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Actinomycin D by inhibition of the caspase-8-mediated apoptotic pathway
BBRC Biochemical and Biophysical Research Communications 344 (2006) 1172–1178 www.elsevier.com/locate/ybbrc
Carbon monoxide protects hepatocytes from TNF-a/Actinomycin D by inhibition of the caspase-8-mediated apoptotic pathway Hoe Suk Kim, Patricia A. Loughran, Peter K. Kim, Timothy R. Billiar, Brian S. Zuckerbraun * Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA Received 22 March 2006 Available online 6 April 2006
Abstract We have previously shown that carbon monoxide (CO) (250 ppm) prevented tumor necrosis factor-a (TNFa)-induced apoptosis and activated the transcription factor NF-jB in hepatocytes both in vivo and in vitro. These studies were conducted to further determine the mechanisms by which CO suppresses apoptotic signaling in TNFa (10 ng/ml) and Actinomycin D (ActD, 200 ng/ml)-treated hepatocytes. Consistent with our previous findings, CO protected against TNFa/ActD-induced cell death, which is in part dependent on NF-jB activation. This was associated with a reduction in mitochondrial damage, a decrease in cytochrome c release, and an inhibition of translocation of Bcl proteins to mitochondria. In conjugation with inhibition of these mitochondrial events, CO also suppressed caspases-8 and -3 cleavage in response to TNFa/ActD. Inhibition of NF-jB activation resulted in diminished CO-induced cFLIP expression and increased caspase-8 cleavage from cells treated with TNFa/ActD. These data indicate that CO interferes with apoptotic signaling at a proximal step. 2006 Published by Elsevier Inc. Keywords: Carbon monoxide (CO); Tumor necrosis factor-a (TNFa); Caspase-8; Nuclear factor-jB (NFjB); Apoptosis
Carbon monoxide (CO) is an endogenous gaseous substance, similar to nitric oxide (NO), which has been shown to have a role in physiologic and pathophysiologic states [1,2]. CO arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. HO cleaves heme molecules to yield biliverdin, CO, and free iron. Others and we have reported in a variety of cell types that CO acts in a cytoprotective manner [3–21]. We have previously reported that CO protects TNFa-induced apoptosis in hepatocytes both in vivo and in vitro [9]. In this study we investigate the possible molecular mechanism by which CO suppresses hepatocyte apoptosis induced by TNFa and Actinomycin D (TNF/ActD). The apoptotic signaling pathways mediated by death receptors and other proapoptotic stimuli have been well characterized. Ligand binding of TNFa and Fas to their *
Corresponding author. Fax: +1 412 647 5959. E-mail address: [email protected] (B.S. Zuckerbraun).
0006-291X/$ - see front matter 2006 Published by Elsevier Inc. doi:10.1016/j.bbrc.2006.03.180
respective receptors initiates recruitment of FADD (FasAssociated Death Domain protein) as well as caspase-8 in its inactive form to form the Death Inducing Signaling Complex (DISC) [22]. Caspase-8 is activated and cleaves the Bcl family protein Bid to its activated form, truncated (tBid). tBid and many other cytosolic Bcl2 members are redistributed from the cytosol to the mitochondria membrane. The relocation of these Bcl2 proteins to the mitochondrial membrane, results in cytochrome c release [23]. Released cytochrome c binds to apoptosis-induced factor (Apaf-1) in the presence of ATP and activates caspases-9 and -3 to induce the major biochemical and morphological changes of apoptosis. Furthermore, activated caspase-8 can activate caspase-3 independently of Bid and the mitochondrial pathway. The proteolytic caspase cascade is associated with a number of specific inhibitors that are upregulated by the activation of the transcription factor nuclear factor jB (NF-jB). The anti-apoptotic effect of NF-jB exerts its
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effects in part by upregulation of inhibitor of apoptosis proteins (IAPs) or FADD-like ICE inhibitory proteins (FLIPs) [24–30]. FLIPs functions similar to procaspase-8 by binding to FADD in a competitive manner, whereas IAP family members inhibit executioner caspases (e.g., caspase-3). The purpose of this investigation was to test the hypothesis that CO protects against TNFa-induced hepatocyte cell death via inhibition of apoptosis signaling. Furthermore, that inhibition of apoptosis by CO is dependent on NF-jB activation and increased anti-apoptotic proteins. Materials and methods Materials. Williams’ medium E, penicillin, streptomycin, L-glutamate, and Hepes were purchased from Life Technologies, Inc (Rockville, MD). Insulin was obtained from Lilly (Indianapolis, IN), and calf serum was purchased from Hyclone Laboratories (Logan, UT). Mouse recombinant TNFa was obtained from R&D Systems (Minneapolis, MN). Antibodies for Bid (R&D Systems) cytochrome c, IjBa (PharMingen; San Diego, CA), Bax (Cell Signlaing Technology; Danvers, MA) Bcl-XL (BD Science; Franklin Lakes, NJ) and b-actin antibody (Sigma) were used. The other antibodies for caspase-8, caspase-3, and XIAP were obtained from StressGen (Victoria, British Columbia, Canada). ECL+Plus was obtained from Amersham Biosciences (Piscataway, NJ), and Supersignal chemiluminescence detection reagents were obtained from Pierce (Rockford, IL). ApoAlert Mitochondrial Membrane Sensor Kit was obtained from BD Science. Unless indicated otherwise, all other chemicals and proteins were purchased from Sigma. Cell culture. Primary mouse hepatocytes were isolated and purified from C57BL/6J and cultured as described previously [9]. Highly purified hepatocytes (>98% purity and >98% viability by trypan blue exclusion) were suspended in Williams medium E supplemented with 10% calf serum, 1 lM insulin, 2 mM L-glutamine, 15 mM Hepes (pH 7.4), 100 U/ml penicillin, and 100 lg/ml streptomycin. The cells were plated on collagencoated tissue culture plates at a density of 2 · 105 cells/well in 12-well plates for cell viability analysis or 5 · 106 cells/100-mm dish for Western blot and enzyme assays. After 18 h preculture, the cells were further cultured with fresh medium containing 5% calf serum and used for experiment. Induction of