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Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA
1 Correspondence: Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Pittsburgh Medical Center, 3459 Fifth Ave., MUH NW 628, Pittsburgh, PA 15213, USA. E-mail: Ryters{at}upmc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: caspase-8 • DISC • hypoxia • reoxygenation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In the lung, the alveolar epithelium represents a direct target of variations in alveolar O2 tension (2)
. Alveolar hypoxia can result from hypoventilation related to central nervous disorders, obstructive airway diseases, or pulmonary edema from heart failure or acute lung injury (2)
. Restitution of O2 to previously hypoxic tissues causes tissue damage associated with increased production of reactive oxygen species (ROS), a hallmark of ischemia/reperfusion (I/R) injury. The hypoxia/reoxygenation (H/R) model, involving a sustained decrease of O2 over many hours, followed by restitution of ambient O2 tension, is often used to simulate the effects of I/R in cell culture.
Numerous studies have suggested that hypoxia can induce apoptosis, dependent on transcriptional activation of apoptotic factors (3)
. Apoptosis serves a critical function in maintaining tissue homeostasis under physiological conditions and may contribute to disease pathogenesis. Cells can initiate the apoptotic process by two known pathways: an intrinsic (mitochondria-dependent) pathway and an extrinsic (receptor-dependent) pathway. Bcl-2 family members act as critical regulators of the intrinsic apoptotic pathway. Some members of this family, such as the anti-apoptotic protein Bcl-XL, predominantly localize to the outer mitochondrial membrane whereas others interact indirectly with mitochondria. In response to diverse stimuli, proapoptotic proteins such as Bax initiate the intrinsic apoptotic pathway by triggering a loss of outer mitochondrial membrane integrity, which releases cytochrome c from the mitochondrial intermembrane space (4)
. Once released to the cytosol, cytochrome c interacts with Apaf-1, which activates caspase-9 and, in turn, its downstream caspase-3, resulting in the morphological features of apoptosis (4)
. Bcl-XL inhibits intrinsic apoptosis by preventing Bax from disrupting outer mitochondrial membrane integrity (5)
.
The extrinsic apoptotic pathway initiates when a death ligand, such as the Fas ligand (FasL), interacts with its cell surface receptor (i.e., Fas), forming a death-inducing signal complex (DISC) (6)
. Activation of Fas triggers its oligomerization and rapid recruitment of FADD (Fas-associated death domain) and caspase-8 to the cytoplasmic death domain of Fas. Activated caspase-8 subsequently cleaves Bid into truncated Bid (tBid), which translocates from the cytosol into the mitochondrial membrane, where it stimulates cytochrome c release and subsequent caspase-9 activation. Thus, the extrinsic and intrinsic apoptotic pathways converge through tBid. The activated caspase-8 can directly activate caspase-3 in some cell types (7)
. Bcl-XL can inhibit extrinsic apoptosis by blocking Bid redistribution downstream of caspase-8 (8)
.
H/R stress can trigger the intrinsic apoptotic cascade in several cell types (9
, 10)
. We previously demonstrated that the induction of apoptosis in mouse lung endothelial cells (MLEC) by H/R treatment involved the activation of Bax-dependent and death receptor-mediated pathways (11)
. In the current study, we found that Bcl-XL inhibited H/R-induced cell death in MLEC by blocking Bax and Bid translocation to the mitochondria. We show that Bcl-XL inhibited caspase-8 cleavage and disrupted DISC formation in the plasma membrane of MLEC subjected to H/R stress. We show for the first time that Bcl-XL may antagonize DISC formation in organelle membranes including the Golgi complex. The inhibitory effect of Bcl-XL on DISC formation in these cellular compartments can protect endothelial cells from the lethal effects of H/R.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation and culture of murine lung endothelial cells (MLEC)
Endothelial cells were isolated with an immunobead protocol as described (12)
. Briefly, mouse lungs were digested in collagenase and filtered through 100 µm cell strainers, centrifuged, and washed twice with medium. Cell suspensions were incubated with a monoclonal antibody (rat anti-mouse) against platelet endothelial cell adhesion molecule-1 (Santa Cruz) for 30 min at 4°C. The cells were washed twice with buffer to remove unbound antibody and resuspended in binding buffer containing washed magnetic beads coated with sheep anti-rat IgG (Dynal Biotech ASA, Oslo, Norway). Attached cells were washed 4 or 5 times in cell culture medium, then digested with trypsin/EDTA to detach the beads. Bead-free cells were centrifuged and resuspended for culture. After two passages, the cells were incubated with fluorescent-labeled di-acetylated LDL, which is taken up only by endothelial cells and macrophages, and sorted to homogeneity by FACS.
