Carbon Monoxide Modulates Fas/Fas Ligand, Caspases, and Bcl-2 Family Proteins via the p38 (cid:1) Mitogen-activated Protein Kinase Pathway during Ischemia-Reperfusion Lung Injury*

Carbon monoxide is protective in ischemia-reperfu-sion organ injury, but the precise mechanisms remain elusive. We have recently shown that low levels of exogenous carbon monoxide (CO) utilize p38 MAPK and attenuate caspase 3 activity to exert an antiapoptotic effect during lung ischemia-reperfusion injury. Our current data demonstrate that CO activates the p38 (cid:1) MAPK isoform and the upstream MAPK kinase MKK3 to modulate Fas/Fas ligand expression; caspases 3, 8, and 9; mitochondrial cytochrome c release; Bcl-2 proteins; and poly(ADP-ribose) polymerase cleavage. We correlate our in vitro findings with in vivo studies using MKK3-defi-cient and Fas-deficient mice. Taken together, our data are the first to demonstrate that CO has an antiapoptotic effect by inhibiting Fas/Fas ligand, caspases, proapoptotic Bcl-2 proteins, and cytochrome c release via the MKK3/p38 (cid:1) paraformaldehyde Blot Analysis— Protein levels of were analyzed by Western blot assays. To verify equivalent sample loading, membranes were stripped with Blot Restore Membrane rejuvenation solution (Chemicon Temecula, CA) and reprobed with anti-total p38 or anti- (cid:2) -tubulin antibody. Isolation of Cytosolic Fraction and Release of Cytochrome c— The cytosolic fraction of PAECs was isolated with Cytochrome c Release Apoptosis assay kit (Oncogene CA). Western blot with mouse anti-cytochrome c monoclonal antibody (On-cogene Research Products) was then performed. To verify equivalent sample loading, membranes were stripped with Blot Restore Membrane rejuvenation solution and reprobed anti- (cid:2) -tubulin antibody. Measurement of Caspase 3, 9 Activity— The activity of caspases 9 was measured with using 8 Colorimetric Activity assay kit respectively. supernatants incubated colorimetric substrate Ac-Asp- Glu-Val-Asp- p -nitroanilide (Ac-DEVD- p NA), N -acetyl-Ile-Glu-Thr-Asp-p -nitroanilide (Ac-IETD- p NA), or Ac-Leu-Glu-His-Asp- p -nitroanilide (Ac-LEHD- p NA) for caspases 3, 8, and 9, respectively. The release of p NA from Ac-DEVD- p NA, Ac-IETD- p NA, or Ac-LEHD- p NA was meas- ured at 405 nm using a spectrophotometer. Plasmid Constructs and Transient Transfections— The p38 (cid:1) MAPK constructs have been described previously (12), and the MKK3 and MKK6 plasmids were obtained from Dr. Jawed Alam. Cells were incu- bated for 6 h with DNA mixtures containing serum-free medium, Fu-GENE 6 reagent (Roche Applied Science), and wild type or dominant negative mutant plasmids. After incubation, cells were cultured for an additional 16 h in complete medium and then exposed to A-R in the presence or absence of 15 ppm CO. We have demonstrated the transfection efficiency of PAECs to exceed 80% using pEGFP transfections and by examining the cells under phase-contrast and fluorescence mi-croscopy as described previously (15). Statistics— Data are analyzed by Student’s test. Significance was accepted at p

Ischemia-reperfusion (I-R) 1 lung injury is a model for oxidant lung injury. There is increasing evidence that apoptosis plays an important role in the pathogenesis of I-R injury in a variety of organs such as brain (1), heart (2), kidney (3), liver (4), and lung (5). More importantly, inhibiting apoptosis during I-R injury is associated with improved survival and organ function (6,7). Unlike necrosis, apoptosis is a regulated cell death process involving specific pathways and cellular components. Therefore, delineating the precise apoptotic mechanisms involved during I-R injury may help optimize future therapies designed to abrogate I-R injury-induced apoptosis.
