From the Department of Chemistry, Bose Institute, 93/1 Acharya Prafulla, Chandra Road, Kolkata 700 009, India
Received for publication, January 27, 2003 , and in revised form, April 24, 2003.
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ABSTRACT |
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INTRODUCTION |
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Apoptosis is an orchestrated suicide program, that among its other functions, enables an organism to remove invading pathogens without causing an inflammatory response. The key morphological alterations of apoptosis are mediated by a family of cysteine proteases called the caspases, which are activated by proteolytic cleavage (1416). Activation of caspases is achieved via two principal signaling pathways, namely the extrinsic and intrinsic death pathways (17, 18). The extrinsic death pathway involves the ligation of death receptors (CD95/Fas; tumor necrosis factor receptor) that leads to the recruitment of adaptor molecules such as Fas-associated death domain (FADD),1 and pro-caspase 8 or procaspase 10 into a death-inducing signaling complex (DISC) (19, 20). Adaptor molecules such as FADD and TRADD activate caspase 8 through proximal interactions between their death effector domains (DED) (21). Activated caspase 8 can then cleave and activate downstream effector caspases such as caspase 3 and caspase 7 (2224). In the intrinsic (or mitochondrial) death pathway, mitochondria release several death-promoting factors including cytochrome c (cyt c) (25), apoptosis-inducing factor (AIF) (26), and Smac (DIABLO) (27, 28) in response to death-inducing signals via the Bcl-2 family of proteins (29). In the cytosol, cyt c interacts with apoptotic protease-activating factor (Apaf-1). On binding cyt c, an ATP/dATP-binding oligomerization domain within Apaf-1 mediates Apaf-1 oligomerization (30, 31). The oligomerized complex (apoptosome) then binds procaspase 9 and facilitates processing of caspase 9 zymogens (32). Activated caspase 9 subsequently activates effector caspases (33).
There is a cross-talk between the extrinsic and intrinsic death pathways. One mechanism for this cross-talk is the cleavage of Bid by caspase 8. Bid is a BH3-domain-only member of the Bcl-2 family of death-regulating proteins (34). The BH3 domain-only family are upstream activators of apoptosis that signal to downstream proapoptotic Bcl-2 family members such as Bax or Bak leading to their oligomerization at the mitochondria. Activated caspase 8 can cleave p22 Bid to generate a p15 active truncated Bid (t-Bid) fragment, which is then targeted to the mitochondria (35). Cleavage of Bid to the mitochondrially active, t-Bid, is a feature of caspase 8-mediated apoptosis induced via death receptors (36, 37) which enables amplification of the apoptotic signal through the mitochondrial release of cyt c (38)
The mitogen-activated protein (MAP) kinase cascade is one of the evolutionarily conserved phosphorylation-regulated protein kinase cascades, which is involved in controlling the decision of cell survival or cell death. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase kinase kinase (MAPKKK) family, which plays a role in stress-induced apoptosis principally through activation of the JNK or the p38 MAP kinase signaling cascades (3942)
Mycobacterium tuberculosis and M. avium are both facultative intracellular bacteria that reside and replicate inside the macrophages of the infected host. The relationship between the course of mycobacterial infection in vivo and the pro- or anti-apoptotic cell signaling within the host is still unclear. However, in vitro studies suggest that induction of apoptosis in macrophages is the host cell strategy to eliminate the invading pathogen, whereas prevention of programmed cell death can result in the possibility of prolonged intracellular survival of mycobacteria. The organism therefore tries to commandeer the host cell signaling machinery to offset cell death (43). The mechanisms of apoptotic cell death in macrophages challenged with mycobacteria are largely unknown, other than reports of apoptotic signaling involving both Fas and TNF receptor (5, 58). The present study was undertaken with the objective of understanding the signaling pathways mediating macrophage apoptosis upon challenge with M. avium. Our results suggest that M. avium-induced apoptosis involves FADD-mediated activation of caspase 8, cleavage of t-Bid, release of cyt c and activation of caspases 9 and 3. Further, we identify the ASK1/p38 signaling pathway as being critical to caspase 8 activation and subsequent M. avium-induced cell death.
