/-) knockout cell lines, demonstrated a significant reduction in Ikappa Balpha tyrosine phosphorylation and NFkappa B activation following pervanadate or H/R treatment. Overexpression of glutathione peroxidase-1 or catalase, but not Mn-SOD or Cu,Zn-SOD, significantly reduced both NFkappa B activation and tyrosine phosphorylation of Ikappa Balpha . In vitro kinase assays further demonstrated that immunoprecipitated c-Src has the capacity to directly phosphorylate GST-Ikappa Balpha and that this Ikappa Balpha kinase activity is significantly reduced by Gpx-1 overexpression. These results suggest that c-Src-dependent tyrosine phosphorylation of Ikappa Balpha and subsequent activation of NFkappa B is controlled by intracellular H2O2 and defines an important redox-regulated pathway for NFkappa B activation following H/R injury that is independent of the IKK complex.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reactive oxygen species (ROS)1 are normal metabolic byproducts and intermediates found in many physiological processes. Three major sources of intracellular ROS include the xanthine/xanthine oxidase system, receptor-coupled NADPH oxidase at the cellular membrane, and the mitochondrial electron transport system (1, 2). ROS have been increasingly recognized as critical components in disease and stress-induced cellular injuries such as ischemia/reperfusion (I/R), UV irradiation, and inflammation. These ROS can lead to direct cellular damage and can also act as intracellular second messengers to modulate signal transduction pathways. One such redox-regulated transcription factor is NFkappa B (3).

NFkappa B family members include p50, p52, p65, and c-RelB, which form homodimeric and heterodimeric transcriptional complexes (4). The activation of NFkappa B is controlled by a family of Ikappa B repressor proteins (Ikappa Balpha , Ikappa Bbeta , and Ikappa Bepsilon ) that sequester NFkappa B in the cytoplasm (4). Phosphorylation-dependent inactivation of Ikappa B proteins leads to the mobilization of NFkappa B to the nucleus where it can act as a transcription factor. These phosphorylation pathways have been most extensively studied for Ikappa Balpha and include two distinct mechanisms involving either serine or tyrosine phosphorylation of Ikappa Balpha . The most comprehensively studied pathway regulating Ikappa Balpha includes phosphorylation on two serine (32 and 36) residues by the Ikappa B kinase complex (IKK) (5). This phosphorylation leads to ubiquitination of Ikappa Balpha at nearby lysine residues and degradation by the proteasome. An alternative, less characterized pathway of NFkappa B activation acts through tyrosine phosphorylation of Ikappa Balpha at residue 42 (6). In contrast to IKK-mediated serine phosphorylation of Ikappa Balpha , tyrosine phosphorylation of Ikappa Balpha is capable of activating NFkappa B in the absence of ubiquitin-dependent degradation of Ikappa Balpha . However, it is presently unclear if IKK and/or the Ikappa Balpha protein-tyrosine kinase (PTK) interactions with Ikappa Balpha are functionally modulated by prior tyrosine or serine phosphorylation of Ikappa Balpha , respectively. Experimental evidence appears to suggest that prior tyrosine phosphorylation of Ikappa Balpha on Tyr-42 may prevent interactions with the IKK complex and inhibit serine phosphorylation on Ser-32/Ser-36 (7). Hence, the existence of reciprocal interactions between IKK- and PTK-mediated phosphorylation of Ikappa Balpha and the net effect on NFkappa B transcriptional activation remains an open question. Although the exact identity of the Ikappa B tyrosine kinase has not yet been demonstrated using in vitro reconstitution assays, both PI 3-kinase and c-Src have been demonstrated to associate with tyrosine phosphorylated Ikappa Balpha in T-cells following pervanadate treatment (8) and bone marrow macrophages (BMMs) following TNFalpha stimulus (9). In addition to pervanadate, H/R has also been shown to induce tyrosine phosphorylation of Ikappa Balpha in T-cells in vitro (6) and following I/R injury to the liver in vivo (10).

The tyrosine kinase p56lck is required for Ikappa Balpha tyrosine phosphorylation and NFkappa B activation in T-lymphocytes following pervanadate treatment (6). Loss of tyrosine kinases p56lck and ZAP-70 in two Jurkat mutants abolished NFkappa B activation and partially suppressed and delayed phosphorylation of Tyr-42 on Ikappa Balpha in response to pervanadate treatment (11). However, this study in T-cells also demonstrated that tyrosine phosphorylation of Ikappa Balpha was not sufficient to activate NFkappa B and suggests that both tyrosine and serine kinases act at multiple levels to dissociate the Ikappa Balpha /NFkappa B complex. Furthermore, tyrosine phosphorylation of Ikappa Balpha is observed in BMMs following TNFalpha treatment, and this phosphorylation requires c-Src activity (9). Given the historical dependence of TNFalpha -mediated activation of NFkappa B on the IKK complex and serine phosphorylation of Ikappa Balpha , the functional involvement of Ikappa Balpha tyrosine phosphorylation in response to TNFalpha appears to be quite unique to BMMs. Furthermore, the vast majority of studies evaluating the importance of Ikappa Balpha tyrosine phosphorylation to date have been performed in hematopoetically derived T-cells or BMMs. Thus, the functional relevance of these systems to epithelial models of ischemia/reperfusion remains an open question. Since c-Src can be directly activated by H2O2 (12), pervanadate (13), hypoxia (14), or hypoxia/reoxygenation (15), its central involvement in ROS-mediated IKK and PTK activation of NFkappa B appears reasonable. It is also recognized that H2O2 is capable of activating both IKK- and PTK-dependent pathways of Ikappa Balpha phosphorylation and NFkappa B activation in T-cells (11).

