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(Circulation Research. 1997;80:336-344.)
© 1997 American Heart Association, Inc.

Articles

Hypoxia/Reoxygenation Stimulates Jun Kinase Activity Through Redox Signaling in Cardiac Myocytes

Keith R. Laderoute, Keith A. Webster

the Department of Cell and Molecular Biology, SRI International, 333 Ravenswood Ave, Menlo Park, Calif.

Correspondence to Dr Keith A. Webster, Department of Molecular and Cellular Pharmacology, Rosenstiel Medical Science Building, 6th Floor, University of Miami, 1600 NW Ave, Miami, FL 33136. E-mail kwebster@chroma.med.miami.edu

	Abstract

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Hypoxia and reoxygenation are principal components of myocardial ischemia and reperfusion and have distinctive effects on the tissue. Both conditions have been associated with inflammation, necrosis, apoptosis, and myocardial infarction. Using a cell culture model of ischemia and reperfusion in which cardiac myocytes were exposed to cycles of hypoxia and reoxygenation, we report here that reoxygenation, but not hypoxia alone, caused sustained 10-fold increases in phosphorylation of the amino-terminal domain of the c-jun transcription factor. The activation was similar to treatments with anisomycin or okadaic acid and correlated with the hypoxia-mediated depression of intracellular glutathione. Reoxygenation-induced c-Jun kinase activity was reduced by preincubating myocytes during the hypoxia phase with the spin-trap agent -phenyl N-tert-butylnitrone or with N-acetylcysteine. The kinase activation was also inhibited by the tyrosine kinase inhibitor genistein but not by other protein kinase inhibitors. These results implicate unquenched reactive oxygen intermediates as the stimulus that initiates a kinase pathway involving the stress-activated protein kinases (JNKs/SAPKs) in reoxygenated cardiac myocytes.

Key Words: ischemia/reperfusion • mitogen-activated protein kinase • stress-activated protein kinase • glutathione • antioxidant

	Introduction

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Intermittent myocardial ischemia imposes two extremes of redox stress on cardiac tissues. During the ischemic phase, hypoxia triggers metabolic and ionic changes,¹ changes in cyclic nucleotide levels and protein kinase activities,^{2 3 4} reduced levels of intracellular redox buffers,^{5 6} and changes in the expression of specific genes including immediate-early stress genes.^{4 7 8 9} Persistent ischemia causes necrosis and infarction and leads to congestive heart failure.^{10 11} Reperfusion of ischemic tissue is associated with additional cell injury caused by ROIs that are generated by reoxygenation. ROIs cause cell damage directly, by oxidation of cellular components, and indirectly, through the activation of inflammation.^{12 13 14} The relative contributions of ischemia or reperfusion phases to cell damage in mild to moderate ischemic heart disease have not been determined. Reperfusion damage to intact hearts, which probably involves both necrosis and apoptosis, can be limited by preexposure to antioxidants or to antibodies that block leukocyte adhesion.^{15 16} The signals and pathways that mediate the responses of CMs to redox stress of this type have not been determined.

Stress- and mitogen-activated protein kinases (JNK/SAPK and MAPK/ERK) constitute pathways of serine, threonine, and tyrosine kinases that relay signals from extracellular stimuli to the cell nucleus (reviewed in References 17 to 20). JNKs/SAPKs were originally identified as serine/threonine kinases that phosphorylate the amino-terminal transactivation domain of the transcription factor c-Jun. MAPKs/ERKs are defined as mitogen-activated protein kinases or extracellular signal–regulated kinases (designated here as MAPKs). Activation of these pathways, through plasma membrane or cytoplasmic receptors, culminates in the translocation of a terminal kinase to the cell nucleus, where the activities of target proteins are modified by phosphorylation, leading to changes in gene expression and other cell functions. Although the JNK/SAPK and MAPK pathways are related, each pathway has distinct activating stimuli, regulatory proteins, protein kinases, and target proteins. Unlike MAPKs, the JNKs/SAPKs are characteristically activated by stress-causing stimuli, including protein synthesis inhibitors, inflammatory cytokines, UV irradiation, osmolarity changes, sodium arsenite, okadaic acid, muscarinic receptor stimulation, and heat shock (reviewed in References 17 and 21). Although the intermediate kinases and terminal targets of the two pathways are distinct, there is cross talk; a number of stimuli induce both pathways, albeit with different potencies and kinetics, and both pathways can be induced by activated Ras.²² MAPK and JNK/SAPK signaling pathways are probably involved in coordinating growth and/or repair responses in the nucleus, cytoplasm, and cytoskeleton and may in some instances be involved in the initiation of apoptosis.^{23 24 25 26}

