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Am J Physiol Heart Circ Physiol 292: H1675-H1684, 2007. First published January 5, 2007; doi:10.1152/ajpheart.01132.2006
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CALL FOR PAPERS
Oxygen Sensing: Life and Death of a Cell

PKC-{alpha} and TAK-1 are intermediates in the activation of c-Jun NH2-terminal kinase by hypoxia-reoxygenation

Donna P. Frazier, Amber Wilson, Christopher J. Dougherty, Huifang Li, Nanette H. Bishopric, and Keith A. Webster

Department of Molecular and Cellular Pharmacology, Vascular Biology Institute, University of Miami School of Medicine, Miami, Florida

Submitted 16 October 2006 ; accepted in final form 1 January 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
c-Jun NH2-terminal kinase (JNK), a member of the MAPK family of protein kinases, is a stress-response kinase that is activated by proinflammatory cytokines and growth factors coupled to membrane receptors or through nonreceptor pathways by stimuli such as heat shock, UV irradiation, protein synthesis inhibitors, and conditions that elevate the levels of reactive oxygen intermediates (ROI). Ischemia followed by reperfusion or hypoxia with reoxygenation represents a condition of high oxidative stress where JNK activation is associated with elevated ROI. We recently demonstrated that the activation of JNK by this condition is initiated by ROI generated by mitochondrial electron transport and involves sequential activation of the proline-rich kinase 2 and the small GTP-binding factors Rac-1 and Cdc42. Here we present evidence that protein kinase C (PKC) and transforming growth factor-beta-activated kinase-1 (TAK-1) are also components of this pathway. Inhibition of PKC with the broad-range inhibitor calphostin C, the PKC-{alpha}/beta-selective inhibitor Go9367, or adenovirus-expressing dominant-negative PKC-{alpha} blocked the phosphorylation of proline-rich kinase 2 and JNK. Reoxygenation activated the mitogen-activated protein kinase kinase kinase, TAK-1, and promoted the formation of a complex containing Rac-1, TAK-1, and JNK but not apoptosis-stimulating kinase-1 or p21-activated kinase-1, which was detected within the first 10 min of reoxygenation. These results identify two new components, PKC and TAK-1, that have not been previously described in this signaling pathway.

cardiac myocyte; mitochondria; proline-rich tyrosine kinase 2; transforming growth factor-beta-activated kinase-1; protein kinase C; hydrogen peroxide

THE MITOGEN-ACTIVATED PROTEIN kinase (MAPK) family comprises three signaling pathways, each with distinct terminal kinases, including the extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and the p38 MAPK (reviewed in Ref. 51). These pathways fulfill fundamental roles in cell growth, cell function, differentiation, and the response to stress by transmitting extracellular signals from the cell membrane to nuclear and cytoplasmic targets. Each MAPK pathway or module contains an evolutionarily conserved three-component cascade of dual-specificity kinases that transmit an activation signal from its source through a phosphoryl relay chain to the final kinase. The three component relay kinases are respectively referred to as MAPK, MAP kinase kinase (MKK), and MAP kinase kinase kinase (MKKK). Multiple individual kinases at the MKKK positions of each module confer differential affinity and selectivity for specific stimuli. In particular, transforming growth factor-beta (TGF-beta)-activated kinase-1 (TAK-1), p-21-activated kinase-1 (PAK-1) and apoptosis-stimulating kinase-1 (ASK-1) have each been linked with MAPK signaling (reviewed in Refs. 37 and 41). Differential responses to any stimuli is channeled by scaffold proteins that physically colocalize specific module components (19, 62, 64). Multiple MKKKs in different modules may be activated by the same stimuli but by different degrees so that the net outcome of any stimulation is a function of the relative activity of each MAPK (29, 34, 35). The downstream targets include transcription factors, structural proteins, enzymes, other kinases, and multiple survival determination factors, each with a specific affinity for each terminal kinase. Activation of the ERK pathway typically confers growth and survival responses, whereas p38 and JNK may promote cell death so that the cellular fate in response to any stimuli is a function of the relative levels of activation of each MAPK (32, 63).

