| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
* Turku Center for Biotechnology, University of Turku and Åbo Akademi University,
Department of Biology, Åbo Akademi University, BioCity, and
Department of Biology, Laboratory of Animal Physiology, University of Turku, Turku, Finland
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The Fas ligand (FasL) (2) and the TNF-related apoptosis-inducing ligand (TRAIL) (3, 4) are members of the TNF family. Both are able to induce rapid apoptosis in potential target cells, the sensitivity of which seems to be regulated by multiple mechanisms. Among the members of the TNF family, TRAIL shows the highest homology with the FasL. TRAIL is a 40-kDa type II transmembrane protein suggested to be involved in many biological processes, such as activation-induced death of lymphocytes (5, 6, 7, 8), T cell-mediated cytotoxicity (9, 10, 11), and maintenance of immune-privileged sites (12), all functions that have been assigned also for the Fas receptor (FasR). While the involvement of FasR in these processes is relatively well established, further investigations are required to determine the exact role of TRAIL in these functions.
TRAIL signaling is mediated and regulated by four distinct receptors: DR4/TRAIL-R1 (13), DR5/TRAIL-R2 (14), decoy receptor (DcR)1/TRAIL-R3 (15), and DcR2/TRAIL-R4 (16), of which the DR4 and DR5 contain functional death domains and are able to induce apoptosis. In contrast, DcR1 and DcR2 act as inhibitory receptors by lacking complete death domains. The elevated expression of DcRs in normally growing tissues could possibly explain why TRAIL induces apoptosis in most transformed but not in normal cells (16, 17).
The apoptotic signaling pathway induced by ligation of the TRAIL receptors (TRAIL-R) is still fairly uncharacterized. Fas-associated death domain (FADD) (18) and caspase-8 (19) have been previously established as important components in the FasR death-inducing signaling complex (DISC) (20). In this sense, both FADD and caspase-8 have also been indicated as crucial elements in the TRAIL-mediated signaling machinery (21, 22, 23). Activation of caspase-8 in the DISC results in activation of downstream caspases and cleavage of cytosolic substrates such as Bid (24). Bid engages a mitochondrial amplification pathway, which has been suggested to be required for induction of apoptosis in some cell types. The cleaved or truncated Bid (tBid) (25) translocates to the mitochondria, where it triggers depolarization of the mitochondria. In concert with the altered mitochondrial membrane potential (MMP), cytochrome c (cyt c) is released to the cytosol, where it forms the apoptosome together with apoptosis protease-activating factor 1 and caspase-9 (26). In turn, caspase-9 can activate downstream caspase-3 or boost the activation of other caspases, such as caspase-8, to complete the mitochondrial amplification loop (reviewed in Ref. 27). The relative importance of the mitochondrial amplification loop in DR-mediated apoptosis is still not fully understood, and both mitochondria-dependent and -independent activation mechanisms have been identified. The available information on the involvement of the mitochondrial activation in TRAIL-R signaling is very scarce.
Suppression of apoptosis has been shown to be of major importance during many physiological as well as pathological processes. Apoptosis can be negatively regulated by inhibitor proteins, such as Bcl family proteins (reviewed in Ref. 28), FLIPs (reviewed in Ref. 29), or inhibitors of apoptosis protein (reviewed in Ref. 30). Another mode of regulation is through expression of DcRs, whose presence has been described for both the Fas (31) and the TRAIL-R system (reviewed in Ref. 32). Finally, protein kinase-mediated signaling has been described as an effective way of directing DR signals (reviewed in Ref. 33). In contrast to regulation by inhibitor proteins and DcRs, phosphorylation-based signaling occurs without requirement of newly synthesized proteins. In this respect, especially the classical mitogen-activated protein kinase (MAPK) signaling pathway has been implicated as a dominant negative regulator of DR-mediated apoptosis. We have observed that MAPK/extracellular signal-regulated kinase (ERK) signaling potently modifies FasR responses (34, 35) and found indications that it is involved in regulating also TRAIL-R responses (36). We have also shown that MAPK/ERK signaling from the TCR is able to protect T cells from FasR-mediated apoptosis (37) before they commit activation-induced cell death (AICD). Therefore, we wanted to test whether this type of regulation could also apply for the TRAIL-Rs. The results of the present study show that the MAPK/ERK pathway in activated Jurkat T cells suppresses TRAIL-mediated apoptosis in a similar fashion as it suppresses FasR-mediated apoptosis. Because little was known about the role of the mitochondrial amplification loop in TRAIL-R-mediated signaling, we paid special attention to clarifying where the inhibition takes place in relation to the proapoptotic mitochondrial signaling sequence. Our results show that MAPK/ERK abrogates the apoptotic signal upstream of the mitochondrial amplification loop by inhibiting initiator caspase activity. This mechanism could especially be involved in regulation of the persistence of peripheral T cell populations.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The human leukemic T cell line Jurkat (clone E6-1) was received from American Type Culture Collection (Manassas, VA). The cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in 5% CO2 in air. The cells were kept at a density of 0.5–1.0 x 106/ml.