Redox events have been directly implicated in the activation of JNKs/SAPKs or MAPKs by UV irradiation, H₂O₂, and cytokines,^{21 26 27 28 29} and redox changes may play a role in the response to other inducers. The JNK/SAPK pathway is activated by ischemia and reperfusion of kidney by a mechanism that correlates with ATP depletion.^{30 31} Modest and transient activations of CM MAPKs have been described in response to hypoxia and reoxygenation in neonatal rats and release from metabolic inhibition in chicks,^{32 33} but the initiating signals were not identified. Inductions of both JNK/SAPK and MAPK were also reported in ischemia/reperfusion models of perfused rat hearts.^{34 35}

In the present article, we show that reoxygenation of hypoxic CMs in culture initiated a potent and sustained activation of c-Jun amino-terminus kinase activity as measured by both substrate-affinity and immunoprecipitation assays. This kinase activity was strongly attenuated by antioxidants and inhibitors of tyrosine kinase. Immunocomplex assays using a polyclonal anti–JNK-2 antibody indicated that the JNK/SAPK family of kinases contributed significantly, although possibly not exclusively, to the JNK/SAPK activity. Our results suggest that this activation, referred to here as JNK/SAPK, is caused by two independent but interacting components: (1) a progressive decline of intracellular GSH during hypoxia and (2) elevated levels of unquenched ROIs generated on resumption of oxidative metabolism.

	Materials and Methods

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Cell Culture
The isolation of CMs from neonatal rat hearts has been described in detail elsewhere.^{4 36} Briefly, hearts from 30 to 40 pups were minced and subjected to serial trypsin digestion to release single cells. After the final digestion, the cells were washed and preplated for 0.5 hours in MEM with 5% fetal calf serum. Nonattached cells were replated in 60-mm Falcon dishes at 4x10⁶ cells per plate in the same medium with 0.1 mmol/L BrdU for 4 days, after which time the BrdU was removed. Cultures were used for experiments after a further 4 or 5 days. The preplating and BrdU steps reduce the background of nonmyocardial cells. These procedures reproducibly generate cultures that contract synchronously at >250 bpm, consistent with the normal beating rate of the intact rat heart.³⁷ Standard conditions of humidified air with 5% CO₂ at 37°C were used for all cultures,^{4 36} except where indicated. Cultures of NMF and C₂C₁₂ skeletal myoblasts have been described previously.^{38 39}

Hypoxia and Reoxygenation
Cultures were placed in serum-free MEM supplemented with GTIB³⁶ 24 hours before exposure to hypoxia (PO₂, 8 to 12 mm Hg). Details of our methods for exposing cells to hypoxia have been described previously.^{4 8} Oxygen was continuously monitored with an oxygen electrode (Controls Katharobic) inside the chamber, and contractility was monitored by edge detection as described previously.^{4 8} After the indicated time periods, plates were removed from the chamber and reoxygenated by exposure to the following conditions: air/5% CO₂, 50% O₂/5% CO₂/balance N₂, or 95% O₂/5% CO₂. Cells were harvested and lysed for protein kinase or GSH assays as described below. Cultures exposed only to hypoxia (without reoxygenation) were lysed under hypoxia using ice-cold deoxygenated buffers. We demonstrated previously that under the conditions of hypoxia used in the present study, ATP levels were maintained at >90% of control levels.⁴ This is because the rate of anaerobic glycolysis by cardiac cultures is induced by >10-fold within 1 hour of hypoxic exposure and because glucose does not become limiting for the duration of the experiments described here (data not shown). Cardiac myocytes retained contractility for the duration of all experiments (data not shown).

Kinase Assays
JNK/SAPK activities in cellular lysates were determined by using substrate affinity or immunocomplex assays with a recombinant GST–c-Jun (1-141) fusion protein essentially as described previously.^{19 40 41} Briefly, plasmid pGEX-2T (generous gift from J. Woodgett, Ontario Cancer Institute, Toronto, Canada) containing GST–c-Jun (1-141) cDNA was expressed as a fusion protein in Escherichia coli BL21(DE3)pLysS cells (Stratagene Cloning Systems). To extract protein, the cells were lysed by three freeze/thaw cycles, followed by sonication in PBST buffer containing 20 mmol/L sodium phosphate (pH 7.0), 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L PMSF, 20 µg/mL leupeptin, 1 mmol/L benzamidine hydrochloride, 100 µmol/L Na₃VO₄, and 50 mmol/L NaF. The fusion proteins were purified from cleared lysates by binding to glutathione-Sepharose 4B beads (Pharmacia Biotech AB) overnight at 4°C, followed by four washes with ice-cold PBST.

