Carbon Monoxide Inhibition of Apoptosis during Ischemia-Reperfusion Lung Injury Is Dependent on the p38 Mitogen-activated Protein Kinase Pathway and Involves Caspase 3*

Xuchen ZhangDagger , Peiying ShanDagger , Leo E. Otterbein§, Jawed Alam||**, Richard A. FlavellDagger Dagger ***, Roger J. Davis§§, Augustine M. K. Choi§¶¶, and Patty J. LeeDagger ||||

From the Dagger  Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven, Connecticut 06520, § Division of Pulmonary, Allergy, and Critical Care, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213, || Department of Molecular Genetics, Alton Ochsner Medical Foundation, New Orleans, Louisiana 70121, Dagger Dagger  Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520, and §§ Program in Molecular Medicine, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

Received for publication, August 17, 2002, and in revised form, October 1, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES

Carbon monoxide (CO), a reaction product of the cytoprotective gene heme oxygenase, has been shown to be protective against organ injury in a variety of models. One potential mechanism whereby CO affords cytoprotection is through its anti-apoptotic properties. Our studies show that low level, exogenous CO attenuates anoxia-reoxygenation (A-R)-induced lung endothelial cell apoptosis. Exposure of primary rat pulmonary artery endothelial cells to minimal levels of CO inhibits apoptosis and enhances phospho-p38 mitogen-activated protein kinase (MAPK) activation in A-R. Transfection of p38alpha dominant negative mutant or inhibition of p38 MAPK activity with SB203580 ablates the anti-apoptotic effects of CO in A-R. CO, through p38 MAPK, indirectly modulates caspase 3. Furthermore, we correlate our in vitro apoptosis model with an in vivo model of A-R by showing that CO can attenuate I-R injury of the lung. Taken together, our data are the first to demonstrate in models of A-R that the anti-apoptotic effects of CO are via modulation of p38 MAPK and caspase 3.

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INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Ischemia-reperfusion (I-R)1 injury in organs or anoxia-reoxygenation (A-R) in cells leads to cell death and severe organ injury. It has been shown that apoptosis is initiated shortly after the onset of ischemia that then continues during reperfusion (1). CO is a reaction product of heme oxygenase activity, the rate-limiting step in the oxidative degradation of heme. Several investigators have used I-R animal models to confirm that increased expression of heme oxygenase-1 or its reaction product CO correlates with improved survival and organ function in brain, kidney, liver, heart, and lung (2-5).

There is increasing evidence that CO, once thought to be an incidental waste product of heme catabolism, is an important signaling molecule and is involved in regulation of vasomotor tone, suppression of platelet aggregation, and anti-inflammatory effects (6, 7). Otterbein et al. (8) show that CO inhibits the expression of lipopolysaccharide-induced pro-inflammatory cytokines such as tumor necrosis factor-alpha , interleukin-1beta , and macrophage inflammatory protein-1beta while simultaneously increasing expression of the anti-inflammatory cytokine interleukin-10. Chapman et al. (9) also show that CO can attenuate aeroallergen-induced inflammation in mice. The cytoprotective effects of CO have also been demonstrated in models of hyperoxia-induced and I-R-induced lung injury (5, 10) as well as in a model of xeno-cardiac transplantation (7). Furthermore, Brouard et al. (11) demonstrate that CO could inhibit tumor necrosis factor-alpha -induced endothelial apoptosis through the activation of the p38 MAPK pathway.

The mitogen-activated protein kinases (MAPKs) are a family of serine-threonine protein kinases that are activated in response to a variety of extracellular stimuli (12). Three major MAPK signaling pathways, which include extracellular signal-regulated protein kinase (ERK1/2), p38 MAPK (p38), and c-Jun NH2-terminal protein kinase (JNK1/2), have been identified in mammalian cells. Separate studies implicate all three MAPK families in regulating apoptosis, but the precise roles of the individual MAPK signaling pathways are still not clear. The activation of MAPKs in I-R injury has been demonstrated in vitro (13, 14) as well as in vivo (15-17). However, the role of each MAPK pathway in I-R-induced apoptosis remains controversial. ERK is generally considered to be a survival mediator involved in the protective effects of growth factors in apoptosis (18, 19), but it has also been reported that induction of apoptosis can be mediated via ERK (20, 21). JNK and p38 MAPKs are usually implicated in the induction of apoptosis and inflammation after exposure to a variety of agents (22-24). However, it is becoming increasingly clear that p38 and JNK MAPK activation may protect against the induction of apoptosis depending upon cell type and inducing agent. Han et al. (25) show that increased p38 levels in hydrogen peroxide-mediated oxidative stress protect rat fibroblast cells from apoptosis. Assefa et al. (26) demonstrate that p38 inhibition over-sensitized HeLa cells to photodynamic therapy-induced apoptosis.

Caspases, a family of specific cysteine proteases, are thought to be critical in the intracellular execution of apoptosis (27). Among the 13 members that have been identified, caspase 3 is pivotal in the effector phase of apoptosis induced by a variety of stimuli. There is accumulating data that caspase inhibition has a significant protective effect in I-R and A-R injury (28-30).

Virtually nothing is known about the anti-apoptotic signaling mechanism(s) utilized by CO in I-R injury. Given that CO has been shown to have protective effects in lung I-R injury (5) as well as anti-apoptotic effects in other models of cell injury, we first examined whether CO exerts an anti-apoptotic effect during I-R and then delineated the role of MAPKs and caspases in mediating the anti-apoptotic effect of CO. Although putative mechanisms such as modulation of the fibrinolytic pathway have been proposed as the basis for CO-mediated protection in lung I-R injury (5), we are the first to describe the anti-apoptotic effect of CO and the role of p38 MAPK and caspase 3 in I-R injury.