hepatocyte death/apoptosis. Cells were treated with 10 ng/ml TNFa and 200 ng/ml Actinomycin D (ActD) to induce cell death. TNFa/ActD treatment has previously been demonstrated to induce cell death, specifically apoptosis, in primary hepatocytes treated with CO, and/or additional pharmacological agents. Cell viability. Cell viability was determined by the crystal violet staining method, as described previously [9]. In brief, cells were stained with 0.5% crystal violet in 30% ethanol and 3% formaldehyde for 10 min at room temperature. Plates were washed four times with tap water. After drying, cells were lysed with 1% SDS solution, and dye uptake was measured at 550 nm using a 96-well plate reader. Cell viability was calculated from relative dye intensity compared with untreated samples. Preparation of cytosolic and mitochondrial protein fractions for the measurement of released cytochrome c and translocation of pro-apoptotic protein. Cells were collected and washed twice in ice-cold PBS, resuspended in S-100 buffer (20 mM Hepes, pH 7.5, 10 mM KCl, 1.9 mM MgCl2, 1 mM EGTA, 1 mM EDTA, mixture of protease inhibitors), and incubated on ice for 20 min. After a 20-min incubation on ice, the cells were homogenized with a Dounce glass homogenizer and a loose pestle (Wheaton, Millville, NJ) for 70 strokes. Cell homogenates were spun at 1000g to remove unbroken cells, nuclei, and heavy membranes. The supernatant was spun again at 12,000g for 20 min to collect the mitochondria-rich (pellet) and cytosolic (supernatant) fractions. The mitochondria-rich fraction was washed once with the extraction buffer, followed by a final resuspension in lysis buffer (150 mM NaCl, 50 mM
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Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA) containing protease inhibitors for Western blot analysis. Western blot analysis. Cells were harvested, washed twice with ice-cold PBS, and resuspended in 20 mM Tris–HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethylsulfonyl fluoride, 5 lg/ml aprotinin, 5 lg/ml pepstatin A, and 1 lg/ml chymostatin). Proteins (30 lg) were separated on SDS–PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 5% nonfat dried milk in Tris-buffered saline and then incubated with primary antibodies for 1 h at room temperature. Blots were developed by peroxidase-conjugated secondary antibody and proteins were visualized by ECL procedures according to the manufacturer’s recommendation. Caspase activity assay. Caspase activity was evaluated by measuring proteolytic cleavage of the chromogenic substrate Ac-IETD-pNA (for caspase-8 activity) as described previously. Ac-IETD-pNA was used as caspase-8 substrate. Briefly, cell lysate (100 lg of protein) was added into buffer A containing 100 lM Ac-IETD-pNA in a final volume of 100 ll. The reaction mixture was incubated at 37 C for 1 h. The increase in absorbance of enzymatically released pNA was measured at 405 nm in a microplate reader. Adenoviral gene transfer. Modified adenoviral vectors carrying an IjB super repressor (provided by D. Brenner, University of North Carolina, Chapel Hill, NC) or b-galactosidase were prepared as described previously [31]. After 18 h of preculture, hepatocytes (3 · 106/6-cm plate) were washed with Hanks’ buffered saline and incubated with adenoviral vector containing either the IjBa or bacterial b-galactosidase (LacZ) cDNA at multiplicity of infection of 1000 virus particles/cell in a volume of 2 ml of Opti-MEM. Following a 2-h infection, the medium was changed to fresh Williams medium E containing 5% calf serum. The infected hepatocytes were recovered overnight prior to changing to fresh medium and subjecting to induction of apoptosis. Electrophoretic mobility shift assays. Nuclear extracts were then prepared and electrophoretic mobility shift assay (EMSA) was performed as described previously [9]. Briefly, a double stranded DNA NF-jB consensus sequence (GGGGACTTTCCC; Santa Cruz Biotechnology) were end-labeled with T4 polynucleotide kinase and [c-32P]ATP and incubated with nuclear proteins (4 lg) at room temperature for 30 min in binding buffer (10 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA (pH 7.5), 5% glycerol, 2 lg of poly(dI-dC), and 5% Nonidet P-40). The proteinÆDNA complexes were loaded onto a 5% native polyacrylamide gel and detected by autoradiography. Immunofluorescence analysis. ApoAlert Mitochondrial Membrane Sensor Kit allows the detection of changes in mitochondrial membrane potential during the early stages of apoptosis. The kit uses a cationic dye (MitoSensor), which fluoresces differently in apoptotic and non-apoptotic cells. In healthy cells, MitoSensor is taken up in the mitochondria, where it forms aggregates exhibiting intense red fluorescence. In apoptotic cells, MitoSensor cannot aggregate in mitochondria; therefore, the dye remains in the cytoplasm where it fluoresces green. Cells were incubated in 1 ml incubation buffer including 1 ll MitoSensor for 15–20 min at 37 C. After fixation of cell with 2% paraformaldehyde, cells were mounted onto microscopic slides using ProLong antifade mounting reagent (Molecular Probes; Eugene, OR). The slides were analyzed by observed using fluorescent microscope. Statistical analysis. Data are presented as means ± SE of the mean of at least three separate experiments. Comparisons were performed using Student’s t test in SigmaStat (SPSS, Chicago, IL). Differences were considered significant at P values 60.05.
Results CO suppresses TNFa/ActD-induced caspase-8 cleavage We first confirmed our previous observations showing that CO suppressed TNFa + Actinomycin D (TNF/ ActD)-induced apoptosis. At an 8-h time point, cell
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Fig. 1. CO protects against TNFa-mediated cell death. Hepatocytes were treated with 250 ppm of carbon monoxide (CO) starting as a 1-h pretreatment and then continued throughout the duration of the experiment. Cells were treated with 10 ng/ml TNFa plus 200 ng/ml Actinomycin D (ActD) for indicated times. Cell viability was determined by crystal violet staining. Results are means ± SE of three independent experiments.