To prepare extracts for kinase assays, cells were washed twice with ice-cold PBS and lysed by homogenizing in buffer containing 50 mmol/L NaF, 100 µmol/L Na₃VO₄, 0.1 mmol/L PMSF, 20 µg/mL aprotinin, 0.5 µg/mL leupeptin, and 1 mmol/L benzamidine. Lysates were centrifuged at 15 000g for 15 minutes at 4°C, and the supernatants were used for the kinase assays. Samples containing 100 µg of protein were mixed with glutathione-Sepharose 4B beads with adducted GST fusion protein and gently rotated at 4°C for 1 hour. During this time, the terminal c-Jun kinase(s) binds to the protein substrate on the beads. The beads were centrifuged at 11 000g for 1 minute at 4°C and washed four times with ice-cold PBST. The beads were incubated in 20 µL of kinase buffer containing 50 mmol/L Tris-HCl (pH 7.5), 10 mmol/L MgCl₂, 100 mmol/L NaF, 1 mmol/L Na₃VO₄, 4 mmol/L ATP, and 1 µCi of [-³²P]ATP (6000 Ci/mmol, Amersham) for 30 minutes at 30°C. The reactions were stopped by adding 40 µL of lysis buffer (4.6% SDS/10% mercaptoethanol/125 mmol/L Tris-HCl, pH 6.8) and boiling for 5 minutes.

Immunocomplex JNK/SAPK and MAPK activities were determined after immunoprecipitation of cleared cell lysates with polyclonal antibodies specific for JNK-1+JNK-2 or ERK-1 (Santa Crux Biotechnology Inc) as described previously.^{20 23 40} Briefly, treated cells were washed twice with ice-cold PBS as described above and lysed in buffer containing 50 mmol/L NaF, 100 µmol/L Na₃VO₄, 0.1 mmol/L PMSF, 20 µg/mL aprotinin, 0.5 µg/mL leupeptin, 1 mmol/L benzamidine-HCl, 10 nmol/L okadaic acid, and 0.1% Triton X-100 at 4°C. Cleared lysates (100 µg protein) were incubated with 2 µL of antisera for 4 hours at 4°C, and the immunocomplexes were recovered by mixing with protein G–Sepharose. Immunocomplex kinase assays were performed as described above using 2 µg of purified GST–c-Jun (1-141) or 20 µg of MBP (Sigma Chemical Co) as substrates.

Kinase reactions were resolved in 12% (GSH–c-Jun) or 15% (MBP) discontinuous SDS-polyacrylamide gels. The gels were stained with Coomassie blue as a second check for protein loading, dried, and exposed to Kodak X-AR film for autoradiography. The GST–c-Jun (1-141) assay usually produced three prominent bands after autoradiography. Western blots of the gels probed with a polyclonal anti–Jun-family antibody (Santa Cruz Biotech, Inc) indicated that phosphorylated GST–c-Jun migrated as a doublet. This is consistent with a previous report describing kinase assays with GST–c-Jun (1-141).¹⁹ A third, faster migrating, unidentified product did not cross-react with the c-Jun antibody in Western blots (not shown). For clarity, figures were trimmed to show only the bands of interest corresponding to the c-Jun amino-terminal peptide(s). Densitometry was performed on autoradiographs using a Lynx video densitometer (Applied Imaging Corp).

Western Blots
For Western blot analyses, cells were lysed with 0.5% SDS and centrifuged at 100 000g for 20 minutes at 4°C. An ECL Western blot kit (Amersham Co) was used with procedures exactly as described by the manufacturer. Briefly, extracts were boiled in 2% SDS/10% glycerol, and equal amounts of protein were separated on 12% polyacrylamide/0.5% SDS gels. The separated proteins were transferred to Hybond-ECL, treated with blocking buffers, and washed exactly as described in the Amersham protocol. Filters were reacted with anti–JNK-2 or anti–ERK-1 antibodies (Santa Cruz Biotech, Inc) and detected by the Amersham ECL method. The ERK-1 antibody cross-reacts with ERK-2, and blots with the JNK-2 antibodies are shown because JNK-1–specific antibodies reacted poorly in this assay (not shown). We do not know whether this was related to poor cross-reaction of the antibody with rat JNK-1 or whether JNK-1 is of low abundance in neonatal rat heart. Duplicate gels were stained with Coomassie blue to check for protein loading.

GSH Assay
Intracellular levels of GSH were measured using a glutathione assay kit (Calbiochem-Novabiochem Corp) exactly as described by the manufacturer. Briefly, monolayers were washed twice with ice-cold PBS, lysed with 6% metaphosphoric acid, and centrifuged at 3000g for 10 minutes. GSH was assayed spectrophotometrically in supernatant fractions using the reagents provided, and concentrations were calculated from a standard curve. All protein determinations were made using the BioRad assay procedure.

Reagents and Inhibitors
Protein kinase inhibitors, including GF 109203X, genistein, and HA-1004, were obtained from Calbiochem Corp. Anisomycin, okadaic acid, NAC, PBN, BSO, and GSH were from Sigma.