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EXPERIMENTAL PROCEDURES |
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Culture of the Murine Macrophage-like Cell Line
J774A-1Media and supplements were purchased from Invitrogen. The
murine macrophage cell line J774A-1 was obtained from the National Centre for
Cell Science, Pune and maintained in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml
penicillin and 100 µg/ml streptomycin in a humidified atmosphere containing
5% CO2. Dishes were washed to remove non-adherent cells. Adherent
cells were 95% viable as determined by trypan blue dye exclusion.
Growth of M. aviumM. avium (NCTC 8562) was obtained from the All India Institute of Medical Sciences New Delhi), grown in Middlebrook 7H9 broth (Difco, Detroit, MI) supplemented with oleic acid-albumin-dextrose (OADC) supplement and 0.04% Tween 80 until mid-log phase, harvested, washed, and resuspended in a small volume of phosphate-buffered saline containing 0.04% (v/v) Tween 80. The suspension was briefly sonicated until no bacterial clumps were visualized by microscopy. The preparation was diluted to a concentration of 2 x 108 CFU/ml in 10% glycerol and stored in aliquots at 80 °C. Freshly thawed aliquots were used no more than once.
Plasmid Constructs and Transient TransfectionsHA-tagged
wild-type ASK1 (ASK1, WT) and a catalytically inactive mutant (K709M) of ASK1
(ASK1, KM) were obtained from Hidenori Ichijo. FLAG-tagged p8 MAPK and its
dominant-negative mutant (p38 (agf)) were obtained from Roger Davis,
University of Massachusetts Medical School, Worcester, MA, and
dominant-negative FADD (FADD (dn)) was from Claudius Vincenz, University of
Michigan, Ann Arbor, MI. 2 x 106 cells were plated in each
well of 6-well plates, and cells were transfected with 2 µg of plasmid
(empty vectors or recombinants) using the Effectene reagent (Qiagen) in
Dulbecco's modified Eagle's medium with 10% fetal bovine serum according to
the manufacturer's instructions. -Galactosidase reporter plasmid was
used to normalize transfection efficiencies.
Infection and Preparation of Cell LysatesJ774A-1 cells were
cultured in 24-well tissue culture plates at 4 x 105 cells
per well and were infected with M. avium at a multiplicity of
infection (MOI) of 10 or as stated under the legends to figures. Cells were
lysed with 1% (v/v) Triton X-100, and the lysate was diluted and plated on
Middlebrook 7H10 agar to quantitate the number of viable intracellular
bacteria. The incubation of J7741 with M. avium at a
bacteria/macrophage ratio of 10:1 for 6 h led to infection of 50% of the
cells. The viability of the control monolayers was monitored during the
experiments by the trypan blue exclusion method. The viability was 95% in
all the experiments described. After incubation with M. avium, the
wells were washed with ice-cold PBS to remove free bacteria. Cells were lysed
with lysis buffer (20 mM Tris-HCl (pH 7.4), 1% (v/v) Nonidet P-40,
10% v/v glycerol, 137 mM NaCl, 20 mM NaF, 1
mM EDTA, 40 mM sodium
-glycerophosphate, 4
µg/ml each of leupeptin, pepstatin, and aprotinin, 1 mM
Na3VO4 1 mM pefabloc, 1 mM
benzamidine) on ice for 15 min. Cell lysates were boiled for 5 min after the
addition of 5x Laemmli sample buffer and subjected to Western blotting.