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Cell Culture-- Rat primary pulmonary endothelial cells (PAEC) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) with 10% fetal bovine serum (Hyclone, Logan, UT) and 100 µg/ml gentamicin (Invitrogen) in a humidified atmosphere of 5% CO2. The PAEC were generously provided by Dr. Troy Stevens (University of Alabama). All data using primary cell cultures were collected before passage 18. For anoxia-reoxygenation, a model previously established (31), cells were exposed to 95% N2, 5% CO2 in a sealed modular chamber (Billup-Rothberg, Del Mar, CA) with continuous monitoring and adjustment to maintain <0.5% O2 (BioSpherix, Redfield, NY) for 24 h. After anoxia, reoxygenation was achieved by changing media and exposing cells to normoxia at 37 °C in a humidified atmosphere of 5% CO2 for 15-60 min. For A-R plus CO, cells were exposed to 15 ppm CO throughout anoxia and reoxygenation. For anoxia plus CO, cells were exposed to 15 ppm CO, 5% CO2 in 95% N2 for 24 h.

Murine Lung Ischemia-Reperfusion Model-- Adult 8-week-old male C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, Maine) for SB203580 and I-R experiments. The caspase 3 null mice have been previously described (32). All mice were housed under pathogen-free conditions for 1 week before experimental exposure. For I-R experiments, after anesthesia with an intraperitoneal injection of urethane (180 mg/kg of body weight), mice were intubated via a tracheostomy and ventilated with a Harvard ventilator (respiratory rate, 120/min; tidal volume, 0.5 ml). A left hilar clamp was placed for 30 min of unilateral ischemia to the left lung, and then the clamp was released for 2 h of reperfusion. For SB203580 administration, mice were given intraperitoneal injections (1 mg/kg body weight) 1 h before I-R, as previously described (33). The Animal Care and Use Committee at Yale University approved this protocol in accordance with the guidelines.

CO Exposure-- CO exposures for cells and animals were performed as previously described (8, 10). For mouse I-R experiments 500 ppm CO was delivered via the ventilator for a 1-h pretreatment and continued throughout the course of I-R. Cells were exposed to 15 ppm CO during A-R using the sealed modular chamber (Billup-Rothberg, Del Mar, CA).

DNA Laddering-- Genomic DNA was isolated from cultured PAEC with the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN), and genomic DNA (20 µg) was electrophoresed on a 2% agarose gel containing ethidium bromide in 0.5× Tris acetate buffer. The gel was then photographed under ultraviolet luminescence.

Terminal Deoxynucleotidyltransferase dUTP Nick End-labeling (TUNEL) Assay-- We used the in situ cell death detection kit and followed the manufacturer's protocol (Roche Molecular Biochemicals). For cultured PAEC, cells were exposed to A-R, washed twice with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100. The slides were then incubated with TUNEL reaction mixture followed by anti-fluorescein conjugated with alkaline phosphatase at 37 °C. Cells were then washed and stained with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate solution before counterstaining with nuclear fast-red. The apoptotic and normal cells were observed under light microscope. Cells with purple nuclei are TUNEL-positive, indicating apoptosis, and cells with red nuclei are normal. 500 cells were counted for each sample, and the numbers of apoptotic cells are expressed as a percentage. For lung tissues, sections of formalin-fixed, paraffin-embedded lung tissue were deparaffinized and rehydrated, rinsed with PBS, and digested with proteinase K (Roche Molecular Biochemicals) at a concentration of 20 µg/ml for 20 min. After PBS washes, sections were incubated with TUNEL reaction mixture at 37 °C for 1 h and then incubated with anti-fluorescein conjugated with alkaline phosphatase at 37 °C for 30 min. Sections were then washed twice with PBS and stained with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrate solution before counterstaining with nuclear fast-red.

Annexin V-FITC Fluorescence-activated Cell Sorter (FACS)-- Using the annexin V-FITC kit from Pharmingen we followed the manufacturer's protocol. Briefly, PAEC were washed with cold PBS and resuspended with binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) before transferring 1 × 105 cells to a 5-ml tube. Then 5 µl of annexin V and 5 µl of propidium iodide were added, and cells were incubated for 15 min in the dark. Binding buffer (400 µl) was then added to each tube and analyzed by flow cytometry.

MAPK Activity-- MAPK activities were measured in immune complex protein kinase assays according to the manufacturer's protocol (Cell Signaling, Beverly, MA). Briefly, after A-R, cells were lysed in cold cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM beta -glycerol phosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Equal amounts of cell lysates were incubated with immobilized phospho-ERK kinase monoclonal antibody, c-Jun fusion protein beads, or immobilized phospho-p38 monoclonal antibody for ERK1/2, JNK1/2, and p38, respectively. After centrifugation, pellets were suspended by kinase buffer (25 mM Tris, pH 7.5, 5 mM beta -glycerol phosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2, and 200 µM ATP) and then immunoprecipitated with specific fusion proteins Elk-1, c-Jun, and activating transcription factor-2 as substrates for phospho-ERK1/2, JNK1/2, and p38, respectively. The activities of phospho-ERK1/2, JNK1/2, and p38 were then measured by Western blot analysis using primary antibodies (rabbit polyclonal phospho-Elk-1, phospho-c-Jun, and phospho-activating transcription factor-2 antibodies, respectively) at 1:1000 dilution followed by horseradish-conjugated anti-rabbit secondary antibody (1:2000). LumiGLO (Cell Signaling, Beverly, MA) reagent was used to detect protein signals.

