Tyrosine Phosphorylation of IκBα Activates NFκB through a Redox-regulated and c-Src-dependent Mechanism Following Hypoxia/Reoxygenation*

NFκB is a critical transcription factor involved in modulating cellular responses to environmental injuries. Tyrosine 42 phosphorylation of IκBα has been shown to mediate NFκB activation following hypoxia/reoxygenation (H/R) or pervanadate treatment. This pathway differs from the canonical proinflammatory pathways, which mediate NFκB activation through serine phosphorylation of IκBα by the IKK complex. In the present study, we investigated the involvement of c-Src in the redox activation of NFκB following H/R or pervanadate treatment. Our results demonstrate that pervanadate or H/R treatment leads to tyrosine phosphorylation of IκBα and NFκB 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 IκBα tyrosine phosphorylation and NFκB 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 NFκB activation and tyrosine phosphorylation of IκBα. In vitro kinase assays further demonstrated that immunoprecipitated c-Src has the capacity to directly phosphorylate GST-IκBα and that this IκBα kinase activity is significantly reduced by Gpx-1 overexpression. These results suggest that c-Src-dependent tyrosine phosphorylation of IκBα and subsequent activation of NFκB is controlled by intracellular H2O2 and defines an important redox-regulated pathway for NFκB activation following H/R injury that is independent of the IKK complex.

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 NFB (3).
NFB family members include p50, p52, p65, and c-RelB, which form homodimeric and heterodimeric transcriptional complexes (4). The activation of NFB is controlled by a family of IB repressor proteins (IB␣, IB␤, and IB⑀) that sequester NFB in the cytoplasm (4). Phosphorylation-dependent inactivation of IB proteins leads to the mobilization of NFB to the nucleus where it can act as a transcription factor. These phosphorylation pathways have been most extensively studied for IB␣ and include two distinct mechanisms involving either serine or tyrosine phosphorylation of IB␣. The most comprehensively studied pathway regulating IB␣ includes phosphorylation on two serine (32 and 36) residues by the IB kinase complex (IKK) (5). This phosphorylation leads to ubiquitination of IB␣ at nearby lysine residues and degradation by the proteasome. An alternative, less characterized pathway of NFB activation acts through tyrosine phosphorylation of IB␣ at residue 42 (6). In contrast to IKK-mediated serine phosphorylation of IB␣, tyrosine phosphorylation of IB␣ is capable of activating NFB in the absence of ubiquitin-dependent degradation of IB␣. However, it is presently unclear if IKK and/or the IB␣ protein-tyrosine kinase (PTK) interactions with IB␣ are functionally modulated by prior tyrosine or serine phosphorylation of IB␣, respectively. Experimental evidence appears to suggest that prior tyrosine phosphorylation of IB␣ 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 PTKmediated phosphorylation of IB␣ and the net effect on NFB transcriptional activation remains an open question. Although the exact identity of the IB 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 IB␣ in T-cells following pervanadate treatment (8) and bone marrow macrophages (BMMs) following TNF␣ stimulus (9). In addition to pervanadate, H/R has also been shown to induce tyrosine phosphorylation of IB␣ in T-cells in vitro (6) and following I/R injury to the liver in vivo (10).
The tyrosine kinase p56 lck is required for IB␣ tyrosine phosphorylation and NFB activation in T-lymphocytes following pervanadate treatment (6). Loss of tyrosine kinases p56 lck and ZAP-70 in two Jurkat mutants abolished NFB activation and partially suppressed and delayed phosphorylation of Tyr-42 on IB␣ in response to pervanadate treatment (11). However, this study in T-cells also demonstrated that tyrosine phosphorylation of IB␣ was not sufficient to activate NFB and suggests that both tyrosine and serine kinases act at multiple levels to dissociate the IB␣/NFB complex. Furthermore, tyrosine phosphorylation of IB␣ is observed in BMMs following TNF␣ treatment, and this phosphorylation requires c-Src activity (9). Given the historical dependence of TNF␣-mediated activation of NFB on the IKK complex and serine phosphorylation of IB␣, the functional involvement of IB␣ tyrosine phosphorylation in response to TNF␣ appears to be quite unique to BMMs. Furthermore, the vast majority of studies evaluating the importance of IB␣ 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 H 2 O 2 (12), pervanadate (13), hypoxia (14), or hypoxia/reoxygenation (15), its central involvement in ROS-mediated IKK and PTK activation of NFB appears reasonable. It is also recognized that H 2 O 2 is capable of activating both IKK-and PTK-dependent pathways of IB␣ phosphorylation and NFB activation in T-cells (11).