Where necessary, J774A-1 cells were first treated with pharmacological
inhibitors or vehicle (Me2SO) alone, prior to incubation with
M. avium. When performing Western blotting for detection of caspases,
6 x 106 cells were pelleted and freeze-thawed thrice in 20
µl of cell extraction buffer (50 mM PIPES/NaOH (pH 6.5), 2
mM EDTA, 0.1% (w/v) CHAPS, 5 mM dithiothreitol, 20
µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1
mM pefabloc). The lysates were centrifuged at 10,000 x
g for 5 min at 4 °C, and the supernatants were collected for
detection of caspases.
Western BlottingProteins were separated on SDS-polyacrylamide gels and then transferred electrophoretically to polyvinylidene difluoride membranes. The blots were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) for 1 h at room temperature and subsequently incubated overnight at 4 °C with primary antibodies in TBS-Tween 20 (1%, v/v) (TBST) with 5% (w/v) bovine serum albumin. Following three washes of 5 min each with TBST, the blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell Signaling Technology) (or appropriate secondary antibody) in blocking buffer for 1 h at room temperature. After three washes with TBST, the blots were developed with BM chemiluminescence reagent (Roche Applied Science) and exposed to x-ray film (Kodak XAR5).
ASK1 Kinase AssayAfter treatment, cells were washed with
ice-cold PBS, and lysed with lysis buffer containing 20 mM Tris-HCl
(pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1%(v/v) Triton X-100, 1% sodium deoxycholate, 0.5 mM sodium
pyrophosphate, 1 mM sodium -glycerophosphate, 1 mM
Na3VO4, and 1 µg/ml leupeptin. Lysates were
centrifuged at 10,000 x g for 10 min at 4 °C. The
supernatant (equivalent to 200 µg of protein) was incubated overnight at 4
°C on a rocker with rabbit polyclonal ASK1 antibody. 10 µl of protein
A/G plus agarose was subsequently added and incubated at 4 °C for an
additional 3 h. The beads were washed twice with lysis buffer and twice with
kinase buffer (25 mM Tris-HCl, pH 7.5, 5 mM
Na-
-glycerophosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4, 10 mM MgCl2). The pellet was washed
once with kinase buffer without protease inhibitors. The beads were then
incubated in 20 µl of kinase buffer in the presence of 0.5 µCi of
[
-32P]ATP (specific activity 3,000 Ci/mmol) with 1 µg of
myelin basic protein (MBP) as substrate at 30 °C for 15 min. The reaction
was stopped by adding protein gel denaturing buffer. After SDS-PAGE and
autoradiography, the band intensities were quantified by densitometric
scanning of the autoradiograms.
Cell Death ELISAJ774A-1 cells were plated (6 x 104 cells/plate) on 96-well plates. Cells were either treated with inhibitors or transfected with different plasmid constructs prior to incubation without or with M. avium. Cell death was detected with the cell death detection ELISA plus kit (Roche Applied Science) according to the manufacturer's protocol.
Caspase Activity AssaysCells (6 x 105) were lysed by incubating with 25 µl of lysis buffer (25 mM HEPES (pH 7.5), 2 mM EDTA, 0.1% (w/v) CHAPS, 5 mM dithiothreitol, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 20 µg/ml leupeptin, 1 mM pefabloc) on ice for 15 min. The supernatant was kept frozen at 70 °C until used. Lysate (20100 µg of protein) was taken in 100 µl of assay buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM dithiothreitol, 10% glycerol, 0.1% CHAPS, and 200 µM substrate. For measuring caspase 3 activity, samples were incubated at 37 °C for 4 h with Ac-DEVD-pNA as substrate, and absorbance at 405 nm was measured. Caspase 8 and 9 activities were measured using the fluorigenic substrates Ac-IETD-AFC and Ac-LEHD-AFC respectively. Fluorescence of the released AFC was measured using excitation and emission wavelengths of 360 and 530 nm, respectively.