Plasmid Constructs and Transient Transfections-- The heme oxygenase-1-overexpressing vector transfection has been previously described (34). Wild type and dominant-negative mutant mammalian p38alpha expression plasmids were generously provided by Roger J. Davis (University of Massachusetts, Worcester, MA) and have been previously described (35). Cells were incubated for 6 h with DNA mixtures containing serum-free media, FuGENE 6 Reagent (Roche Molecular Biochemicals), and wild type or dominant-negative mutant p38alpha plasmids. After incubation, cells were cultured for an additional 16 h in complete medium and then exposed to 24 h of anoxia alone or 24 h of anoxia plus CO.

Caspase 3 Activity-- The activity of caspase 3 was measured by the colorimetric assay with CaspACE assay system (Promega, Madison, WI). Briefly, after anoxia, in the presence or absence of CO, cells were washed twice with ice-cold PBS and resuspended in cell lysis buffer. Cell lysates were incubated with colorimetric substrate, acetyl-Asp-Glu-Val-Asp-7-amino-p-nitroanilide. The release of p-nitroanilide from acetyl-Asp-Glu-Val-Asp-7-amino-p-nitroanilide was measured at 405 nm using a spectrophotometer.

Chemicals-- Tin protoporphyrin was purchased from Frontier Scientific, Logan, UT. Benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), benzyloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe) fluoromethyl ketone (Z-DQMD-FMK), and acetyl-Try-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-CMK) were purchased from Calbiochem.

Statistics-- Data are expressed as mean ± S.E. from three independent experiments and were analyzed with one-way analysis of variance. Significant difference was accepted at p < 0.05.

    RESULTS
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ABSTRACT
INTRODUCTION
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CO Attenuates A-R-induced PAEC Apoptosis-- We focused our in vitro studies on the PAEC because endothelial cells have a central role in vascular responses to A-R injury in the lung. To determine whether A-R induces apoptosis, genomic DNA was isolated from PAEC after 24 h of anoxia alone or anoxia followed by a time course of reoxygenation and analyzed for DNA laddering. Fig. 1, panel A, shows marked DNA laddering by 24 h of anoxia (lane 2) compared with room air control (lane 1), and this laddering persists up to 8 h of reoxygenation (lanes 3-5), which disappeared by 24 h of reoxygenation (lane 6). In contrast (panel B), when cells were exposed to anoxia or A-R in the presence of 15 ppm CO, which is significantly lower than the documented safe levels of 50 ppm (36, 37), laddering was significantly attenuated (lanes 8-11). To confirm our DNA laddering assay and the anti-apoptotic effect of CO, we used TUNEL, FACS, and caspase 3 activity assays as outlined below. Fig. 2a shows increased TUNEL staining in PAEC exposed to 24 h of anoxia alone or 24 h of anoxia followed by 30 min of reoxygenation (panels B and C, respectively). There is a marked diminution of TUNEL staining when cells are exposed to exogenous CO during anoxia alone or A-R (panels D and E, respectively). Fig. 2b is a graphical quantitation of TUNEL staining that shows that CO significantly decreases PAEC TUNEL staining from 14.3 ± 0.6 to 5.4 ± 0.8% during anoxia alone and from 13.8 ± 1.1 to 5.5 ± 0.6% during A-R. Of note, our observation that the majority of cells do not undergo apoptosis in A-R is consistent with that of a number of other investigators utilizing different cell types. Calf pulmonary artery endothelial cells show 25% apoptosis after 24 h of hypoxia (38), whereas human umbilical vein endothelial cells and epidermoid cells exhibit <5% apoptosis after 24 h of anoxia (39, 40). The degree of apoptosis in response to A-R injury is likely dependent upon cell type, the degree/duration of the injury, cytokine milieu, activation of stress response genes, the relative abundance of pro- and anti-apoptotic proteins, and levels of death receptor expression. Human umbilical vein endothelial cells, for instance, exhibited increased apoptosis from <5% at 24 h of anoxia to 35% at 48 h of anoxia (39), whereas epidermoid cells increased from <5% to 15% apoptosis by 48 h of anoxia (40). In addition, there is likely induction of various cytoprotective proteins during hypoxia, such as heme oxygenase-1, Ref-1, and vascular endothelial growth factor, which may attenuate the degree of cell death observed (38, 41, 42). In addition, the observation that a majority of cells do not undergo apoptosis during A-R lung injury as well as other forms of acute lung injury also correlates with in vivo observations. Lung sections from animals exposed to I-R, hyperoxia, or lipopolysaccharide show rates of apoptosis to be less than 20% (1, 43, 44).


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Fig. 1.   CO attenuates A-R-induced DNA laddering in PAEC. Genomic DNA was isolated from PAEC exposed to 24 h of anoxia alone or anoxia followed by a time course of reoxygenation in the presence or absence of CO. DNA fragmentation was analyzed on a 2% agarose gel. A, lane 1, RA; lane 2, 24 h of anoxia; lanes 3-6, 24 h of anoxia followed by reoxygenation for 30 min or 1, 8, or 24 h, respectively. B, lane 7, room air control; lane 8, 24 h of anoxia in the presence of CO; lanes 9-12, 24 h of anoxia followed by reoxygenation for 30 min or 1, 8, or 24 h, respectively, in the presence of CO. The data are representative of three independent experiments.


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Fig. 2.   CO attenuates apoptosis of PAEC after A-R. a, TUNEL staining of PAEC after A-R. PAEC grown on a 4-well slide were exposed to 24 h of anoxia alone or anoxia followed by reoxygenation in the presence or absence CO. A, RA; B, 24 h of anoxia; C, 24 h of anoxia and then reoxygenation for 30 min; D, 24 h of anoxia in the presence of CO; E, 24 h of anoxia and then reoxygenation for 30 min in the presence of CO. Arrows denote TUNEL-positive cells. The data are representative of three independent experiments. b, quantitation of TUNEL staining after A-R. Bar 1, RA; bar 2, 24 h of anoxia; bar 3, 24 h of anoxia and then reoxygenation for 30 min; bar 4, 24 h anoxia in the presence of CO; bar 5, 24 h of anoxia and then reoxygenation for 30 min in the presence of CO. Data are shown as mean ± S.E. of three independent experiments. *, p < 0.01 compared with RA (bar 1); #, p < 0.01 compared with 24 h of anoxia (bar 2). c, CO attenuates apoptosis after anoxia as assessed by FACS assay. Flow cytometry was used to detect apoptosis after anoxia in the presence or absence CO by annexin V-FITC FACS as described under "Experimental Procedures." The data are representative of three independent experiments.