Jurkat T cells were incubated at a density of 1 x 106/ml with TRAIL (100 ng/ml; Alexis, Läuflingen, Switzerland) along with 2 µg/ml cross-linking FLAG-tagged Ab M2 (Sigma-Aldrich, St. Louis, MO) or 100 ng/ml agonistic anti-human FasR IgM Ab (MBL, Watertown, MA) for the indicated time periods in the absence or presence of 100 µg/ml immobilized OKT3 (R.W. Johnson Pharmaceutical Institute, Bassersdorf, Switzerland) as described earlier (37), 20 nM tetradecanoyl phorbol acetate (TPA; Sigma-Aldrich), 30 µM PD 98059 (Calbiochem, La Jolla, CA), or 5 µM cycloheximide (CHX; Sigma-Aldrich).
Analysis of phosphatidylserine exposure
To detect phosphatidylserine exposure by flow cytometry, Jurkat T cells were washed once with PBS and incubated for 10 min in 400 µl binding buffer (2.5 mM HEPES/NaOH (pH 7.4), 35 mM NaCl, 0.625 mM CaCl2) with 1 µl annexin V-FITC (Alexis) and analyzed on a FACScan flow cytometer (BD Biosciences, San Jose, CA) or viewed under a RMB epifluorescence microscope (Leica, Deerfield, IL).
Immunoblotting techniques used
Immunoblotting was performed by lysing cells in Laemmli sample buffer and then resolving the proteins on a 12.5% SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) probed with the specific Ab to ERK2 (BD Transduction Laboratories, Lexington, KY), phospho-ERK1/2 (New England Biolabs, Boston, MA), caspase-8 (a kind gift from P. Krammer, German Cancer Research Center, Heidelberg, Germany) (38), Bid (Santa Cruz Biotechnology, Santa Cruz, CA), Hsc70 (StressGen Biotechnologies, Victoria, British Columbia, Canada), or actin (Sigma-Aldrich), followed by coupling to the appropriate HRP-conjugated secondary Abs and visualization with the ECL system (Amersham, Little Chalfont, U.K.)
Transfection studies
Cells were transiently transfected by electroporation (220 V, 975 µF) in 400 µl of OptiMeM (Life Technologies, Rockville, MD) and allowed to rest for 48 h before treatments. The DNA constructs used were pMCL-HA-MKK1-K97 M and pMCL-HA-MKK1-S218E/S222, encoding for hemagglutinin (HA)-tagged dominant negative and constitutively active forms of MAPK kinase 1 (MKK1). The plasmid was a kind gift from N. Ahn (University of Colorado, Boulder, CO). Mock transfections were conducted using a pIRES-EGFP plasmid (Clontech Laboratories, Palo Alto, CA). For detection of transfected cells, the cells were fixed with 3% formaldehyde in PBS and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). After washing and blocking, cells were incubated with 10 µg/ml of a monoclonal HA-specific Ab (12CA5; Boehringer Mannheim, Mannheim, Germany) followed by incubation with FITC-conjugated anti-mouse secondary Ab and 10 mg/ml Hoechst 33342 (Molecular Probes, Eugene, OR). Bid-GFP transfection studies were conducted as mentioned above. The plasmid was a kind gift from G. J. Gores (Mayo Clinic, Rochester, MN) (39). For detection of apoptotic nuclei, cells were labeled with Hoechst 33342. Cells were finally mounted in 50% glycerol and viewed under a Leica RMB epifluorescence microscope.
Measurement of MMP by confocal microscope
To measure MMP, Jurkat T cells were equilibrated with 50 nM tetramethyl rhodamine methyl ester (TMRM; Molecular Probes) in RPMI 1640 medium supplemented with 25 mM HEPES (pH 7.2) for 1 h at 37°C in the dark. Subsequently, TRAIL was added to the equilibration medium. Leica TCS SP confocal microscope with 63x NA 1.4 oil immersion planapochromat objective was used to collect TMRM and transmission images at given time points. Red fluorescence of TMRM was imaged by using 568 nm excitation light from argon/krypton laser and emitted light was collected through 575–705 nm.