Isolation of Cytosol and Mitochondrial Fractions for Detection of Cyt c
and BidAfter treatments, 18 x 106 cells were
washed with ice-cold PBS, and cell pellets were resuspended in 400 µl of
resuspension buffer (20 mM HEPES (pH 7.5), 10 mM KCl,
1.5 mM MgCl2, 1 mM EDTA, 1 mM
EGTA, 1 mM pefabloc, 10 µg/ml leupeptin, 10 µg/ml aprotinin,
150 mM sucrose). The cells were broken with 20 passages through a
26-gauge needle, and the homogenate was centrifuged at 750 x g
for 10 min, to remove nuclei and unbroken cells. The mitochondrial pellet was
obtained by centrifugation at 10,000 x g for 15 min and
resuspended in 40 µl of resuspension buffer (20 mM Tris-HCl (pH
7.4), 100 mM NaCl, 1 mM pefabloc, 10 µg/ml leupeptin,
10 µg/ml aprotinin). The supernatant was centrifuged at 100,000 x
g for 60 min at 4 °C. The supernatant obtained was the cytosolic
fraction. Cytosolic and mitochondrial fractions (20 µg of protein) were
used for quantification of cyt c and Bid, respectively. Prior to
Western blotting, protein concentrations were assayed and all samples were
normalized to equal protein concentrations. Blots were stripped and reprobed
for -actin expression.
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RESULTS |
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M. avium-induced Macrophage Cell DeathTaking into account previous reports that M. avium induces apoptosis of macrophages, and our results showing M. avium-induced ASK1 activation, we asked the question whether ASK1-dependent signaling is linked to death of J774A-1 macrophages challenged with M. avium. M. avium induced macrophage cell death in a time-dependent manner (Fig. 2, panel A) as measured using histone ELISA. Transfection of cells with catalytically inactive ASK1 abrogated M. avium-induced cell death (Fig. 2, panel B). Dominant-negative p38 MAP kinase exerted a similar effect, supporting the view that cell death was dependent on ASK1/p38 MAP kinase signaling. This was further supported by the fact that SB203580 could inhibit M. avium-induced cell death in a dose-dependent manner (Fig. 2, panel C), whereas U0126, an inhibitor of ERK 1/2 MAP kinase was without effect. In order to evaluate the role of FADD-mediated signaling in M. avium-induced apoptosis, cells were transfected with dominant-negative FADD (FADD(dn)) prior to challenge with M. avium. FADD (dn) abrogated M. avium-induced cell death (Fig. 2, panel B). FADD was not upstream of ASK1/p38 MAP kinase signaling since M. avium-induced ASK1 and p38 MAP kinase activation (assessed by probing Western blots with phospho-ASK1 and phospho-p38-specific antibodies) occurred even in FADD (dn)-transfected cells (Fig. 1, panel E). Cell death was also inhibited by treatment with NAC (Fig. 2, panel C). It is presently unclear how NAC regulates ASK1 activation and cell death in this case. ROS may oxidize thioredoxin thus dissociating it from ASK1 leading to ASK1 activation (46). It is also possible that a sequential signaling pathway links ROS production to ASK1 activation.
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Members of the caspase family are crucial mediators of apoptosis. Therefore the role of caspases, in M. avium-mediated cell death was evaluated using specific inhibitors for different caspases. As evident from Fig. 2 (panel D), the involvement of caspases 8, 9, and 3 was evident from the death-inhibitory effects z-IETD-FMK, z-LEHD-FMK, and z-DEVD-FMK, respectively.