Oxidative stress is perhaps the most common environmental stress to be imposed on biological systems and may result from direct exposure to extracellular oxidizing agents such as H2O2 and menadione (1, 54) or secondary to other primary stimuli such as UV irradiation or hyperglycemia (12, 23). Pathologically, the most frequent oxidative stress occurs when tissues are subject to ischemia-reperfusion, a condition that is most often associated with vascular disease (28, 49, 58). ERK, JNK, and p38 MAPKs are all activated in cardiac myocytes or neuronal cells exposed to H2O2 treatment or after exposure to hypoxia and reoxygenation (26, 42, 49, 60). JNK is strongly induced by oxidative stress in cultured cardiac myocytes and in intact hearts (2526) and may be a critical determinant of reperfusion injury in the myocardium as well as in the brain (8, 20). We previously reported that signals originating in the mitochondria linked to calcium flux provide the initiating stimuli for JNK activation in cardiac myocytes subjected to hypoxia-reoxygenation (13). The pathway includes the proline-rich kinase 2 (Pyk2) and the small G proteins Rac-1 and Cdc42. These studies demonstrated a link between mitochondria and JNK activation but did not identify the nature of the communication between calcium and Pyk2, the most upstream kinase so far identified in this pathway.

PKC is a family of serine/threonine kinases, which presently consist of at least 11 isozymes (reviewed in Refs. 16 and 10). These isozymes have been classified into three groups according to their structure and cofactor regulation. Conventional PKCs ({alpha}, beta1, beta2, and {gamma}) are activated by calcium or diacylglycerol (DAG); novel PKCs ({delta}, {epsilon}, {sigma}, {phi}, µ) are independent of calcium but responsive to DAG. Atypical PKCs are independent of calcium and DAG but require membrane phospholipids for activation. Different PKC isoforms may participate in both receptor and nonreceptor-mediated activation of JNK, ERK, and p38 in a manner that is context, time, and tissue dependent (reviewed in Ref. 46). JNK activation through heterotrimeric G{alpha}q receptors stimulated by angiotensin II and endothelin-1 have been described in detail (22, 30, 36, 48). Both involve Pyk2, and the endothelin-1 pathway requires PKC-{epsilon} (33). JNK activation by {alpha}1-adrenergic stimulation of PC12 cells on the other hand is both calcium and PKC independent (4). The PKC isozymes required for the activation of JNK by phorbol esters are also tissue specific. In myeloid leukemia cells, phorbol esters activate JNK through PKC-beta but in small-cell, human lung cancer cells, the same stimulus that involves PKC-{alpha} and -{epsilon} (27).

Neonatal rat ventricular myocytes express one conventional PKC isoform (PKC-{alpha}), two novel isoforms (PKC-{delta} and -{epsilon}), and one atypical isoform (PKC-{lambda}) (17). The conventional and novel isoforms are all activated by hypoxia and/or reoxygenation in cardiac myocytes. Because PKC can mediate the phosphorylation of Pyk2 in a calcium-dependent manner, we investigated whether PKC activation is necessary for the activation of Pyk2 and JNK by reoxygenation. We report here that the activation of both Pyk2 and JNK by reoxygenation was indeed blocked by a broad-spectrum PKC inhibitor, a PKC-{alpha} selective inhibitor, and by adenoviral vector expressing dominant-negative PKC-{alpha} but not dominant-negative PKC-{delta}. Downstream of this interaction we present evidence that TAK-1 is activated and binds to a complex that includes a MKK, JNK, and Rac-1. The results identify PKC-{alpha} and TAK-1 as components of the signaling pathway linking reoxygenation to JNK. Furthermore, we found that H2O2 stimulates JNK by a pathway distinct from reoxygenation that involves ASK-1 instead of TAK-1 (50, 52).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Antibodies to JNK-1 and -2, TAK-1, ASK-1, PAK-1, and hemagglutinin (HA) were from Santa Cruz Biotech (Santa Cruz, CA). Antibodies against ERK, phospho-JNK, and phospho-Pyk2 were from New England Biolabs (Beverly, MA). Hoechst 33342 and propidium iodide dyes were from Calbiochem (La Jolla, CA). Recombinant adenoviral vectors including dominant-negative (dn)JNK, constitutively active (ca)MEKK-1, dnPKC-{alpha}, dnPKC-{delta}, and GFP have been described previously (14, 65). Wild-type (wt)TAK-1 was a gift from Dr. Anton M. Jetton, (Cell Biology Section, National Institute of Environmental Health Sciences, Bethesda, MD) and the kinase-negative (kn) forms of TAK-1, pSRa-HA-TAK-1 and pSRa-HA-knTAK-1 (44), were gifts from Dr. Eisuke Nishida (Department of Genetics and Molecular Biology, Kyoto University, Sakyo-ku, Japan). Plasmid-encoding HA-JNK (35) was a gift from Dr. Silvio Gutkind (Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, Bethesda, MD). Inhibitors calphostin C and Go9367 were from Calbiochem.