Cell culture and treatments
The MLEC were cultured in DMEM containing 10% fetal bovine serum (FBS), 6.32 g/L of HEPES, and 3.3 mL of EC growth supplements, in humidified incubators at 37°C. For adenoviral infections, cells were grown to 30% confluence and changed to serum-free medium containing 106 CPU/mL of an adenoviral vector inserted with Bcl-XL- or LacZ. Infected cells were incubated for 3 h, then restored to normal DMEM medium containing 10% FCS for an additional 2 days incubation. For hypoxia treatment, the MLEC were grown to 90% confluence, changed to fresh medium, and transferred to a COY anaerobic chamber (COY Laboratory Products Inc., Ann Arbor, MI, USA) with 95% N2, 5% CO2 for 24 h. After hypoxia incubation, cells were restored to 95% air, 5% CO2 for various reoxygenation intervals.
Cell death determination
Cell viability was determined by Trypan blue exclusion analysis (Life Technologies, Grand Island, NY, USA) and expressed as the percentage of dead (blue-stained) cells.
Cell fractionation
For mitochondrial isolation, at various times after exposure to H/R, MLEC were harvested in 0.05% digitonin in an extraction buffer containing 50 mM HEPES, pH 7.5, 50 mM KCl, 5 mM DGTA, and 2 mM MgCl2 with protease inhibitors. The cell extracts were spun at 700 x g for 10 min. The supernatants were transferred to new tubes, then centrifuged again at 14,000 x g at 4°C for 20 min. The resulting supernatants were removed and the pellets retained for Western blot.
For isolation of plasma membrane, MLEC were harvested and homogenized in MBS (25 mM 2-(N-morpholino)-ethanesulfonic acid, pH 6.5, 0.15 M NaCl) containing 1% Triton X-100. Homogenates were adjusted to 40% sucrose by the addition of 2 mL of 80% sucrose prepared in MBS and placed at the bottom of an ultracentrifuge tube. A 5–30% discontinuous sucrose gradient was formed above (4 mL of 5% sucrose/ 4 mL of 30% sucrose, both in MBS lacking detergent) and centrifuged at 39,000 rpm for 18 h in an SW41 rotor (Beckman Instruments, Palo Alto, CA, USA). A band at the interface of 5% and 30% sucrose was collected and used for IP and Western blot or to measure alkaline phosphatase activity.
The Golgi complex was isolated using sucrose density gradient centrifugation, as described elsewhere (13)
. After washing with PBS, the cells were harvested in G buffer (10 mM Tris-HCI, 0.25 M sucrose, 2 mM MgCI2, pH 7.4) containing 10 mM CaCI2 and proteinase inhibitors. The cells were disrupted with 20 strokes in a Potter-type homogenizer. The homogenate was centrifuged at 2500 x g for 10 min and the pellet was discarded. The resulting postnuclear supernatant was harvested and the sucrose concentration adjusted to 1.4 M final concentration. This suspension was loaded onto thebottom of an ultracentrifuge tube and overlaid in succession with 1.2 M, 1.0 M, and 0.8 M of sucrose in G buffer. Samples were then centrifuged at 95,000 x g for 2.5 h. Two bands from the top, representing 0.8/1.0 and 1.0/1.2 M interfaces, were carefully removed and diluted with G buffer without sucrose, collected by centrifugation at 80,000 x g for 30 min, and used for the experiments.