In the present study, we sought to investigate the involvement of c-Src in the redox-mediated activation of NFkappa B activation following H/R or pervanadate treatments in an epithelial cell line (HeLa cells). Since both IKK-dependent and independent pathways of NFkappa B activation have been associated with c-Src activation, we used a number of adenoviral vectors expressing dominant mutants of IKKalpha , IKKbeta , and Ikappa Balpha to selectively test for serine or tyrosine Ikappa Balpha phosphorylation-dependent transcriptional activation of NFkappa B. In contrast to previous studies, we have utilized an NFkappa B-responsive luciferase reporter gene to directly assess changes in the transcriptional activation of NFkappa B. Since the association of tyrosine-phosphorylated Ikappa Balpha with PI 3-kinase has been suggested in proposed models to alter the transcriptional properties of NFkappa B dimers (8, 11), direct functional assessment of activation may be more informative than assessing DNA binding. Triple knockout cell lines (c-Src-/-, Fyn-/-, Yes-/-) with and without c-Src were also used to confirm the dependence of Ikappa Balpha tyrosine phosphorylation on c-Src. Furthermore, recombinant adenoviral vectors expressing various ROS scavengers were used to test whether activation of these pathways contained redox-sensitive components. Results from these studies indicate that tyrosine phosphorylation of Ikappa Balpha and NFkappa B activation is mediated through redox activation of c-Src.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Adenoviral Vectors-- Several E1-deleted recombinant adenoviral vectors were used to modulate the NFkappa B pathway and assay for transcriptional induction of NFkappa B. Previously described vectors included the dominant negative mutants Ad.IKKalpha (KM) (16), Ad.IKKbeta (KA) (16), Ad.Ikappa Balpha (S32A/S36A) (17), and the NFkappa B-responsive luciferase reporter vector Ad.NFkappa Bluc (16). Ad.BglII was used as an empty vector control (16). Ad.Ikappa Balpha (Y42F), which expresses the Y42F mutant form of Ikappa Balpha , was generated by cloning the previously described Ikappa Balpha Y42F cDNA (6) into pAd.CMVlink (18). All adenoviral vectors, except for Ad.NFkappa Bluc, used the CMV enhancer/promoter to express the transgene. Recombinant adenovirus was purified by two rounds of CsCl centrifugation and desalted prior to use as described (19).

Cell Culture, Adenoviral Transduction, and Treatments-- HeLa, SYF, and SYF+c-Src cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 100 µg/ml penicillin and streptomycin. For the tyrosine phosphorylation assays, HeLa cells were transduced with Ad.IKKalpha (KM), Ad.IKKbeta (KA), Ad.Ikappa Balpha (S32A/S36A), Ad.Ikappa Balpha (Y42F), or Ad.BglII at a multiplicity of infection (MOI) equal to 1000 particles/cell. For the NFkappa B luciferase reporter assay, HeLa cells were co-infected with Ad.NFkappa BLuc at an MOI = 500 particles/cell and Ad.IKKalpha (KM), Ad.IKKbeta (KA), Ad.Ikappa Balpha (S32A/S36A), or Ad.Ikappa Balpha (Y42F) at an MOI = 1000 particles/cell. Luciferase reporter assays in SYF and SYF+c-Src cells were performed following infection with Ad.NFkappa BLuc alone at an MOI = 500 particles/cell. Adenoviral infections were performed for 2 h in DMEM without FBS followed by the addition of an equal volume of 20% FBS, DMEM and continued incubation for 22 h. Virus-containing media was replaced at 24 h post-infection with 10% FBS/DMEM. Typically, experiments were initiated at 24 h post-transduction. Experimental methods used to induce NFkappa B were performed according to the following protocols.

Pervanadate Treatment-- Sodium orthovanadate was prepared fresh in water at a concentration of 500 mM. 40 µl of sodium orthovanadate and 5 µl of 30% (w/w) H2O2 was then added to 455 µl phosphate-buffered saline. This mixture was incubated for 5 min at room temperature prior to the addition of catalase (200 µg/ml) to remove the excess H2O2. The pervanadate solution (final concentration 40 mM) was further incubated for 5 min at room temperature, immediately diluted in DMEM and applied to cells. Cells were harvested at 6 h post-pervanadate treatments for NFkappa B activation using luciferase assays or as indicated. Control cells were fed with identical fresh medium that was devoid of pervanadate.

Hypoxia/Reoxygenation-- DMEM (devoid of glucose or FBS) (Invitrogen) equilibrated in 95% N2, 5% CO2 or 95% O2, 5% CO2 was used as hypoxia and reoxygenation medium, respectively. Cells were covered with minimal hypoxia medium and incubated at 37 °C for 5 h in an airtight chamber equilibrated with 5% CO2 and 95% N2. The medium was then replaced with a minimal amount of reoxygenation medium and incubated further at 37 °C in a chamber flushed with 5% CO2 and 95% O2. Cells were harvested 6 h after reoxygenation for NFkappa B activation luciferase assays. Control cells were fed with fresh medium at identical times as the hypoxia/reoxygenation samples, but were exposed to 5% CO2 in atmospheric oxygen.

TNFalpha Treatment-- Mouse recombinant TNFalpha (R&D systems, Minneapolis, MN) was diluted in fresh DMEM medium (10 ng/ml final concentration) and applied to cells at the time of treatment. Cells remained exposed to TNFalpha until they were harvested at 6 h post-stimulation for NFkappa B activation luciferase assays. Control cells were fed at the time of treatment with fresh DMEM medium without TNFalpha .

Western Blotting and Ikappa Balpha Phosphorylation Assays-- Cells were lysed in RIPA buffer (0.15 M NaCl, 50 mM Tris pH 7.2, 1% deoxycholate, 1% Triton X-100, 0.1% SDS), and the protein concentration was determined using a Bio-Rad protein assay (Bio-Rad, Hercules, CA). 5 µg of cell lysate was resolved on a 10% SDS-PAGE and then transferred to nitrocellulose membrane using previously described protocols (18). Ikappa Balpha protein levels were determined by Western blot analysis using an anti-Ikappa Balpha monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). To evaluate Ikappa Balpha tyrosine phosphorylation, 200 µg of cell lysate was immunoprecipitated using 2 µg of Ikappa Balpha antibody (Santa Cruz Biotechnology) followed by Western blot analysis using antiphosphotyrosine antibody (Santa Cruz Biotechnology) and standard protocols (10). Phosphorylated forms of c-Src or total c-Src were detected using anti-c-SrcPY416, anti-c-SrcPY139, and anti-c-Src antibodies (Santa Cruz Biotechnology).