cyt c immunofluorescence analysis by confocal microscope
For immunofluorescence analysis, Jurkat cells were centrifuged onto glass coverslips, washed with PBS, and fixed with 3% paraformaldehyde. Subsequently, cells were permeabilized with 0.5% Triton X-100/PBS for 10 min at room temperature. After blocking with normal goat serum (GS), samples were incubated with mouse anti-cyt c, (clone 6H2.B4, 1:150 in PBS/0.01% Triton X-100 with 1.5% GS; BD PharMingen, San Diego, CA) for 2 h in a humidified dark chamber at 37°C. After three washes with PBS/0.01% Triton X-100, samples were incubated with Alexa 488-conjugated goat anti-mouse IgG (1:150 in PBS/0.01% Triton X-100 with 1.5% GS; Molecular Probes) for 45 min in a dark chamber. After three washes with PBS/0.01% Triton X-100, nuclei of the cells were counterstained with 0.1 µg/ml 4',6'-diamidino-2-phenylindile hydrochloride (DAPI) and coverslips were mounted on microscope slides in 80% glycerol in PBS. cyt c release and nuclear morphology of the cells were imaged by Leica TCS SP MP confocal microscope with 63x NA 1.4 oil immersion planapochromat objective. Alexa 488 fluorescence was excited by using a 488-nm excitation line from argon/krypton laser and emission window was set at 492–560 nm. DAPI fluorescence was imaged by using a 780-nm excitation light from Ti-Sapphire (Tsunami; Spectra Physics, Mountain View, CA) laser and emission light was recorded through 400–490 nm.
Surface expression analysis of DR4 and DR5
A total of 0.5 x 106 cells were treated with TPA for the indicated time points with or without TRAIL. After washing, cells were blocked for 30 min with 1% BSA in PBS. Cells were then incubated with 1 µg of Abs to DR4 or DR5 (Alexis) in 1% BSA in PBS for 30 min followed by washing with PBS. Finally, cells were incubated with Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes) for 30 min. After washes cells were analyzed on a FACScan flow cytometer. Only secondary Ab was used as a control.
TRAIL-R immunoprecipitation and DISC analysis
A total of 2 x 108 Jurkat cells per sample were left untreated or pretreated with 20 nM TPA in a 37°C water bath at cell densities between 1 and 2 x 106/ml. After 15 min cells were pelleted at 500 x g for 7 min and resuspended in 1 ml prewarmed RPMI medium. To stimulate TRAIL-Rs, 1 µg FLAG-tagged recombinant human soluble TRAIL (Alexis) and 2 µg anti-FLAG monoclonal M2 Ab (Sigma-Aldrich) were added to the cell suspension. Cells were incubated in a 37°C water bath for 15 min and the reaction was stopped by adding 10 ml of ice-cold PBS to the cell suspension. Cells were pelleted, washed with ice-cold PBS, and lysed in 1 ml lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 0.2% Nonidet P-40, 0.1% deoxycholate, and complete protease inhibitor mixture (Roche, Basel, Switzerland) for 30 min on ice. The cell debris was removed by centrifugation at 15,000 x g for 15 min at 4°C. The amount of protein was determined by Bradford assay and an equal amount of protein from each sample was precleared with 50 µl of Sepharose-CL-4B for 2 h at 4°C. A total of 5 µg of monoclonal anti-DR5 and 2.5 µg monoclonal anti-DR4 (Alexis) were added to samples and immunoprecipitated with 15 µl protein G beads (Amersham) for 2.5 h at 4°C. Beads were washed six times in 1 ml lysis buffer, resuspended in 3x Laemmli sample buffer, and boiled for 3 min. About one-third of immunoprecipitation samples and 20–50 µg protein from cell lysates were analyzed by 12.5 or 10% SDS-PAGE. Western blot was performed with anti-DR5 (Alexis), anti-FADD (BD Transduction Laboratories), caspase-8 (C15 caspase-8 Ab, a kind gift from P. Krammer, German Cancer Research Center), and anti-FLIP (Alexis) as described above.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To study whether MAPK/ERK activation is able to modulate
TRAIL-induced apoptosis of Jurkat T cells, we pretreated cells with two
known MAPK/ERK activators (34, 37), the phorbol ester TPA
or OKT3, the latter of which is an agonistic Ab to CD3 of the TCR
complex. Our results show that pretreatment with both TPA and OKT3
suppresses TRAIL-induced apoptosis (Fig. 1
, A–C). Apoptosis
was measured by flow cytometric analysis of phosphatidylserine exposure
on the cell membrane with annexin V conjugated to FITC (Fig. 1, B and C). Incubation with TRAIL alone induced
rapid apoptosis in the cells. After 2 h almost 50% of the cells
were apoptotic, whereas pretreatment with TPA or OKT3 efficiently
suppressed TRAIL-induced apoptosis, as indicated by decreased
phosphatidylserine exposure (Fig. 1, A and B) and
DNA fragmentation (data not shown). Furthermore, the T cell activator
OKT3 was able to suppress TRAIL-induced apoptosis for at least 12
h (Fig. 1D). These results on inhibition of TRAIL-induced
apoptosis by MAPK/ERK activators correspond well to our previous
results showing that MAPK/ERK signaling is an effective inhibitor of
FasR-mediated apoptosis (34), as well as our data showing
that activated T cells stay insensitive to FasR-mediated apoptosis as
long as their MAPK/ERK activity is elevated (37).