Fig. 2. CO prevents increases in TNFa-induced caspase-8 activity. Hepatocytes were treated with or without CO, including a 1-h pretreatment. Cells were further treated with TNFa/ActD and protein was harvested at 6 h. Caspase-8 protease activity was evaluated by measuring proteolytic cleavage of the chromogenic substrate Ac-IETD-pNA using a colorimetric assay. Activity assays are consistent with Western blot results demonstrating decreased activity of caspase-8 in the CO-treated cells (P < 0.05). Results are means ± SE of three separate experiments.
viability in hepatocytes treated with TNFa/ActD was decreased by about 50% as compared to control cells (Fig. 1). CO treatment (250 ppm) almost completely blocked this TNF/ActD-induced cell death. TNFa-mediated apoptosis requires activation of apical caspase-8 and subsequent cleavage of Bid, which leads to mitochondria damage. To determine whether CO suppresses caspase-8 activity, enzymatic activity of caspase-8 was measured in cytosolic extracts from TNFa/ActD-treated hepatocytes by a colorimetric assay using the caspase-8-specific substrate IETD-pNA. Treatment of cells with TNFa/ActD increased caspase-8 activity twofold at 6 h compared with that of the untreated control cells. The increase in caspase-8 activity was significantly suppressed by treatment with CO (Fig. 2). CO inhibits caspase-8 mediated down stream apoptotic signaling Given the effects on caspase-8, the effects of CO on the activation of the pro-apoptotic protein Bid were investigated. Activated caspase-8 is known to cleave cytosolic p22 Bid at its amino terminus and generate a p15 truncated Bid (tBid), which then translocates to the mitochondria. TNFa/ActD treatment resulted in increased cleavage of Bid, while CO treatment inhibited proteolytic fragmentation of the pro-apoptotic protein (Fig. 3). Similarly CO treatment blocked the cleavage of caspase-3. XIAP has been shown to be a direct inhibitor of caspases-3 and -7 and to interfere with the Bax/cytochrome c pathway by inhibiting caspase-9. Moreover, the resulting XIAP cleavage by TNF-mediated apoptotic signaling pathway, although capable of inhibiting caspase-3 and caspase-7, is less potent than full-length XIAP [24,25]. These studies
Fig. 3. CO inhibits caspase-8 mediated activation of down stream apoptotic signaling molecules. TNFa/ActD increased cleavage of Bid, caspase-3, and XIAP. CO treatment decreased cleavage of these proteins.
demonstrate that CO suppressed the TNFa/ActD-induced cleavage of XIAP (Fig. 3). CO inhibits the translocation of the Bcl-2 family members Bcl-XL, tBid, and Bax to mitochondria Cytosolic Bcl-XL, tBid, and Bax translocate to the mitochondria during apoptosis, resulting in caspase-3 activation by mitochondrial apoptotic events including release of mitochondrial cytochrome c into the cytosol. We examined the effect of CO on the translocation of the Bcl-2 family members Bcl-XL, tBid, and Bax to the mitochondria following treatment with TNFa/ActD at a 6-h time point. The levels of tBid, Bax, and Bcl-XL significantly increased
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Fig. 5. CO up-regulates expression of anti-apoptotic protein (Bcl-XL) and down-regulates pro-apoptotic protein (Bax). Western blots of Bcl-XL and Bax from whole lysates were examined. The level of Bcl-XL decreased from TNFa/ActD treated cells, while those of Bax increased. CO suppressed the decreasing Bcl-XL or increasing Bax in TNFa/ActDmediated apoptotic cells.
tein in hepatocytes treated with TNFa/ActD in the absence or presence of CO for various time periods. Bcl-XL expression was decreased, whereas Bax was increased in cells treated with TNFa/ActD (Fig. 5).
Fig. 4. CO inhibits mitochondrial translocation of proapoptotic protein and maintains mitochondrial membrane potential. (A) Western blot of mitochondrial fractions of Bid, XIAP, Bcl-XL, and Bax were examined. CO inhibited translocation of these proteins to the mitochondria. (B) CO preserved mitochondrial membrane potential in TNFa/ActD treated cells. While cells treated with TNFa/ActD had a decrease in Wm (fluoresce green), CO treatment maintained a normal Wm (fluoresces red).
in the mitochondrial fraction in TNF/ActD treated cells. CO exposure suppressed mitochondrial translocation of these Bcl-2 family members as well as release of mitochondrial cytochrome c from TNFa/ActD-treated cells (Fig. 4A). To further examine the effects of CO on reduction in apoptosis-associated mitochondrial events the mitochondrial transmembrane potential (Wm) was accessed using a mitochondria specific probe that aggregates to fluoresce red in the mitochondria. In apoptotic cells, mitochondrial membrane potential is decreased, and the probe remains a monomer in the cytoplasm and fluoresces green. While cells treated with TNFa/ActD had a decrease in Wm (fluoresce green), CO treatment maintained a normal Wm (fluoresces red; Fig. 4B). Taken together, these results indicate that CO protects against mitochondrial injury and disruption of Wm in the response to TNFa/ActD.
CO protects the hepatocyte against TNFa/ActD via NF-jBdependent cFLIP modulation NF-jB has a role in the liver as part of a pro-survival signaling pathway. Our previous study demonstrated that the anti-apoptotic effect of CO in protecting hepatocytes against TNFa/ActD injury is dependent on NF-jB activation [9]. We further hypothesize that the anti-apoptotic effects of CO associated with NF-jB activity is via the regulation of NF-jB-dependent anti-apoptotic genes. In order to test this hypothesis we utilized adenoviral gene transfer of IjBa (AdIjBaSR) to prevent activation of NF-jB, and assayed for changes in apoptotic and anti-apoptotic proteins. The effectiveness of AdIjBaSR was investigated by Western blot analysis for IjBa protein and EMSA for NF-jB nuclear binding (Figs. 6A and B). cFLIP is a NFjB regulated anti-apoptotic gene. CO increased levels of cFLIP, however, this was reversed by inhibition of NFjB in TNFa/ActD treated cells. Corresponding to this, over-expression of IjBa led to increased cleavage of caspases-3 and -8 in CO exposed TNFa/ActD treated cells (Fig. 6C). This correlates with data of viability (Fig. 6D). These data suggest that the anti-apoptotic effect of CO against TNFa/ActD is mediated in part through the upregulation of NF-jB-dependent anti-apoptotic proteins, such as cFLIP.