Statistical Analysis
Significance tests were performed by t test using the ANOVA program and InStat 2.00 software for Macintosh computers (GraphPad Software). Data are presented as mean±SEM.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Exposure of CMs to hypoxia for 16 hours followed by reoxygenation resulted in a strong induction of amino-terminal Jun kinase activity measured by the substrate affinity binding assay with Jun (1-141) as the substrate (Fig 1A). The activity was induced within 10 minutes and remained induced for at least 40 minutes after reoxygenation. The level of induction by reoxygenation under these conditions was similar to that caused by treating cells for 1 hour with anisomycin (lane 6); in three experiments, the phosphorylation of Jun (1-141) was increased 13±3.3-fold over the aerobic level by hypoxia/reoxygenation (95% O₂) and 10.6±3.9-fold with anisomycin. Protein synthesis inhibitors have been shown previously to be strong activators of the JNK/SAPK pathway, but not of the MAPK pathway.^{17 19 20}

View larger version (17K): [in this window] [in a new window]   Figure 1. Activation of Jun kinase by hypoxia/reoxygenation in CMs. CMs and NMFs were isolated and cultured as described in "Materials and Methods." After isolation, CMs were incubated for 6 days in serum-containing medium before transferring to serum-free MEM with GTIB; NMFs were grown to confluence before transferring to MEM with GTIB. Cultures were serum-starved for 24 hours and then placed under hypoxia or maintained under aerobic conditions. After an additional 16 hours of hypoxia, cells were either lysed directly under hypoxia for kinase assays (panel A, HX lane) or transferred to a humidified chamber preequilibrated with 95% O2/5% CO2 at 37°C. Aerobic cultures were either lysed directly or treated with anisomycin (10 µg/mL) for 1 hour before lysis. Reoxygenated cultures (panel A, Re-Ox) were removed at the indicated times and lysed for kinase assays. In panel B, CMs were removed from hypoxia after 30 minutes and lysed, or they were treated for 16 hours with hypoxia and reoxygenated for 2 hours with air+5% CO2 (20%), 50% O2/5% CO2/balance N2, or 95% O2/5% CO2. In panel C, NMFs were lysed in air, subjected to 16 hours of hypoxia (HX), reoxygenated with 95% O2 for 1 hour (Re-Ox), or treated with anisomycin (10 µg/mL for 1 hour). In panel D, CMs were exposed to hypoxia for 16 hours and reoxygenated with 50% O2/5% CO2/balance N2 for the indicated time periods before harvest and lysis. Genistein (750 µmol/L) (lane 6) was added to myocytes after 15.5 hours of exposure to hypoxia and 30 minutes before reoxygenation with 50% O2 (treatment with 300 µmol/L genistein had the same effect; not shown). Okadaic acid (100 nmol/L) (lane 7) was added to aerobic myocytes, and the cells were lysed after 1 hour. Kinase assays were by the substrate affinity assay, as described in "Materials and Methods." All data are representative of at least three experiments.

To test the effects of hypoxia alone and of different oxygen tensions during reoxygenation, myocyte cultures were exposed to short- and long-term hypoxia and to chronic (16-hour) hypoxia followed by exposure to 21% O₂ (air), 50% O₂, or 95% O₂, and Jun (1-141) kinase activity was again determined as in Fig 1A. These results are shown in Fig 1B. A brief exposure to hypoxia did not affect the kinase activity, whereas chronic exposure (16 hours or longer) caused the activity to decrease slightly. Relative to untreated controls, Jun kinase activity was induced 6.4-fold by reoxygenation in air, and there were additional increments with the higher oxygen tensions. Therefore, the activation of Jun kinases in this model requires reoxygenation, and the magnitude of induction appears to be related to oxygen tension.

To determine whether hypoxia/reoxygenation induced Jun kinase activity in other cells, NMFs and C₂C₁₂ myotubes were cultured as described in "Materials and Methods" and subjected to identical conditions of 16 hours of hypoxia and reoxygenation with 95% O₂. Fig 1C shows results with NMFs. There was no detectable increase over basal aerobic Jun kinase activity, although the activity was strongly induced by anisomycin (similar results were obtained using C₂C₁₂ myotubes or myoblasts; data not shown). These results suggest that CMs were more sensitive to the activation of Jun kinases by hypoxia/reoxygenation.

Because elevated oxygen mediated a slightly larger Jun kinase induction than air alone, we chose to use 50% O₂ for further experiments to characterize the effect. Fig 1D shows that reoxygenation-induced Jun kinase activity remained elevated at least 4 hours after reoxygenation with 50% O₂, indicating a sustained effect. This figure also shows the effects of treatments with genistein and okadaic acid. Genistein is a tyrosine kinase–selective inhibitor that blocks kinase pathways requiring this activity.⁴² It will probably inhibit both SAPK and MAPK pathways at the dual-specificity (MEK/SEK) step; okadaic acid selectively inhibits protein phosphatases 1 and A2, causing a general activation of serine/threonine kinases, and is a potent activator of SAPKs.^{17 21 43} Pretreatment of myocytes with >300 µmol/L genistein added under conditions of hypoxia 30 minutes before reoxygenation completely eliminated Jun kinase activity (n=3). The inhibition was dose dependent, with a half-maximal inhibition (IC₅₀) of 100 µmol/L (not shown). Previous studies, including our own, have shown that this concentration of genistein selectively inhibits tyrosine kinases and is not toxic, at least during the short-term treatments used here.^{39 42 44} As controls for these experiments, cultures were also pretreated with the PKA and PKC inhibitor HA 1004 (50 µmol/L) and the PKC-selective inhibitor GF 109203X (1 µmol/L). These treatments had no effect on the activation of Jun kinase by hypoxia, even though both inhibitors suppressed the PMA-mediated induction of MAPK-MBP phosphorylation in CMs by >70% (n=2, data not shown).