|
C), thus indicating that the protective
effect of these compounds is MAPK/ERK dependent. Also, in agreement
with previous observations on the FasR (34), the
protective effect was protein synthesis independent as the
MAPK/ERK-mediated protection was equally efficient in the presence of
CHX (Fig. 1C). To verify that OKT3 and TPA stimulation
induces phosphorylation of MAPK/ERK, we analyzed the activation of
MAPK/ERK by immunoblotting with an Ab that recognizes phosphorylated
MAPK/ERK (Fig. 2). Incubation with TPA
and OKT3 induced an increase in MAPK/ERK activity, which was inhibited
by pretreatment with PD 98059 (Fig. 2), correlating well with the
relative inhibition of apoptosis observed in Fig. 1C. The
results further support that the protective effect of TPA and OKT3 is
MAPK/ERK dependent.
|
The suppressive role of MAPK/ERK in TRAIL-induced apoptosis was
verified by transient transfections with HA-tagged constitutively
active and dominant negative mutants of MKK1 before treatment with
TRAIL. The transfected cells were then visualized by immunofluorescence
labeling of HA and by DNA labeling with Hoechst 33342 to identify the
nuclear morphology of the cells (Fig. 3).
Constitutively active MKK1 (MKK1-CA) rendered the cells insensitive to
apoptosis induced by TRAIL, while this effect was lost when cells were
transfected with the dominant negative mutant. Mock transfections with
green fluorescent protein did not affect the number of apoptotic
cells.
|
Sensitization to DR-mediated apoptosis could be modulated by
altered surface expression of the receptors. To rule out the
possibility of surface receptor down-regulation, we analyzed the
relative amount of DR4 and DR5 on the surface of Jurkat T cells. Jurkat
T cells were immunofluorescence labeled with mAbs to the two respective
receptors and analyzed by flow cytometry. The results show that
predominantly DR5 is expressed on Jurkat T cells and that MAPK/ERK
activation does not affect the relative number of receptors on the cell
surface after treatment with TPA for up to 2 h (Fig. 4). The amount of DR4 and DR5 on the cell
surface did not change in the presence of TRAIL.
|
To study at what level in the apoptotic activation machinery the
inhibitory effect of MAPK/ERK is targeted, we started by analyzing
whether the mitochondrial amplification loop is affected. To assess
this question, we analyzed changes in the MMP after treatment with
TRAIL alone or after pretreatment with TPA. While the cells that were
treated with TRAIL alone lost their MMP, cells pretreated with TPA were
not affected at this level (Fig. 5A). Also, in FasR-mediated
apoptosis the MMP was lost (data not shown). The FasR-mediated decrease
in MMP was also abolished by the TPA-mediated activation of MAPK/ERK
(data not shown). To show that this was a MAPK/ERK-dependent effect, we
pretreated the cells with the MKK1 inhibitor PD 98059. Inhibition of
MAPK/ERK reinduced the loss in MMP and accelerated apoptosis. Because
decreased MMP has been suggested to cause release of cyt c
to the cytosol (40), it was to be expected that cyt
c release would also be affected by activation of MAPK/ERK.
Jurkat T cells treated with TRAIL alone or pretreated with TPA were
immunolabeled for cyt c and viewed under a microscope. In
control cells cyt c was located in the mitochondria, which
can be seen as clusters next to the nucleus in Fig. 5B. In
apoptotic Jurkat T cells, treated with TRAIL alone for 2 h,
TRAIL-caused release of cyt c from the mitochondria to the
cytosol was clearly visible. When cells were pretreated with the
MAPK/ERK activator TPA, the release of cyt c was inhibited.
Also in this case, treatment with PD 98059 reversed this inhibition and
allowed the cells to undergo apoptosis. Also, FasR-mediated cyt
c release was inhibited (data not shown). Treatment with TPA
or PD alone did not affect the release of cyt c.
|
C). After 2 h in the
presence of TRAIL all Bid-GFP in apoptotic cells was located in
clusters near the nucleus that could be referred to as mitochondria.
When the transfected cells were pretreated with TPA, Bid-GFP was no
longer detected in clusters and apoptosis was inhibited. Instead, Bid
was diffusely located in the cytosol similarly to control cells.