Cell culture. All procedures involving animals were performed in accordance with institutional guidelines for the care and use of animals and approved by the institutional ethics committee. Methods for primary culture of neonatal rat cardiac myocytes have been previously described (55, 56). In brief, enriched cultures of myocyte and nonmyocyte cells were obtained from 1- to 2-day-old neonatal rats by stepwise trypsin dissociation and plated at a density of 4 x 106/60-mm dish or on two-well glass dishes (Nunc) at a density of 4 x 105 cells/cm2 in minimal essential medium supplemented with 5% fetal calf serum, penicillin, and streptomycin (MEM + 5% FCS). After 3–5 days, cells were rinsed three times in MEM and transferred to a defined serum-free DMEM or MEM supplemented with transferrin, vitamin B12, and insulin. The final cultures contained >95% cardiac myocytes contracting at >200 beats/min. Bromodeoxyuridine (0.1 mM) was included in the media for the first 3 days after plating to inhibit fibroblast growth.

Hypoxia and reoxygenation. Details of our methods for exposing cells to hypoxia have been described previously (5, 13, 57). Oxygen and pH were continuously monitored by using appropriate electrodes, and contractility was monitored by edge detection as described (55, 56). The medium oxygen concentration was maintained at <10 mmHg. For reoxygenation, plates were removed from the chamber and reoxygenated by replacing the medium with oxygenated medium or by gentle mixing without media change and incubated under 21% O2 (air-5% CO2).

Kinase assays. Our methods for the assay of JNK activity have been described in detail elsewhere (13, 14, 57), and the MKKK assays (TAK-1, ASK-1, and PAK-1) were modifications of these procedures using the appropriate antibodies and substrates as indicated in the figure legends. Briefly, myocytes were lysed in 150 µl of ice-cold lysis buffer with protease inhibitors; equal amounts of precleared extracts were incubated on ice with 6 µl of antibody (anti-JNK1/JNK2, anti-ERK, anti-HA, anti-TAK-1, anti-ASK-1, and anti-PAK-1) and protein A agarose beads. The beads were pelleted and washed twice with lysis buffer and resuspended in kinase buffer containing (in mM) 25 HEPES (pH 7.4), 25 MgCl2, 2 DTT, 0.1 sodium vanadate, and 25 beta-glycerophosphate with 5 µg of purified c-Jun141 fusion protein, 5 µg myelin basic protein or 5 µg MKKK substrate, and ATP with 10 µCi 32P-ATP. Samples were incubated at 30°C for 30 min. Reactions were electrophoresed on 12% SDS-polyacrylamide gels as described previously (25, 57). Kinase activity was quantitated by analyzing densitometry of fragments on digitized images using NIH Image 1.60 with Adobe Photoshop 4.0.

Subcellular fractionation. Cardiac myocytes were fractionated as described previously (25); briefly, cells were washed with PBS, incubated in hypotonic lysis buffer [containing (in mM) 100 mannitol, 10 Tris, 5 MgCl2, 1 EGTA, and 1 DTT and Roche Complete Protease Inhibitor Cocktail], and lysed by 50 strokes of a Dounce homogenizer with a tight-fitting pestle. Unbroken cells and nuclei were pelleted at 1,000 g for 5 min. The mitochondrial-enriched fraction was pelleted at 12,000 g for 20 min. The cytoplasmic fraction was obtained by centrifuging supernatants at 100,000 g for 30 min and collecting the supernatant. Mitochondrial pellets were washed one time in hypotonic lysis buffer and resuspended in radioimmunoprecipitation assay lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and Roche Complete Protease Inhibitor Cocktail in PBS).

Western blot and immunoprecipitation analyses. Detailed procedures for Western blot analyses have been described previously (13, 14, 25). Briefly, cultures were harvested in ice-cold lysis buffer [50 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and 50 mM NaF] with freshly added 1 mM Na3VO4, 0.5 mM DTT, 1 mM PMSF, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. Equal amounts of protein from cleared lysates were fractionated on 12% or 15% SDS-polyacrylamide gels and electroblotted to nitrocellulose (Bio-Rad). Blots were stained with Ponceau red to monitor the transfer of proteins. Membranes were blocked for 1 h at room temperature with 5% nonfat milk in TBS (in mM: 25 Tris, 137 NaCl, and 2.7 KCl) containing 0.05% Tween-20, and incubated with specific antibodies for 2–4 h in the same buffer. After being washed, the blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody and visualized by using enhanced chemiluminescence (Pierce). Immunoprecipitation assays followed the same procedures, except that cleared lysates were preincubated with specific antibodies (JNK and TAK-1) and complexes separated using protein G agarose before gel electrophoresis (45).

