From the Departments of Cardiovascular Pharmacology,
Molecular Recognition,
Toxicology,
and ** Bone and Cartilage, SmithKline Beecham Pharmaceuticals, King of
Prussia, Pennsylvania 19046, the §§ Department
of Environmental Health, University of Washington, Seattle, Washington
98195, and ¶ Human Genome Sciences, Inc.,
Rockville, Maryland 20850
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ABSTRACT |
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TL1 is a recently discovered novel member of the
tumor necrosis factor (TNF) cytokine family. TL1 is abundantly
expressed in endothelial cells, but its function is not known. The
present study was undertaken to explore whether TL1 induces apoptosis in endothelial cells and, if so, to explore its mechanism of action. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to
TL1 showed morphological (including ultrastructural) and biochemical features characteristic of apoptosis. TL1-induced apoptosis in BPAEC
was a time- and concentration-dependent process
(EC50 = 72 ng/ml). The effect of TL1 was not
inhibited by soluble TNF receptors 1 or 2. TL1 up-regulated Fas
expression in BPAEC at 8 and 24 h after treatment, and
significantly activated stress-activated protein kinase (SAPK) and p38
mitogen-activated protein kinase (p38 MAPK). The peak activities of
SAPK and p38 MAPK in TL1-treated BPAEC were increased by 9- and 4-fold,
respectively. TL1-induced apoptosis in the BPAEC was reduced by
expression of a dominant-interfering mutant of c-Jun (62.8%,
p < 0.05) or by a specific p38 inhibitor, SB203580
(1-10 µM) dose-dependently. TL1 also
activated caspases in BPAEC, and TL1-induced apoptosis in BPAEC was
significantly attenuated by the caspase inhibitor,
ZVAD-fluromethyl-ketone. The major component activated by TL1 in BPAEC
was caspase-3, which was based on substrate specificity and
immunocytochemical analysis. These findings suggest that TL1 may act as
an autocrine factor to induce apoptosis in endothelial cells via
activation of multiple signaling pathways, including stress protein
kinases as well as certain caspases.
TL1 is a novel protein with a molecular mass of 22 kDa identified
recently by searching the Human Genome Sciences cDNA data base. TL1
is a type II membrane protein and exhibits about 30% sequence homology
to human tumor necrosis factor Materials
Ac-YVAD-AMC and Ac-DEVD-AMC were purchased from American Peptide
(Sunnyvale, CA). ZVAD-fmk and Ac-YVAD-CHO were obtained from Enzyme
Systems (Dublin, CA) and Peptides International (Louisville, KY),
respectively. Ac-DQMD-AMC, Ac-LEED-AMC, Ac-VETD-AMC, and anti-p38 MAPK
monoclonal antibodies were provided by SmithKline Beecham (SB)
Pharmaceuticals (King of Prussia, PA). Ac-IETD-AMC and mouse-anti-human
JNK monoclonal antibodies were purchased from Biomol Research
Laboratories (Plymouth Meeting, PA) and PharMingen (San Diego, CA),
respectively. Mouse soluble TNF receptor 1 (sTNFR1) and TNF receptor 2 (sTNFR2) were obtained from R&D Systems (Minneapolis, MN).
Expression and Purification of Bacterially Expressed TL1
An expression vector containing a 24-amino acid deletion from
the N-terminal sequence of the predicted full-length protein (GenBankTM
accession number AF039390) was constructed. The DNA sequence encoding
TL1 was amplified using polymerase chain reaction oligonucleotide
primers specific to the coding sequence of the TL1 gene. Additional
nucleotides containing restriction sites to facilitate cloning
were added to the 5' and 3' sequences, respectively. The 5'
oligonucleotide 5'-GCGCCATGGTGAGACAAACTCCCACA -3' contained a
NcoI restriction site, followed by 18 nucleotides of
corresponding coding sequence. The 3' primer has the sequence
5'-CGCAAGCTTCTATAGTAAGAAGGCTCC-3' containing a HindIII
restriction site followed by 18 nucleotides complementary to the last
15 nucleotides of the coding sequence and the stop codon. The amplified
TL1 DNA was cloned into vector pQE60 (Qiagen) after digestion with
restriction enzymes and ligation. The ligation mixture was transformed
into competent Escherichia coli cells (strain M15/rep4).