In the present study, we sought to investigate the involvement of c-Src in the redox-mediated activation of NFB activation following H/R or pervanadate treatments in an epithelial cell line (HeLa cells). Since both IKK-dependent and independent pathways of NFB activation have been associated with c-Src activation, we used a number of adenoviral vectors expressing dominant mutants of IKK␣, IKK␤, and IB␣ to selectively test for serine or tyrosine IB␣ phosphorylation-dependent transcriptional activation of NFB. In contrast to previous studies, we have utilized an NFB-responsive luciferase reporter gene to directly assess changes in the transcriptional activation of NFB. Since the association of tyrosine-phosphorylated IB␣ with PI 3-kinase has been suggested in proposed models to alter the transcriptional properties of NFB dimers (8,11), direct functional assessment of activation may be more informative than assessing DNA binding. Triple knockout cell lines (c-SrcϪ/Ϫ, FynϪ/Ϫ, YesϪ/Ϫ) with and without c-Src were also used to confirm the dependence of IB␣ 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 IB␣ and NFB activation is mediated through redox activation of c-Src.
Cell Culture, Adenoviral Transduction, and Treatments-HeLa, SYF, and SYFϩc-Src cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 100 g/ml penicillin and streptomycin. For the tyrosine phosphorylation assays, HeLa cells were transduced with Ad.IKK␣(KM), Ad.IKK␤(KA), Ad.IB␣(S32A/S36A), Ad.IB␣(Y42F), or Ad.BglII at a multiplicity of infection (MOI) equal to 1000 particles/cell. For the NFB luciferase reporter assay, HeLa cells were co-infected with Ad.NFBLuc at an MOI ϭ 500 particles/cell and Ad.IKK␣(KM), Ad.IKK␤(KA), Ad.IB␣(S32A/S36A), or Ad.IB␣(Y42F) at an MOI ϭ 1000 particles/cell. Luciferase reporter assays in SYF and SYFϩc-Src cells were performed following infection with Ad.NFBLuc alone at an MOI ϭ 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 NFB were performed according to the following protocols.
Pervanadate Treatment-Sodium orthovanadate was prepared fresh in water at a concentration of 500 mM. 40 l of sodium orthovanadate and 5 l of 30% (w/w) H 2 O 2 was then added to 455 l phosphatebuffered saline. This mixture was incubated for 5 min at room temperature prior to the addition of catalase (200 g/ml) to remove the excess H 2 O 2 . 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 NFB 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% N 2 , 5% CO 2 or 95% O 2 , 5% CO 2 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% CO 2 and 95% N 2 . The medium was then replaced with a minimal amount of reoxygenation medium and incubated further at 37°C in a chamber flushed with 5% CO 2 and 95% O 2 . Cells were harvested 6 h after reoxygenation for NFB activation luciferase assays. Control cells were fed with fresh medium at identical times as the hypoxia/reoxygenation samples, but were exposed to 5% CO 2 in atmospheric oxygen.
TNF␣ Treatment-Mouse recombinant TNF␣ (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 TNF␣ until they were harvested at 6 h post-stimulation for NFB activation luciferase assays. Control cells were fed at the time of treatment with fresh DMEM medium without TNF␣.
Western Blotting and IB␣ 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 g of cell lysate was resolved on a 10% SDS-PAGE and then transferred to nitrocellulose membrane using previously described protocols (18). IB␣ protein levels were determined by Western blot analysis using an anti-IB␣ monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). To evaluate IB␣ tyrosine phosphorylation, 200 g of cell lysate was immunoprecipitated using 2 g of IB␣ 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).