A 100 µL aliquot of each fraction (membrane or Golgi) was used to measure alkaline phosphatase activity. After direct addition of substrate solution (p-nitrophenyl phosphate; R&D Systems, Minneapolis, MN, USA), followed by 30 min incubation at room temperature, the absorbance at 405 nm was measured.
For nuclei isolation, MLEC were washed twice with cold PBS and scraped with a rubber policeman in the same buffer. Nuclear protein extracts were prepared as described (14)
.
Western blot analysis
Proteins were isolated from the culture of MLEC with RIPA buffer (1xPBS, 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.1 mg/mL PMSF, 30 µL/mL aprotinin, 1 mM sodium orthovanadate). For IP, 1 µg of anti-Fas antibody was added to 500 µg of total protein in 500 µL, rotated for 2 h at 4°C, then incubated with 20 µL of protein A-sucrose beads (Santa Cruz Biotechnology) for another 2 h, spun down at 500 x g, and washed three times with RIPA buffer. Then 20 µL of loading buffer (100 mM Tris-HCl, 200 mM DTT, 4% SDS, 0.2% bromphenol blue, 20% glycerol) was added. For SDS-PAGE, samples containing equal amounts of protein were boiled in the loading buffer and separated on SDS-PAGE, followed by transfer to polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk and stained with primary antibodies for 2 h at the optimal concentrations. After five washes in PBS with 0.2% Tween 20, the horseradish peroxidase-conjugated secondary antibody was applied and the blot was developed with ECL reagents (Amersham Biosciences, Piscataway, NJ, USA).
Statistical analysis
All values are expressed as means ±SE. Statistical significance was determined by Student’s t test and a value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). We investigated the effect of H/R treatment on cell death in MLEC, and the regulatory role of Bcl-XL, using Trypan blue exclusion as an assay for cell death. MLEC were infected with adenovirus containing LacZ or Bcl-XL for 3 days, exposed to hypoxia (95% N2, 5% CO2) for 24 h, then restored to normoxic culture conditions (95% air, 5% CO2) for various reoxygenation intervals. As shown in Fig. 1B
, Bcl-XL expression significantly protected MLEC from cell death after 6 h of reoxygenation relative to the LacZ-infected control. MLEC (LacZ or Bcl-XL) maintained under continuous hypoxia for up to 72 h, exhibited a similar level of cell death (data not shown) as observed for the initial 24 h hypoxia interval with 0 h reoxygenation (Fig. 1B
).
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Next we tested the hypothesis that cell death induced by H/R occurred by apoptosis, a caspase-dependent event. After H/R treatment, the activated form of caspase-9 (p37) appeared during the reoxygenation phase (12–24 h) in control (LacZ-infected) cells (Fig. 1C
). Bcl-XL expression prevented the appearance of activated caspase-9 until 48 h of reoxygenation (Fig. 1C
).
Bcl-XL protected against H/R-induced cell death in MLEC by inhibiting the activation and mitochondrial translocation of Bax
After H/R treatment of MLEC, cytosolic Bax levels were high during the reoxygenation phase and did not change as a function of Bcl-XL expression (Fig. 2
A). During exposure to H/R, MLEC infected with Bcl-XL displayed diminished levels of Bax protein in the mitochondrial fraction, relative to the high levels observed in mitochondria from control (LacZ-infected) cells (Fig. 2B
). Cell lysates were immunoprecipitated with the anti-Bax monoclonal antibody (6A7), which specifically recognizes a conformational change in Bax protein associated with its activation (15)
. Bcl-XL expression inhibited the activation of Bax during reoxygenation (Fig. 2C
). Total cellular Bax levels are shown for reference (Fig. 2D
), and remained unchanged by Bcl-XL expression. The results show that Bcl-XL can inhibit H/R-induced Bax activation and mitochondrial translocation.