The heme oxygenase-1/carbon monoxide (CO) system has been shown to provide significant protection against hyperoxic lung injury (8), transplant rejection (9), vascular injury (10), and most recently arteriosclerotic lesions associated with chronic graft rejection (11). CO, a reaction product of heme oxygenase-1 activity, has been shown to have potent anti-inflammatory, antiproliferative, and antiapoptotic effects and thereby confers, at least in part, the cytoprotective effects of heme oxygenase-1. Furthermore the mitogen-activated protein kinase (MAPK) pathway, specifically p38 MAPK, appears to mediate the biologic effects of CO (12,13). We have recently shown that low levels of exogenous CO can suppress I-R-induced apoptosis in pulmonary endothelial cells and mouse lungs through p38 MAPK activation and caspase 3 activity inhibition (12).
However, if CO is to have potential as a therapeutic agent, more precise identification of CO-modulated targets will be necessary. Given our previous data showing that the antiapoptotic effects of CO in I-R lung injury is likely through caspase modulation as well as p38 MAPK activation (12), we extended our investigations to characterize the precise antiapoptotic pathways and the specific p38 MAPK isoform modulated by CO. We show that CO inhibits Fas/Fas ligand (FasL) expression and subsequent activation of caspases 3, 8, and 9; poly-(ADP-ribose) polymerase (PARP) cleavage; and mitochondrial cytochrome c release. In addition, CO differentially modulates the pro-and antiapoptotic members of the Bcl-2 family proteins. Furthermore all these effects of CO depend upon p38 MAPK activation, specifically p38␣ MAPK and the upstream MAPK kinase MKK3. We correlate our endothelial cell findings to mouse lungs subjected to I-R by using MKK3-deficient (MKK3 Ϫ/Ϫ ) and Fas receptor-deficient (Fas Ϫ/Ϫ ) mice. Taken together, our data are the first to demonstrate in cell and mouse models that the antiapoptotic effects of CO are dependent on the down-regulation of Fas/FasL expression, caspase activity, and modulation of Bcl-2 proteins via the MKK3/p38␣ MAPK pathway.
Cell Culture and CO Exposure-Rat primary pulmonary artery endothelial cells (PAECs) were generously provided by Dr. Troy Stevens (University of Alabama, Birmingham, AL) and were exposed to anoxiareoxygenation (A-R) in the presence or absence of CO according to our previous methods (12).
Apoptosis Assays-An annexin V-FITC kit (BD Pharmingen) was used to detect the apoptosis of PAECs, and a terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the apoptosis of lung tissues by using the in situ cell death detection kit (Roche Applied Science) as detailed previously (12).
Flow Cytometric Analysis of Cell Surface Fas and FasL Expression-Pulmonary artery endothelial cell surface expression of Fas and FasL was analyzed using a flow cytometer (BD Biosciences) and Cellquest software. In brief, cells were detached using trypsin; washed twice in cold PBS; pelleted; suspended in PBS containing Fas (1:100 dilution), FasL (1:100 dilution), or control rat IgG (1:100 dilution) antibody; and incubated on ice for 45 min. The cells were washed twice with cold PBS, resuspended in PBS containing anti-rat-FITC (1:50 dilution) antibody, and incubated on ice for 45 min. After two washes with PBS, cells were fixed in 1% paraformaldehyde and subjected to flow cytometry analysis.
Isolation of Cytosolic Fraction and Release of Cytochrome c-The cytosolic fraction of PAECs was isolated with Cytochrome c Release Apoptosis assay kit (Oncogene Research Products, San Diego, CA). Western blot with mouse anti-cytochrome c monoclonal antibody (Oncogene Research Products) was then performed. To verify equivalent sample loading, membranes were stripped with Blot Restore Membrane rejuvenation solution and reprobed with anti-␤-tubulin antibody.
Plasmid Constructs and Transient Transfections-The p38␣ MAPK constructs have been described previously (12), and the MKK3 and MKK6 plasmids were obtained from Dr. Jawed Alam. Cells were incubated for 6 h with DNA mixtures containing serum-free medium, Fu-GENE 6 reagent (Roche Applied Science), and wild type or dominant negative mutant plasmids. After incubation, cells were cultured for an additional 16 h in complete medium and then exposed to A-R in the presence or absence of 15 ppm CO. We have demonstrated the transfection efficiency of PAECs to exceed 80% using pEGFP transfections and by examining the cells under phase-contrast and fluorescence microscopy as described previously (15).
Statistics-Data are expressed as mean Ϯ S.E. and analyzed by Student's t test. Significance was accepted at p Ͻ 0.05.