NFkappa B Activation Assays-- NFkappa B transcriptional activity was evaluated using an Ad.NFkappa BLuc reporter vector as previously described (16). Briefly, cells were infected with Ad.NFkappa BLuc at an MOI of 500 particles/cell 24 h prior to TNF-alpha , pervanadate, or H/R treatment. 5 µg of total protein from each sample was assayed for luciferase activity using manufacturer's protocols (Promega, Madison, WI) in a luminometer as previously reported (16). Luciferase activity was assessed as relative light units and used as an indicator for the transcription induction of NFkappa B. To assess potential global changes in transcription induced by each type of environmental stimuli, that were not dependent on NFkappa B, several experiments were performed normalizing changes in Ad.NFkappa BLuc expression to that seen with a control Ad.CMVLacZ vector (20). In these studies both Ad.NFkappa BLuc and Ad.CMVLacZ were co-infected into cells for each of the conditions examined (MOI = 500 particles/cell for each vector) 24 h prior to TNF-alpha , pervanadate, or H/R treatment. Luciferase activity was then assessed using 5 µg of lysate as described above, and beta -galactosidase activity was quantified with 5 µg of lysate using a previously described protocol (21). Luciferase activity was then normalized for beta -galactosidase expression in reference to the Ad.BglII infected (no injury) control. Electrophoretic mobility shift assays for NFkappa B DNA binding were performed as previously described using a 32P-labeled NFkappa B oligonucleotide probe (18).

In Vitro Kinase Assays-- Two types of in vitro kinase assays (radioactive and non-radioactive) were used to evaluate the ability of immunoprecipitated c-Src or IKKbeta to phosphorylate GST-Ikappa Balpha in vitro following different environmental stimuli. For radioactive in vitro kinase assays, HeLa, SYF, or SYF+c-Src cells were washed in ice-cold PBS and lysed in 1 ml of ice-cold RIPA buffer (0.15 M NaCl, 50 mM Tris, pH 7.2, 1% deoxycholate, 1% Triton X-100, 0.1% SDS) followed by centrifugation at 10,000 rpm for 10 min at 4 °C. The protein concentration was then determined using a Bio-Rad protein assay (Bio-Rad, Hercules, CA). 500 µg of protein was immunoprecipitated with anti-c-Src or anti-IKKbeta antibodies (Santa Cruz Biotechnology) and protein A-agarose beads. 1 µg of GST-IkBalpha protein (Santa Cruz Biotechnology) was then added to washed protein A pellets in the presence of 10 µl of kinase buffer (40 mM Hepes, 1 mM beta -glycerophosphate, 1 mM nitrophenolphosphate, 1 mM Na3VO4, 10 mM MgCl2, 2 mM dithiothreitol, 0.3 mM cold ATP, and 10 µCi of [gamma -32P]ATP) and incubated at 30 °C for 30 min. The reaction was terminated by the addition of protein-loading buffer (with SDS) and boiled at 98 °C for 5 min. Samples were then centrifuged to remove the agarose beads, and the supernatant was loaded onto a 10% SDS-PAGE gel. After electrophoresis, proteins were transferred to nitrocellulose membrane (which reduces the background of free [gamma -32P]ATP) and exposed to x-ray film. Non-radioactive in vitro kinase assays were performed to directly evaluate the extent of tyrosine phosphorylation of GST-Ikappa Balpha by immunoprecipitated c-Src or IKKbeta . These in vitro kinase assays were performed identical to the protocol described above except for the omission of [gamma -32P]ATP. In vitro labeled GST-Ikappa Balpha samples were then evaluated by Western blotting for the extent of tyrosine phosphorylation using antiphosphotyrosine antibody (Santa Cruz Biotechnology).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transcriptional Activation of NFkappa B Following Pervanadate or H/R Treatment Requires Ikappa Balpha Tyr-42 Phosphorylation and Is Independent of the IKK Complex and Ikappa Balpha Serine Phosphorylation-- NFkappa B activation can occur through at least two mechanisms that control Ikappa Balpha phosphorylation on either tyrosine 42 or serine 32/36. Proinflammatory stimuli such as TNFalpha are well suited to activate NFkappa B through the Ikappa B kinase complex (IKK) that mediates serine phosphorylation of Ikappa Balpha and ubiquitin-dependent degradation of Ikappa Balpha . In contrast, NFkappa B activation in the liver following ischemia/reperfusion (I/R) injury (10), and in T-cells following H/R (6), occurs in the absence of Ikappa Balpha degradation and is associated with an increase in tyrosine phosphorylation of Ikappa Balpha . To better define the mechanisms involved in NFkappa B activation following I/R injury, we developed an in vitro epithelial cell line model system capable of modulating NFkappa B activity through tyrosine or serine phosphorylation of Ikappa Balpha following H/R, pervanadate, or TNFalpha treatments.