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Bcl-XL protected against H/R-induced cell death in MLEC by inhibiting DISC formation and Fas trafficking in the extrinsic apoptotic pathway
H/R stress may stimulate the expression of FasL, which can trigger the extrinsic (Fas-dependent) apoptotic pathway (16
, 17)
. We examined the status of apoptotic mediators starting with DISC formation, the most proximal event in the Fas apoptotic pathway. Exposure to H/R triggered the time-dependent activation of caspase-8 in MLEC during the reoxygenation phase (Fig. 3
A). Expression of Bcl-XL inhibited the activation of caspase-8 at early time points during reoxygenation (12–24 h) (Fig. 3A
).
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Plasma membrane-containing fractions were isolated from MLEC subjected to H/R treatments. Anti-Fas immunoprecipitation revealed an interaction of caspase-8 with Fas (Fig. 3B
), demonstrating DISC formation in both Bcl-XL and LacZ-infected cells subjected to H/R treatment. DISC formation was elevated in both cell lines at two reoxygenation time points (3 h, 6 h), whereas there was no apparent DISC formation in either cell line under normoxic conditions (absence of proapoptotic stimuli) (Fig. 3B
). DISC formation was markedly diminished in the Bcl-XL-infected cells relative to that observed in LacZ-infected cells. According to immunoblot analysis, total Fas levels were unaltered as a function of cell type or treatment and were used as a loading control. Caveolin-1, a marker of plasma membrane lipid microdomains (18)
, was strongly expressed in all plasma membrane fractions and did not modulate as a function of cell type or treatment. The Golgi complex-associated protein (GRASP65), a Golgi-specific marker (19)
, was virtually undetectable in the isolated plasma membrane fractions (Fig. 3B
).
Bcl-XL expression differentially modulates the subcellular localization of the DISC
After translation of Fas, cells can express Fas intracellularly and at the cell surface. Cytoplasmic Fas localizes to the Golgi complex (20)
, a series of functionally distinct processing compartments that plays a central role in the biosynthesis and secretion of macromolecules in eukaryotic cells (21)
. We therefore examined the subcellular localization of the DISC in MLEC in response to H/R treatment and the regulatory role of Bcl-XL. MLEC infected with Bcl-XL or LacZ were subjected to hypoxia (24 h), followed by reoxygenation (6 h), then fractionated to isolate nuclei, Golgi complex, or mitochondria. Immunoprecipitation with anti-Fas and immunoblotting with caspase-8 revealed that the level of DISC formation in the Golgi complex and nucleus was diminished in Bcl-XL-expressing cells relative to LacZ-infected cells (Fig. 4
A). Conversely, more DISC formed in mitochondria from Bcl-XL-expressing cells subjected to H/R, relative to LacZ-infected cells (Fig. 4A
). The purity of the Golgi fractions was assessed by immunoblot analysis of marker proteins. The Golgi fraction exhibited a strong expression of GRASP65, which did not modulate as a function of cell type or treatment condition (Fig. 4B
). There was no detectable cytochrome c, a marker of mitochondria, in the Golgi fraction (Fig. 4B
). The purity of the Golgi fraction was assessed by measuring alkaline phosphatase activity. Golgi fractions displayed an average A405 of 0.127 ± 0.022 comparable to negative control values from protein-free reactions (0.123±0.012). Positive control (membrane extract) displayed average values of A405 of 2.105 ± 0.015.
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A family of coiled-coil proteins that includes GRASP65 maintains the Golgi structure by forming an exoskeleton or Golgi matrix. After its translation, GRASP65 directly localizes to the Golgi membrane without assembly on the endoplasmic reticulum (ER) or subsequent vesicular transport to the Golgi apparatus (19)
. Immunoprecipitation with anti-GRASP65, followed by immunoblotting with caspase-8 or Fas, revealed an association between GRASP65 and both FAS and caspase-8 in control (LacZ-infected) MLEC subjected to H/R treatment (Fig. 4C
). Bcl-XL expression significantly decreased the association of GRASP65 with either caspase-8 or Fas relative to that observed in control cells (Fig. 4C
). Total GRASP65 levels, as assessed by immunoblotting, did not modulate as a function of cell type and were used as the loading control (Fig. 4C
).