CO increases total expression of Bcl-XL while suppressing those of Bax
Discussion
The Bcl-2 family, comprised of both pro-apoptotic and anti-apoptotic members, constitutes a critical intracellular checkpoint for apoptosis within a common cell death pathway. The balance in expression of Bcl-2 family members is crucial in determining the fate of cells that undergo apoptosis [23]. Pro-apoptotic Bax is required for death signaling through mitochondria in response to TNFa. We investigated the effects of CO on the expression of Bcl-2 family pro-
Increased expression of HO-1, with subsequent increased generation of CO is part of a physiological response to injury by which many cells are protected from undergoing apoptosis. We previously demonstrated that exogenous administration of CO dramatically inhibited TNFa-mediated liver injury in vivo and hepatocyte cell death in vitro. Additionally, the hepatoprotective effect of CO is dependent on NF-jB activation. These
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Fig. 6. CO inhibits caspase-8 cleavage by increase of cFLIP expression via NF-jB. Hepatocytes underwent adenoviral gene transfer of LacZ (control) or IjB-super repressor (AdIjBaSR). (A) Western blot analysis of native and mutated IjBa was investigated after 24 h of adenoviral gene transfer of AdIjBaSR. (B) Electrophoretic mobility shift assay for NF-jB was performed with CO treatment and treatment with AdIjBaSR. AdLacZ was used as a transfection control and TNFa (10 ng) was used as a positive control. CO-induced NF-jB binding was markedly diminished by AdIjBaSR. (C) Twenty-four hours after gene transfer, hepatocytes were treated with TNFa/ActD with or without CO and protein was harvested at 6 h or viability was assayed at 12 h. cFLIP protein levels were maintained by CO in the LacZ group, however, these levels were diminished by inhibition of NF-jB activation in the TNFa/ActD treated group. Furthermore, inhibition of NF-jB activation led to increased cleavage of caspases-3 and -8. (D) Protection against TNFa/ActD-induced cell death by apoptosis was diminished by inhibition of NF-jB activation. TNFa/ActD induced significant cell death compared to LacZ controls (P < 0.01). CO treatment significantly protected against TNFa/ActDinduced death (*P < 0.05 compared to TNFa/ActD-treated LacZ control). However, inhibition of NF-jB activation by adenoviral gene transfer of IjB super-repressor significantly reversed protection by CO (#P < 0.05 compared to TNFa/ActD and CO-treated LacZ group).
data show that CO decreases activation of apoptotic proteins, including proximal caspase-8, and increases levels of anti-apoptotic proteins including XIAP and cFLIP. Furthermore, changes in cFLIP are dependent on NF-kB activation.
It is well described that the most proximal caspase activated following interaction of TNFa with its receptor is caspase-8 in various types of cells including hepatocytes [32]. Activated caspase-8 may in turn either activate effector caspases (i.e., caspase-3) or cleave Bid. The activated form of Bid triggers apoptosis by mitochondrial translocation. In the mitochondria, translocation of pro-apoptotic Bcl-2 family members such as Bax forms membrane channels by opening the permeability transition pore, leading to cytochrome c release and caspase activation [33]. Caspase-8 also directly activates caspase-3 independent of mitochondria, but this occurs slowly in hepatocytes. These data demonstrate changes at all of these levels, indicating that the CO may have an effect at multiple levels including proximal to caspase-8. Induction of HO-1 or delivery of CO was shown to protect mice from liver injury or lung injury by modulation of caspase or Bcl-2 family via MAPK pathway [21,34–36]. In these models of apoptotic liver injury, damage can be prevented by CO-mediated caspase modulation. We found that CO-mediated protection is accompanied by significant reduction of caspase-8 activity and inhibition of translocation of Bid and Bax a to mitochondria, which is associated cytochrome c release and subsequent mitochondria damage.These data further support that CO-mediated protection operates by activating NF-jB, which in the presence of an inflammatory stimulus leads to the up-regulation of NF-jB-dependent antiapoptotic genes [36,37]. The role of NF-jB in the liver as part of a pro-survival signaling pathway has been studied [32,38]. Activation of the transcription factor NF-jB is a major effector of the inducible resistance to death receptor-mediated apoptosis by modulating antiapoptotic genes [26]. Over-expressing NF-jBinduced proteins, FLIP or c-IAP, has been shown to be lead to a blockade of caspase-8 activation, resulting in increased resistance to Fas ligand (FasL) or TNFa. CO has been reported to be cytoprotective by modulation of NF-jB-dependent genes from several types of cells, yet there is no report that CO modulates caspase-8 through regulation of NF-jB. Given FLIP is induced by NF-jB, we considered the possibility that CO can modulate FLIP expression by NF-jB. Once NF-jB activation is inhibited, as by over-expression of its natural inhibitor IjBa, CO-induced FLIP expression was inhibited from cells during TNFa/ActD-apoptosis. Taken together, CO showed strong anti-apoptotic effects by modulation of cFLIP expression, at least in part by a NF-jB-dependent mechanism. In summary, the present study demonstrates that CO protects hepatocytes against TNFa/ActD by inhibiting caspase-8-mediated apoptotic pathway. CO-mediated antiapoptotic effects are associated with a decrease in the proteolytic fragment of caspases-8 and-3, as well as Bid. In addition, CO suppressed the translocation of Bid, Bax and Bcl-XL to mitochondria, resulting in inhibition of cytochrome c. Furthermore, the total level of anti-apoptotic proteins Bcl-XL or XIAP increased, whereas those of the proapoptotic protein Bax decreased. CO modulated