To establish that NFkappa B activation following H/R occurs through a selective pathway involving tyrosine phosphorylation of Ikappa Balpha that is independent of the IKK complex, we utilized several dominant negative mutants to modulate IKK activation and Ikappa Balpha phosphorylation. NFkappa B transcriptional activity was evaluated using a recombinant adenoviral reporter vector (Ad.NFkappa BLuc) expressing the NFkappa B-inducible luciferase gene. As expected and previously reported in epithelial cell lines, the transcriptional induction of NFkappa B following TNFalpha treatment was significantly inhibited (p < 0.001) by expression of Ad.IKKbeta (KA), Ad.IKKalpha (KM), or Ad.Ikappa Balpha (S32A/S36A) in comparison to Ad.BglII (empty vector control)-transduced cells (Fig. 1A). No inhibition in TNFalpha -induced NFkappa B activation was seen following expression of Ad.Ikappa Balpha Y42F. These results confirm the functionality of our vectors to inhibit IKK-mediated TNFalpha activation of NFkappa B and demonstrate a lack of functional involvement of Ikappa Balpha Y42 phosphorylation under these conditions. In contrast to findings with TNFalpha , Ikappa Balpha (Y42F) expression significantly inhibited NFkappa B transcriptional activation following pervanadate (p < 0.001) or H/R (p < 0.001) treatments (Fig. 1, A and B). No significant alterations in pervanadate or H/R-mediated activation of NFkappa B was seen following infection with Ad.IKKbeta (KA), Ad.IKKalpha (KM), or Ad.Ikappa Balpha (S32A/S36A) mutant vectors. Furthermore, when the induction of NFkappa B-mediated luciferase expression was normalized to changes in expression of an irrelevant internal control LacZ transgene under the control of the CMV promoter, the patterns and changes for each of the environmental stimuli and dominant mutants tested were not significantly altered (Fig. 1, A-C). These data demonstrate that global changes in the overall transcriptional state of cells cannot account for the specific alterations induced by the various dominant mutants for a given stimulus.


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Fig. 1.   NFkappa B activation following pervanadate or H/R treatment is inhibited by Ikappa Balpha (Y42F) but not Ikappa Balpha (S32A/S36A) or IKK-dominant mutants. HeLa cells were co-infected with Ad.Ikappa Balpha (Y42F), Ad.Ikappa Balpha (S32A/S36A), Ad.IKKalpha (KM), Ad.IKKbeta (KA), or Ad.BglII (MOI of 1000 particles/cell) together with Ad.NFkappa BLuc (MOI of 500 particles/cell) 24 h before experimental analysis. Cells were treated with TNF-alpha (10 ng/ml) (A), pervanadate (100 µM) (B), or H/R (5 h of hypoxia, 6 h of reoxygenation) (C). Whole cell extracts were harvested 6 h following treatments, normalized for total protein content, and evaluated for NFkappa B transcriptional activity using a luciferase assay. NFkappa B transcriptional activity was determined by the mean relative luciferase activity (RLA) (± S.E., n = 6). D, electrophoretic mobility shift assays for NFkappa B DNA binding in nuclear extracts harvested from HeLa cells following TNFalpha (10 ng/ml, 1 h), pervanadate (50 µM, 1 h), or H/R (5 h of hypoxia, 3 h of reoxygenation) treatments. Exposure times are not equivalent for each treatment condition. E, HeLa cells were treated with TNFalpha (10 ng/ml) for 30 min, pervanadate (100 µM) for 30 min, or H/R (5 h of hypoxia, 30 min of reoxygenation). IKKbeta was immunoprecipitated with anti-IKKbeta antibody from 500 µg of protein lysate. The ability of immunoprecipitated IKKbeta to directly phosphorylate GST-Ikappa Balpha fusion protein was evaluated in the presence of [gamma 32 -P]ATP in vitro.

To confirm that changes in transcriptional activation of NFkappa B mirrored those seen in DNA binding, electrophoretic mobility shift assays were performed for each of the various stimuli. These results shown in Fig. 1D confirm that NFkappa B transcriptional activation is accompanied by increased DNA binding in nuclear extracts. Cumulatively, our results evaluating IKK and Ikappa Balpha mutants suggest that IKK-mediated serine 32/36 phosphorylation of Ikappa Balpha does not play a significant role in regulating NFkappa B following pervanadate or H/R stimuli in our HeLa cell line model. To directly evaluate whether TNF-alpha imparts selective activation of the IKK complex not observed following H/R or pervanadate treatments, we performed in vitro kinase assays with immunoprecipitated IKKbeta to directly evaluate IKK activation and ability to phosphorylate GST-Ikappa Balpha following each of these stimuli. Results from this analysis are shown in Fig. 1E and demonstrate that TNF-alpha treatment stimulates higher levels of IKK activity as compared with H/R and pervanadate treatments. However, activation of IKK was also observed at lower levels following both H/R and pervanadate treatments, suggesting that some overlap in signaling may exist. This apparent overlap may be due to pervanadate and H/R activation of cytokines, which restimulate cells through the IKK pathway. These findings substantiate the small non-significant, but observed, partial inhibition of NFkappa B transcriptional activation by IKK mutants seen following H/R and pervanadate treatments.

Src Inhibitor pp2 Blocks NFkappa B Activation and Ikappa Balpha Tyrosine Phosphorylation Following Pervanadate or H/R Treatment-- Our results in the HeLa cell model have established that NFkappa B activation following H/R or pervanadate treatment is independent of IKK and serine phosphorylation of Ikappa Balpha . We next sought to evaluate candidate upstream factors capable of mediating tyrosine phosphorylation of Ikappa Balpha and subsequent NFkappa B activation. Src family kinases are widely recognized for their importance in regulating stress response genes in response to redox-regulated stimuli such as H/R (15, 22). Furthermore, it has been reported that c-Src activity was necessary for TNFalpha -induced tyrosine phosphorylation of Ikappa Balpha in BMMs (9). Given the lack of a functional requirement for Ikappa Balpha tyrosine phosphorylation in the transcriptional induction of NFkappa B following TNFalpha in our epithelial cell line model, we investigated whether c-Src might also play a role in NFkappa B activation following H/R or pervanadate treatment.