Analysis of apoptosis. DNA fragmentation analyses (ladders) and quantitative apoptosis assays were implemented exactly as described previously (14, 25, 57). Apoptotic cardiac myocytes were scored for morphological evidence of apoptosis or necrosis after staining by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, Hoechst 33342, or propidium iodide, combined with immunostaining of sarcomeric myosin.

Adenoviral infections. Cardiac myocyte cultures were placed in serum-free DMEM-medium 199 (4:1), containing glucose, transferrin, insulin, and vitamin B12, 24 h before infection. Myocytes were infected at 5 plaque-forming units/cell 24 h before experiments as described previously (14), and media was replaced after 12 h.

Statistical analysis. Results are expressed as means ± SE. Differences between means were evaluated by two-tailed Student's t-test. ANOVA was carried out by using InStat 2.0 statistical software for Macintosh.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Activation of Pyk2 and JNK by reoxygenation is blocked by PKC inhibitors. Our previous results indicated that the activation of JNK by reoxygenation required Pyk2 phosphorylation and was blocked by calcium chelators and calcium channel blockers (13). This indicates a calcium-dependent step as part of the initiating stimulus for Pyk2 and JNK activation by reoxygenation. Conventional and novel PKC isoforms are activated by hypoxia and/or reoxygenation of cardiac myocytes; therefore, we asked whether PKC inhibitors blocked Pyk2 and JNK activation. Calphostin C is a broad spectrum PKC inhibitor that inhibits DAG binding to PKC, thereby inhibiting all conventional and novel PKC isoforms. As shown in Fig. 1A, calphostin C significantly blocked the activation of both Pyk2 and JNK by reoxygenation without affecting the protein levels. Calphostin C also reduced Pyk2 and JNK activity in the presence of PMA, but JNK activation by anisomycin was not affected. Calphostin C stimulated JNK activity in cardiac myocytes subjected to aerobic but not hypoxic incubation. This effect has not been reported previously and may reflect the activation of a stress pathway that requires respiring cells. There was no parallel induction of Pyk2 by calphostin C, suggesting that this mechanism of JNK activation is distinct from that mediated by hypoxia-reoxygenation. Quantification of these results is shown in Fig. 1B. To further define the role of PKC isoforms, cardiac myocytes were pretreated with the conventional PKC-{alpha} selective inhibitor Go6976. These results are shown in Fig. 2. Go6976 also strongly inhibited both Pyk2 and JNK activation by hypoxia-reoxygenation. Pyk2 activation by reoxygenation was significantly inhibited at all time points after 5 min, and Go6976 also blocked Pyk2 activation by PMA as expected. JNK activation by reoxygenation or PMA was similarly inhibited by Go6976, but the activation by anisomycin was not affected.


Figure 1
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  		Fig. 1. Effects of calphostin C on proline-rich tyrosine kinase 2 (Pyk2) and JNK activity by hypoxia-reoxygenation. A: contracting cardiac myocytes were cultured under air or hypoxic (Hypox) conditions or reoxygenated (Reox) as described in METHODS. Calphostin C at a concentration of 1 µM was added 1 h before cell isolation of aerobic and hypoxic cultures or 10 min before reoxygenation. In parallel, cardiac myocytes were treated with anisomycin (100 nM) for 1 h or with PMA for 30 min as positive controls for JNK and Pyk2 phosphorylation, respectively. The cell lysates were harvested and analyzed by Western blot with phospho (p)-Pyk2, p-JNK1/2, Pyk2, and JNK1/2 antibodies. B: quantification from 3 separate experiments; p-Pyk2 levels are normalized to PMA; p-JNK levels are normalized to anisomycin. Shown are 5 min, 10 min, 30 min, and 1 h Reox. *P < 0.05 and **P < 0.01, comparing calphostin C treated with untreated. Black bars, control; gray bars, calphostin C.

 

Figure 2
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  		Fig. 2. Effects of Go6976 on Pyk2 and JNK activity by hypoxia-reoxygenation. A: conditions were identical to those described in Fig. 1, except that cardiac myocytes were treated with Go6976 (1 µM) instead of calphostin C. B: quantification from 4 separate experiments. Shown are 5 min, 10 min, 30 min, 1 h, and 2 h Reox. *P < 0.05 and **P < 0.01, comparing Go6976 treated with untreated. Black bars, control; gray bars, Go6976.