NFB Activation Assays-NFB transcriptional activity was evaluated using an Ad.NFBLuc reporter vector as previously described (16). Briefly, cells were infected with Ad.NFBLuc at an MOI of 500 particles/cell 24 h prior to TNF-␣, pervanadate, or H/R treatment. 5 g 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 NFB. To assess potential global changes in transcription induced by each type of environmental stimuli, that were not dependent on NFB, several experiments were performed normalizing changes in Ad.NFBLuc expression to that seen with a control Ad.CMVLacZ vector (20). In these studies both Ad.NFBLuc and Ad.CMVLacZ were coinfected into cells for each of the conditions examined (MOI ϭ 500 particles/cell for each vector) 24 h prior to TNF-␣, pervanadate, or H/R treatment. Luciferase activity was then assessed using 5 g of lysate as described above, and ␤-galactosidase activity was quantified with 5 g of lysate using a previously described protocol (21). Luciferase activity was then normalized for ␤-galactosidase expression in reference to the Ad.BglII infected (no injury) control. Electrophoretic mobility shift assays for NFB DNA binding were performed as previously described using a 32 P-labeled NFB 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 immu-noprecipitated c-Src or IKK␤ to phosphorylate GST-IB␣ in vitro following different environmental stimuli. For radioactive in vitro kinase assays, HeLa, SYF, or SYFϩc-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 g of protein was immunoprecipitated with anti-c-Src or anti-IKK␤ antibodies (Santa Cruz Biotechnology) and protein A-agarose beads. 1 g of GST-IkB␣ protein (Santa Cruz Biotechnology) was then added to washed protein A pellets in the presence of 10 l of kinase buffer (40 mM Hepes, 1 mM ␤-glycerophosphate, 1 mM nitrophenolphosphate, 1 mM Na 3 VO 4 , 10 mM MgCl 2 , 2 mM dithiothreitol, 0.3 mM cold ATP, and 10 Ci of [␥-32 P]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 [␥-32 P]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-IB␣ by immunoprecipitated c-Src or IKK␤. These in vitro kinase assays were performed identical to the protocol described above except for the omission of [␥-32 P]ATP. In vitro labeled GST-IB␣ samples were then evaluated by Western blotting for the extent of tyrosine phosphorylation using antiphosphotyrosine antibody (Santa Cruz Biotechnology).

Transcriptional Activation of NFB Following Pervanadate or H/R Treatment Requires IB␣ Tyr-42 Phosphorylation and Is
Independent of the IKK Complex and IB␣ Serine Phosphorylation-NFB activation can occur through at least two mechanisms that control IB␣ phosphorylation on either tyrosine 42 or serine 32/36. Proinflammatory stimuli such as TNF␣ are well suited to activate NFB through the IB kinase complex (IKK) that mediates serine phosphorylation of IB␣ and ubiquitin-dependent degradation of IB␣. In contrast, NFB activation in the liver following ischemia/reperfusion (I/R) injury (10), and in T-cells following H/R (6), occurs in the absence of IB␣ degradation and is associated with an increase in tyrosine phosphorylation of IB␣. To better define the mechanisms involved in NFB activation following I/R injury, we developed an in vitro epithelial cell line model system capable of modulating NFB activity through tyrosine or serine phosphorylation of IB␣ following H/R, pervanadate, or TNF␣ treatments.