These results demonstrate a strong induction of Jun kinase activity by hypoxia and reoxygenation as assessed by the substrate affinity-binding assay. Because this assay may involve multiple kinases that phosphorylate the N-terminal sites on c-Jun (1-141), we repeated the kinase measurements using immunoprecipitated kinases and either Jun (1-141) or MBP for substrate, as described in "Materials and Methods." Antibodies -JNK-1+-JNK-2 and -ERK-1 were used to discriminate between JNKs/SAPKs and ERKs/MAPKs, respectively, in the assays. Fig 2, left, shows representative results from these analyses. Cells undergoing hypoxia/reoxygenation (50% O₂) experienced a 4.9-fold increase of Jun (1-141) phosphorylation after 1 hour of reoxygenation compared with aerobic cells using protein precipitated with the JNK antibodies (mean of three separate experiments). This activation was similar in magnitude to that induced by treatments with anisomycin (lane 7), and the activity returned to basal levels after 10 hours of reoxygenation, consistent with the activity measured by substrate affinity assays (see Fig 4 below). There was no difference in the phosphorylation of MBP by -ERK-1–precipitated protein from the aerobic control or hypoxia/reoxygenated myocytes (Fig 2, bottom left). In both assays, prolonged (16-hour) hypoxia caused an apparent small loss of kinase activity. The positive controls in these experiments, anisomycin and serum stimulation, both mediated strong inductions of the respective kinase activity. These experiments indicate that JNK-2, but probably not ERK/MAPK, contributes significantly, but possibly not exclusively, to the Jun (1-141) kinase activity stimulated by hypoxia/reoxygenation.

View larger version (27K): [in this window] [in a new window]   Figure 2. Immunokinase and Western blot analyses of JNK/SAPK and ERK/MAPK in CMs. Immunocomplex kinase assays (left) and Western blots (right) of CM cell lysates using anti-JNK or anti–ERK-1 antibodies were carried out as described in "Materials and Methods." In the top left panel, CMs were lysed after no treatment (air); treatment for 16 hours under hypoxia (HX); treatment for 16 hours under conditions of hypoxia and reoxygenation (Re-ox) with 50% O2/5% CO2/balance N2 for 1, 6, and 10 hours as indicated; or after treatment for 1 hour with 10 µg/mL anisomycin. The treatments were the same in the lower left panel, except that myocytes were exposed to 20% serum for 1 hour in lane 1. Similarly, in the right panels, CMs were untreated (air) or exposed to HX for 20 hours, 95% O2/5% CO2 for 1 hour, or HX and Re-ox for 20 hours at 0.5, 1, 4, or 8 hours of 50% O2/5% CO2/balance N2 as indicated. Results are representative of at least two separate experiments for each panel.

View larger version (22K): [in this window] [in a new window]   Figure 4. Correlations between intracellular GSH levels and Jun kinase activation by hypoxia/reoxygenation. CMs were isolated, cultured, and exposed to hypoxia as described in the legend for Fig 1. On the left, for bars 2 through 7, CMs were exposed to hypoxia (HX) for the indicated times and reoxygenated with 50% O2/5% CO2/balance N2 for 2 hours before lysis; for bars 8 through 10, cultures were exposed to 16 hours of HX followed by the indicated period of reoxygenation (ReOX) with 50% O2/5% CO2/balance N2. For the BSO column, aerobic myocytes were treated with 50 µmol/L BSO for 16 hours and oxygenated with 95% O2 for 2 hours, and then the cells were lysed. All kinase assays were by the substrate affinity procedure as described in "Materials and Methods." Fold stimulations were relative to the aerobic controls. On the right, culture plates were lysed and removed from the HX chamber at the indicated times, and GSH was measured in the cell extracts as described in "Materials and Methods." Similarly, for bars 8 through 13, cultures were subjected to 16 hours of HX and then transferred to 50% O2/5% CO2/balance N2 for the indicated times and removed for GSH assays. BSO treatment was as described for the left panel.