Consistent with the activation of c-Src following H/R or pervanadate treatment, we observed an increase in both Tyr-416- and Tyr-139-phosphorylated forms of activated c-Src (Fig. 2). H/R treatment demonstrated a greater increase in both phosphorylated forms while pervanadate treatment more selectively increased the Tyr-416-phosphorylated form of c-Src. These findings suggest that indeed c-Src is activated by both pervanadate or H/R treatment and is consistent with the previously reported redox-mediated involvement in the activation of c-Src (15). To assign functional importance to c-Src in the tyrosine phosphorylation of Ikappa Balpha and subsequent activation of NFkappa B, we next evaluated the effect of the pp2 c-Src inhibitor. Pretreatment of HeLa cells with pp2 significantly inhibited both pervanadate- and H/R-induced NFkappa B activation (p < 0.001) (Fig. 3A). Furthermore, the level of tyrosine phosphorylation of Ikappa Balpha following pervanadate or H/R treatment was also significantly reduced in the presence of pp2. These results are consistent with the hypothesis that c-Src is functionally required for both Ikappa Balpha tyrosine phosphorylation and subsequent activation of NFkappa B.


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Fig. 2.   Activation of c-Src following pervanadate or H/R treatment. HeLa cells were treated with pervanadate (100 µM) (A) for 15, 30, and 45 min or hypoxic media (B) (95% N2, 5% CO2) for 5 h followed by reoxygenation media (95% O2, 5% CO2) for 15, 30, and 45 min. Both untreated and treated samples were harvested at the indicated time points into lysis buffer, and 5 µg of total protein was separated on a 10% SDS-PAGE. Western blots were evaluated for c-Src tyrosine phosphorylation using two phosphospecific antibodies that recognize phospho-Tyr-416 and phospho-Tyr-139 of activated c-Src. The extent of c-Src phosphorylation is referenced to the total level of c-Src in the sample using an anti-c-Src antibody that recognized both phosphorylated and unphosphorylated forms of c-Src.


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Fig. 3.   Inhibition of c-Src activation blocks NFkappa B transcriptional activation and Ikappa Balpha tyrosine phosphorylation following pervanadate or H/R treatment. A, HeLa cells were transduced with Ad.NFkappa BLuc (MOI of 500 particles/cell) 24 h prior to the initiation of experimental treatment. Cells were then exposed to fresh media with and without pp2 (10 µM) for 30 min, followed by treatment with pervanadate (100 µM) for 6 h or H/R (hypoxic media; 5% N2, 5% CO2) for 5 h followed by reoxygenation media (95% O2, 5% CO2) for 6 h. pp2 inhibitor was continually present during pervanadate and H/R treatments. Whole cell extracts were harvested into lysis buffer, normalized for total protein content, and evaluated for NFkappa B activation using a luciferase assay. Results depict the mean (± S.E., n = 6) relative luciferase activity (RLA). HeLa cells were treated with pervanadate (100 µM) (B) for 30 min or hypoxic media (95% N2, 5% CO2) (C) for 5 h followed by reoxygenation media (95% O2, 5% CO2) for 30 min. One group was pretreated with pp2 (10 µM) for 30 min prior to pervanadate or H/R. Inhibitor (pp2) was continually present during the treatments. Cell lysates were harvested and 200 µg of total protein was immunoprecipitated with anti-Ikappa Balpha antibody followed by Western blotting with an antiphosphotyrosine antibody or anti-Ikappa Balpha antibody.

Tyrosine Phosphorylation of Ikappa Balpha and NFkappa B Activation Is Significantly Reduced in a c-Src, Fyn, and Yes Triple Knockout Cell Line Following H/R or Pervanadate Treatment-- The importance of c-Src in mediating tyrosine phosphorylation of Ikappa Balpha and NFkappa B activation was further investigated using a knockout cell line deficient for Src family kinases Src, Fyn, and Yes. These kinases have similar redundant functions and thus, the knockout of a single gene will not completely abolish their activity. In SYF cells, Src, Yes, and Fyn have been all knocked out to establish null Src mutant activity (23). In SYF+src cell lines, the c-Src activity was reintroduced into the SYF background. Thus, by comparing these two cell lines, one can elucidate c-Src function.

Results evaluating the SYF cell line demonstrated a complete loss of pervanadate- and H/R-induced NFkappa B transcriptional activation in comparison to SYF+src cells (p < 0.001) (Fig. 4A). In contrast, there was no significant difference in TNFalpha -mediated induction of NFkappa B in either of these two cell lines. These results suggest that c-Src activity is required for NFkappa B pathways involving tyrosine, but not serine-mediated phosphorylation of Ikappa Balpha . To conclusively address the requirement for c-Src activity to mediate tyrosine phosphorylation of Ikappa Balpha , we next evaluated the extent of Ikappa Balpha tyrosine phosphorylation in both SYF and SYF+src cells following pervanadate or H/R treatment. These studies demonstrated that Ikappa Balpha tyrosine phosphorylation was significantly reduced in SYF following pervanadate and completely blocked following H/R as compared with SYF+src cells (Fig. 4, B and C). Given the previous demonstration of p56Lck function in the activation of Ikappa Balpha tyrosine phosphorylation following pervanadate treatment (11), the residual phosphorylation seen in our c-Src, Fyn, and Yes knockout cell lines may be due to redundant Lck function. However, our studies evaluating NFkappa B activation following pervanadate treatment suggest that this residual phosphorylation may not be functionally active. In contrast, our studies evaluating H/R demonstrate for the first time that Ikappa Balpha tyrosine phosphorylation and NFkappa B activation can be completely blocked in c-Src, Fyn, and Yes knockout cells and fully restored by c-Src activity alone. These findings suggest that other Src family kinases (i.e. Lck, Lyn, etc.) play a minor role in mediating the activation of this pathway in epithelial cells following H/R.