Pretreatment with PD 98059 abolished the inhibition of Bid-GFP
translocation (Fig. 5C) as well as subsequent apoptosis
(Fig. 1C). Again, PD or TPA had no effect alone. Taken together these results show that TRAIL-induced apoptosis is directed toward the mitochondrial amplification loop and that MAPK/ERK signaling protects Jurkat T cells from apoptosis by inhibiting any dysregulation of the mitochondria by turning off the mitochondrial amplification loop.
MAPK/ERK activation suppresses the cleavage of Bid and caspase-8
To examine whether the protective effect of MAPK/ERK signaling
would occur at the level of the DISC, we analyzed how the cleavage of
initiator caspase-8 was affected in cells treated with TPA before
incubation with TRAIL. The results in Fig. 6A show that the cleavage of
caspase-8 to the active 18-kDa fragment is markedly reduced in cells
pretreated with TPA. Also, processing to the intermediate 42/43-kDa
fragments is reduced in the presence of TPA. Caspase-8 has previously
been shown to activate Bid by cleavage to a 15-kDa tBid fragment
(25). Therefore, we wanted to examine whether activation
of Jurkat T cells suppresses the cleavage of Bid to its active
proapoptotic 15-kDa fragment. As expected, its cleavage was reduced
after preincubation of the cells with TPA (Fig. 6B). This
inhibition is likely to prevent the advancing of the apoptotic signal
to the mitochondrial amplification loop, which has been implicated to
be necessary for FasR-mediated apoptosis in type II cells
(42).
|
Activation of MAPK/ERK does not affect the recruitment of FADD and caspase-8 to the TRAIL-DISC
To examine whether the observed protection mediated by MAPK/ERK
activation could be located at the very early stages of DR signaling,
we immunoprecipitated the TRAIL-DISC to analyze the assembly of the
adapter proteins recruited to the DISC. Because Jurkat T cells have
been indicated to be type II cells, only a moderate amount of DISC is
formed after receptor activation, also reflected in our experiments by
the relative low levels of both FADD and caspase-8 coimmunoprecipitated
with DR4 and DR5 after TRAIL stimulation (Fig. 7A). To control successful
immunoprecipitation, the presence of DR5 in the immunoprecipitates was
detected in the immunoprecipitated samples but not in the protein G
control devoid of immunoprecipitating Abs. In our study we did not
detect any changes in the amount of FADD or caspase-8 recruited to the
DISC after pretreatment with TPA (Fig. 7A). Although the
overall caspase-8 cleavage is reduced by MAPK/ERK activation (Fig. 6A), the amount of caspase-8 cleaved at the DISC is equal in
the presence of TPA. Furthermore, we did not detect any changes in the
recruitment of cFLIP to the DISC upon TPA stimulation (data not shown).
To verify that MAPK/ERK was active during the same experiment, we also
analyzed the cell lysates for phosphorylated ERK1/2. Active ERK1/2
could be detected only in the presence of TPA (Fig. 7B).
Furthermore, to verify the protecting effect of TPA under the same
conditions, cells were further incubated at 37°C and later monitored
for apoptosis. The cells pretreated with TPA were still protected from
TRAIL-induced apoptosis after several hours (data not shown). Together
these experiments show that the assembly of the TRAIL-DISC is not
affected by the elevated MAPK/ERK activity as earlier shown also for
the FasR-DISC (37).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In T cells both FasR and FasL are up-regulated upon T cell activation and both participate later in the attenuation of immune responses, to avoid formation of autoreactive T cells (reviewed in Ref. 46). This modulation occurs through AICD, where T cells kill themselves or their neighboring cells, supposedly by activating the FasR. However, T cells are insensitive to FasR-mediated apoptosis immediately after activation, although they express both FasR and FasL. This insensitivity is important in order for the activated T cells to fulfill their task of killing the target cell. It has also been shown that TRAIL is up-regulated immediately after T cell activation (47, 48) and that TRAIL later participates in the down-regulation of immune responses. While it is still quite controversial whether TRAIL is involved in AICD of human peripheral T lymphocytes in vivo (5, 7, 8), there are results indicating that AICD of Jurkat T cells (8) as well as T cells derived from HIV patients (6) involves TRAIL. There are also indications that TRAIL would inhibit cell cycle progression, thereby arresting the T cells so they can be killed by other ligands, such as FasL (49). Therefore, it is important that recently activated T cells stay insensitive to both FasL and TRAIL during the early phase of activation so that the cells can fulfill their task of killing their target cells without being sensitive to their own death ligands.