To distinguish between activation of kinase activity and changes of the individual protein kinase levels during hypoxia and reoxygenation, we estimated JNK-2 and ERK levels by Western blots using cell extracts after the various treatments. Fig 2, right, shows representative blots of these experiments (n=3). Staining with the JNK-2 antibody produced a prominent specific band that migrated with a molecular size of 54 kD, the reported size of JNK-2²⁰ ; staining with anti-ERK revealed characteristic p42 and p44 protein bands corresponding to ERK-1 and ERK-2, respectively. There were no discernible changes in any of these proteins caused by chronic hypoxia, high oxygen, or up to 10 hours of reoxygenation. Staining of blots with a JNK-1 antibody under the same conditions generated a very weak signal that was not influenced by treatments (not shown). Therefore, enhanced production of these kinases during the treatments was probably not a factor in the increased kinase activity.

To investigate the nature of the initiating stimulus for reoxygenation-induced activation of JNKs/SAPKs, CMs were treated with the antioxidants PBN or NAC during the hypoxic phase immediately preceding reoxygenation (described in the Fig 3 legend). A representative result is shown in Fig 3A. In three experiments, the spin-trap antioxidant PBN reduced JNK/SAPK activation by 82±23% relative to control levels, whereas vehicle (DMSO) was without effect (data not shown). Similarly, pretreatment of cultures with NAC (lane 5) reduced JNK/SAPK activation by 56±17% relative to control (n=3). These experiments support an involvement of ROIs in the activation of JNK/SAPK by hypoxia/reoxygenation. Pretreatment of CMs with either PBN or NAC had no effect on the activation of JNK/SAPK by anisomycin (Fig 3B). Therefore, these antioxidants neutralized a step in JNK/SAPK activation that was at least partially selective for the reoxygenation stimuli without effecting the potential for JNK/SAPK activation by another pathway.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effects of antioxidants and BSO on Jun kinase activity in CMs. CMs were isolated and cultured as described in the legend for Fig 1. In panel A, myocytes remained aerobic (air) or were subjected to 16 hours of hypoxia (HX) or 16 hours of HX and 1 hour of reoxygenation (Re-Ox) with 50% O2/5% CO2/balance N2 as indicated. In lanes 4 and 5, myocytes were treated under HX, respectively, with 10 mmol/L PBN dissolved in DMSO added 30 minutes before Re-Ox or 10 mmol/L NAC, pH 7.2, added 3 hours before reoxygenation. In panel B, cultures of aerobic myocytes were untreated (lane 1, air), treated with 10 µg anisomycin for 1 hour (lane 2), treated with 10 mmol/L PBN dissolved in DMSO 30 minutes before anisomycin (lane 3), or treated with 10 mmol/L NAC, pH 7.2, added 3 hours before anisomycin, as described above. In panel C, cultures of aerobic myocytes were untreated (lane 1, air), treated with 50 µmol/L BSO for 16 hours (lane 2), exposed to 95% O2 for 2 hours (lane 3, Air-Ox), or treated with 50 µmol/L BSO for 16 hours and then transferred to 95% O2 for 2 hours (lane 4, BSO-Ox). In panels A and C, substrate affinity kinase assays were used; in panel B, the immunocomplex assay was used; each was as described in "Materials and Methods." Results are representative of three experiments.

The results described above demonstrate that JNK/SAPK activation by hypoxia and reoxygenation could be blocked by antioxidants; this suggests that ROIs may initiate the signaling pathway. Increased levels of ROIs after ischemia/reperfusion have been described previously.^{45 46} ROIs will be elevated in cells or tissues either if they are generated at higher than normal levels or if the intracellular redox buffering capacity is decreased. Because hypoxia may cause a decrease in GSH, the major redox buffer in most cells,^{5 6} we tested whether the effects of hypoxia could be mimicked by artificially lowering GSH with the -glutamylcysteine synthetase inhibitor BSO.⁴⁷ Myocyte cultures were exposed to 50 µmol/L BSO under normal oxygen for 16 hours, conditions that we have previously shown to markedly reduce intracellular thiol levels.⁴⁷ Treated and untreated cultures were transferred to high (95%) oxygen as described above for reoxygenation of hypoxic cells. After 2 hours, the cells were lysed and assayed for JNK/SAPK activity. As can be seen in Fig 3C JNK/SAPK activity was elevated slightly by treatment with BSO alone (lane 2) and was strongly activated by the shift to high oxygen. Transferring cultures to high oxygen without pretreatment with BSO did not result in a strong JNK/SAPK activation. These experiments indicate that strong activation of the JNK/SAPK pathway required both elevated oxygen and compromised GSH.