CO Exerts an Antiapoptotic Effect through the p38␣ Isoform of p38 MAPK and the Upstream MAPK Kinase MKK3 in PAECs during A-R-
We have previously shown that CO activates p38 MAPK and that the inhibition of p38 MAPK using SB203580 ablates the antiapoptotic effect of CO (12). Despite increased p38 MAPK activity during reperfusion, apoptosis still occurs, and this is likely due to the fact that apoptosis is initiated during anoxia; unless p38 MAPK is activated to sufficient levels during anoxia, there is no attenuation of apoptosis during the reperfusion phase (12). In Fig. 1A we show a time course of when p38 activation occurs in the presence and absence of CO in PAECs. CO increases phospho-p38 levels during anoxia after 8 h, but p38 activation is maximal at 24 h. Of note, in the absence of CO, pulmonary artery endothelial cell death is maximal after 24 h of anoxia and is maintained during 30 min to 8 h of reoxygenation (15). Therefore, in subsequent assays we use 24 h of anoxia and 24 h of anoxia followed by 1 h of reoxygenation as the time points of interest in PAECs. There are four known isoforms of p38 MAPK (␣, ␤, ␥, and ␦). SB203580 specifically inhibits p38␣ and p38␤ but has no effects on p38␥ and p38␦ (16). Therefore, in Fig. 1B we investigated whether CO activated p38␣ and/or p38␤ during A-R. CO activated p38 MAPK, the p38␣ isoform, and the MAPK kinase(s) upstream of p38 MAPK, MKK3/6, but not p38␤ (data not shown) during A-R. There are no specific antibodies to MKK3 and MKK6 individually to the best of our knowledge. To delineate the specific roles of p38␣, MKK3, and MKK6 in mediating the antiapoptotic effect of CO during A-R, we performed transient transfection experiments with dominant negative mutant (DNM) plasmids of p38␣, MKK3, and MKK6. CO was unable to inhibit A-R-induced apoptosis in PAECs transfected with p38␣ or MKK3 DNM plasmids ( Fig. 1, C and D). However, CO still attenuated A-R-induced apoptosis in PAECs transfected with wild type p38␣ (12), wild type MKK3, wild type MKK6, or MKK6 DNM (data not shown). Of note, the background level of cell death due to transfection of p38 DNM or MKK3 DNM in room air was similar to that in room air alone. These data indicate that CO exerts its antiapoptotic effect by modulating the MKK3/p38␣ MAPK pathway, and the ability of SB203580 to attenuate the antiapoptotic effect of CO is likely due to the inhibition of p38␣ (since p38␤ is not involved in our model).

Inhibition of Fas/FasL or Modulation of the MKK3/p38 MAPK Pathway by CO Attenuates I-R-induced Apoptosis in Vitro (PAECs) and in Vivo (Mouse Lung) during I-R-The binding of
FasL to the Fas receptor is a prototypic signal for apoptosis, and therefore we investigated whether FasL inhibition can attenuate A-R-induced apoptosis in PAECs. Pretreatment of PAECs with a blocking antibody to FasL decreased apoptosis to levels similar to those of cells treated with exogenous CO (Fig. 2, A and B). In addition, 1 h of pretreatment with 10 M SB203580, a specific inhibitor of p38 MAPK, attenuated the antiapoptotic effect of CO. Pretreatment with SB203580 or anti-FasL in room air showed basal levels of cell death similar to those in room air alone. Lung  in vivo (mouse lung). A, flow cytometry analysis of apoptosis after PAECs were exposed to room air control (RA), 24 h of anoxia (24A), or 24 h of anoxia followed by 1 h of reoxygenation (24A/1R) in the presence or absence of CO, SB203580 (SB, a specific p38 MAPK inhibitor), or a I-R injury is an in vivo correlate of A-R injury in pulmonary cells. Similar to our in vitro data, our in vivo data confirm that CO has an antiapoptotic effect during I-R that is mediated by MKK3/p38 MAPK. In Fig. 2C, panel b, we show that wild type mice subjected to lung I-R exhibited increased TUNEL staining throughout the lung compared with that in naïve mice (panel a). Exogenous CO significantly attenuated I-R-induced TUNEL staining (panel d).