 
Dominant-negative PKC isoforms block Pyk2 and JNK activation. These results implicate the PKC-{alpha} isoform in the pathway of Pyk2 and JNK activation by hypoxia-reoxygenation as well as by phorbol ester. This result is consistent with several reports that have placed PKC upstream of Pyk2 in response to different stimuli (2, 3, 7, 18). To confirm the role of PKC-{alpha}, cardiac myocytes were infected with adenoviral vectors expressing dominant-negative forms of PKC-{alpha}, PKC-{delta}, or GFP (control). Each PKC mutant contains a lysine to arginine point mutation in the ATP-binding domain that renders them incapable of phosphorylating downstream substrates but competent to compete with the endogenous proteins. Myocytes were infected with 5 plaque-forming units virus/cell, a concentration that mediated 78% infection but did not alter morphology or contractility, estimated microscopically using an edge detection system motion analysis (55, 56). Results from these experiments are shown in Figs. 3 and 4. Treatment with adenovirus (Ad)-dnPKC-{alpha} (Fig. 3) significantly inhibited reoxygenation-mediated Pyk2 and JNK activity at both time points examined. PMA-mediated kinase activation was also blocked by Ad-dn-PKC-{alpha}, but there was no effect on JNK activation by anisomycin. Infection with Ad-dnPKC-{delta} did not inhibit Pyk2 or JNK activation by reoxygenation or PMA (Fig. 4). These results further support a role of PKC in the activation of Pyk2 and JNK by hypoxia-reoxygenation and implicate PKC-{alpha}.


Figure 3
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  		Fig. 3. Inhibition of Pyk2 and JNK by dominant-negative (dn) PKC-{alpha}. Top: cardiac myocytes were infected with adenovirus (Ad)-GFP or Ad-dnPKC{alpha}, as described in METHODS, and subjected to aerobic, hypoxic, or reoxygenation conditions as in Figs. 1 and 2. Cell lysates were analyzed by Western blot analysis for p-JNK, p-Pyk2, total JNK, and total Pyk2. Bottom: quantification from 3 separate experiments. Shown are 10 min and 1 h Reox. *P < 0.05 and **P < 0.01, comparing Ad-dnPKC treated with Ad-GFP. Black bars, Ad-GFP; gray bars, Ad-PKC-{alpha}-kinase dead (KD).

 

Figure 4
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  		Fig. 4. Effect of dominant-negative PKC-{delta}. Top: cardiac myocytes were infected with Ad-GFP or Ad-dnPKC{delta} and subjected to aerobic, hypoxic, or reoxygenation conditions. Cell lysates were then analyzed by Western blot analysis for p-JNK, p-Pyk2, total JNK, and total Pyk2. Bottom: quantification from 3 separate experiments. Changes were not significant.

 
Subcellular distribution of JNK, Pyk2, and PKC. PKC enzymes receive localization signals from anchoring proteins, including receptors for activated C-kinase, A-kinase-anchoring proteins, inhibitory proteins, and substrates in addition to lipids and calcium (6, 38). Activation involves a transient translocation of PKC from the soluble to the mitochondrial fraction followed by further translocation of activated kinases to reach substrates in different subcellular compartments. To determine the subcellular compartmentalization of the hypoxia-reoxygenation signaling components, cardiac myocytes were exposed to air, hypoxia, or hypoxia and reoxygenation, and the cells were fractionated. Total protein and phosphorylated proteins were analyzed by Western blot analysis of mitochondrial and soluble subcellular fractions as described in METHODS. A representative blot is shown in Fig. 5. In agreement with previous reports (17, 27), we found that PKC was present in soluble and mitochondrial fractions with greater amounts in the mitochondrial fraction under all conditions. There was a small apparent shift in the distribution of PKC-{delta} from the mitochondrial to the soluble fraction during hypoxia, in agreement with Goldberg et al. (17), but no major detectable change in the distribution of PKC-{alpha}. JNK and Pyk2 were present in soluble and mitochondrial fractions, and there was no detectable change in the distribution of either protein in response to treatments. Despite the presence of a significant amount of Pyk2 in the cytoplasm, phospho-Pyk2 was almost exclusively in the membrane fraction after reoxygenation. In sharp contrast, phospho-JNK was present predominantly in the soluble fraction. These observations are consistent with a signaling pathway in which Pyk2 is phosphorylated in association with membranes, perhaps the mitochondria and perhaps with PKC-{alpha}. Phospho-JNK may translocate to the soluble cell fraction after phosphorylation in the mitochondrial fraction, or it may be phosphorylated within a complex in the cytoplasm (see Fig. 6).