Given that PAEC apoptosis, as evidenced by DNA laddering and TUNEL staining, was initiated during anoxia and CO had similar anti-apoptotic effects during anoxia alone or A-R, we used 24 h of anoxia alone for subsequent apoptosis assays. Fig. 2c is a FACS assay confirming that CO significantly attenuates anoxia-induced PAEC apoptosis. Because DNA laddering correlated with TUNEL and FACS findings in PAEC, we demonstrated cellular apoptosis by DNA laddering in subsequent studies. To further confirm the presence of apoptosis in our model of A-R, we examined whether caspase inhibition attenuates anoxia-induced DNA laddering in our PAEC. Fig. 3A demonstrates that the broad caspase inhibitor Z-VAD-FMK (50 µM) indeed blocks anoxia-induced laddering. Given that Z-VAD-FMK is a broad caspase inhibitor that includes caspases 1, 3, 4, and 7 (45), we sought to delineate the role of specific caspases. Inhibiting caspase 3 with Z-DQMD-FMK (50 µM) rather than inhibiting caspase 1 and 4 with Ac-YVAD-CMK (50 µM) appears to be more effective in preventing anoxia-induced laddering (Fig. 3, A-C). However, to our knowledge a caspase 7-specific inhibitor is not yet available, and thus, caspase 7 is difficult to evaluate individually. Fig. 3D shows the graphical quantitation of the mean FACS results in the presence of CO or caspase inhibitors. Again, there is a significant decrease in anoxia-induced apoptosis in the presence of CO, the broad caspase inhibitor Z-VAD-FMK, or the caspase 3-specific inhibitor Z-DQMD-FMK. The specificity of the respective caspase inhibitors has been previously validated and published (46-48). Thus far, it appears that CO is able to attenuate A-R-induced apoptosis, as detected by DNA laddering, TUNEL, and FACS, and this apoptosis is caspase-dependent. Our next series of studies, as outlined below, evaluate the potential mechanisms of the anti-apoptotic effect of CO.


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Fig. 3.   Caspase inhibition, specifically caspase 3 inhibition, attenuates anoxia-induced DNA laddering in PAEC. Genomic DNA was isolated from PAEC exposed to 24 h of anoxia alone or 24 h of anoxia in the presence or absence of caspase inhibitors. DNA fragmentation was analyzed on a 2% agarose gel. A, lane 1, RA; lane 2, 24 h of anoxia (A); lane 3, pretreatment with the broad caspase inhibitor (Broad), Z-VAD-FMK (50 µM), then 24 h of anoxia; lane 4, pretreatment with a caspase 3-specific inhibitor (Casp 3), Z-DQMD-FMK (50 µM), then 24 h of anoxia. B, given that Z-VAD-FMK inhibits caspases 1 and 4 in addition to caspase 3, we investigated whether pretreatment with caspase 1, 4 inhibitor (Casp 1,4) with Ac-YVAD-CMK (50 µM) also attenuated anoxia-induced DNA laddering. Lane 1, RA; lane 2, 24 h of anoxia; lane 3, 24 h of anoxia in the presence of CO; lane 4, pretreatment with Ac-YVAD-CMK, a caspase 1,4 inhibitor. C, FACS assay was used to quantitate the degree of apoptosis in the presence or absence of CO or caspase inhibitors. PAEC were exposed to RA only, 24 h of anoxia, 24 h of anoxia in the presence of CO, or pretreatment with various caspase inhibitors, Z-VAD-FMK, Z-DQMD-FMK, or Ac-YVAD-CMK, and then 24 h of anoxia. The data are representative of three independent experiments. D, graphical quantitation of the mean FACS results ± S.E. from three independent FACS experiments. *, p < 0.01 compared with RA; #, p < 0.01 compared with 24 h of anoxia. FL1, FITC detector; PI, propidium iodide; FL2, phycoerythrin fluorescence detector.

The Anti-apoptotic Effect of CO Is Not Mediated by Nitric Oxide, Guanylate Cyclase/cGMP, Heme Oxygenase, or MEK (MAP or ERK Kinase)/ERK1/2-- Most vascular functions attributed to CO have been linked to its ability to bind guanylate cyclase and generate cGMP (6, 49). Because cGMP can regulate apoptosis (50), we tested the role of guanylate cyclase and cGMP in the anti-apoptotic effect of CO during anoxia. When PAEC were exposed to 24 h of anoxia plus CO (15 ppm), there was no increase in the generation of cGMP as assessed by immunoassay (data not shown). Pretreatment with 50 µM ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one), a chemical inhibitor of guanylate cyclase, did not suppress the anti-apoptotic effect of CO during anoxia (Fig. 4, lane 5). Given our observation that CO exerts an anti-apoptotic effect through a cGMP-independent pathway and the fact that CO can bind the heme moiety of nitric-oxide synthase and thereby modulate the production of nitric oxide (NO) (51), we tested the role of NO and nitric-oxide synthase in the anti-apoptotic effect of CO during anoxia. PAEC exposed to 24 h of anoxia plus CO (15 ppm) did not exhibit increased NO production (data not shown). In addition, CO was able to rescue PAEC pretreated with 10 µM NG-nitro-L-arginine methyl ester, a selective inhibitor of nitric-oxide synthase, from anoxia-induced apoptosis as seen in Fig. 4, lane 4. Similar to data from other laboratories, we demonstrated that CO has an anti-apoptotic effect independent of heme oxygenase-1 activity. In Fig. 4, lane 6, we show that despite pretreatment with tin protoporphyrin (SnPP), a selective HO activity inhibitor, exogenous CO attenuated anoxia-induced laddering. Given previous publications implicating MAPKs, specifically p38 MAPK, in the biologic effects of CO (8, 11), we used chemical inhibitors of p38 and ERK1/2 in Fig. 4, lanes 7 and 8, respectively. SB203580 (SB), a selective p38 activity inhibitor, abrogated the anti-laddering effect of CO (lane 7), whereas an inhibitor of ERK1/2, PD98059 (PD), had no effect. This implicated p38 MAPK as an important mediator of the anti-apoptotic effect of CO.