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Fig. 4.   Tyrosine phosphorylation of Ikappa Balpha and transcriptional activation of NFkappa B are significantly reduced in c-Src knockout cell lines. A, SYF cells or SYF+src cells were transduced with Ad.NFkappa BLuc (MOI of 500 particles/cell) 24 h prior to initiated experiments. Cells were treated with pervanadate (50 µM) for 6 h or H/R (5 h of hypoxia, 6 h of reoxygenation), harvested into lysis buffer, normalized for total protein content, and subjected to luciferase assays. NFkappa B transcriptional activation was evaluated as the relative luciferase activity. Results depict the mean (± S.E., n = 6) relative luciferase activity (RLA). SYF or SYF+src cells were treated with pervanadate (50 µM) (B) for 15, 30, and 45 min or hypoxic media (95% N2, 5% CO2) (C) for 5 h followed by reoxygenation media (95% O2, 5% CO2) for 15, 30, and 45 min. Both untreated and treated cell lysates were harvested at the indicated time points, and 200 µg of total protein was immunoprecipitated with anti-Ikappa Balpha antibody, followed by Western blot analysis with an antiphosphotyrosine antibody or anti-Ikappa Balpha antibody.

c-Src Phosphorylates IkBalpha in Vitro-- Having demonstrated that c-Src activity is required for tyrosine phosphorylation and NFkappa B activation, we tested whether c-Src could be the tyrosine kinase that is directly responsible for tyrosine phosphorylation of Ikappa Balpha . We used an in vitro kinase assay to evaluate c-Src tyrosine kinase activity in SYF cells or SYF+c-src cells following 30 min of pervanadate treatment. Our results presented in Fig. 5A demonstrate that immunoprecipitated c-Src from untreated SYF+c-src cells has the ability to phosphorylate a GST-Ikappa Balpha fusion protein. Furthermore, as anticipated, the extent of GST-Ikappa Balpha phosphorylation is significantly increased following PV treatment. Similar assays using SYF cell lysates demonstrated no significant GST-Ikappa Balpha phosphorylation at baseline, or following pervanadate treatment, and serve as negative controls for the specificity of c-Src immunoprecipitation and kinase function. To conclusively demonstrate that c-Src tyrosine phosphorylates GST-Ikappa Balpha , we performed cold in vitro kinase assays and evaluated the phosphorylated GST-Ikappa Balpha substrate by Western blotting with antiphosphotyrosine antibody. These results demonstrated that both H/R and pervanadate treatments of HeLa cells activates the ability of immunoprecipitated c-Src to tyrosine phosphorylate Ikappa Balpha (Fig. 5B). Furthermore, when similar assays were performed using immunoprecipitated IKKbeta , no increase in tyrosine phosphorylation of Ikappa Balpha was observed over baseline untreated controls. Cumulatively, these results suggest that c-Src activation following H/R and pervanadate treatment is required for tyrosine phosphorylation of IkBalpha . They also suggest that c-Src is likely the direct tyrosine kinase responsible for this phosphorylation event. However, we cannot rule out the possibility that other tyrosine kinases associated with c-Src are not also involved in tyrosine phosphorylation of IkBalpha .


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Fig. 5.   c-Src phosphorylates Ikappa Balpha in vitro. A, SYF cells or SYF+src cells were treated with pervanadate (50 µM) for 30 min and then harvested in RIPA buffer. c-Src was immunoprecipitated with anti-c-Src antibody from 500 µg of protein lysate. The ability of immunoprecipitated c-Src to directly phosphorylate GST-Ikappa Balpha fusion protein was then evaluated in the presence of [gamma -32P]ATP in vitro. Labeled GST-Ikappa Balpha fusion protein was detected by SDS-PAGE and autoradiography. B, HeLa cells were treated with pervanadate (100 µM, 30 min), or H/R (5 h of hypoxia, 30 min of reoxygenation) and evaluated for c-Src activity using a cold in vitro kinase assay. c-Src or IKKbeta was immunoprecipitated with anti-c-Src or anti-IKKbeta antibody from 500 µg of protein lysate. The ability of immunoprecipitated c-Src or IKKbeta to directly tyrosine-phosphorylate GST-Ikappa Balpha fusion protein was evaluated by Western blotting with antiphosphotyrosine antibody. Immunoreactivity was detected by ECL and autoradiography.

Overexpression of Gpx-1 or Catalase, but Not Mn-SOD or Cu,Zn-SOD, Inhibits Tyrosine Phosphorylation of Ikappa Balpha and NFkappa B Activation Following Pervanadate or H/R-- Activation of NFkappa B is widely recognized to be dependent on the redox environment within the cell. In the context of IKK-mediated activation of NFkappa B, ROS have been demonstrated to be a critical component in the activation of both IKKbeta (16) and IKKalpha (24) subunits of the IKK complex following environmental stimuli. Moreover, H2O2 has been shown to activate tyrosine phosphorylation of Ikappa Balpha in T-cells in a manner similar to pervanadate (11). Given the fact that H2O2 has been shown to activate c-Src (25) and the observed dependence of Ikappa Balpha tyrosine phosphorylation and NFkappa B transcriptional activation on c-Src activity in our H/R models, we next sought to investigate whether ROS were a signal component of Ikappa Balpha tyrosine phosphorylation following H/R.

To investigate the redox-dependence of NFkappa B transcriptional activation following pervanadate or H/R treatment, we manipulated the intracellular redox environment using a set of recombinant adenoviruses that encoded various ROS-scavenging enzymes. These included Ad.Catalase or Ad.GPx-1 vectors that degrade H2O2, and Ad.Mn-SOD or Ad.Cu,Zn-SOD vectors that dismutate superoxide anion (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>) into H2O2. Using our in vitro model, we analyzed the role of these antioxidant enzymes in regulating Ikappa Balpha tyrosine phosphorylation and NFkappa B transcriptional activation. Results from these studies demonstrated a significant inhibition (p < 0.001) in both pervanadate (Fig. 6A) and H/R (Fig. 6C) induction of NFkappa B transcriptional activity following expression of GPx-1 or catalase. Consistent with this NFkappa B activation data, tyrosine phosphorylation of Ikappa Balpha following pervanadate or H/R treatments was also significantly inhibited by GPx-1 or catalase overexpression (Fig. 6, B and D). In contrast, overexpression of either Cu,Zn-SOD or Mn-SOD failed to alter Ikappa B tyrosine phosphorylation or NFkappa B activation. These findings suggest that H2O2 is an important redox component in NFkappa B activation mediated through Ikappa B-alpha tyrosine phosphorylation.