In the presence of a specific p38 MAPK inhibitor, SB203580, or in the genetic absence of MKK3, CO had little effect (panels e and f, respectively). Of note, similar to our cell data, CO retained its antiapoptotic effect in MKK6-deficient mice (data not shown). Furthermore we confirm our in vitro data by showing that Fas Ϫ/Ϫ mice do not exhibit I-R-induced lung apoptosis, suggesting that the Fas pathway may potentially be a mechanism of I-R-induced apoptosis (panel c).

CO Decreases Fas and FasL Expression through the MKK3/ p38 MAPK Pathway in Vitro and in Vivo during I-R-In
PAECs and mouse lung, we illustrated that the antiapoptotic effect of CO is dependent upon MKK3/p38 MAPK and that Fas/FasL inhibition also has a profound antiapoptotic effect. Therefore, we hypothesized that CO exerts an antiapoptotic effect during I-R by modulating the Fas/FasL pathway through MKK3/p38 MAPK. Anoxia alone or A-R increased Fas expression, which was significantly decreased in the presence of CO (Fig. 3A, lanes 2-5). Furthermore CO-mediated attenuation of Fas expression was ablated in the presence of a specific p38 MAPK inhibitor, SB203580 (lanes 7 and 8). Similar results were obtained for FasL expression in PAECs (Fig. 3B). Cells treated with SB203580 in room air (lane 6) showed basal levels of Fas and FasL expression similar to those in room air alone. In Fig. 3C, lane 3, we confirmed our data in vivo by showing that CO decreased I-R-induced Fas/FasL expression in lung tissue. However, CO could not decrease Fas/FasL expression in wild type mice treated with a specific p38 MAPK inhibitor, SB203580, or MKK3 Ϫ/Ϫ mice subjected to lung I-R (Fig. 3C, lanes 4 and 7, respectively). Naïve MKK3 Ϫ/Ϫ mice exhibited basal levels of Fas and FasL expression (Fig. 3C, lane 5). These data indicate that CO can inhibit the expression of Fas/FasL and that this effect depends upon MKK3/p38 MAPK in both cells and mouse lung during I-R injury.
CO Inhibits the Activity of Caspases 3, 8, and 9 through the MKK3/p38 MAPK Pathway in Vitro and in Vivo during I-R-We have previously shown that CO inhibits caspase 3 activity via p38 MAPK and that this contributes to the antiapoptotic effect of CO in PAECs during A-R (12). Our current studies investigate the roles of other caspases and potential downstream targets in the antiapoptotic effects of CO. Caspase 3 activation is regulated by at least two pathways, the "mitochondrial pathway," which involves the release of cytochrome c from the mitochondria into the cytosol and subsequent caspase 9 and caspase 3 activation, and/or receptor-mediated pathways, such as Fas/FasL binding, which lead to caspase 8 and then caspase 3 activation (17). Activated caspase 3 then cleaves substrates, such as PARP, leading to DNA fragmentation and apoptosis. Therefore, in the next series of studies we determined whether CO modulates caspases 3, 8, and 9, PARP, and cytochrome c release. In Fig. 4A, lanes 4 and 5, we first show that CO can effectively attenuate A-R-induced caspase 3, 8, and 9 activation in PAECs during A-R. In addition, pretreatment with SB203580 ablated the ability of CO to inhibit A-R-induced caspase activation, suggesting that CO depends upon p38 MAPK to modulate caspases during A-R in PAECs (lanes 6 and 7). We have already shown that CO decreases Fas/FasL expression through MKK3/p38 MAPK (Fig. 3), and given that blocking FasL also effectively diminishes A-R-induced caspase activities (Fig. 4A, lanes 8 and 9), we postulated a potential sequence of events, namely, that CO activates MKK3/p38␣ MAPK, leading to decreased Fas/FasL expression and a subsequent decrease in the activity of caspases 3, 8, and 9. Although it appears that CO retains some ability to decrease caspase 3 activity despite treatment with SB203580 (Fig. 4A, lanes 6 and 7), there was no statistical difference between lanes 2 and 3 and lanes 6 and 7. We certainly recognize that there may be other pathways aside from p38 MAPK that are involved; however, at this juncture it is beyond the scope of our studies. Our in vivo data corroborates that CO attenuates caspase 3, 8, and 9 activity through MKK3/p38 MAPK during lung I-R. CO was unable to decrease caspase activity in mouse lungs that were pretreated with SB203580, a p38 MAPK inhibitor, or that were MKK3deficient (Fig. 4B).