MAPK/ERK signaling modulates apoptosis induced by TRAIL
Because our previous results indicate that the insensitivity of activated T cells to FasR-mediated apoptosis depends on MAPK/ERK activation (34, 37), we examined whether MAPK/ERK signaling could suppress TRAIL-induced apoptosis during early phases of T cell activation. Our results show that activated Jurkat T cells display similar kinetics of MAPK/ERK activation and insensitivity to TRAIL, as was shown with the FasR (37). Our results indicate that MAPK/ERK signaling mediates a protective signal to both FasR-mediated and TRAIL-induced apoptosis during these early phases of T cell activation. This protective signal is then turned off at the end of the immune response to allow AICD and attenuation of the immune response. There is one report indicating that activation of protein kinase C (PKC) can protect cells from TRAIL-induced apoptosis independently of MAPK/ERK (50). This study indicated that PKC activity is mainly responsible for the observed protection from TRAIL-induced apoptosis upon TPA treatment. Differences in the experimental setup could explain the different outcome of the experiments. It is also difficult to separate these two pathways at the level of PKC, because it is an upstream regulator of MAPK/ERK, especially by using only pharmacological signaling inhibitors and activators, the principal approach in the above-mentioned study. There is also the distinct possibility that PKC and MAPK/ERK act as separate signaling entities regulating DR responses. However, while it is plausible that PKC and possibly other signaling modulators regulate FasR and TRAIL-R sensitivity, our results undoubtedly show that the MAPK/ERK signaling pathway can function as a single dominant regulator of TRAIL responses.
The MAPK/ERK-mediated protection is independent of protein synthesis and does not alter the relative amount of DR4 or DR5 on the cell surface
It is well known that several cell lines can be sensitized to DR-mediated apoptosis by pretreatment with protein synthesis inhibitors (36). This raises the possibility that the MAPK/ERK-mediated effect could be protein synthesis dependent. However, our previous results have demonstrated that MAPK/ERK-mediated suppression of FasR-mediated apoptosis is not protein synthesis dependent (34, 35, 36). The results presented in this work show that the same is true for MAPK/ERK-mediated suppression of TRAIL-induced apoptosis. Thus, a high MAPK/ERK activity is sufficient to trigger the protective effect. Direct modulation by phosphorylation-based signaling is beneficial to quickly modulate TRAIL sensitivity in situations with rapidly fluctuating conditions, such as cell growth and differentiation. This type of rapid protein synthesis-independent regulation is likely to act in concert with various regulatory proteins (e.g., FLIP). Once such a regulatory protein has been produced, it will yield a more long-term and stable protection or modulation.
There are many possible targets for a direct signaling-mediated modulation of the DR signal. A protein synthesis-independent signal-based mechanism that regulates TNF-R1 sequestration has been reported. In this case, MAPK/ERK signaling was shown to phosphorylate TNF-R1 directly, thereby causing the internalization of the receptor from the surface of the cell to the cytosol and inhibition of its cytotoxic ability (51, 52). However, our study excludes the possibility of TRAIL-R internalization or down-regulation as a MAPK/ERK target, because the surface expression of the DR4 and DR5 was not altered by the activation of MAPK/ERK.
It is tempting to speculate that the protective action of MAPK/ERK would be mediated by direct or indirect phosphorylation of a DISC component. Phosphorylation of FADD has previously been indicated (53, 54). However, our results show that MAPK/ERK activation does not alter the binding of FADD or caspase-8 to the TRAIL-DISC. Therefore, it is unlikely that MAPK/ERK activation would affect the assembly of the DISC by phosphorylation of a DISC protein. Recent findings also show that FasR, DR4, and DR5 are not phosphorylated by active MAPK/ERK or by TPA (55). It seems that finding the potential MAPK/ERK target responsible for conveying the observed protection will require detailed phosphoprotein analysis far beyond the DISC.
MAPK/ERK activation inhibits the processing of caspase-8 and Bid, thereby turning off the mitochondrial amplification loop
To resolve whether the cells still maintain their normal functions, it is of great interest to know whether MAPK/ERK suppresses TRAIL-induced apoptosis at the same stage of the signaling pathway as it does in FasR-mediated apoptosis. Similarly to the FasR (34, 37), MAPK/ERK signaling seemed to suppress TRAIL-induced apoptosis before activation of caspase-8 and Bid. It has been shown that TRAIL induces caspase-8-mediated cleavage of Bid in other cell systems (56, 57, 58). Because MAPK/ERK signaling appeared to inhibit activation of both caspase-8 and Bid, we wanted to see whether inhibition of this upstream activator suppressed all traces of downstream mitochondrial activation. It has not yet been clear whether the mitochondrial pathway is involved in TRAIL-induced apoptosis. The results in this work show that Bid is rapidly cleaved with simultaneous translocation of tBid in TRAIL-stimulated cells. MAPK/ERK activation inhibits both cleavage and translocation of tBid from the cytoplasm to the mitochondria, as well as the observed loss in MMP and release of cyt c to the cytosol, after stimulation of the FasR and TRAIL-Rs. Normally during apoptosis, released cyt c binds to apoptosis protease-activating factor 1 in the cytosol to form the apoptosome where caspase-9 is activated. Caspase-9 accelerates the cleavage of caspase-8 by the proposed mitochondrial amplification loop suggested in type II cells (42). The results presented in this work show that MAPK/ERK signaling completely abrogates the engagement of mitochondria during TRAIL-induced apoptosis. Furthermore, our results indicate that the activation of TRAIL-induced cyt c release is truly dependent on the cleavage of Bid, because there were no traces of cyt c release when Bid cleavage was inhibited. Taken together, all of our results demonstrate that the inhibition of the apoptotic TRAIL signal occurs at the very proximal stages of apoptotic signaling. Inhibition at the site of death signal initiation would be a favorable way to abrogate the death signal, because the cell can thus avoid any partial damage and survive unaffected.