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Fig. 6.   Overexpression of Gpx-1 or catalase, but not Mn-SOD or Cu,Zn-SOD, inhibits NFkappa B activation and tyrosine phosphorylation of Ikappa Balpha following pervanadate or H/R treatment. HeLa cells were co-infected with Ad.BglII, Ad.Gpx-1, Ad.Catalase, Ad.Cu,Zn-SOD, or Ad.Mn-SOD (MOI of 1000 particles/cell) together with Ad.NFkappa BLuc (MOI of 500 particles/cell) for 24 h prior to initiating experimental treatments described below. Cells were treated with per- vanadate (100 µM) (A) for 6 h or H/R (5 h of hypoxia/6 h of reoxygenation) (C). Whole cell extracts were normalized for total protein content and subjected to luciferase assays. NFkappa B transcriptional activity was assessed as the mean relative luciferase activity (RLA) (± S.E., n = 6). For evaluation of Ikappa Balpha phosphorylation, cells were treated with pervanadate (100 µM) (B) for 30 min or H/R (6 h of hypoxia/30 min of reoxygenation) (D). Both untreated and treated samples were harvested into lysis buffer, and 200 µg of total protein was immunoprecipitated with anti-Ikappa Balpha antibody followed by Western blot analysis with an antiphosphotyrosine antibody or anti-Ikappa Balpha antibody.

GPx-1 Overexpression Reduces c-Src Kinase Activity-- Having demonstrated that Gpx-1 overexpression is able to reduce NFkappa B activity as well as tyrosine phosphorylation of Ikappa Balpha , we next investigated whether GPx-1 expression acts to directly inhibit activation of c-Src using an in vitro kinase assay. Results from these experiments in HeLa cells demonstrated a significant inhibition in the ability of c-Src to phosphorylate GST-Ikappa Balpha following pervanadate treatment in the presence of Ad.GPx-1 infection as compared with Ad.BglII-infected control (Fig. 7). Furthermore, in this assay, transient treatment with 1 mM H2O2 for 30 min also significantly activated c-Src kinase function as previously demonstrated. In summary, our data demonstrate that intracellular hydrogen peroxide (or hydroxyl radical products) mediates NFkappa B activation through regulation of c-Src-dependent Ikappa Balpha tyrosine phosphorylation. Overexpression of H2O2 scavengers is able to efficiently reduce c-Src kinase activity, Ikappa Balpha tyrosine phosphorylation, and NFkappa B activation following H/R or pervanadate injury.


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Fig. 7.   Overexpression of GPx-1 reduces c-Src kinase activity following pervanadate treatment. HeLa cells were infected with Ad.BglII or Ad.Gpx-1 (MOI of 1000 particles/cell) for 24 h prior to initiating experimental treatments. Cells were then treated with pervanadate (100 µM) or hydrogen peroxide (1 mM) for 30 min and then lysed in RIPA buffer. c-Src was immunoprecipitated with anti-c-Src antibody from 500 µg of protein lysate. The ability of immunoprecipitated c-Src to directly phosphorylate GST-Ikappa Balpha fusion protein was then evaluated in the presence of [gamma -32P]ATP in vitro.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The physiologic significance of Ikappa Balpha tyrosine phosphorylation in mediating NFkappa B transcriptional activation has remained one of the poorly understood aspects of this well-studied transcription factor. To date, all studies evaluating the functional regulation of Ikappa Balpha tyrosine phosphorylation and NFkappa B activation have been performed in T-cells or BMMs. Although the tyrosine phosphatase inhibitor pervanadate has been shown to be a significant activator of this pathway, natural physiologic stimuli that induce Ikappa Balpha tyrosine phosphorylation have remained elusive. However, TNFalpha treatment of BMMs has recently been shown to activate NFkappa B recruitment to the nucleus in an Ikappa Balpha tyrosine phosphorylation-dependent manner (9). This TNFalpha -induced pathway of NFkappa B activation in BMMs appears to differ significantly from the classical IKK-dependent pathway involving serine phosphorylation of Ikappa Balpha that is active in other epithelial-derived cell types. Other non-hematopoetic systems have increasingly demonstrated components of Ikappa Balpha tyrosine phosphorylation in models of in vitro and in vivo injury (10, 26, 27). The unique fingerprint of this pathway appears to be the ability of an Ikappa Balpha PTK to activate NFkappa B in the absence of Ikappa Balpha proteolytic degradation. In the present study, we have sought to clarify several issues regarding the involvement of this Ikappa Balpha PTK pathway in mediating NFkappa B transcriptional activation in epithelial-derived cells following H/R.

Using comparative cell line models to evaluate both Ikappa Balpha serine and tyrosine phosphorylation-dependent pathways of NFkappa B transcriptional activation, our studies have further characterized an IKK-independent pathway that regulates NFkappa B through c-Src activation. Studies using Ikappa Balpha phosphorylation mutants and SYF knockout cells demonstrated that c-Src-mediated transcriptional activation of NFkappa B functionally requires tyrosine, but not serine, phosphorylation of Ikappa Balpha . Using this HeLa cell model system, we found no functional requirement for Ikappa Balpha tyrosine phosphorylation in the transcriptional activation of NFkappa B following TNFalpha stimulus and no evidence for tyrosine phosphorylation of Ikappa Balpha following TNFalpha treatment. Furthermore, our studies evaluating IKK-dominant mutants demonstrate, for the first time, the lack of IKK involvement in PTK-mediated pathways of NFkappa B transcriptional activation following pervanadate or H/R treatments, while confirming the selective activation of NFkappa B by TNFalpha as mediated through serine phosphorylation of Ikappa Balpha . These functional studies evaluating the transcriptional activation of NFkappa B following three independent stimuli suggest that little overlap, if any, exists in IKK and PTK pathways controlling the Ikappa Balpha ·NFkappa B complex.