Caspase 8 Activation Is Upstream of Caspases 9 and 3 during Anoxia-We next attempted to delineate a general order of the caspases using caspase-specific inhibitors. The specificity of the caspase inhibitors have been previously validated (18). When PAECs were pretreated with Z-DQMD-FMK, a caspase 3-specific inhibitor, during anoxia, only caspase 3 activity was attenuated, indicating that caspase 3 was downstream of caspases 8 and 9 (Fig. 4C, lane 3). When PAECs were pretreated with Z-IETD-FMK, a caspase 8-specific inhibitor, during anoxia, all three caspases were inhibited (Fig. 4C, lane 4). When PAECs were pretreated with Z-LEHD-FMK, a caspase 9-specific inhibitor, caspase 8 activity was not affected, whereas caspases 3 and 9 were inhibited (Fig. 4C, lane 5). The data indicated that the sequence of caspase activation was 8, 9, and then 3 in PAECs during anoxia. Of note, inhibiting any of the caspases (8, 9, or 3) significantly attenuated A-R-induced apoptosis in PAECs (data not shown).
CO Inhibits PARP Cleavage through MKK3/p38 MAPK in Vitro and in Vivo during I-R-Caspase 3 activation results in PARP cleavage and subsequent DNA fragmentation and apoptosis (19). In Fig. 5A we show that CO inhibits PARP cleavage via MKK3/p38 MAPK in PAECs and mouse lung. We confirmed that PARP cleavage is downstream of caspases 8, 9, and 3 by showing that pretreatment with either caspase 3-, 8-, or 9-specific inhibitors all diminished PARP cleavage in PAECs during anoxia (Fig. 5B).

CO Inhibits Bid Cleavage through the MKK3/p38 MAPK Pathway in Vitro and in Vivo during I-R-Caspase 8 cleaves
Bid, a Bcl-2 homology 3 domain-containing proapoptotic Bcl-2 family protein, into its active, truncated form with subsequent translocation to the mitochondria where it induces the release of cytochrome c in a manner that is 500-fold more potent than Bax, another proapoptotic Bcl-2 protein (20,21). We demonstrated that caspase 8 activity was significantly increased during I-R and that CO attenuated caspase 8 activity via MKK3/p38 MAPK in Fig. 4. We then illustrated that A-R can induce the cleavage of the precursor form of Bid ( Fig. 6A, upper bands) to the active, truncated form tBid (lower bands) and that CO can inhibit Bid cleavage via p38 MAPK in PAECs (Fig. 6A, top panel, lanes 4 -7). Our in vivo data in Fig. 6A, bottom panel, correlate with our in vitro data. We next confirmed that caspase 8, rather than caspase 3 or 9, is responsible for Bid cleavage during anoxia in PAECs (Fig.  6B). Inhibition of caspase 8 activity with Z-IETD-FMK inhibited anoxia-induced Bid cleavage (Fig. 6B, lane 4), whereas inhibiting caspase 3 or 9 had no effect (Fig. 6B, lanes 3 and 5,  respectively).