Figure 5
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  		Fig. 5. Subcellular distribution of protein kinases during treatments. Cardiac myocytes were exposed to aerobic, hypoxic, and reoxygenation conditions as described in METHODS and Figs. 1–4. Cultures were harvested at various time points in hypotonic lysis buffer, subjected to dounce homogenization, and fractionated by differential centrifugation. The whole cell/nuclear fraction was discarded, and the heavy (mitochondrial) membrane and soluble (cytoplasmic) fractions were analyzed by Western blot analysis and probed with antibodies to PKC-{alpha}, PKC-{delta}, JNK, Pyk2, p-JNK, and p-Pyk2. The blot was also probed with the mitochondrial-localizing enzyme cytochrome oxidase subunit IV (COX IV) to determine fractional purity. Results are representative of 2 separate experiments.

 

Figure 6
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  		Fig. 6. Activation of transforming growth factor-beta-activated kinase-1 (TAK-1) by hypoxia (Hx)-heoxygenation. A: cardiac myocytes were subjected to hypoxia-reoxygenation, H2O2 (30 min, 100 µM), or PMA (1 h, 100 nM) as indicated and lysates were subjected to immunoprecipitation (IP) with anti-apoptosis-stimulating kinase-1 (ASK-1), anti-p21-activated kinase-1 (PAK-1), or anti-TAK-1 antibodies, and kinase activity was measured using mitogen-activated protein (MAP) kinase kinase (MKK)6 as substrate, as described in METHODS. Right: quantification. *P < 0.05 compared with hypoxia control (n = 3 experiments). B: lysates from treated cardiac myocytes were subjected to IP with anti-TAK-1 or anti-PAK-1 antibody, and proteins were analyzed by Western blot analysis using anti-JNK or ERK antibodies as described in METHODS. Squares, TAK-1 IP, JNK blot; closed circles, PAK-1 IP, JNK blot; open circles, PAK-1 IP, ERK blot. TAK-1 binding to JNK after 5 and 10 min of reoxygenation was significantly greater than the air control (P < 0.05; n = 3 experiments). There was no significant change in the binding of JNK to PAK-1 during reoxygenation (n = 3 experiments).

 
TAK-1 is activated by reoxygenation. We recently reported that reoxygenation activated the small G protein Rac-1, and the activation of JNK was dependent on activated Rac-1, thus confirming the presence of this intermediate in the signaling pathway (13, 26). We next investigated the nature of the kinase(s) that interact with Rac-1 in this pathway. TAK-1, PAK-1, and ASK-1 have all been linked with the stress-activated JNK cascade (44, 52). TAK-1 is the major MKKK downstream of Rac-1 in the activation of JNK by ceramide, inflammatory cytokines, and sorbitol (44); PAK-1 is downstream of Rac-1 in the signaling pathway initiated by ANG II (36); and ASK-1 is activated by H2O2 (52). To determine whether any of these kinases were activated in cardiac myocytes by reoxygenation, pull-down assays and coimmunoprecipitation were used. Lysates from untreated cardiac myocytes and myocytes subjected to hypoxia-reoxygenation or H2O2 were immunoprecipitated with anti-PAK-1, anti-TAK-1, or anti-ASK-1 antibodies, and the precipitated proteins were assayed for kinase activity using MKK6 substrate as described in METHODS. As shown in Fig. 6, PAK-1 and TAK-1 were activated by reoxygenation but not by H2O2 treatment. ASK-1 was not activated by reoxygenation but was strongly activated by H2O2 treatment. In Fig. 6B, cell lysates were immunoprecipitated with either anti-TAK-1 or anti-PAK-1 antibodies, and Western blots were probed with anti-JNK or -ERK antibodies. After reoxygenation, TAK-1 but not PAK-1 became associated with JNK. PAK-1 selectively bound to an ERK-containing complex (Fig. 3B, bottom). These results suggest that TAK-1 is recruited to a reoxygenation-activated complex that includes MKK as well as JNK.

JNK activation is blocked by dnTAK-1. To further investigate the role of TAK-1 in this pathway, cardiac myocytes were cotransfected with HA-JNK and TAK-1 mutants with dominant-negative or transactivating functions as described previously (13). Transfected cultures were exposed to hypoxia-reoxygenation (Fig. 7), and the activity of HA-JNK was measured. JNK activation by reoxygenation was blocked equally by dnJNK or dnTAK-1. Cotransfection with caMEKK1 or wtTAK-1, included as controls, did not effect HA-JNK activation by reoxygenation; both of these vectors activated JNK in aerobic controls (not shown). In agreement with previous reports, the activation of JNK by anisomycin was not inhibited by dnTAK-1 (not shown) (9). Quantification of these effects is shown in Fig. 7B. These results support the results of immunoprecipitation assays, both implicating TAK-1 in the pathway of reoxygenation-mediated JNK activation.