To establish that NFB activation following H/R occurs through a selective pathway involving tyrosine phosphorylation of IB␣ that is independent of the IKK complex, we utilized several dominant negative mutants to modulate IKK activation and IB␣ phosphorylation. NFB transcriptional activity was evaluated using a recombinant adenoviral reporter vector (Ad.NFBLuc) expressing the NFB-inducible luciferase gene. As expected and previously reported in epithelial cell lines, the transcriptional induction of NFB following TNF␣ treatment was significantly inhibited (p Ͻ 0.001) by expression of Ad.IKK␤(KA), Ad.IKK␣(KM), or Ad.IB␣(S32A/ S36A) in comparison to Ad.BglII (empty vector control)-transduced cells (Fig. 1A). No inhibition in TNF␣-induced NFB activation was seen following expression of Ad.IB␣Y42F. These results confirm the functionality of our vectors to inhibit IKK-mediated TNF␣ activation of NFB and demonstrate a lack of functional involvement of IB␣ Y42 phosphorylation under these conditions. In contrast to findings with TNF␣, I〉␣(Y42F) expression significantly inhibited NF〉 transcriptional activation following pervanadate (p Ͻ 0.001) or H/R (p Ͻ 0.001) treatments (Fig. 1, A and B). No significant alterations in pervanadate or H/R-mediated activation of NFB was seen following infection with Ad.IKK␤(KA), Ad.IKK␣(KM), or Ad.I〉␣(S32A/S36A) mutant vectors. Furthermore, when the induction of NFB-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.
To confirm that changes in transcriptional activation of NF〉 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 NFB transcriptional activation is accompanied by increased DNA binding in nuclear extracts. Cumulatively, our results evaluating IKK and IB␣ mutants suggest that IKK-mediated serine 32/36 phosphorylation of IB␣ does not play a significant role in regulating NFB following pervanadate or H/R stimuli in our HeLa cell line model. To directly evaluate whether TNF-␣ imparts selective activation of the IKK complex not observed following H/R or pervanadate treatments, we performed in vitro kinase assays with immunoprecipitated IKK␤ to directly evaluate IKK activation and ability to phosphorylate GST-IB␣ following each of these stimuli. Results from this analysis are shown in Fig. 1E and demonstrate that TNF-␣ 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 NFB transcriptional activation by IKK mutants seen following H/R and pervanadate treatments.
Src Inhibitor pp2 Blocks NFB Activation and IB␣ Tyrosine Phosphorylation Following Pervanadate or H/R Treatment-Our results in the HeLa cell model have established that NFB activation following H/R or pervanadate treatment is independent of IKK and serine phosphorylation of IB␣. We next sought to evaluate candidate upstream factors capable of mediating tyrosine phosphorylation of IB␣ and subsequent NFB 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 TNF␣-induced tyrosine phosphorylation of IB␣ in BMMs (9). Given the lack of a functional requirement for IB␣ tyrosine phosphorylation in the transcriptional induction of NFB following TNF␣ in our epithelial cell line model, we investigated whether c-Src might also play a role in NFB 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-416and 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 IB␣ and subsequent activation of NFB, 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 NFB activation (p Ͻ 0.001) (Fig. 3A). Furthermore, the level of tyrosine phosphorylation of IB␣ 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 IB␣ tyrosine phosphorylation and subsequent activation of NFB.
Tyrosine Phosphorylation of IB␣ and NFB 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 IB␣ and NFB 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

FIG. 2. Activation of c-Src following pervanadate or H/R treatment.