CO Inhibits A-R-induced Cytochrome c Release, and This Precedes Caspase 9 and 3 Inhibition-Cleaved
Bid can translocate to the mitochondria and induce cytochrome c release, which then leads to caspase 9 and 3 activation (21). We found that CO can attenuate A-R-induced cytochrome c release (Fig.  7A, lanes 4 and 5) and that this is through p38 MAPK activity (Fig. 7B, lane 7). FasL inhibition also attenuates cytochrome c release (Fig. 7A, lanes 6 and 7). When we pretreated PAECs with Z-IETD-FMK, a caspase 8-specific inhibitor, cytosolic cytochrome c levels were decreased during anoxia (Fig. 7B, lane  4), whereas caspase 3 and 9 inhibition had no effect (Fig. 7B,  lanes 3 and 5, respectively). These data in conjunction with our data showing that CO modulates caspase 8 via MKK3/p38 MAPK (Fig. 4) indicate that CO inhibits cytochrome c through p38 MAPK activation and caspase 8 inhibition, which precedes caspase 9 and 3 inhibition. Of note, CO does not modulate Bax (see below). Fig. 8, top panel, we show that A-R decreases the endogenous levels of the antiapoptotic proteins Bcl-2 and Bcl-X L in PAECs. CO inhibits the A-R-induced decrease in Bcl-2 and Bcl-X L expression but depends upon p38 MAPK activity as shown by the effects of SB203580 pretreatment (Fig.  8, top panel, lanes 6 and 7). A-R and CO had no effects on the expression of the proapoptotic Bax. Our in vivo data also strongly support our in vitro results by showing that CO can increase the expression of Bcl-2 and Bcl-XL during lung I-R and that the effect of CO is dependent upon the MKK3/p38 pathway (Fig. 8, bottom panel). CO was unable to increase Bcl-2 and Bcl-XL expression in mice that were pretreated with a specific p38 MAPK inhibitor, SB203580 (lane 4), or that were deficient in MKK3 (lane 6) during I-R. Of note, inhibiting FasL with anti-FasL antibody had the same effect on cleaved Bid, Bcl-2, and Bcl-X L expression as CO had in PAECs during A-R (data not shown).

DISCUSSION
There is significant evidence for the pivotal role of apoptosis in the pathogenesis of I-R organ injury (6,7,22). Strategies to attenuate I-R-induced apoptosis will expand the currently limited therapeutic options. We have found that CO acts as a potent regulator of apoptosis and may be one mechanism whereby the induction of heme oxygenase-1, the enzyme responsible for more than 90% of endogenous CO generation, affords significant cytoprotection against a variety of noxious stimuli such as lung I-R injury. We previously demonstrated that exogenous administration of 15 ppm CO, an extremely low level significantly below accepted safety standards, dramatically inhibited A-R-induced apoptosis and that the antiapoptotic effect of CO is mediated via p38 MAPK and involves caspase 3 (12). Our current studies more precisely delineate potential mechanisms utilized by CO. We show that exogenous CO activates MKK3/6 and p38␣ MAPK, but not p38␤ MAPK, during A-R in PAECs. The upstream MAPK kinases MKK3 and MKK6 are thought to be the predominant kinases responsible for activating p38 MAPK. Differences in the relative contribution of these protein kinases in activating p38 MAPK are attributed to the p38 MAPK isoform involved, the stimuli, and cell type. For instance, MKK3 is the major activator of p38 MAPK in PC-12 cells exposed to osmotic stress, while MKK6 is the dominant activator of p38 MAPK in epithelial cells exposed to osmotic stress, tumor necrosis factor-␣, and interleukin-1 and in monocytes stimulated by bacterial lipopolysaccharide (23)(24)(25). Furthermore it also has been reported in transformed FIG. 4. CO inhibits the activity of caspases 3, 8, and 9 through MKK3/ p38 MAPK during I-R in vitro and in vivo. A, caspase activity in PAECs was detected as described under "Experimental Procedures" after cells were exposed to CO in the presence or absence of SB203580, a specific inhibitor of p38 MAPK, or in the presence of a FasL-blocking antibody (anti-FasL) during A-R. The mean caspase activity from three independent experiments Ϯ S.E. is shown. *, p Ͻ 0.05 compared with room air control. B, caspase activity in lung lysates from wild type and MKK3 Ϫ/Ϫ mice was detected after mice were subjected to lung I-R in the presence or absence of CO or SB203580. *, p Ͻ 0.05 compared with naïve. C, caspase activity in PAECs was detected after pretreatment with a caspase 3-specific inhibitor (Z-DQMD), a caspase HeLa and COS-7 cells that MKK3 activates p38␣ and p38␥, while MKK6 activates p38␣, p38␤, and p38␥ (26). The different signal transduction pathways initiated by various stimuli leading to the activation of different p38 MAPK isoforms by one or more specific MAPK kinases may account for stimulus-specific and cell-specific responses.