Figure 7
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  		Fig. 7. Selective effects of interfering mutants on JNK activation by reoxygenation. A: representative kinase assays from cardiac myocyte cultures cotransfected with hemagglutinin (HA)-JNK and an empty pcDNA3 vector or with the different kinase mutants as indicated. Myocytes were cotransfected with HA-JNK (5 µg) and the indicated interfering mutant kinase (2 µg), and JNK assays were performed after aerobic incubation or after hypoxia-reoxygenation as described in METHODS. HA-JNK activity was assayed after IP using anti-HA as described in METHODS, and total HA-JNK was detected by Western blot analysis. B: quantification of HA-JNK activation. ca-MEKK1, constitutively active MAP kinase kinase kinase 1; C, control. *dnJNK and dnTAK-1 significantly inhibited HA-JNK activity and dnJNK significantly inhibited HA-JNK activation by wild-type (WT) TAK-1 (P < 0.05; n = 3 experiments for both).

 
JNK activation is protective in this model, and blocking JNK activity with kinase-dead mutants increases cell death (13, 14). To determine whether interference of JNK activation by inhibition of TAK-1 affected the death pathway, we measured apoptosis in reoxygenated myocytes after infection with dnTAK-1 or wtTAK-1. In agreement with our previous report, we found that dnTAK-1 increased apoptosis caused by hypoxia-reoxygenation from 28 ± 3.7 to 46 ± 10.2% (P < 0.01, n = 4), whereas wtTAK-1 did not effect the level of apoptosis (data not shown). These results also support a role for TAK-1 in the pathway of JNK activation by reoxygenation.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We present evidence for the presence of two new intermediates, PKC-{alpha} and TAK-1, in the pathway of JNK activation by reoxygenation in cardiac myocytes. A requirement for conventional PKC isoforms was implicated by the sensitivity of the pathway to intracellular calcium modulators and the PKC-selective inhibitor calphostin C. By blocking the binding of DAG to PKC, calphostin C inhibits all conventional and novel PKC isoforms. Because neonatal cardiac myocytes express predominantly the PKC-{alpha} conventional isoform and the PKC-{delta} and -{epsilon} novel isoforms, these are probably the principal targets for calphostin C in these cells. Only the conventional isoforms require both calcium and DAG. Inhibition of the pathway by Go6976 further implicates PKC-{alpha} because this inhibitor is selective for the conventional PKC isoforms and PKD (PKC-{lambda}). Inhibition by dominant-negative PKC-{alpha} but not PKC-{delta} is also consistent with a selective role for PKC-{alpha} in this pathway. In these analyses, PMA and anisomycin were used as positive controls for Pyk2 and JNK activation, respectively. PMA-mediated JNK activation is cell-type specific (27). In our experiments, the activation of Pyk2 and JNK by PMA was weaker than that mediated by reoxygenation, suggesting that PKC-{alpha} is only part of the signaling pathway and that other factors are required for full activation. The activation of JNK by anisomycin was not affected by inhibitors of PKC, confirming our previous report that different pathways are responsible for the activation of JNK by reoxygenation and anisomycin. Although PKC-{alpha} is implicated, our results do not eliminate other intermediate kinases, such as PKC-{epsilon} or atypical PKC isoforms. Both PKC-{alpha} and -{epsilon} have been implicated in the activation of JNK by PMA in cancer cells (27).

Calcium flux is a critical element of the reoxygenation-mediated signaling pathway in cardiac myocytes. The pathway requires contractility and calcium flux, as illustrated by the sensitivity to the calcium channel blocker nifedipine, and the dependence of Pyk2 and JNK activation on active contractility at the time of reoxygenation (13, 14). In our model of chronic hypoxia, respiration and oxidative phosphorylation are inhibited but ATP levels are maintained by anaerobic glycolysis (55, 56). Calcium accumulates slowly during hypoxia but rapidly during reoxygenation when electron flow is resumed (11, 31). Intracellular calcium levels have been reported to increase approximately fivefold during hypoxia-reoxygenation (43). Our results suggest that this increase of intracellular calcium is a component of the signal that activates the Pyk2-JNK pathway, possibly by contributing to the activation of PKC-{alpha}.