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Carbon monoxide protects hepatocytes from TNF-a/Actinomycin D by inhibition of the caspase-8-mediated apoptotic pathway Hoe Suk Kim, Patricia A. Loughran, Peter K. Kim, Timothy R. Billiar, Brian S. Zuckerbraun * Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA Received 22 March 2006 Available online 6 April 2006
Abstract We have previously shown that carbon monoxide (CO) (250 ppm) prevented tumor necrosis factor-a (TNFa)-induced apoptosis and activated the transcription factor NF-jB in hepatocytes both in vivo and in vitro. These studies were conducted to further determine the mechanisms by which CO suppresses apoptotic signaling in TNFa (10 ng/ml) and Actinomycin D (ActD, 200 ng/ml)-treated hepatocytes. Consistent with our previous findings, CO protected against TNFa/ActD-induced cell death, which is in part dependent on NF-jB activation. This was associated with a reduction in mitochondrial damage, a decrease in cytochrome c release, and an inhibition of translocation of Bcl proteins to mitochondria. In conjugation with inhibition of these mitochondrial events, CO also suppressed caspases-8 and -3 cleavage in response to TNFa/ActD. Inhibition of NF-jB activation resulted in diminished CO-induced cFLIP expression and increased caspase-8 cleavage from cells treated with TNFa/ActD. These data indicate that CO interferes with apoptotic signaling at a proximal step. 2006 Published by Elsevier Inc. Keywords: Carbon monoxide (CO); Tumor necrosis factor-a (TNFa); Caspase-8; Nuclear factor-jB (NFjB); Apoptosis
Carbon monoxide (CO) is an endogenous gaseous substance, similar to nitric oxide (NO), which has been shown to have a role in physiologic and pathophysiologic states [1,2]. CO arises in biological systems during the oxidative catabolism of heme by the heme oxygenase (HO) enzymes. HO cleaves heme molecules to yield biliverdin, CO, and free iron. Others and we have reported in a variety of cell types that CO acts in a cytoprotective manner [3–21]. We have previously reported that CO protects TNFa-induced apoptosis in hepatocytes both in vivo and in vitro [9]. In this study we investigate the possible molecular mechanism by which CO suppresses hepatocyte apoptosis induced by TNFa and Actinomycin D (TNF/ActD). The apoptotic signaling pathways mediated by death receptors and other proapoptotic stimuli have been well characterized. Ligand binding of TNFa and Fas to their *
Corresponding author. Fax: +1 412 647 5959. E-mail address: [email protected] (B.S. Zuckerbraun).
0006-291X/$ - see front matter 2006 Published by Elsevier Inc. doi:10.1016/j.bbrc.2006.03.180
respective receptors initiates recruitment of FADD (FasAssociated Death Domain protein) as well as caspase-8 in its inactive form to form the Death Inducing Signaling Complex (DISC) [22]. Caspase-8 is activated and cleaves the Bcl family protein Bid to its activated form, truncated (tBid). tBid and many other cytosolic Bcl2 members are redistributed from the cytosol to the mitochondria membrane. The relocation of these Bcl2 proteins to the mitochondrial membrane, results in cytochrome c release [23]. Released cytochrome c binds to apoptosis-induced factor (Apaf-1) in the presence of ATP and activates caspases-9 and -3 to induce the major biochemical and morphological changes of apoptosis. Furthermore, activated caspase-8 can activate caspase-3 independently of Bid and the mitochondrial pathway. The proteolytic caspase cascade is associated with a number of specific inhibitors that are upregulated by the activation of the transcription factor nuclear factor jB (NF-jB). The anti-apoptotic effect of NF-jB exerts its
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effects in part by upregulation of inhibitor of apoptosis proteins (IAPs) or FADD-like ICE inhibitory proteins (FLIPs) [24–30]. FLIPs functions similar to procaspase-8 by binding to FADD in a competitive manner, whereas IAP family members inhibit executioner caspases (e.g., caspase-3). The purpose of this investigation was to test the hypothesis that CO protects against TNFa-induced hepatocyte cell death via inhibition of apoptosis signaling. Furthermore, that inhibition of apoptosis by CO is dependent on NF-jB activation and increased anti-apoptotic proteins. Materials and methods Materials. Williams’ medium E, penicillin, streptomycin, L-glutamate, and Hepes were purchased from Life Technologies, Inc (Rockville, MD). Insulin was obtained from Lilly (Indianapolis, IN), and calf serum was purchased from Hyclone Laboratories (Logan, UT). Mouse recombinant TNFa was obtained from R&D Systems (Minneapolis, MN). Antibodies for Bid (R&D Systems) cytochrome c, IjBa (PharMingen; San Diego, CA), Bax (Cell Signlaing Technology; Danvers, MA) Bcl-XL (BD Science; Franklin Lakes, NJ) and b-actin antibody (Sigma) were used. The other antibodies for caspase-8, caspase-3, and XIAP were obtained from StressGen (Victoria, British Columbia, Canada). ECL+Plus was obtained from Amersham Biosciences (Piscataway, NJ), and Supersignal chemiluminescence detection reagents were obtained from Pierce (Rockford, IL). ApoAlert Mitochondrial Membrane Sensor Kit was obtained from BD Science. Unless indicated otherwise, all other chemicals and proteins were purchased from Sigma. Cell culture. Primary mouse hepatocytes were isolated and purified from C57BL/6J and cultured as described previously [9]. Highly purified hepatocytes (>98% purity and >98% viability by trypan blue exclusion) were suspended in Williams medium E supplemented with 10% calf serum, 1 lM insulin, 2 mM L-glutamine, 15 mM Hepes (pH 7.4), 100 U/ml penicillin, and 100 lg/ml streptomycin. The cells were plated on collagencoated tissue culture plates at a density of 2 · 105 cells/well in 12-well plates for cell viability analysis or 5 · 106 cells/100-mm dish for Western blot and enzyme assays. After 18 h preculture, the cells were further cultured with fresh medium containing 5% calf serum and used for experiment. Induction of hepatocyte death/apoptosis. Cells were treated with 10 ng/ml TNFa and 200 ng/ml Actinomycin D (ActD) to induce cell death. TNFa/ActD treatment has previously been