HeLa cells were treated with pervanadate (100 M) (A) for 15, 30, and 45 min or hypoxic media (B) (95% N 2 , 5% CO 2 ) for 5 h followed by reoxygenation media (95% O 2 , 5% CO 2 ) for 15, 30, and 45 min. Both untreated and treated samples were harvested at the indicated time points into lysis buffer, and 5 g 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. out to establish null Src mutant activity (23). In SYFϩsrc 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 NFB transcriptional activation in comparison to SYFϩsrc cells (p Ͻ 0.001) (Fig. 4A). In contrast, there was no significant difference in TNF␣-mediated induction of NFB in either of these two cell lines. These results suggest that c-Src activity is required for NFB pathways involving tyrosine, but not serine-mediated phosphorylation of IB␣. To conclusively address the requirement for c-Src activity to mediate tyrosine phosphorylation of IB␣, we next evaluated the extent of IB␣ tyrosine phosphorylation in both SYF and SYFϩsrc cells following pervanadate or H/R treatment. These studies demonstrated that IB␣ tyrosine phosphorylation was significantly reduced in SYF following pervanadate and completely blocked following H/R as com-pared with SYFϩsrc cells (Fig. 4, B and C). Given the previous demonstration of p56 Lck function in the activation of IB␣ 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 NFB 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 IB␣ tyrosine phosphorylation and NFB activation can be completely blocked in c-Src, Fyn, and Yes knockout cells and fully restored

FIG. 3. Inhibition of c-Src activation blocks NFB transcriptional activation and IB␣ tyrosine phosphorylation following pervanadate or H/R treatment. A, HeLa cells were transduced with
Ad.NFBLuc (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 M) for 30 min, followed by treatment with pervanadate (100 M) for 6 h or H/R (hypoxic media; 5% N 2 , 5% CO 2 ) for 5 h followed by reoxygenation media (95% O 2 , 5% CO 2 ) 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 NFB activation using a luciferase assay. Results depict the mean (Ϯ S.E., n ϭ 6) relative luciferase activity (RLA). HeLa cells were treated with pervanadate (100 M) (B) for 30 min or hypoxic media (95% N 2 , 5% CO 2 ) (C) for 5 h followed by reoxygenation media (95% O 2 , 5% CO 2 ) for 30 min. One group was pretreated with pp2 (10 M) for 30 min prior to pervanadate or H/R. Inhibitor (pp2) was continually present during the treatments. Cell lysates were harvested and 200 g of total protein was immunoprecipitated with anti-IB␣ antibody followed by Western blotting with an antiphosphotyrosine antibody or anti-IB␣ antibody.

FIG. 4. Tyrosine phosphorylation of IB␣ and transcriptional activation of NFB are significantly reduced in c-Src knockout cell lines. A, SYF cells or SYFϩsrc cells were transduced with
Ad.NFBLuc (MOI of 500 particles/cell) 24 h prior to initiated experiments. Cells were treated with pervanadate (50 M) 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. NFB transcriptional activation was evaluated as the relative luciferase activity. Results depict the mean (Ϯ S.E., n ϭ 6) relative luciferase activity (RLA). SYF or SYFϩsrc cells were treated with pervanadate (50 M) (B) for 15, 30, and 45 min or hypoxic media (95% N 2 , 5% CO 2 ) (C) for 5 h followed by reoxygenation media (95% O 2 , 5% CO 2 ) for 15, 30, and 45 min. Both untreated and treated cell lysates were harvested at the indicated time points, and 200 g of total protein was immunoprecipitated with anti-IB␣ antibody, followed by Western blot analysis with an antiphosphotyrosine antibody or anti-IB␣ antibody. 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. c-Src Phosphorylates IkB␣ in Vitro-Having demonstrated that c-Src activity is required for tyrosine phosphorylation and NFB activation, we tested whether c-Src could be the tyrosine kinase that is directly responsible for tyrosine phosphorylation of IB␣. We used an in vitro kinase assay to evaluate c-Src tyrosine kinase activity in SYF cells or SYFϩc-src cells following 30 min of pervanadate treatment. Our results presented in Fig. 5A demonstrate that immunoprecipitated c-Src from untreated SYFϩc-src cells has the ability to phosphorylate a GST-IB␣ fusion protein. Furthermore, as anticipated, the extent of GST-IB␣ phosphorylation is significantly increased following PV treatment. Similar assays using SYF cell lysates demonstrated no significant GST-IB␣ 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-IB␣, we performed cold in vitro kinase assays and evaluated the phosphorylated GST-IB␣ 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 IB␣ (Fig.  5B). Furthermore, when similar assays were performed using immunoprecipitated IKK␤, no increase in tyrosine phosphorylation of IB␣ was observed over baseline untreated controls. Cumulatively, these results suggest that c-Src activation fol-lowing H/R and pervanadate treatment is required for tyrosine phosphorylation of IkB␣. 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 IkB␣.