If JNK/SAPK is activated by ROIs that escape GSH scavenging, then the intracellular GSH levels of hypoxic cells should correlate with reoxygenation-mediated JNK/SAPK activation. To test for this correlation, reoxygenation-induced Jun kinase activity was measured at a series of time points during hypoxia and during an extended reoxygenation phase. GSH levels were measured in parallel. Fig 4 shows that the loss of intracellular GSH during hypoxia correlated closely with JNK/SAPK activation by reoxygenation. At the early hypoxia exposure times, there was no significant change in GSH, and JNKs/SAPKs were not activated by reoxygenation. Similarly, when GSH levels began to recover at later times in the reoxygenation phase, JNK/SAPK activity decreased. These results suggest that the drop of intracellular GSH is required for JNK/SAPK activation by reoxygenation. Cells that were not compromised in this respect did not activate JNKs/SAPKs in response to hypoxia or reoxygenation. Furthermore, JNK/SAPK activation was sustained during reoxygenation until GSH was replenished. These results suggest that unquenched ROIs generated by reoxygenation of CMs in a redox-compromised state mediate a sustained activation of JNK/SAPK. The activation is sustained until cellular antioxidant buffers are replenished.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stress Pathways Associated With Hypoxia and Reoxygenation
Alternating extremes of redox state created by hypoxia and reoxygenation are the primary causes of stress and ensuing tissue damage in ischemia-related diseases. Both the ischemic/hypoxic and the reperfusion/reoxygenation phases are probably associated with distinct signal transduction pathways. During hypoxia, oxidative reactions are inhibited, there is a shift to a more reduced intracellular environment, and a multifactorial stress response occurs involving modulations of protein kinase activities and changes in the expression of specific genes.^{8 9 48 49 50 51} Reoxygenation is associated with an immediate resumption of oxidative functions and an associated increase of ROIs.^{5 14 45 46} The mechanisms underlying the elevated ROIs are not well established, although there are several potential sources.^{5 12 14 15}

Activation of SAPK and Associated Pathways by ROIs
An induction of the JNK/SAPK pathway by ischemia and reperfusion leading to phosphorylation of c-Jun and ATF2 has been described previously in rat kidney tubular epithelial cells.^{30 31} By analogy with a chemical hypoxia model, the authors reasoned that depletion of ATP was the likely stimulus for kinase activation in these studies. Two additional recent reports described transient activations of both JNK/SAPK and MAPK activity by experimental ischemia and reperfusion in Langendorff-perfused rat heart models.^{34 35} In the former study, ROIs were implicated as initiating the cascades, and JNK/SAPK, but not MAPK, activation required the presence of free calcium in the perfusate.³⁴ The latter study reported activation of p38/RK MAPK by global ischemia and JNK/SAPK activation only after reperfusion.³⁵ Other recent studies also described transitory weak activations of MAPK by hypoxia or hypoxia/reoxygenation in rat CMs or by release from metabolic inhibition in chick myocytes. The precise nature of the initiating signal was not addressed in these studies.^{32 33}

In the present study, we have demonstrated that reoxygenation of chronically hypoxic CMs causes a potent and sustained induction of c-Jun N-terminal phosphorylation, a major component of which was due to induction of the JNK/SAPK cascade. The activity is referred to as JNK/SAPK activity, although kinases in addition to JNK/SAPK may be involved in this model. The inductions were not accompanied by changes of JNK/SAPK or MAPK protein levels. There was no effect of hypoxia alone on JNK/SAPK activity, and ATP depletion was not associated with the induction.⁴ Therefore, ATP depletion is not an obligatory part of the signaling pathway for JNK/SAPK activation by hypoxia and reoxygenation. Rather, the results implicate a direct oxidative component similar to that involved with UV irradiation, cytokine stimulation, or peroxide treatments.^{26 27 28 29 32 34} Several results support the direct involvement of ROIs in this response: (1) there was a graded response to increased oxygen tension during reoxygenation; (2) treatment of cultures with BSO for 16 hours followed by exposure to high oxygen mimicked the effects of hypoxia and reoxygenation on JNK/SAPK activation; (3) there was a close reciprocal correlation between intracellular GSH levels and JNK/SAPK, but not MAPK, activation during reoxygenation; and (4) the induction of JNK/SAPK activity was strongly repressed by pretreating myocytes immediately before reoxygenation with either the spin-trap reagent PBN or the GSH precursor NAC.

BSO is a highly specific competitive inhibitor of -glutamylcysteine synthase, the rate-limiting enzyme in the GSH biosynthetic pathway. It has no other documented cellular effects. As shown in Fig 4, BSO treatment caused about the same drop in cellular GSH as exposures to hypoxia for >8 hours. The observation that BSO pretreatment was as effective as hypoxia in the conditioning of myocytes for reoxygenation/activation of JNK/SAPK activity suggests that reduction in GSH level was the principal, and perhaps sole, contributing factor of the hypoxic phase. We found that hypoxia alone had no effect at any stage on JNK/SAPK activity in CMs. In addition, when myocytes were subjected to hypoxic incubations of <4 hours, which suppressed oxidative phosphorylation but did not affect the GSH levels (see "Materials and Methods" and References 4, 8, and 52), reoxygenation did not activate JNK/SAPK. Therefore, fluctuations in oxygen levels or respiratory chain activity, without changing GSH, were not sufficient to activate the pathway. Consistent with the critical role of GSH levels, JNK/SAPK activation during reoxygenation subsided only when GSH was replenished after >4 hours of reoxygenation. The decline of GSH is probably caused by inhibition by hypoxia of GSH biosynthetic enzymes.^{5 6}