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Fig. 4.   The anti-apoptotic effect of CO is not mediated by nitric oxide, guanylate cyclase/cGMP, heme oxygenase, or MEK (MAP or ERK kinase)/ERK1/2. Genomic DNA was isolated from PAEC treated with 24 h of anoxia and the indicated chemicals in the presence or absence of CO. DNA fragmentation was analyzed on a 2% agarose gel. Lane 1, RA; lane 2, 24 h of anoxia; lane 3, 24 h of anoxia in the presence of CO; lane 4, pretreatment with 10 µM NG-nitro-L-arginine methyl ester (LNAME) for 1 h and then 24 h of anoxia in the presence of CO; lane 5, pretreatment with 50 µM ODQ (1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one) for 1 h and then 24 h of anoxia in the presence of CO; lane 6, pretreatment with 50 µM tin protoporphyrin (SnPP) for 1 h and then 24 h of anoxia in the presence of CO; lane 7, pretreatment with 5 µM SB203580 for 1 h and then 24 h of anoxia in the presence of CO; lane 8, pretreatment with 10 µM PD98059 for 1 h and then 24 h of anoxia in the presence of CO. The data are representative of three independent experiments.

CO Inhibited A-R-induced Activation of ERK1/2 and JNK1/2 but Increased the Activation of p38 during A-R-- We tested MAPK activation in the presence and absence of CO during A-R using MAPK activity assays. Fig. 5 shows that CO effectively diminished ERK1/2 and JNK1/2 activity during reoxygenation but, interestingly, increased p38 activation during 15 min to 1 h of reoxygenation. There is minimal MAPK activation during anoxia alone, which is why, initially, CO was used only during reoxygenation for MAPK assays.


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Fig. 5.   CO inhibits A-R-induced activation of ERK1/2 and JNK1/2 MAPKs but increases p38 MAPK activity. PAEC were exposed to 24 h of anoxia alone or anoxia followed by a time course of reoxygenation in the presence or absence of CO. Cell lysates were analyzed for MAPK activation using MAPK activity assays as described under "Experimental Procedures." The data are representative of three independent experiments.

CO Increases the Activation of p38 during Anoxia Alone-- Given that inhibition of p38 activity attenuated the anti-apoptotic effect of CO (Fig. 4, lane 7) and CO increased p38 activity during reoxygenation (Fig. 5), we hypothesized that CO may exert an anti-apoptotic effect via p38 MAPK. If this were the case, exogenous CO should increase p38 activity, as measured by activating transcription factor-2 phosphorylation during anoxia alone, which is when apoptosis is initiated. Fig. 6 shows that CO (15 ppm) activates p38 during anoxia alone that is blocked by SB203580 (5 µM), a p38-specific inhibitor. We also tested for ERK1/2 and JNK1/2 activation during anoxia in the presence of CO and found none (data not shown).


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Fig. 6.   CO increases p38 MAPK activity during anoxia alone. We confirmed that CO increases p38 activity not only during the reoxygenation phase of A-R, as shown in Fig. 5, but also during anoxia alone, which is when apoptosis is initiated. PAEC were exposed to 24 h of anoxia in the presence or absence of CO. SB203580 (5 µM) pretreatment was used to inhibit p38 activity. Lane 1, RA; lane 2, 24 h of anoxia; lane 3, 24 h of anoxia in the presence of CO; lane 4, SB203580 pretreatment (SB) followed by 24 h of anoxia in the presence of CO. The data are representative of three independent experiments.

The Anti-apoptotic Effect of CO Is Mediated through the p38 MAPK Pathway-- Thus far we know that 1) CO activates p38 during anoxia alone (Fig. 6) and A-R (Fig. 5), and 2) the anti-apoptotic effect of CO was suppressed by pretreatment with the p38-specific inhibitor SB20358 but not the MEK/ERK1/2 inhibitor PD98059 (Fig. 4). The specific role of p38 was further confirmed by transfection experiments. In Fig. 7, PAEC were transfected with wild type (Wt) or DNM of p38alpha and then exposed to 24 h anoxia in the presence or absence of CO. As expected, both Wt and DNM PAEC exhibited DNA laddering in anoxia. However, CO is able to rescue the Wt p38alpha cells from anoxia-induced apoptosis, whereas CO cannot rescue the p38alpha DNM PAEC. Alternatively, PAEC transfected with ERK1/2 or JNK1/2 DNMs still showed decreased DNA laddering in the presence of anoxia and CO (data not shown), again demonstrating that p38 MAPK is necessary for the anti-apoptotic effect of CO in PAEC during anoxia.