Expression of the TL1 protein was induced by
isopropyl-1-thio- Cell Cultures
BPAEC were obtained from the American Type Culture Collection
(Rockville, MD). The cells were grown in Dulbecco's modified Eagle's
medium supplemented with 10% heat-inactivated fetal calf serum in a
humidified environment of 5% CO2, 95% air at 37 °C as
described previously (12). Cells at a subconfluent density were used.
Before experiments, the medium was changed to Dulbecco's modified
Eagle's medium containing 2% fetal calf serum. BPAEC from passages
17-20 were used in all studies.
Morphological Assessment and Quantification of Apoptosis
To quantify cells undergoing apoptosis, cell monolayers were
fixed and stained with Hoechst 33324 (Molecular Probes, Eugene, OR) as
described previously (12). The morphological features of apoptosis
(cell shrinkage, chromatin condensation, blebbing, and fragmentation)
were monitored by fluorescence microscopy. Transmission electron
microscopy study was done as reported previously (12).
DNA Fragmentation Analysis
DNA Ladder--
Cells treated with vehicle or TL1 were lysed in
lysis buffer containing 100 mM NaCl, 10 mM
Tris·HCl, pH 8.0, 2.5 mM EDTA, 0.5% SDS, and 100 µg/ml
protein kinase K. The lysates were incubated at 55 °C for 16 h.
After incubation, the lysates were gently extracted three times with
phenol/chloroform/isoamyl alcohol, precipitated in ethanol, treated
with DNase-free RNase, re-extracted, and precipitated again as
described previously (12). DNA electrophoresis was carried out in 1.8%
agarose gels containing ethidium bromide, and DNA fragmentations were
visualized under ultraviolet light.
In Situ End Labeling (TUNEL)--
BPAEC were cultured in
two-chamber slides (Nunc) and treated with TL1 for 8-24 h. In
situ detection of apoptotic cells was performed by using terminal
deoxyribonucleotide transferase-mediated dUTP nick end labeling with an
ApopTag in situ apoptosis detection kit (Oncor) following
the manufacturer's recommendation.
Stress-activated Protein Kinase (SAPK/JNK) Assay
SAPK activity was measured using GST-c-Jun-(1-81) as bound to
glutathione-Sepharose 4B as described previously (12, 13).
p38 MAPK Assay
The cell lysates were immuno-precipitated with anti-p38 MAKP
antibody bound to protein A-agarose for 4 h at 4 °C. The beads were washed with lysis buffer and then with kinase buffer as described previously (14). The immune complex kinase assay was initiated by the
addition of 25 µl of kinase buffer containing 2 µg of GST-ATF2 and
50 µM [ In Vitro Transfection of Dominant-interfering Mutant of c-Jun in
BPAEC
The cells were plated in two-chamber slides. The cells were
cotransfected with 0.5 µg/ml Pegfp-c-1 (CLONTECH)
(15) as a fluorescent marker of transfected cells together with 1 µg/ml amounts of either the empty cloning vector pCDNA1 (control)
or the dominant-interfering c-Jun mutant pCDNA1-Flg Caspase Activity Assay
The cells were treated with vehicle or TL1 for a time period
indicated in the figure legends. Caspase activity assays were performed
with five different substrates under the condition optimal for each
caspase as reported previously (13). Levels of released 7-amino-4-methylcoumarin (AMC) were measured with a Cytofluor-4000 fluorescent plate reader (Perseptive Biosystems) at excitation and
emission wavelengths of 360 and 460 nm, respectively.