The involvement of ROIs as initiators of the JNK/SAPK-reoxygenation response is further supported by the demonstration that either the spin-trap reagent PBN or the GSH precursor NAC effectively blocked the activity. Previous NMR studies of cardiac cells in vitro and in vivo have demonstrated that PBN complexes with and neutralizes superoxide, hydrogen peroxide, and hydroxyl radicals liberated by myocardial reperfusion.⁴⁵ These studies have demonstrated that PBN infusion affords significant cardioprotection against ischemia/reperfusion damage in a whole-animal model. PBN at the same concentration as described in Reference 45 also blocked the induction of JNK/SAPK activity in reoxygenated myocytes, presumably by interception and quenching of ROIs. In further support of this conclusion, NAC, a well-documented antioxidant, was also an effective inhibitor of JNK/SAPK activation by reoxygenation.

Pretreatment of cells with NAC has been previously reported to suppress the induction of JNK/SAPK activity by UV irradiation,²⁷ the induction of MAPK activity by H₂O₂,²⁸ and, recently, the induction of SAPK activation by cytokines.²⁹ These studies support a central role for ROIs in the activation of Jun kinase, SAPK, and MAPK by a diverse set of extracellular stimuli. Therefore, ROIs appear to be multifunctional second messengers involved in stress-signaling pathways, possibly in the determination of cell fate,^{24 26 28} and in the regulation of specific transcription factor activities.^{12 47 53}

Relevance to Ischemia and Reperfusion
The activation of JNK/SAPK in CMs by hypoxia/reoxygenation was at least partially specific for CMs, because the same conditions of hypoxia and reoxygenation did not activate the kinases in skeletal myotubes or in primary cardiac fibroblasts. The specificity may be related to the high metabolic turnover of rapidly contracting myocytes or other cardiac-specific factor(s). Although it is not possible to directly extrapolate our results to myocardial ischemia and reperfusion in vivo, previous reports described inductions of both JNK/SAPKs and MAPK in Langendorff-perfused models of ischemia and reperfusion,^{9 34} and other studies have demonstrated that ischemia and reperfusion strongly induce the immediate early genes c-fos and c-jun in the porcine heart.^{54 55} JNK/SAPK activation could be part of this activation of activator protein-1 in vivo. Presumably, any condition that causes reductions in redox buffering concurrently with active oxidative metabolism could initiate this kinase cascade. Although GSH levels fell gradually during hypoxia in our cultured myocyte model, the levels may decrease more rapidly in whole hearts subjected to repeated ischemia and reperfusion.⁵⁶

We have previously shown that PKC is involved in the induction of c-jun by hypoxia alone.⁸ Therefore, the immediate-early gene response to redox stress in CMs appears to involve activation of at least three distinct protein kinase pathways: PKC during the hypoxic phase and JNKs/SAPKs and MAPKs during reoxygenation. At the present time, it is not clear whether the activation of these pathways by redox stress affects CM survival. In agreement with previous reports, we found that hypoxia alone caused an increase in the rate of apoptosis and that this was increased further by reoxygenation (authors' unpublished data, 1996). There is evidence from other cells that the ERK/SAPK pathways are involved in defensive and/or survival responses.^{24 25 26 27 34}

	Selected Abbreviations and Acronyms

 

BrdU = bromodeoxyuridine BSO = L-buthionine-[S,R]-sulfoximine CM = cardiac myocyte DMSO = dimethyl sulfoxide ERK = extracellular signal–regulated (protein) kinase GSH = reduced glutathione GTIB = glucose, transferrin, insulin, and vitamin B12–supplemented MEM JNK = c-Jun N-terminal protein kinase MAPK = mitogen-activated protein kinase MBP = myelin basic protein NAC = N-acetylcysteine NMF = nonmuscle cardiac fibroblasts PBN = -phenyl N-tert-butylnitrone PKA, PKC = protein kinases A and C PMSF = phenylmethylsulfonyl fluoride ROI = reactive oxygen intermediate SAPK = stress-activated protein kinase

	Acknowledgments

 
This study was supported by grants HL-44578 (Dr Webster) and CA-57333 (Dr Laderoute) from the National Institutes of Health and by the Cigarette and Tobacco Surtax of the State of California through the Tobacco-Related Disease Research Program of the University of California, grant 1RT-402 (Dr Webster). We would like to thank Daryl Discher, Holly Mendonca, and Joy Calaoagan for their excellent technical contributions and Nanette Bishopric for discussions and critique of the manuscript.

Received July 16, 1996; accepted December 5, 1996.

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