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Fig. 7.   The anti-apoptotic effect of CO is mediated through the p38 MAPK pathway. PAEC were transfected with Wt or DNM p38alpha plasmids and then exposed to 24 h of anoxia in the presence or absence of CO. Genomic DNA was isolated, and DNA fragmentation was analyzed on a 2% agarose gel. Lane 1, RA; lane 2, 24 h of anoxia; lane 3, 24 h of anoxia in the presence of CO; lane 4, PAEC transfected with Wt p38alpha were exposed to 24 h of anoxia; lane 5, PAEC transfected with DNM p38alpha were exposed to 24 h of anoxia; lane 6, PAEC transfected with Wt p38alpha were exposed to 24 h of anoxia in the presence of CO; lane 7, PAEC transfected with DNM p38alpha were exposed to 24 h of anoxia in the presence of CO. The data are representative of three independent experiments.

CO Has an Anti-apoptotic Effect in Vivo during Lung I-R-- Lung I-R is an in vivo correlate to the in vitro A-R model (52). In Fig. 8a, lungs were removed from mice after 0 (naive mice, panels A and E) or 30 min of lung ischemia alone (panels B and F) or 30 min of ischemia followed by 2 h of reperfusion in the absence of CO (panels C and G) or the presence of CO (panels D and H). There is a marked increase in TUNEL-positive cells involving most cells types (arrows indicate TUNEL-positive cells) after 30 min of lung ischemia alone (panels B and F) and after I-R (panels D and H) compared with naive mice. The initiation of TUNEL staining in the lung as early as 30 min of ischemia is consistent with the published observations of other investigators who have noted increased organ TUNEL staining after 30 min of ischemia (53). Exogenous CO during I-R effectively attenuated lung apoptosis as seen in panels D and H. Of note, exogenous CO also attenuated lung apoptosis induced by ischemia alone (Fig. 9, panel B). We recognize that TUNEL-positive staining can be indicative of general cell damage and death rather than apoptosis. Therefore, we confirmed the importance of apoptosis in our I-R model and confirmed our in vitro caspase 3 inhibition data shown in Fig. 3 by assessing the importance of caspase 3 in vivo. Fig. 8b shows that caspase 3 null (-/-) mice have significantly less TUNEL staining after I-R, similar to mice treated with CO during I-R, compared with the wild type littermate controls. This implies that caspase 3 activity, a pivotal effector protease for apoptosis, is necessary for I-R-induced cell death.


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Fig. 8.   a, CO blocks I-R-induced apoptosis in vivo. To correlate our in vitro findings in vivo, mice were subjected to lung ischemia-reperfusion injury as described under "Experimental Procedures." Lung specimens were processed for TUNEL staining. A and E, naive (10× and 20×, respectively); B and F, 30-min lung ischemia (10× and 20×, respectively); C and G, 30-min ischemia and 2-h reperfusion (10× and 20×, respectively); D and H, 30-min ischemia and 2-h reperfusion in the presence of CO (10× and 20×, respectively). Arrows indicate TUNEL staining. The data are representative of three mice. b, caspase 3 -/- mice do not exhibit I-R-induced lung apoptosis. We subjected caspase 3 null mice (-/-) and their wild type littermate controls (caspase 3 +/+) to lung I-R as described under "Experimental Procedures," and lung sections were processed for TUNEL staining. The upper panels represent 10× original magnification. The lower panels represent 20× original magnification. The arrows indicate cells positive for TUNEL staining. The data are representative of three mice.


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Fig. 9.   The anti-apoptotic effect of CO is mediated through the p38 MAPK pathway in vivo. Mice were subjected to lung ischemia-reperfusion injury as described under "Experimental Procedures," and lung specimens were processed for TUNEL staining. A, 30 min of ischemia (I); B, 30-min ischemia in the presence of CO; C, pretreatment with SB203580, a specific inhibitor of p38, and then 30-min of ischemia in the presence of CO; D, naïve; E, 30 min of ischemia and 2 h of reperfusion; F, 30 min of ischemia and 2 h of reperfusion in the presence of CO; G, pretreatment with SB203580, then 30 min of ischemia and 2 h of reperfusion in the presence of CO. All panels represent 20× original magnification. The data are representative of three mice.

The Anti-apoptotic Effect of CO in Vivo Is via the p38 MAPK Pathway during Lung I-R-- Given that p38 MAPK is important in the anti-apoptotic effect of CO in PAEC, we determined the role of p38 in vivo by systemically administering SB203580, a p38 specific inhibitor, before subjecting mice to lung I-R. Fig. 9, panel C, demonstrates that CO cannot attenuate ischemia-induced lung apoptosis in the presence of SB203580. Similarly, SB203580 also blocks the anti-apoptotic effect of CO during I-R (panel G). The TUNEL data are supported by PAEC data in Fig. 10 that show SB203580 also inhibits the ability of CO to modulate an apoptotic event upstream of TUNEL staining, namely caspase 3 activity.


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Fig. 10.   CO modulates caspase 3 activity in anoxia via p38 MAPK. Caspase 3 activity in PAEC was detected as described under "Experimental Procedures" after cells were exposed to 1) RA, 2) 24 h of anoxia, 3) 24 h of anoxia in the presence of CO, 4) pretreatment with 5 µM SB203580 (SB) and then 24 h of anoxia in the presence of CO, 5) PAEC transfected with Wt p38alpha were exposed to 24 h of anoxia in the presence of CO, or 6) PAEC transfected with DNM p38alpha were exposed to 24 h of anoxia in the presence of CO. The graph represents the mean ± S.E. of three independent experiments. *, p < 0.05 compared with RA; #, p < 0.05 compared with 24 h of anoxia.