Caspase Activities in M. avium-challenged J774A-1 MacrophagesWestern blot analysis for cleaved, active caspases was undertaken to determine caspase activation. Consistent with the effect of caspase inhibitors on M. avium-induced cell death, M. avium was found to activate the executioner caspase 3 (Fig. 3, panel A) and the initiator caspase 9 (Fig. 3, panel E) in a time-dependent manner as evidenced by detection of the cleaved, activated form of the caspases by Western blotting. Caspase 3 activation was also supported by the detection of the cleaved form of the caspase 3 substrate PARP in M. avium-challenged macrophages (Fig. 3, panel C). The generation of cleaved caspase 3 could be blocked by pretreatment with the caspase 3-specific inhibitor z-DEVD-FMK as well as the caspase 9-specific inhibitor z-LEHD-FMK (Fig. 3, panel B). z-DEVD-FMK could also block the generation of cleaved PARP in M. avium-challenged macrophages (Fig. 3, panel C). Generation of cleaved caspase 3 and cleaved caspase 9 could be blocked by transfection of cells with ASK1(KM) (Fig. 3, panels D and F, respectively). Generation of cleaved caspase 9 could also be blocked by pretreating cells with the caspase 8-specific inhibitor z-IETD-FMK (Fig. 3, panel E). These data supported the role of ASK1 as well as caspase 8 in the activation of caspase 9 and subsequent activation of caspase 3 in M. avium-challenged macrophages. The role of caspase 8 as an initiator of M. avium-induced death signaling was supported by Western blotting analysis of pro- and active caspase 8. Cleaved caspase 8 was detected in M. avium-challenged macrophages (Fig. 3, panel G). This could be blocked in cells pretreated with the caspase 8-specific inhibitor z-IETD-FMK but not with the caspase 3-specific inhibitor z-DEVD-FMK. M. avium-induced caspase 8 activation was also blocked by pretreatment with the p38 MAP kinase inhibitor SB203580 (Fig. 3, panel G) suggesting a role of p38 MAP kinase in caspase 8 activation. The role of FADD-mediated signaling was suggested by the fact that caspase 8 activation was blocked in cells transfected with FADD (dn) (Fig. 3, panel H). The data obtained from Western blotting was further supported by caspase activity assays. M. avium-induced activation of caspase 9 was abrogated in cells transfected with ASK1(KM) or pretreated with the caspase 8 inhibtor z-IETD-FMK (Fig. 4, panel A). Caspase 3 activation could also be abrogated in cells transfected with ASK1(KM) or pretreated with the caspase 8 inhibtor z-IETD-FMK or the caspase 9 inhibtor z-LEHD-FMK (Fig. 4, panel B). Transfection with FADD (dn) blocked caspase 8 activation (Fig. 4, panel C). Western blotting and activity assays therefore suggested that M. avium-triggered caspase 8 activation likely involves FADD-dependent signaling. Of particular interest was the observation that caspase 8 activation was blocked in cells transfected with ASK1(KM) (Fig. 4, panel E). The p38 MAP kinase inhibitor SB203580 also inhibited caspase 8 activation in a dose-dependent manner both in untransfected (Fig. 4, panel D) as well as in ASK1-transfected (Fig. 4, panel E) cells. These results suggested that ASK1/p38 MAP kinase and FADD signaling lead to caspase 8 activation.
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Generation of Truncated Bid and Release of Cyt cThe activation of caspase 9 in M. avium-challenged macrophages suggested a role of the mitochondrial pathway in M. avium-induced cell death. Taking into consideration that caspase 8 was also activated in M. avium-treated macrophages it appeared likely that caspase 8 activation was linked to the mitochondrial death pathway. Linkage of caspase 8 to mitochondrial death signaling occurs through the caspase 8-mediated cleavage of Bid and generation of t-Bid, which translocates to the mitochondria and promotes the release of cyt c. We tested whether M. avium-induced caspase 8 activation leads to signaling to the mitochondria by the generation of t-Bid. M. avium-treated macrophages showed time-dependent increase of t-Bid in the mitochondrial fraction (Fig. 5, panel A, upper blot). Generation of t-Bid was abrogated in caspase 8 inhibitor-treated- or in FADD(dn)-transfected cells (Fig. 5, panel A, middle and lower blots), suggesting that t-Bid formation is dependent both on caspase 8 activation and on FADD. Concomitant with this, cyt c was detected in the cytosol in a time-dependent manner although the content of actin remained the same (Fig. 5, panel B). Death signaling originating from FADD-dependent caspase 8 activation was likely amplified by mitochondrial cyt c release since pretreatment with the caspase 8 inhibitor z-IETD-FMK (Fig. 5, panel B) or transfection with FADD (dn) (Fig. 5, panel C) abrogated M. avium-induced cyt c release. Cyt c release was also blocked in cells transfected with dominant-negative p38 MAP kinase (agf) or ASK(KM) (Fig. 5, panel C), suggesting a role of ASK1/p38 MAP kinase signaling not only in caspase 8 activation but also in mitochondrial cyt c release in M. avium-challenged macrophages.