Overexpression of Gpx-1 or Catalase, but Not Mn-SOD or Cu,Zn-SOD, Inhibits Tyrosine Phosphorylation of IB␣ and NFB Activation Following Pervanadate or H/R-Activation of NFB is widely recognized to be dependent on the redox environment within the cell. In the context of IKK-mediated activation of NFB, ROS have been demonstrated to be a critical component in the activation of both IKK␤ (16) and IKK␣ (24) subunits of the IKK complex following environmental stimuli. Moreover, H 2 O 2 has been shown to activate tyrosine phosphorylation of IB␣ in T-cells in a manner similar to pervanadate (11). Given the fact that H 2 O 2 has been shown to activate c-Src (25) and the observed dependence of IB␣ tyrosine phosphorylation and NFB transcriptional activation on c-Src activity in our H/R models, we next sought to investigate whether ROS were a signal component of IB␣ tyrosine phosphorylation following H/R.
To investigate the redox-dependence of NFB 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 H 2 O 2, and Ad.Mn-SOD or Ad.Cu,Zn-SOD vectors that dismutate superoxide anion (O 2 . ) into H 2 O 2 . Using our in vitro model, we analyzed the role of these antioxidant enzymes in regulating IB␣ tyrosine phosphorylation and NFB transcriptional activation. Results from these studies demonstrated a significant inhibition (p Ͻ 0.001) in both pervanadate (Fig.  6A) and H/R (Fig. 6C) induction of NFB transcriptional activity following expression of GPx-1 or catalase. Consistent with this NFB activation data, tyrosine phosphorylation of IB␣ 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 IB tyrosine phosphorylation or NFB activation. These findings suggest that H 2 O 2 is an important redox component in NFB activation mediated through IB-␣ tyrosine phosphorylation.

GPx-1 Overexpression Reduces c-Src Kinase
Activity-Having demonstrated that Gpx-1 overexpression is able to reduce NFB activity as well as tyrosine phosphorylation of IB␣, 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-IB␣ 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 H 2 O 2 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 NFB activation through regulation of c-Src-dependent IB␣ tyrosine phosphorylation. Overexpression of H 2 O 2 scavengers is able to efficiently reduce c-Src kinase activity, IB␣ tyrosine phosphorylation, and NFB activation following H/R or pervanadate injury. DISCUSSION The physiologic significance of IB␣ tyrosine phosphorylation in mediating NFB transcriptional activation has remained one of the poorly understood aspects of this well-stud- and evaluated for c-Src activity using a cold in vitro kinase assay. c-Src or IKK␤ was immunoprecipitated with anti-c-Src or anti-IKK␤ antibody from 500 g of protein lysate. The ability of immunoprecipitated c-Src or IKK␤ to directly tyrosine-phosphorylate GST-IB␣ fusion protein was evaluated by Western blotting with antiphosphotyrosine antibody. Immunoreactivity was detected by ECL and autoradiography.
ied transcription factor. To date, all studies evaluating the functional regulation of IB␣ tyrosine phosphorylation and NFB 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 IB␣ tyrosine phosphorylation have remained elusive. However, TNF␣ treatment of BMMs has recently been shown to activate NFB recruitment to the nucleus in an IB␣ tyrosine phosphorylation-dependent manner (9). This TNF␣-induced pathway of NFB activation in BMMs appears to differ significantly from the classical IKKdependent pathway involving serine phosphorylation of IB␣ that is active in other epithelial-derived cell types. Other nonhematopoetic systems have increasingly demonstrated components of IB␣ 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 IB␣ PTK to activate NFB in the absence of IB␣ proteolytic degradation. In the present study, we have sought to clarify several issues regarding the involvement of this IB␣ PTK pathway in mediating NFB transcriptional activation in epithelial-derived cells following H/R.