CO Modulates Caspase 3 Activity through p38 MAPK-- Given that both caspase 3 inhibition (chemically in cells and genetically in caspase 3 null mice) and CO administration attenuated apoptosis, we hypothesized that apoptosis induced by A-R or I-R involves caspase 3 activation and, furthermore, that the anti-apoptotic effect of CO at least in part may be associated with caspase 3 inhibition. Caspase 3 does not contain a heme moiety, and therefore, it is unlikely to be a direct target for CO; however, CO may modulate key mediators upstream of caspase 3 such as cytochrome oxidases present in mitochondria, which do contain heme moieties and are important in cell death responses. These issues will be the focus of future studies. Fig. 10 shows that anoxia (24A) significantly increased caspase 3 activity that was effectively decreased by CO. Furthermore, in Fig. 10 we show that the inhibition of p38 with either SB203580 or p38alpha DNM transfection attenuated the ability of CO to decrease anoxia-induced caspase 3 activity. Collectively, this suggests that CO modulates anoxia-induced DNA laddering, TUNEL staining, and caspase activation via p38 MAPK. Our results demonstrate for the first time that caspase 3 activation plays an important role in PAEC and mouse lung apoptosis during I-R and that the anti-apoptotic effect of CO may involve, at least indirectly, caspase 3 activity inhibition.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

I-R injury in the lung is of clinical importance in patients after hemorrhagic or cardiogenic shock, lung surgery, and lung transplantation. It is well established that apoptosis occurs in I-R injury of the lung, heart, kidney, intestine, and brain (2-5, 54). Furthermore, inhibiting apoptosis is cytoprotective and promotes organ survival after I-R injury (29, 30, 55). This suggests a pivotal role for apoptosis in the pathogenesis of I-R injury. Our current in vitro and in vivo results show that apoptosis is initiated in lung endothelial cells (PAEC) and mouse lungs during anoxia or ischemia, respectively, and continues during the reoxygenation or reperfusion phase. We also demonstrated that exogenous administration of 15 ppm CO, an extremely low level that is significantly below accepted safety standards, dramatically inhibits anoxia-induced apoptosis. Of note, previous publications documenting the protective effects of CO in cells use CO in the 10,000 ppm (1%) range, 1,000 times higher than our studies, which has important implications for the future therapeutic applications of exogenous CO. In addition, for the first time, we show that the anti-apoptotic effect of CO in PAEC anoxia or lung ischemia is mediated via p38 MAPK and involves caspase 3.

CO is a product of heme oxygenase-1 catalysis of cellular hemoprotein degradation. Although there is accumulating data for the protective functions of both heme oxygenase-1 and CO in I-R, the precise mechanisms and signaling pathways remain elusive. Our present study suggests that the anti-apoptotic function of CO via p38 MAPK and potentially caspase 3 may be one mechanism whereby CO is protective in I-R injury. Although we would predict that there must be modulation of a heme-containing protein(s) that is responsible for the protective effects, they remain elusive at this time and are the focus of ongoing studies. Suffice it to say that the signaling pathways described here and by others continue to support the role of p38 in the cytoprotective response of CO. Several potential mechanisms of the biologic effects of CO have been recognized. Recently, researchers found that CO protects against lung I-R injury via cGMP (5). However, Amersi et al. (56) show that CO-mediated cytoprotective effects against hepatic I-R injury were mediated via p38 MAPK. We tested whether CO had an anti-apoptotic effect in lung I-R, a phenomenon that has not been previously described, and examined the potential signaling pathways utilized by CO in eliciting these effects. In addition to the guanylate cyclase/cGMP pathway, the nitric-oxide synthase/NO system is a potential candidate. Both guanylate cyclase and nitric-oxide synthase have a heme moiety, and CO is a potent heme binder. Furthermore, other laboratories found that the NO/nitric-oxide synthase system can modulate I-R-induced apoptosis in cardiac myocytes and human endothelial cells (57, 58). When PAEC were exposed to anoxia in the presence of CO, there was no increase in cGMP or NO generation (data not shown). In addition, inhibiting guanylate cyclase or nitric-oxide synthase activity did not suppress the anti-apoptotic effect of CO. However, chemical or genetic inhibition of p38 MAPK ablated the anti-apoptotic effect of CO, indicating that the anti-apoptotic effect of CO in lung I-R is p38 MAPK-dependent.

The p38 MAPK pathway has been implicated in promoting cell survival as well as cell death (26). The p38 MAPKs are widely expressed in many tissues and are activated by dual phosphorylation on Thr and Tyr within the motif Thr-Gly-Tyr. The p38 MAPKs include the isoforms p38alpha , p38beta , p38gamma , and p38delta (59). The expression of multiple p38 MAPK isoforms in mammalian tissues suggests that these MAPKs may differ in their physiological function. The pyridinyl imidazole compound SB203580 selectively targets p38alpha and p38beta activation by competitive binding to the ATP pocket site. Interestingly, p38alpha and p38beta are inhibited by SB203580, whereas p38gamma , p38delta , and other MAPKs such as ERK1/2 and JNK1/2 are not (60). To the best of our knowledge, there are no known chemical inhibitors of p38gamma or p38delta . SB203580, presumably through p38 inhibition, has been shown to have a wide range of biologic effects when administered in vivo, including a potent anti-inflammatory effect in rats and mice with IC50 values of 15-25 mg/kg (61). Although there is a general agreement that p38 is activated by A-R or I-R, it is unclear whether p38 activation is protective or detrimental during A-R or I-R. The literature presents both pro-apoptotic and anti-apoptotic effects of p38 activation that are likely a reflection of cell type, different inducers, and potentially the differential modulation of each of the p38 isoforms. In cardiac myocyte apoptosis, for instance, the alpha  isoform promotes, whereas the beta  isoform prevents, apoptosis (62, 63). Our data show that CO activates p38 activity while down-regulating ERK1/2 and JNK1/2 activities during A-R. The fact that SB203580 inhibits the anti-apoptotic effects induced by CO in cells and mouse lungs invokes the involvement of p38alpha and/or p38beta . Although we use a p38alpha DNM in our CO studies, we recognize that we cannot at this juncture attribute the anti-apoptotic effect of CO solely to p38alpha , given that there are close structural relationships between p38alpha and -beta (64), and the biologic effect of p38alpha DNM may extend beyond inhibition of p38alpha alone, an aspect of DNM biology that is yet not well understood. The fact that there is an increase in p38 activity during reoxygenation (Fig. 5) and yet there is apoptosis during reoxygenation (Figs. 1 and 2) in the absence of CO may indicate that either there are insufficient levels of p38 generated during reoxygenation alone or that CO may up-regulate other anti-apoptotic isoforms of p38, such as p38beta , which has been shown to be anti-apoptotic (63). It is beyond the scope of the current study to delineate the relative contributions of the specific p38 isoforms in CO-mediated protection against apoptosis, but this is certainly an important focus for future studies.