Several similarities and differences between our present studies and those in BMMs are worth noting. First, pervanadate appears to be a universal activator of NFkappa B-requiring Ikappa Balpha tyrosine phosphorylation, with consistent results observed in HeLa cells, T-cells, and BMMs. Second, unlike HeLa cells, treatment of BMMs with TNFalpha results in significant activation of NFkappa B in a manner dependent on Ikappa Balpha phosphorylation of tyrosine 42 (9). This difference underscores the importance of cell type-specific dependences in the activation of NFkappa B and Ikappa Balpha protein tyrosine kinases. Third, our current studies are the first to directly evaluate the transcriptional activation of NFkappa B using an NFkappa B-responsive reporter gene. To date, all assays for NFkappa B activation following stimulation of Ikappa Balpha tyrosine phosphorylation had been performed using NFkappa B DNA binding.

Intracellular production of ROS has been implicated in the regulation of numerous signal transduction cascades and in the activation of NFkappa B following ischemia/reperfusion injury (1, 28). Furthermore, c-Src can be directly activated by hydrogen peroxide treatment (12, 25), and stimuli such as angiotensin II can induce c-Src activity, which is inhibited by antioxidants (29). The association of c-Src activation following H/R (15) has also suggested that c-Src may act as a redox sensor in the activation of NFkappa B. However, other pathways of NFkappa B activation involving pro-inflammatory stimuli such as TNFalpha and LPS also have redox-sensitive activation components, which are associated with the IKK complex (16, 24). These studies have suggested that superoxide formation may be the primary initiating ROS involved in activation of the IKK complex. Hence, the pathways for redox activation of NFkappa B are quite diverse and likely regulated by the spatial relationship of both specific ROS and the signaling components involved. Our findings in the present study have shed additional light on the redox diversity of NFkappa B activation involving tyrosine phosphorylation of Ikappa Balpha . The demonstration that both Gpx-1 and catalase, but not Mn-SOD or Cu,Zn-SOD, are capable of inhibiting H/R or pervanadate-induced Ikappa Balpha tyrosine phosphorylation and NFkappa B activation, suggests a preference for H2O2 and/or hydroxyl radicals (as a product of H2O2) in the activation of the Ikappa Balpha tyrosine kinase. These findings are consistent with previous reports demonstrating the direct activation of Ikappa Balpha tyrosine phosphorylation by H2O2 in T-cells (11).

Recent evidence has suggested that the p85 subunit of PI3-kinase associates with tyrosine 42 phosphorylated Ikappa Balpha but not with unphosphorylated Ikappa Balpha (8). The catalytic p110 subunit also appears to be critical in the activation of NFkappa B. The function of p110 may involve phosphorylation of NFkappa B and/or dissociation of the Ikappa Balpha : NFkappa B complex. Since both PI 3-kinase and c-Src have been shown to associate with one another (30) and both maintain redox-sensitive components in their activation (31-33), it is plausible that c-Src may act on this PI 3-kinase complex to mediate NFkappa B activation. It has been previously demonstrated that c-Src is at least partially required for Ikappa Balpha tyrosine phosphorylation in BMMs following TNFalpha treatment (9). However, these studies performed in c-Src (-/-) BMMs demonstrated a delay only in the induction of p50/p65 heterodimers in the nucleus following the TNFalpha treatment. In comparison to our present studies using SYF cells with a c-Src, Fyn, and Yes, knockout background, we find a more complete block in both Ikappa Balpha tyrosine phosphorylation and NFkappa B transcriptional activation following both H/R and pervanadate treatment than previously described. Furthermore this block was completely reversed by reconstitution of only c-Src activity. These findings highlight the functional redundancy of Src family kinases and conclusively demonstrate that c-Src is fully capable of mediating NFkappa B activation through Ikappa Balpha tyrosine phosphorylation. Our studies, for the first time, have also successfully reconstituted Ikappa Balpha tyrosine phosphorylation with immunoprecipitated c-Src in an in vitro kinase assay. Furthermore, the activity of c-Src Ikappa Balpha tyrosine kinase activity was modulated in response to H/R and pervanadate treatments in a redox-dependent fashion. These findings provide the most conclusive evidence to date that c-Src is able to directly tyrosine phosphorylate Ikappa Balpha and that this phosphorylation event is required for NFkappa B activation following H/R or pervanadate treatments. The physiologic relevance of redundant Src family kinases in the activation of NFkappa B still remains unclear. However, the redox-regulated mechanisms that control activation of NFkappa B by Src family kinases may be a particularly relevant therapeutic target for organ damage following I/R injury.

    ACKNOWLEDGEMENTS

We thank NIDDK, National Institutes of Health funding of the DERC for tissue culture supplies. We thank Dr. J. F. Peyron (Pasteur, France) for providing the Ikappa Balpha (Y42F) cDNA. We gratefully acknowledge the editorial assistance of Dr. Reitu S. Agrawal and Kevin Wyne.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants P50 HL60316 (to G. H.) and DK51315 (to J. F. E.) and the Center for Gene Therapy funded by NIDDK (P30 DK54759).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Dept. of Anatomy and Cell Biology, University of Iowa, Rm. 1-111 BSB, 51 Newton Rd., Iowa City, IA 52242-1109. Tel.: 319-335-7753; Fax: 319-335-7198; E-mail: john-engelhardt@uiowa.edu.

Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M206718200

    ABBREVIATIONS

The abbreviations used are: ROS, reactive oxygen species; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; RIPA, radioimmune precipitation assay buffer; MOI, multiplicity of infection; PI, phosphatidylinositol; GST, glutathione S-transferase; PTK, protein tyrosine kinase; CMV, cytomegalovirus; H/R, hypoxia/reoxygenation; SOD, superoxide dismutase; BMM, bone marrow macrophages; I/R, ischemia/reperfusion.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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