We show in our I-R model that in the presence of p38␣ DNM or MKK3 DNM (in PAECs) or MKK3 deficiency (in mice), CO can no longer attenuate I-R-induced apoptosis. Notably MKK6 DNM transfection in cells or MKK6 deficiency in mice had no effect on the antiapoptotic effect of CO. The literature presents both proapoptotic and antiapoptotic effects of p38 MAPK activation that are likely a reflection of cell type, different inducers, and, potentially, the differential modulation of each of the different p38 MAPK isoforms. Although p38␣ MAPK is generally thought to be proapoptotic, there are recent reports that p38␣ MAPK, but not p38␤ MAPK, can inhibit the apoptotic death of differentiating neurons (27). The p38 MAPK isoforms are likely coupled to distinct upstream signal transduction pathways. This would enable activation of specific p38 MAPK isoforms in response to a variety of stimuli. Alternatively the p38 MAPK isoforms may have different downstream targets, which would allow coupling of the various p38 MAPK isoforms to specific biologic responses. Thus, the differential activation of the p38 MAPK isoforms can facilitate cell type-and stimulus-specific cellular responses and may account for the multiple actions of p38 MAPK, thereby highlighting the importance of precisely identifying the p38 MAPK isoforms and upstream modulators involved. Our data indicate that the antiapoptotic effect of CO, in our models of endothelial cell and lung I-R, is dependent upon MKK3/p38␣ MAPK pathways.
The Fas (CD95)/Fas ligand (CD95L) system is a key regulator of apoptosis. Binding of Fas by its ligand FasL can induce caspase 8 activation and lead to the activation of downstream caspases followed by cleavage of key regulatory proteins, such as PARP, and ultimately result in apoptosis (28 -30). The Fas/ FasL system was up-regulated in myocytes during hypoxia, ischemia, and I-R (31, 32). Kitamura et al. (33) found that increased Fas/FasL expression in lung tissues after lipopolysaccharide injury played a critical role in lung injury and that the proper regulation of the Fas/FasL system was important for the potential treatment of acute lung injury or acute respiratory distress syndrome. Ke et al. (34) showed that heme oxygenase-1 gene transfer prevented Fas/FasL-mediated apoptosis and significantly prolonged allogeneic orthotopic liver transplantation survival. We show that I-R lung injury increases Fas/FasL expression in PAECs and lung tissues, which can then be modulated by CO. The administration of a blocking FasL antibody in cells or Fas deficiency in mice decreased apoptosis to levels found in CO-treated cells and animals. Moreover, CO modulates Fas/FasL and subsequent downstream effectors via p38␣ MAPK and MKK3 in lung I-R. CO could not attenuate Fas and FasL expression and the downstream effectors in the presence of p38 MAPK inhibition or MKK3 deficiency.
We also show that CO differentially modulates pro-and antiapoptotic Bcl-2 family members through the MKK3/p38 MAPK pathway. I-R injury decreases antiapoptotic protein levels (Bcl-2 and Bcl-X L ), but CO maintains the levels of Bcl-2 and Bcl-X L while decreasing levels of the proapoptotic cleaved Bid during I-R. Bid has been implicated in the tumor necrosis factor and Fas death signal pathways (21,35). The precise mechanism through which Bid is proteolytically activated in PAECs and lung during I-R is unclear at the present time. However, in other cells and tissues, several intracellular molecules have been proposed to be the activators of Bid, including caspase 8, granzyme B, and caspase 3 (20,21,36,37). The Fas/caspase 8 pathway has been reported to be the most efficient mechanism for Bid cleavage in various cell types and could be the major pathway for Bid cleavage in our model. We show that Fas/FasL expression and caspase 8 activity are sig- FIG. 5. CO inhibits PARP cleavage through MKK3/p38 MAPK during I-R in vitro and in vivo. A, top, PAECs were exposed to A-R in the presence or absence of CO or SB203580, a specific p38 MAPK inhibitor, and cell lysates were analyzed for PARP cleavage by immunoblotting with anti-cleaved PARP antibody as described under "Experimental Procedures." A, bottom, wild type or MKK3 Ϫ/Ϫ mice were subjected to lung I-R in the presence or absence of CO or SB203580, and lung lysates were analyzed for PARP cleavage by immunoblotting with anticleaved antibody. B, PAECs were exposed to 24 h of anoxia in the presence or ab- nificantly increased during I-R and that the blockade of Fas/ FasL during A-R also attenuated Bid cleavage. Furthermore caspases 9 and 3 have no effect on Bid cleavage. CO exposure can inhibit cleavage of Bid during I-R but is dependent upon p38 MAPK/MKK3 activity in cells and mouse lung. There is no change in the proapoptotic protein level of Bax in PAECs during A-R with or without CO exposure. We also demonstrate that CO attenuates the apoptotic events downstream of Bid cleavage, namely cytochrome c release, caspase 9 and 3 activation, and finally PARP cleavage. We confirm that the aforementioned events are mediated by CO through MKK3/p38␣ MAPK and Fas/FasL modulation.