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DISCUSSION |
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The diverse signals associated with apoptosis converge upon the activation of procaspases which play a central role in the initiation and execution of apoptosis. In the present study we demonstrate that caspases 3, 8, and 9 are activated after challenge of macrophages with M. avium and that these are all required for mycobacteria-induced death of J774A-1 macrophages. Inhibition of any one of these caspases with caspase-specific peptide inhibitors was sufficient to block cell death.
Interestingly, we observed that the stress-activated MAP kinase kinase kinase ASK1 is activated in M. avium-challenged macrophages. NAC inhibited M. avium-induced cell death by inhibiting the activation of ASK1. However, it remains to be elucidated whether the effect of NAC was due to ROS-triggered sequential signaling leading to ASK1 activation or due to dissociation of thioredoxin from ASK1. That ASK1 plays a central role in innate immunity against M. avium was supported by the observation that a catalytically inactive mutant of ASK1 could block M. avium-induced cell death. p38 MAP kinase appeared likely to be the downstream effector of ASK1, since ASK1 activated p38 MAP kinase and a dominant-negative mutant of p38 MAP kinase, or the p38 MAP kinase-specific inhibitor SB203580 could block M. avium-induced cell death. The link between ASK1/p38 MAP kinase signaling and caspase activation was evident from the observation that a catalytically inactive mutant of ASK1 could prevent activation of caspases 8, 9, and 3. Treatment with SB203580 was also associated with the inhibition of caspase 8 activation. Our results suggest a role of ASK1/p38 signaling in caspase 8 activation. The release of cyt c from the mitochondria could be blocked by caspase 8 inhibitor, suggesting that the death signal triggered by caspase 8 activation is amplified by the mitochondrial release of cyt c. Cyt c release could also be blocked in p38 (agf)-transfected cells suggesting that ASK1/p38 MAP kinase signaling plays a crucial role not only in activation of caspase 8 but also in downstream events leading to the release of cyt c and activation of caspases 9 and 3 (Fig. 6). Caspase 3 inhibition did not affect the activation of caspase 8 ruling out caspase 3 as the mediator of caspase 8 activation.
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In order to examine the role of FADD in M. avium-induced signaling leading to cell death, we transfected cells with FADD (dn) and examined downstream events. The role of FADD-dependent signaling was demonstrated by the abrogation of M. avium-induced caspase 8 activation and cell death in FADD (dn)-transfected cells. However, FADD was not upstream of ASK1/p38 MAP kinase activation since FADD (dn) transfection did not affect M. avium-induced ASK1 and p38 MAP kinase activation. Since death signals are in several instances amplified through the mitochondrial pathway by the caspase 8-mediated cleavage of Bid and its translocation to the mitochondria, we analyzed the status of Bid in the mitochondrial fractions of M. avium-challenged macrophages. Western blotting showed an increase in the fraction of t-Bid in the mitochondria as a function of time of challenge with M. avium, whereas caspase 8 inhibitor or FADD(dn) transfection could block the generation of t-Bid in the mitochondrial fraction, suggesting that FADD-dependent, and caspase 8-mediated Bid cleavage activates the mitochondrial death pathway in this case. This was supported by the concomitant detection of cyt c in the cytosolic fraction.