demonstrated to induce cell death, specifically apoptosis, in primary hepatocytes treated with CO, and/or additional pharmacological agents. Cell viability. Cell viability was determined by the crystal violet staining method, as described previously [9]. In brief, cells were stained with 0.5% crystal violet in 30% ethanol and 3% formaldehyde for 10 min at room temperature. Plates were washed four times with tap water. After drying, cells were lysed with 1% SDS solution, and dye uptake was measured at 550 nm using a 96-well plate reader. Cell viability was calculated from relative dye intensity compared with untreated samples. Preparation of cytosolic and mitochondrial protein fractions for the measurement of released cytochrome c and translocation of pro-apoptotic protein. Cells were collected and washed twice in ice-cold PBS, resuspended in S-100 buffer (20 mM Hepes, pH 7.5, 10 mM KCl, 1.9 mM MgCl2, 1 mM EGTA, 1 mM EDTA, mixture of protease inhibitors), and incubated on ice for 20 min. After a 20-min incubation on ice, the cells were homogenized with a Dounce glass homogenizer and a loose pestle (Wheaton, Millville, NJ) for 70 strokes. Cell homogenates were spun at 1000g to remove unbroken cells, nuclei, and heavy membranes. The supernatant was spun again at 12,000g for 20 min to collect the mitochondria-rich (pellet) and cytosolic (supernatant) fractions. The mitochondria-rich fraction was washed once with the extraction buffer, followed by a final resuspension in lysis buffer (150 mM NaCl, 50 mM
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Tris–HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EGTA) containing protease inhibitors for Western blot analysis. Western blot analysis. Cells were harvested, washed twice with ice-cold PBS, and resuspended in 20 mM Tris–HCl buffer (pH 7.4) containing a protease inhibitor mixture (0.1 mM phenylmethylsulfonyl fluoride, 5 lg/ml aprotinin, 5 lg/ml pepstatin A, and 1 lg/ml chymostatin). Proteins (30 lg) were separated on SDS–PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 5% nonfat dried milk in Tris-buffered saline and then incubated with primary antibodies for 1 h at room temperature. Blots were developed by peroxidase-conjugated secondary antibody and proteins were visualized by ECL procedures according to the manufacturer’s recommendation. Caspase activity assay. Caspase activity was evaluated by measuring proteolytic cleavage of the chromogenic substrate Ac-IETD-pNA (for caspase-8 activity) as described previously. Ac-IETD-pNA was used as caspase-8 substrate. Briefly, cell lysate (100 lg of protein) was added into buffer A containing 100 lM Ac-IETD-pNA in a final volume of 100 ll. The reaction mixture was incubated at 37 C for 1 h. The increase in absorbance of enzymatically released pNA was measured at 405 nm in a microplate reader. Adenoviral gene transfer. Modified adenoviral vectors carrying an IjB super repressor (provided by D. Brenner, University of North Carolina, Chapel Hill, NC) or b-galactosidase were prepared as described previously [31]. After 18 h of preculture, hepatocytes (3 · 106/6-cm plate) were washed with Hanks’ buffered saline and incubated with adenoviral vector containing either the IjBa or bacterial b-galactosidase (LacZ) cDNA at multiplicity of infection of 1000 virus particles/cell in a volume of 2 ml of Opti-MEM. Following a 2-h infection, the medium was changed to fresh Williams medium E containing 5% calf serum. The infected hepatocytes were recovered overnight prior to changing to fresh medium and subjecting to induction of apoptosis. Electrophoretic mobility shift assays. Nuclear extracts were then prepared and electrophoretic mobility shift assay (EMSA) was performed as described previously [9]. Briefly, a double stranded DNA NF-jB consensus sequence (GGGGACTTTCCC; Santa Cruz Biotechnology) were end-labeled with T4 polynucleotide kinase and [c-32P]ATP and incubated with nuclear proteins (4 lg) at room temperature for 30 min in binding buffer (10 mM Tris–HCl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, 1 mM EDTA (pH 7.5), 5% glycerol, 2 lg of poly(dI-dC), and 5% Nonidet P-40). The proteinÆDNA complexes were loaded onto a 5% native polyacrylamide gel and detected by autoradiography. Immunofluorescence analysis. ApoAlert Mitochondrial Membrane Sensor Kit allows the detection of changes in mitochondrial membrane potential during the early stages of apoptosis. The kit uses a cationic dye (MitoSensor), which fluoresces differently in apoptotic and non-apoptotic cells. In healthy cells, MitoSensor is taken up in the mitochondria, where it forms aggregates exhibiting intense red fluorescence. In apoptotic cells, MitoSensor cannot aggregate in mitochondria; therefore, the dye remains in the cytoplasm where it fluoresces green. Cells were incubated in 1 ml incubation buffer including 1 ll MitoSensor for 15–20 min at 37 C. After fixation of cell with 2% paraformaldehyde, cells were mounted onto microscopic slides using ProLong antifade mounting reagent (Molecular Probes; Eugene, OR). The slides were analyzed by observed using fluorescent microscope. Statistical analysis. Data are presented as means ± SE of the mean of at least three separate experiments. Comparisons were performed using Student’s t test in SigmaStat (SPSS, Chicago, IL). Differences were considered significant at P values 60.05.
Results CO suppresses TNFa/ActD-induced caspase-8 cleavage We first confirmed our previous observations showing that CO suppressed TNFa + Actinomycin D (TNF/ ActD)-induced apoptosis. At an 8-h time point, cell
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Fig. 1. CO protects against TNFa-mediated cell death. Hepatocytes were treated with 250 ppm of carbon monoxide (CO) starting as a 1-h pretreatment and then continued throughout the duration of the experiment. Cells were treated with 10 ng/ml TNFa plus 200 ng/ml Actinomycin D (ActD) for indicated times. Cell viability was determined by crystal violet staining. Results are means ± SE of three independent experiments.