In summary, our observations that FasL inhibition can reproduce the effects of CO on Bcl-2 proteins, cytochrome c release, and PARP in conjunction with our data showing that CO-modulated Fas/FasL expression is downstream of MKK3/ p38␣ MAPK suggest that a likely progression of CO-mediated antiapoptotic signaling is first MKK3/p38␣ MAPK activation, then Fas/FasL down-regulation followed by decreased caspase 8 activity, then differential modulation of pro-and antiapoptotic Bcl-2 proteins, and finally decreased cytochrome c release and cleaved PARP expression. Furthermore we are the first to validate these cellular mechanisms of CO in vivo when we demonstrate that Fas-deficient mice do not exhibit I-R-induced apoptosis and that p38 MAPK inhibition or MKK3 deficiency in mice ablates the ability of CO to modulate Fas/FasL and downstream apoptosis effectors.
FIG. 6. CO attenuates proapoptotic Bid cleavage through MKK3/p38 MAPK activation during I-R in vitro and in vivo. A, top, PAECs were exposed to A-R in the presence or absence of CO or SB203580, a specific p38 MAPK inhibitor, and cell lysates were analyzed for Bid cleavage (tBid) by immunoblotting with anti-Bid antibody as described under "Experimental Procedures." A, bottom, wild type or MKK3 Ϫ/Ϫ mice were subjected to lung I-R in the presence or absence of CO or SB203580, and lung lysates were analyzed for Bid cleavage by immunoblotting with anti-Bid antibody. B, PAECs were exposed to 24 h of anoxia in the presence or absence of caspase 3 (Z-DQMD)-, 8 (Z-IETD)-, or 9 (Z-LEHD)-specific inhibitors and then analyzed for Bid cleavage by immunoblotting with anti-Bid antibody. ␤-Tubulin was used as a loading control for all immunoblots. The data are representative of three independent experiments. RA, room air control; 24A, 24 h of anoxia; 1R, 24 h of anoxia followed by 1 h of reoxygenation; SB, SB203580; I/R, I-R; CO/I/R, I-R in the presence of CO.

FIG. 7. CO attenuates cytochrome c release during A-R in
PAECs. The cytosolic fraction of PAECs was isolated and analyzed for cytochrome c release by immunoblotting with anti-cytochrome c antibody as described under "Experimental Procedures." A, PAECs were exposed to A-R in the presence or absence of CO and then analyzed for cytochrome c release. B, PAECs were exposed to 24 h or anoxia in the presence or absence of SB203580, a specific p38 MAPK inhibitor, or caspase 3 (Z-DQMD)-, 8 (Z-IETD)-, or 9 (Z-LEHD)-specific inhibitors and then analyzed for cytochrome c release. ␤-Tubulin was used as a loading control for all immunoblots. The data are representative of three independent experiments. RA, room air control; 24A, 24 h of anoxia; 1R, 24 h of anoxia followed by 1 h of reoxygenation; SB, SB203580.
FIG. 8. CO increases antiapoptotic Bcl-2 family proteins through MKK3/p38 MAPK activation during I-R in vitro and in vivo. Top, PAECs were exposed to A-R in the presence or absence of CO or SB203580, a specific p38 MAPK inhibitor, and then analyzed for Bax, Bcl-X L , and Bcl-2 by immunoblotting with the respective antibodies as described under "Experimental Procedures." Bottom, wild type or MKK3 Ϫ/Ϫ mice were subjected to lung I-R in the presence or absence of CO or SB203580, and lung lysates were analyzed for Bax, Bcl-X L , and Bcl-2 by immunoblotting with the respective antibodies. ␤-Tubulin was used as a loading control for all immunoblots. The data are representative of three independent experiments. RA, room air control; 24A, 24 h of anoxia; 1R, 24 h of anoxia followed by 1 h of reoxygenation; SB, SB203580; I/R, I-R; CO/I/R, I-R in the presence of CO.