The elevated MAPK/ERK signaling after activation of T cells enters and inhibits the DR pathway, thereby allowing the cells to live long enough to fulfill their tasks. When the T cells are no longer needed, the MAPK/ERK activity and the levels of inhibitory proteins decrease as a consequence of insufficient activating signals, thereby allowing the cells to die by AICD. In future studies it will be of great importance to determine the molecular mechanisms and targets underlying the MAPK/ERK-mediated inhibition of apoptotic signaling in both TRAIL-induced and FasR-mediated apoptosis. Defining these targets will have great potential in treatments of various disorders related to the functions of these receptors.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Current address: Department of Cell Biology, Max-Planck-Institut for Biochemistry, D-82152, Martinsried, Germany.
3 Address correspondence and reprint requests to Dr. John E. Eriksson, Department of Biology, Laboratory of Animal Physiology, Science Building 1, University of Turku, FIN-20014 Turku, Finland. E-mail address: john.eriksson{at}utu.fi
4 Abbreviations used in this paper: AICD, activation-induced cell death; CHX, cycloheximide; cyt c, cytochrome c; DAPI, 4',6'-diamidino-2-phenylindile hydrochloride; DcR, decoy receptor; DISC, death-inducing signaling complex; DR, death receptor; ERK, extracellular signal-regulated kinase; FADD, Fas-associated death domain; FasL, Fas ligand; FasR, Fas receptor; GS, goat serum; HA, hemagglutinin; MAPK, mitogen-activated protein kinase; MKK1, MAPK kinase 1; MMP, mitochondrial membrane potential; PKC, protein kinase C; tBid, truncated Bid; TPA, tetradecanoyl phorbol acetate; TMRM, tetramethyl rhodamine methyl ester; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, TRAIL receptor.
Received for publication September 18, 2001. Accepted for publication June 26, 2002.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
converting enzyme-like protease involvement in Fas-induced and activation-induced peripheral blood T cell apoptosis in HIV infection: TNF-related apoptosis-inducing ligand can mediate activation-induced T cell death in HIV infection. J. Exp. Med. 186:1365.
B and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain. Immunity 7:813.[Medline]
This article has been cited by other articles:
|
|
 |
|
  D. Chiron, C. Pellat-Deceunynck, M. Maillasson, R. Bataille, and G. Jego Phosphorothioate-Modified TLR9 Ligands Protect Cancer Cells against TRAIL-Induced Apoptosis J. Immunol., October 1, 2009; 183(7): 4371 - 4377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Guicciardi and G. J. Gores Life and death by death receptors FASEB J, June 1, 2009; 23(6): 1625 - 1637. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-H. Yoon, M.-J. Kim, M.-T. Park, J.-Y. Byun, Y.-H. Choi, H.-S. Yoo, Y.-M. Lee, J.-W. Hyun, and S.-J. Lee Activation of p38 Mitogen-Activated Protein Kinase Is Required for Death Receptor-Independent Caspase-8 Activation and Cell Death in Response to Sphingosine Mol. Cancer Res., March 1, 2009; 7(3): 361 - 370. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Voisin, A. El Firar, C. Rouyer-Fessard, V. Gratio, and M. Laburthe A hallmark of immunoreceptor, the tyrosine-based inhibitory motif ITIM, is present in the G protein-coupled receptor OX1R for orexins and drives apoptosis: a novel mechanism FASEB J, June 1, 2008; 22(6): 1993 - 2002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-H. Liao, S.-M. Hsu, and P.-H. Huang ARMS Depletion Facilitates UV Irradiation Induced Apoptotic Cell Death in Melanoma Cancer Res., December 15, 2007; 67(24): 11547 - 11556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. O'Brien, M. A. Richardson, S. F. Mehrbod, D. T. Berg, B. Gerlitz, A. Gupta, and B. W. Grinnell Activated Protein C Decreases Tumor Necrosis Factor Related Apoptosis-Inducing Ligand by an EPCR- Independent Mechanism Involving Egr-1/Erk-1/2 Activation Arterioscler Thromb Vasc Biol, December 1, 2007; 27(12): 2634 - 2641. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. V. Richter and D. J. Topham The {alpha}1beta1 Integrin and TNF Receptor II Protect Airway CD8+ Effector T Cells from Apoptosis during Influenza Infection J. Immunol., October 15, 2007; 179(8): 5054 - 5063. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Baritaki, A. Katsman, D. Chatterjee, K. C. Yeung, D. A. Spandidos, and B. Bonavida Regulation of Tumor Cell Sensitivity to TRAIL-Induced Apoptosis by the Metastatic Suppressor Raf Kinase Inhibitor Protein via Yin Yang 1 Inhibition and Death Receptor 5 Up-Regulation J. Immunol., October 15, 2007; 179(8): 5441 - 5453. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. E. Guicciardi, S. F. Bronk, N. W. Werneburg, and G. J. Gores cFLIPL prevents TRAIL-induced apoptosis of hepatocellular carcinoma cells by inhibiting the lysosomal pathway of apoptosis Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1337 - G1346. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Meinander, T. S. Soderstrom, A. Kaunisto, M. Poukkula, L. Sistonen, and J. E. Eriksson Fever-Like Hyperthermia Controls T Lymphocyte Persistence by Inducing Degradation of Cellular FLIPshort J. Immunol., March 15, 2007; 178(6): 3944 - 3953. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nekrasova, C. Shive, Y. Gao, K. Kawamura, R. Guardia, G. Landreth, and T. G. Forsthuber ERK1-Deficient Mice Show Normal T Cell Effector Function and Are Highly Susceptible to Experimental Autoimmune Encephalomyelitis J. Immunol., August 15, 2005; 175(4): 2374 - 2380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bhattacharya, R. M. Ray, and L. R. Johnson Prevention of TNF-{alpha}-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2 Am J Physiol Gastrointest Liver Physiol, March 1, 2004; 286(3): G479 - G490. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Fernandez, A. M. Ramos, P. Sancho, D. Amran, E. de Blas, and P. Aller 12-O-Tetradecanoylphorbol-13-acetate May Both Potentiate and Decrease the Generation of Apoptosis by the Antileukemic Agent Arsenic Trioxide in Human Promonocytic Cells: REGULATION BY EXTRACELLULAR SIGNAL-REGULATED PROTEIN KINASES AND GLUTATHIONE J. Biol. Chem., January 30, 2004; 279(5): 3877 - 3884. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-T. Park, J.-A Choi, M.-J. Kim, H.-D. Um, S. Bae, C.-M. Kang, C.-K. Cho, S. Kang, H. Y. Chung, Y.-S. Lee, et al. Suppression of Extracellular Signal-related Kinase and Activation of p38 MAPK Are Two Critical Events Leading to Caspase-8- and Mitochondria-mediated Cell Death in Phytosphingosine-treated Human Cancer Cells J. Biol. Chem., December 12, 2003; 278(50): 50624 - 50634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zanin-Zhorov, R. Hershkoviz, I. Hecht, L. Cahalon, and O. Lider Fibronectin-Associated Fas Ligand Rapidly Induces Opposing and Time-Dependent Effects on the Activation and Apoptosis of T Cells J. Immunol., December 1, 2003; 171(11): 5882 - 5889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Gober, C. C. Smith, K. Ueda, J. A. Toretsky, and L. Aurelian Forced Expression of the H11 Heat Shock Protein Can Be Regulated by DNA Methylation and Trigger Apoptosis in Human Cells J. Biol. Chem., September 26, 2003; 278(39): 37600 - 37609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Jia, C. Yu, M. Rahmani, G. Krystal, E. A. Sausville, P. Dent, and S. Grant Synergistic antileukemic interactions between 17-AAG and UCN-01 involve interruption of RAF/MEK- and AKT-related pathways Blood, September 1, 2003; 102(5): 1824 - 1832. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Zhang, J. Hirahashi, X. Cullere, and T. N. Mayadas Elucidation of Molecular Events Leading to Neutrophil Apoptosis following Phagocytosis: CROSS-TALK BETWEEN CASPASE 8, REACTIVE OXYGEN SPECIES, AND MAPK/ERK ACTIVATION J. Biol. Chem., August 1, 2003; 278(31): 28443 - 28454. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ibata-Ombetta, T. Idziorek, P.-A. Trinel, D. Poulain, and T. Jouault Candida albicans Phospholipomannan Promotes Survival of Phagocytosed Yeasts through Modulation of Bad Phosphorylation and Macrophage Apoptosis J. Biol. Chem., April 4, 2003; 278(15): 13086 - 13093. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