Fig. 2. CO prevents increases in TNFa-induced caspase-8 activity. Hepatocytes were treated with or without CO, including a 1-h pretreatment. Cells were further treated with TNFa/ActD and protein was harvested at 6 h. Caspase-8 protease activity was evaluated by measuring proteolytic cleavage of the chromogenic substrate Ac-IETD-pNA using a colorimetric assay. Activity assays are consistent with Western blot results demonstrating decreased activity of caspase-8 in the CO-treated cells (P < 0.05). Results are means ± SE of three separate experiments.
viability in hepatocytes treated with TNFa/ActD was decreased by about 50% as compared to control cells (Fig. 1). CO treatment (250 ppm) almost completely blocked this TNF/ActD-induced cell death. TNFa-mediated apoptosis requires activation of apical caspase-8 and subsequent cleavage of Bid, which leads to mitochondria damage. To determine whether CO suppresses caspase-8 activity, enzymatic activity of caspase-8 was measured in cytosolic extracts from TNFa/ActD-treated hepatocytes by a colorimetric assay using the caspase-8-specific substrate IETD-pNA. Treatment of cells with TNFa/ActD increased caspase-8 activity twofold at 6 h compared with that of the untreated control cells. The increase in caspase-8 activity was significantly suppressed by treatment with CO (Fig. 2). CO inhibits caspase-8 mediated down stream apoptotic signaling Given the effects on caspase-8, the effects of CO on the activation of the pro-apoptotic protein Bid were investigated. Activated caspase-8 is known to cleave cytosolic p22 Bid at its amino terminus and generate a p15 truncated Bid (tBid), which then translocates to the mitochondria. TNFa/ActD treatment resulted in increased cleavage of Bid, while CO treatment inhibited proteolytic fragmentation of the pro-apoptotic protein (Fig. 3). Similarly CO treatment blocked the cleavage of caspase-3. XIAP has been shown to be a direct inhibitor of caspases-3 and -7 and to interfere with the Bax/cytochrome c pathway by inhibiting caspase-9. Moreover, the resulting XIAP cleavage by TNF-mediated apoptotic signaling pathway, although capable of inhibiting caspase-3 and caspase-7, is less potent than full-length XIAP [24,25]. These studies
Fig. 3. CO inhibits caspase-8 mediated activation of down stream apoptotic signaling molecules. TNFa/ActD increased cleavage of Bid, caspase-3, and XIAP. CO treatment decreased cleavage of these proteins.
demonstrate that CO suppressed the TNFa/ActD-induced cleavage of XIAP (Fig. 3). CO inhibits the translocation of the Bcl-2 family members Bcl-XL, tBid, and Bax to mitochondria Cytosolic Bcl-XL, tBid, and Bax translocate to the mitochondria during apoptosis, resulting in caspase-3 activation by mitochondrial apoptotic events including release of mitochondrial cytochrome c into the cytosol. We examined the effect of CO on the translocation of the Bcl-2 family members Bcl-XL, tBid, and Bax to the mitochondria following treatment with TNFa/ActD at a 6-h time point. The levels of tBid, Bax, and Bcl-XL significantly increased
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Fig. 5. CO up-regulates expression of anti-apoptotic protein (Bcl-XL) and down-regulates pro-apoptotic protein (Bax). Western blots of Bcl-XL and Bax from whole lysates were examined. The level of Bcl-XL decreased from TNFa/ActD treated cells, while those of Bax increased. CO suppressed the decreasing Bcl-XL or increasing Bax in TNFa/ActDmediated apoptotic cells.
tein in hepatocytes treated with TNFa/ActD in the absence or presence of CO for various time periods. Bcl-XL expression was decreased, whereas Bax was increased in cells treated with TNFa/ActD (Fig. 5).
Fig. 4. CO inhibits mitochondrial translocation of proapoptotic protein and maintains mitochondrial membrane potential. (A) Western blot of mitochondrial fractions of Bid, XIAP, Bcl-XL, and Bax were examined. CO inhibited translocation of these proteins to the mitochondria. (B) CO preserved mitochondrial membrane potential in TNFa/ActD treated cells. While cells treated with TNFa/ActD had a decrease in Wm (fluoresce green), CO treatment maintained a normal Wm (fluoresces red).
in the mitochondrial fraction in TNF/ActD treated cells. CO exposure suppressed mitochondrial translocation of these Bcl-2 family members as well as release of mitochondrial cytochrome c from TNFa/ActD-treated cells (Fig. 4A). To further examine the effects of CO on reduction in apoptosis-associated mitochondrial events the mitochondrial transmembrane potential (Wm) was accessed using a mitochondria specific probe that aggregates to fluoresce red in the mitochondria. In apoptotic cells, mitochondrial membrane potential is decreased, and the probe remains a monomer in the cytoplasm and fluoresces green. While cells treated with TNFa/ActD had a decrease in Wm (fluoresce green), CO treatment maintained a normal Wm (fluoresces red; Fig. 4B). Taken together, these results indicate that CO protects against mitochondrial injury and disruption of Wm in the response to TNFa/ActD.
CO protects the hepatocyte against TNFa/ActD via NF-jBdependent cFLIP modulation NF-jB has a role in the liver as part of a pro-survival signaling pathway. Our previous study demonstrated that the anti-apoptotic effect of CO in protecting hepatocytes against TNFa/ActD injury is dependent on NF-jB activation [9]. We further hypothesize that the anti-apoptotic effects of CO associated with NF-jB activity is via the regulation of NF-jB-dependent anti-apoptotic genes. In order to test this hypothesis we utilized adenoviral gene transfer of IjBa (AdIjBaSR) to prevent activation of NF-jB, and assayed for changes in apoptotic and anti-apoptotic proteins. The effectiveness of AdIjBaSR was investigated by Western blot analysis for IjBa protein and EMSA for NF-jB nuclear binding (Figs. 6A and B). cFLIP is a NFjB regulated anti-apoptotic gene. CO increased levels of cFLIP, however, this was reversed by inhibition of NFjB in TNFa/ActD treated cells. Corresponding to this, over-expression of IjBa led to increased cleavage of caspases-3 and -8 in CO exposed TNFa/ActD treated cells (Fig. 6C). This correlates with data of viability (Fig. 6D). These data suggest that the anti-apoptotic effect of CO against TNFa/ActD is mediated in part through the upregulation of NF-jB-dependent anti-apoptotic proteins, such as cFLIP.