Our results provide new insight into the role of ASK1 signaling in cell death in macrophages and differ from the results obtained with mink lung epithelial cells where ASK1-induced apoptosis has been reported to occur through mitochondria-dependent caspase activation in a caspase 8-independent manner (51). Similar to our observations Raoul et al. (52) have demonstrated the involvement of ASK1 and p38 MAP kinase together with the classical FADD/caspase-8 pathway in the Fas-triggered death of embryonic motor neurons, whereas Gilot et al. (53) have demonstrated that hepatoprotection results from both inhibition of the caspase cascade and prevention of ASK1-JNK/p38 MAP kinase signaling.
We hypothesize that upon M. avium challenge, FADD-mediated caspase 8 activation occurs downstream of ASK1/p38 MAP kinase signaling (Fig. 6). ASK1/p38 MAP kinase signaling also leads to the release of cyt c. Caspase 8-mediated cleavage of Bid and its translocation to the mitochondria is probably associated with the release of cyt c leading to the formation of an apoptosome, activation of caspase 9 and finally to the execution of apoptosis mediated by activation of caspase 3. Critical to the activation of caspase 8 is the formation of the DISC. Since ASK1 activates caspase 8, it is possible that ASK1 exerts its effects upstream of the mitochondria probably at the level of the formation of the DISC. Progression of the apoptotic signal depends on an interplay of protein-protein interactions at different levels. Among these, cellular FLICE-like inhibitory protein (c-FLIP) prevents FADD-mediated apoptotic transduction through inhibition of caspase 8 maturation (5456). It appears possible that ASK1 acts at the level of regulating c-FLIP activity. This could be at the level of synthesis or degradation of c-FLIP or through its phosphorylation (57) as suggested in recent reports. On the other hand, ASK1/p38 MAP kinase signaling could also possibly regulate caspase 8 activity by phosphorylating caspase 8 itself. However, to the best of our knowledge, there are to date no reports of phosphorylation-dependent activation of caspases. It is also possible that ASK1/p38 MAP kinase signaling could lead to increased death receptor ligand expression as documented in the case of p38 MAP kinase-dependent overexpression of Fas ligand in intestinal epithelial cells undergoing anoikis (48). Testing of these possibilities is the subject of a separate investigation. The importance of the present investigation lies in the fact that it is the first elucidation of a signaling pathway through which ASK1 plays a central role in innate immunity and therefore in the containment of bacterial infection. It also one of the few recent reports of the ASK1-dependent regulation of caspase 8 activation.
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FOOTNOTES |
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To whom correspondence may be addressed. Fax: 91-33-23506790; E-mail:
mani18{at}hotmail.com.
To whom correspondence may be addressed. E-mail:
joyoti{at}vsnl.com.
1 The abbreviations used are: FADD, Fas-associated death domain; ASK1,
apoptosis signal-regulating kinase 1; MAP, mitogen-activated protein; DED,
death effector domains; cyt c, cytochrome c; t-Bid,
truncated Bid; DISC, death-inducing signaling complex; NAC,
N-acetylcysteine; AFC, 7-amino-4-trifluromethyl coumarin; FMK,
fluoromethyl ketone; z-DEVD-FMK,
z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-FMK;
z-LEHD-FMK, z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F; z-IETD-FMK,
z-lle-Glu(OMe)-Thr-Asp(OMe)-FMK; Ac-DEVD-pNA,
Ac-Asp-Glu-Val-Asp-pNA; Ac-LEHD-AFC, Ac-Leu-Glu-His-Asp-AFC;
Ac-IETD-AFC, Ac-lle-Glu(OMe)-Thr-Asp(OMe)-AFC; MOI, multiplicity of infection;
Me2SO, dimethylsulfoxide; PIPES, 1,4-piperazinediethanesulfonic
acid; CHAPS, 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonic acid;
MBP, myelin basic protein; PBS, phosphate-buffered saline.
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ACKNOWLEDGMENTS |
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REFERENCES |
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