Caspases play important roles in apoptosis signaling and effector mechanisms. All caspases share protein sequence homology and key catalytic and substrate-recognition amino acids and are expressed as proenzymes (46). Despite the 13 mammalian caspases identified (named 1-13), little is known regarding specific biological roles and interrelationships of these enzymes. Furthermore, even less is known about caspase regulation or inactivation. The facts that caspase precursors are constitutively expressed in cells and apoptosis can be quickly induced by multiple causative agents indicate that caspase regulation is extremely intricate and complex (65, 66). Caspase 3, one of three known effector caspases that mediate the final degradation steps, is thought to be a crucial component of the cell death machinery (26). In some models, caspase 3 is necessary and sufficient to execute apoptosis (65). We demonstrate that A-R in cells and I-R in lungs are caspase 3-dependent models. Caspase 3 is activated by A-R injury and inhibition of caspase 3 in PAEC and mice attenuates apoptosis. In addition, we show that CO can prevent A-R-induced caspase activation and I-R-induced TUNEL staining, which provide further evidence for CO as an anti-apoptotic molecule. Cross-talk between p38 MAPK and caspase signaling pathways has been previously reported in Fas-activated apoptosis (67). However, it is unclear as to whether p38 is upstream of caspases or vice versa (26, 67). We show that CO-induced p38 activation is likely upstream of CO-modulated caspase activity, and we are actively investigating its precise mechanisms. Given that the phosphorylation of caspases has not yet been described, direct interaction between p38 and caspase 3 seems unlikely. Rather, we speculate that any causal relationships between p38 activity and caspase 3 (de)activation are indirect, involving intermediate steps or components such as modulation of cytochrome c release. Vantieghem and co-workers (68) show that cytochrome c release is one of the earliest events in hypericin-induced apoptosis and involves caspase 3 activation in HeLa cells. Thus far, our current studies show that caspase 3 activation is a key mediator of A-R-induced cell death and that caspase 3 is downstream of p38 MAPK activation. Our future studies will address whether CO modulates upstream components of caspase 3 activation or cytochrome c release via p38 MAPK in A-R injury.

Furthermore, there are other potential downstream p38 MAPK targets of CO-mediated cytoprotection independent of caspase 3 activation. Brouard et al. (69) recently demonstrated that heme oxygenase-1/CO cooperates with NF-kappa B-dependent anti-apoptotic genes (c-IAP2 and A1) to protect against tumor necrosis factor-alpha -mediated endothelial cell apoptosis. However, CO does not appear to activate NF-kappa B directly but rather requires basal NF-kappa B activity to suppress tumor necrosis factor-alpha -mediated apoptosis. The p38 MAPK activity assays we used in the current studies show that CO increases activating transcription factor-2 phosphorylation, which can then modulate many genes. Other potential p38 MAPK targets include other known substrates such as the transcription factors CHOP and Elk1 or other kinases such as mitogen- and stress-activated protein kinase-1 and MAPK-activated protein, which then exert pleiotropic biologic effects depending on cell type and stimulus. At this point, however, CO modulation of these proteins via p38 MAPK is still speculative but warrants future detailed studies. In conclusion, our studies are the first to demonstrate that CO not only has an anti-apoptotic effect in I-R injury, thereby affording cellular protection, but also utilizes p38 MAPK and caspase 3 in exerting its anti-apoptotic effect both in vitro and in vivo during I-R injury.

    FOOTNOTES

* 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.

Supported by American Heart Association Grant 0160332U and by a Pfizer Atorvastatin Research Award.

** Supported by National Institutes of Health (NIH) Grant DK-43135.

*** An investigator of the Howard Hughes Medical Institute.

¶¶ Supported in part by NIH Grants HL-55330, AI-42365, and HL-60234 and by an American Heart Association Established Investigator Award (EIA).

|||| Supported by NIH Grant HL-04034 and by the American Lung Association of Connecticut. To whom correspondence should be addressed: Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, 333 Cedar St., LCI 105, New Haven, CT 06520. Tel.: 203-785-5877; Fax: 203-785-3826; E-mail: patty.lee@yale.edu.

Published, JBC Papers in Press, October 23, 2002, DOI 10.1074/jbc.M208419200

    ABBREVIATIONS

The abbreviations used are: I-R, ischemia-reperfusion; A-R, anoxia-reoxygenation; MAPK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated protein kinase; JNK1/2, c-Jun NH2-terminal protein kinase; PAEC, primary pulmonary artery endothelial cells; TUNEL, terminal deoxynucleotidyltransferase dUTP nick end-labeling; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorter; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; Z-DQMD-FMK, benzyloxycarbonyl-Asp(OMe)-Gln-Met-Asp(OMe) fluoromethyl ketone; Ac-YVAD-CMK, acetyl-Try-Val-Ala-Asp-chloromethyl ketone; DNM, dominant negative mutant; Wt, wild type; RA, room air; MEK, MAP or ERK kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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