Bcl-XL inhibition of Bid cleavage
We demonstrated in Fig. 3A
that caspase-8, which lies downstream of Fas, was activated in H/R-treated MLEC during the reoxygenation phase. Caspase-8 and Fas play a role in the activation of Bid, a proapoptotic Bcl-2 family protein, a substrate of caspase-8 (22)
. MLEC were infected with adenovirus containing Bcl-XL or LacZ and subjected to hypoxia (24 h). After 6 h of reoxygenation, the truncated form of Bid (p15) appeared in control (LacZ-infected) cells but not in Bcl-XL-infected cells (Fig. 5
).
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Bcl-XL stimulated FLIP expression during reoxygenation
Fas-mediated-apoptosis can be regulated by other DED-containing molecules, such as FLIP (Flice-like inhibitory protein), which resembles caspase-8. FLIP can inhibit caspase-8 activity in other cell types (23)
. MLEC expressing Bcl-XL, when subjected to H/R, up-regulated FLIP expression during the reoxygenation phase, relative to control cells expressing LacZ (Fig. 6
). These results suggest another possible mechanism by which Bcl-XL can down-regulate caspase-8.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, suggesting that up-regulation of Bcl-XL may participate in protective ischemic preconditioning. We demonstrated that H/R caused a time-dependent loss of cell viability in MLEC associated with the activation of caspase-9. In this model, we observed that Bcl-XL protected against H/R-induced cell death, associated with an inhibition of caspase-9 activation (Fig. 1)
.
It is well known that BclXL protects cells from apoptosis induced by a variety of stimuli, but the underlying mechanisms remain incompletely understood. Bcl-XL preserves mitochondrial integrity and prevents the subsequent release of apoptogenic molecules such as cytochrome c (25)
. Several Bcl-2 family members, such as Bax and Bid, which are activated by diverse proapoptotic stimuli, including hypoxia (9)
, translocate to the mitochondrial membrane, where they homo-oligomerize and form a multmeric pore that facilitates the initial efflux of cytochrome c (26)
. Overexpression of Bcl-XL reportedly confers protection upon mitochondria, rendering it more difficult for such stimuli to induce permeability transition pore opening (27)
. A functional role of Bcl-2 family proteins as ion channels has recently been described (28)
. Bcl-XL may form small ion channels that assume a mostly closed conformation, selective for the passage of cations; whereas the proapoptotic protein Bax tends to form larger channels, which assume a mostly open conformation selective for the passage of anions (28)
.
Bcl-XL can inhibit the intrinsic apoptotic pathway by preventing the activation of Bax. Furthermore, Bcl-XL blocks Bax translocation, possibly as a secondary consequence of its inhibition of Bax activation, which is a prerequisite for translocation (29)
. Under normal conditions, Bax is constrained from targeting membrane sites, including mitochondria. In the absence of a death signal, Bax adopts a conformation in which its carboxyl-terminal transmembrane signal-anchor domain cannot insert into membranes. The restriction on BAX targeting, dependent on the NH2-terminal domain, is relieved by a proapoptotic stimulus, allowing the carboxyl terminus signal-anchor of Bax to insert into the mitochondrial membrane (30)
. The mitochondrial translocation of Bax triggers mitochondrial permeability transition (31)
. Bcl-XL can sequester BH3 domain-only proteins (such as Bid, Bim, and Bad) in stable mitochondrial complexes, preventing the activation of Bax (29)
. We demonstrated that Bcl-XL blocked Bax activation in response to H/R stress (Fig. 2C
). Bcl-XL has been reported to block Bax mitochondrial translocation (32)
, which is consistent with the results shown here (Fig. 2B
).