Previous work has linked TAK-1 with JNK activation by ceramide, proinflammatroy cytokines (IL-1), and UV irradiation (44). Using immunoprecipitation kinase assays, we found that reoxygenation activated TAK-1 and PAK-1. After reoxygenation, TAK-1 but not PAK-1 became physically associated with a protein complex that included JNK. In support of a direct role of TAK-1, dnTAK-1 eliminated JNK activation. The kinetics of TAK-1 association with the JNK protein complex after reoxygenation and the selectivity for TAK-1 over ASK-1 and PAK-1 are consistent with a primary role for TAK-1 as the MKKK component of this pathway.

The signaling pathway for JNK activation by hypoxia-reoxygenation shares intermediates with both receptor and nonreceptor pathways. It parallels pathways activated by ANG II and UV irradiation in the components Pyk2 and Rac-1 and to ceramide and proinflammatory cytokines in TAK-1 at the MKKK position (44). The reoxygenation pathway differs in a number of respects from that mediated by H2O2 where the signal is channeled through Src, growth-factor receptor-bound protein 2-associated binder-1 (Gab1), and ASK-1 and may involve stimulation of the TNF-{alpha} receptor (21, 39, 52). Our studies confirm that ASK-1 is activated by H2O2 but not by reoxygenation. The activation of distinct signaling pathways by reoxygenation and H2O2 treatment may be related to the source of the signal as well as the nature of the reactive oxygen species. H2O2 treatment delivers extracellular oxidative stress to the receptor side of the plasma membrane, whereas oxidative stress from reoxygenation is initiated in the mitochondria and involves superoxide production by the mitochondrial electron transport chain (see Fig. 8). The absence of detectable ASK-1 activation by reoxygenation or TAK-1 activation by H2O2 suggests that there is little cross talk between these signaling pathways and that reoxygenation may not generate significant cytosolic H2O2. The activation of JNK by both H2O2 and reoxygenation involves PKC; however, PKC-{lambda} (PKD) may be the essential component in the H2O2 pathway, whereas it is PKC-{alpha} that is in the reoxygenation pathway (66).


Figure 8
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  		Fig. 8. Distinct components for JNK activation by reoxygenation and H2O2. Steps in the activation of JNK by treatment of cardiac myocytes with H2O2 include stimulation of the TNF-{alpha} receptor (TNF{alpha}R) and TRAF2 at the plasma membrane (PM) followed by sequential phosphoryl transfers between PKC, Src, growth-factor receptor-bound protein 2-associated binder-1 (Gab1), Ras, ASK-1, MKK4 or -6, and JNK (see Refs. 21, 39, 60). The Ras-binding proteins Raf-1, Rac-1, and Cdc42 may also be involved. The reoxygenation signal is initiated in the mitochondria and requires coupled electron transport and calcium (13). PKC-{alpha} is the most upstream kinase, identified in this study, and is directly or indirectly required to activate Pyk2, stimulating the recruitment of Rac-1 and/or Cdc42. The latter mediates activation of TAK-1, MKK4/6, and JNK that are physically linked by a scaffold (black bar). Cal-C, calphostin C; anti-ox, antioxidant; Traf2, TNF receptor-associated factor 2; Nif, nifedipine.

 
Oxidative stress is generated internally or externally by a diverse set of conditions, including UV irradiation, hyperglycemia, H2O2, hypoxia, reperfusion, and exposure to various xenobiotics (53). These stresses activate the MAPK modules differentially in a stimulus and cell-specific manner. The level of activation of each pathway is determined primarily by a selection of the initial kinases and G proteins determined in turn by the species, magnitude, and cellular location of reactive oxygen. A second level of regulation involves the assembly of intermediates on a scaffold to bring signaling molecules together in a stimulus-specific manner (15, 40, 47, 61). Our studies suggest that reoxygenation favors the formation of a scaffold that includes TAK-1, MKK4/6, and JNK and excludes PAK-1.

Reoxygenation associated with ischemia-reperfusion may be the most common cause of severe oxidative stress experienced by mammalian tissues (59). JNK activation by reperfusion has been described in multiple tissues, and JNK has been assigned positive and negative survival roles (14, 24). The present study confirms our previous report that JNK activation can be protective and that blocking JNK activity at multiple points of the pathway interferes with JNK activation and promotes apoptotic cell death.


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This study was supported by National Heart, Lung, and Blood Institute Grants HL-44578 (to K. A. Webster) and HL-07109 (to N. H. Bishopric) and the Walter G. Ross endowed Chair in Vascular Biology (to K. A. Webster).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. A. Webster, Dept. of Molecular and Cellular Pharmacology and Vascular Biology Inst., Univ. of Miami School of Medicine, 1600 NW 10th Ave., RMSB 6038, Miami, FL 33136 (e-mail: kwebster{at}med.miami.edu)

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.


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