CO increases total expression of Bcl-XL while suppressing those of Bax
Discussion
The Bcl-2 family, comprised of both pro-apoptotic and anti-apoptotic members, constitutes a critical intracellular checkpoint for apoptosis within a common cell death pathway. The balance in expression of Bcl-2 family members is crucial in determining the fate of cells that undergo apoptosis [23]. Pro-apoptotic Bax is required for death signaling through mitochondria in response to TNFa. We investigated the effects of CO on the expression of Bcl-2 family pro-
Increased expression of HO-1, with subsequent increased generation of CO is part of a physiological response to injury by which many cells are protected from undergoing apoptosis. We previously demonstrated that exogenous administration of CO dramatically inhibited TNFa-mediated liver injury in vivo and hepatocyte cell death in vitro. Additionally, the hepatoprotective effect of CO is dependent on NF-jB activation. These
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Fig. 6. CO inhibits caspase-8 cleavage by increase of cFLIP expression via NF-jB. Hepatocytes underwent adenoviral gene transfer of LacZ (control) or IjB-super repressor (AdIjBaSR). (A) Western blot analysis of native and mutated IjBa was investigated after 24 h of adenoviral gene transfer of AdIjBaSR. (B) Electrophoretic mobility shift assay for NF-jB was performed with CO treatment and treatment with AdIjBaSR. AdLacZ was used as a transfection control and TNFa (10 ng) was used as a positive control. CO-induced NF-jB binding was markedly diminished by AdIjBaSR. (C) Twenty-four hours after gene transfer, hepatocytes were treated with TNFa/ActD with or without CO and protein was harvested at 6 h or viability was assayed at 12 h. cFLIP protein levels were maintained by CO in the LacZ group, however, these levels were diminished by inhibition of NF-jB activation in the TNFa/ActD treated group. Furthermore, inhibition of NF-jB activation led to increased cleavage of caspases-3 and -8. (D) Protection against TNFa/ActD-induced cell death by apoptosis was diminished by inhibition of NF-jB activation. TNFa/ActD induced significant cell death compared to LacZ controls (P < 0.01). CO treatment significantly protected against TNFa/ActDinduced death (*P < 0.05 compared to TNFa/ActD-treated LacZ control). However, inhibition of NF-jB activation by adenoviral gene transfer of IjB super-repressor significantly reversed protection by CO (#P < 0.05 compared to TNFa/ActD and CO-treated LacZ group).
data show that CO decreases activation of apoptotic proteins, including proximal caspase-8, and increases levels of anti-apoptotic proteins including XIAP and cFLIP. Furthermore, changes in cFLIP are dependent on NF-kB activation.
It is well described that the most proximal caspase activated following interaction of TNFa with its receptor is caspase-8 in various types of cells including hepatocytes [32]. Activated caspase-8 may in turn either activate effector caspases (i.e., caspase-3) or cleave Bid. The activated form of Bid triggers apoptosis by mitochondrial translocation. In the mitochondria, translocation of pro-apoptotic Bcl-2 family members such as Bax forms membrane channels by opening the permeability transition pore, leading to cytochrome c release and caspase activation [33]. Caspase-8 also directly activates caspase-3 independent of mitochondria, but this occurs slowly in hepatocytes. These data demonstrate changes at all of these levels, indicating that the CO may have an effect at multiple levels including proximal to caspase-8. Induction of HO-1 or delivery of CO was shown to protect mice from liver injury or lung injury by modulation of caspase or Bcl-2 family via MAPK pathway [21,34–36]. In these models of apoptotic liver injury, damage can be prevented by CO-mediated caspase modulation. We found that CO-mediated protection is accompanied by significant reduction of caspase-8 activity and inhibition of translocation of Bid and Bax a to mitochondria, which is associated cytochrome c release and subsequent mitochondria damage.These data further support that CO-mediated protection operates by activating NF-jB, which in the presence of an inflammatory stimulus leads to the up-regulation of NF-jB-dependent antiapoptotic genes [36,37]. The role of NF-jB in the liver as part of a pro-survival signaling pathway has been studied [32,38]. Activation of the transcription factor NF-jB is a major effector of the inducible resistance to death receptor-mediated apoptosis by modulating antiapoptotic genes [26]. Over-expressing NF-jBinduced proteins, FLIP or c-IAP, has been shown to be lead to a blockade of caspase-8 activation, resulting in increased resistance to Fas ligand (FasL) or TNFa. CO has been reported to be cytoprotective by modulation of NF-jB-dependent genes from several types of cells, yet there is no report that CO modulates caspase-8 through regulation of NF-jB. Given FLIP is induced by NF-jB, we considered the possibility that CO can modulate FLIP expression by NF-jB. Once NF-jB activation is inhibited, as by over-expression of its natural inhibitor IjBa, CO-induced FLIP expression was inhibited from cells during TNFa/ActD-apoptosis. Taken together, CO showed strong anti-apoptotic effects by modulation of cFLIP expression, at least in part by a NF-jB-dependent mechanism. In summary, the present study demonstrates that CO protects hepatocytes against TNFa/ActD by inhibiting caspase-8-mediated apoptotic pathway. CO-mediated antiapoptotic effects are associated with a decrease in the proteolytic fragment of caspases-8 and-3, as well as Bid. In addition, CO suppressed the translocation of Bid, Bax and Bcl-XL to mitochondria, resulting in inhibition of cytochrome c. Furthermore, the total level of anti-apoptotic proteins Bcl-XL or XIAP increased, whereas those of the proapoptotic protein Bax decreased. CO modulated
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