Bcl-XL can inhibit extrinsic apoptosis by suppressing DISC formation and associated caspase-8 activation. Earlier studies have reported that Bcl-XL cannot block the activation of Fas during FasL-induced apoptosis (33)
. It has been reported that Bcl-XL functions downstream of caspase-8 to inhibit Fas-induced apoptosis in MCF7 breast carcinoma cells (34)
. Whereas Bcl-XL failed to block caspase-8 cleavage in MCF7 cells, Bcl-XL inhibited anti-CD95-induced apoptosis in this cell type (35)
. We found that Bcl-XL blocked caspase-8 cleavage in MLEC subjected to H/R treatment in a time-dependent manner (Fig. 3A
). We have demonstrated that Bcl-XL disrupted DISC formation in the plasma membrane fraction of MLEC as a function of H/R treatment (Fig. 3B
). These results indicate that DISC formation in the plasma membrane is necessary and sufficient to initiate Fas-mediated apoptosis and critically important for caspase-8 cleavage and activity (Fig. 3)
. We show that Bcl-XL can retain the caspase-8-associated DISC in the mitochondria (Fig. 4A
), where caspase-8 activation by the DISC is inefficient (35)
.
We have demonstrated that Fas and caspase-8 can associate with the Golgi complex-associated protein GRASP65. This interaction apparently sequesters the DISC to the Golgi apparatus, which would precede its transfer to the plasma membrane where caspase-8 could be cleaved and activated efficiently. We demonstrated an association between GRASP65 and DISC components Fas and caspase-8 in MLEC subjected to H/R stress. We showed that Bcl-XL disrupted the associations of GRASP65 with caspase-8 and Fas in the Golgi (Fig. 4B
).
Inhibition of extrinsic apoptosis by Bcl-XL may involve the inhibition of Bid activity. We demonstrated the time-dependent cleavage of Bid as a function of H/R treatment in MLEC (Fig. 5)
, which is consistent with previous results. The caspase-8 dependent cleavage of Bid leading to cytochrome c release was observed in a model of focal cerebral ischemia in mice (36)
. Also consistent with previous studies (37)
, we demonstrate the inhibition of Bid cleavage by Bcl-XL expression in MLEC (Fig. 5)
.
The activated form of Bid (tBid) can induce cytochrome c release from mitochondria by two possible mechanisms dependent on association with either Bak or Bax (38)
. Bid assists in the auto-oligomerization of Bak (39)
. Bid associates with the activated form of Bax and facilitates the translocation and insertion of Bax into the mitochondrial membrane to form functional oligomers, leading to cytochrome c release (40
41
42)
. Bid does not necessarily require Bax to induce cytochrome c release (42)
. However, tBid cannot release cytochrome c from rat liver mitochondria, which lack both Bax and Bak, suggesting that tBid cannot act independently of both Bax and Bak. The maximum amount of cytochrome c release from the mitochondria occurs in presence of both Bax and Bak (38)
. It remains unclear whether Bcl-XL blocks Bid cleavage directly by an unknown mechanism or acts strictly by limiting the availability of active caspase-8 by promoting its mitochondrial sequestration.
Another role of Bcl-XL might be to up-regulate endogenous inhibitors of caspase-8 such as FLIP. We show that Bcl-XL overexpressing MLEC up-regulated FLIP during the course of H/R treatment (Fig. 6)
. FLIP has been shown to directly inhibit apoptosis by blocking caspase-8 activation in the DISC and may activate both NF-
B and Erk-dependent survival pathways (23)
. We have observed that Bcl-XL can activate p38 MAPK and JNK signaling pathways in MLEC (data not shown).
In summary, H/R induces mouse lung endothelial cell death by activating extrinsic (Fas/caspase-8/Bid) -dependent and intrinsic (Bax/mitochondria) -dependent apoptotic signaling pathways (Fig. 7
). Bcl-XL, an anti-apoptotic member of Bcl-2 family, can inhibit both apoptotic pathways in the context of H/R stress. Bcl-XL inhibits Bax activation and blocks Bax redistribution. Bcl-XL inactivates caspase-8 by disrupting DISC formation in the plasma membrane, Golgi complex, and nucleus. Bcl-XL retains the DISC in mitochondria where caspase-8 is inactivated. Bcl-XL breaks the physical association of Fas and caspase-8 with GRASP65, a Golgi apparatus-related protein. These results suggest that Bcl-XL down-regulates the transfer of DISC to the plasma membrane by the Golgi component, at the same time diverting the DISC formation to the mitochondria.
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ACKNOWLEDGMENTS |
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Received for publication April 9, 2004. Accepted for publication August 17, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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