Using comparative cell line models to evaluate both IB␣ serine and tyrosine phosphorylation-dependent pathways of NFB transcriptional activation, our studies have further characterized an IKK-independent pathway that regulates NFB through c-Src activation. Studies using IB␣ phosphorylation mutants and SYF knockout cells demonstrated that c-Src-mediated transcriptional activation of NFB functionally requires tyrosine, but not serine, phosphorylation of IB␣. Using this HeLa cell model system, we found no functional requirement for IB␣ tyrosine phosphorylation in the transcriptional activation of NFB following TNF␣ stimulus and no evidence for tyrosine phosphorylation of IB␣ following TNF␣ treatment. Furthermore, our studies evaluating IKK-dominant mutants demonstrate, for the first time, the lack of IKK involvement in PTK-mediated pathways of NFB transcriptional activation vanadate (100 M) (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. NFB transcriptional activity was assessed as the mean relative luciferase activity (RLA) (Ϯ S.E., n ϭ 6). For evaluation of I〉␣ phosphorylation, cells were treated with pervanadate (100 M) (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 g of total protein was immunoprecipitated with anti-IB␣ antibody followed by Western blot analysis with an antiphosphotyrosine antibody or anti-IB␣ antibody. following pervanadate or H/R treatments, while confirming the selective activation of NFB by TNF␣ as mediated through serine phosphorylation of IB␣. These functional studies evaluating the transcriptional activation of NFB following three independent stimuli suggest that little overlap, if any, exists in IKK and PTK pathways controlling the IB␣⅐NFB 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 NFB-requiring IB␣ tyrosine phosphorylation, with consistent results observed in HeLa cells, T-cells, and BMMs. Second, unlike HeLa cells, treatment of BMMs with TNF␣ results in significant activation of NFB in a manner dependent on IB␣ phosphorylation of tyrosine 42 (9). This difference underscores the importance of cell type-specific dependences in the activation of NFB and IB␣ protein tyrosine kinases. Third, our current studies are the first to directly evaluate the transcriptional activation of NFB using an NFB-responsive reporter gene. To date, all assays for NFB activation following stimulation of IB␣ tyrosine phosphorylation had been performed using NFB DNA binding.
Intracellular production of ROS has been implicated in the regulation of numerous signal transduction cascades and in the activation of NF〉 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 NFB. However, other pathways of NFB activation involving pro-inflammatory stimuli such as TNF␣ 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 NFB 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 NFB activation involving tyrosine phosphorylation of IB␣. 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 IB␣ tyrosine phosphorylation and NFB activation, suggests a preference for H 2 O 2 and/or hydroxyl radicals (as a product of H 2 O 2 ) in the activation of the IB␣ tyrosine kinase. These findings are consistent with previous reports demonstrating the direct activation of IB␣ tyrosine phosphorylation by H 2 O 2 in T-cells (11).
Recent evidence has suggested that the p85 subunit of PI3kinase associates with tyrosine 42 phosphorylated IB␣ but not with unphosphorylated IB␣ (8). The catalytic p110 subunit also appears to be critical in the activation of NFB. The function of p110 may involve phosphorylation of NFB and/or dissociation of the IB␣: NFB 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)(32)(33), it is plausible that c-Src may act on this PI 3-kinase complex to mediate NFB activation. It has been previously demonstrated that c-Src is at least partially required for IB␣ tyrosine phosphorylation in BMMs following TNF␣ treatment (9). However, these studies performed in c-Src (Ϫ/Ϫ) BMMs demonstrated a delay only in the induction of p50/p65 heterodimers in the nucleus following the TNF␣ 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 IB␣ tyrosine phosphorylation and NFB 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 NFB activation through IB␣ tyrosine phosphorylation. Our studies, for the first time, have also successfully reconstituted IB␣ tyrosine phosphorylation with immunoprecipitated c-Src in an in vitro kinase assay. Furthermore, the activity of c-Src IB␣ 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 IB␣ and that this phosphorylation event is required for NFB activation following H/R or pervanadate treatments. The physiologic relevance of redundant Src family kinases in the activation of NFB still remains unclear. However, the redox-regulated mechanisms that control activation of NFB by Src family kinases may be a particularly relevant therapeutic target for organ damage following I/R injury.