Immunohistochemical Analysis for Fas, Bcl-2, and Caspase-3
Expression
The cells were cultured in two-chamber slides. After treatment
with vehicle or TL1, the cells were fixed with 4% paraformaldehyde for
30 min at 4 °C and then changed to cold PBS. The cells were treated
with 0.2% Triton X-100 for 40 min at 4 °C and washed with cold PBS,
and then nonspecific immunoglobulin binding sites were blocked with
normal goat serum (Vector Laboratories) for 1 h at room
temperature. The cell samples were incubated with the primary antibody:
mouse anti-human Fas (Upstate Biotechnology), mouse anti-human Bcl-2
(DAKO), or rabbit anti-human CPP32 p17 peptide polyclonal antisera
(provided by Dr. K. Kikly, SmithKline Beecham), for 1 h at room
temperature. As a negative control, the cell samples were incubated
with nonimmune IgG (for Bcl-2 and CPP32) or IgM (for Fas) instead of
the primary antibody. After incubation with the primary antibody, cells
were washed with PBS and then incubated for 30 min with a secondary
antibody conjugated to fluorescein isothiocyanate. Cells were washed,
treated with Vectashield mounting medium (Vector Laboratories), and
viewed by fluorescence microscopy (Olympus IX70).
Statistical Analysis
All values in the text and figures are represented as mean ± S.E. of n independent experiments. Statistical evaluation
was performed by using one-way analysis of variance. Differences with a
value of p < 0.05 were considered significant.
Expression and Purification of Bacterially Expressed TL1--
The
predicted amino acid sequence of TL1 shares 20-30% homology to other
members of the TNF family as reported previously (1). Hydrophobicity
analysis of the protein predicts a hydrophobic region of 10-12 amino
acid near the N terminus of the protein following 10-15
non-hydrophobic amino acids and a membrane-anchoring region at the
C-terminal region, suggesting that TL1 is a characteristic type II
membrane-bound protein. Therefore, we constructed an expression vector
containing a 24-amino acid deletion from the N-terminal sequence of
TL1. The recombinant TL1 was expressed, purified, and subjected to
N-terminal sequencing analysis. The purity of the protein was >90%,
which was determined by SDS-PAGE.
TL1 Induces Apoptosis in BPAEC--
BPAEC exposed to TL1 shrunk
and retracted from their neighboring cells, and their cytoplasma
appeared condensed. Cells stained with Hoechst 33324 and assessed by
fluorescence microscopy demonstrated condensed chromatin, fragmented
nuclei, and blebbing of the plasma membrane (Fig.
1A, panel
B). The study with transmission electron microscopy revealed
that TL1-treated BPAEC displayed morphologic alterations characteristic
of apoptosis including condensation of chromatin and appearance of
apoptotic bodies (Fig. 1B, panel b). The characteristic degradation of DNA into
oligonucleosomal-length fragmentation was observed when the cells were
exposed to TL1 (30-300 ng/ml) for 24 h (Fig. 1C). DNA
fragments in situ were further visualized by the TUNEL
method (Fig. 1D, panel B). A
considerable fraction of endothelial cells treated with TL1 showed
positive staining; no positively stained cells were found in the
vehicle-treated cultures (Fig. 1D, panel
A).
TL1-induced endothelial cell apoptosis was a time- and
concentration-dependent process with an EC50 value
of 72 ng/ml (Fig. 2). A significant
increase in the number of cells with apoptotic morphology was apparent
6-8 h after exposure of the cells to TL1. Under similar conditions,
TNF- Effects of sTNFR1 and sTNFR2 on TL1-induced Apoptosis in
BPAEC--
Neither sTNFR1 nor sTNFR2 at 30 µg/ml showed effect on
TL1 (0.1 µg/ml)-induced apoptosis in BPAEC. Under the same condition TNF Regulation of Fas and Bcl-2 Expression in Endothelial Cells by
TL1--
Immunocytochemical analysis of Fas and Bcl-2 proteins was
determined at 8 and 24 h after treatment with TL1. Fas antigen was not detectable at resting BPAEC (Fig.
3A). However, a significant number of cells expressing Fas receptor were detected at 8 and 24 h after stimulation (Fig. 3, B and C). When mouse
IgM was substituted for the primary antibody, positive Fas
immunoreactivity was not detected. In contrast, Bcl-2 expression was
not detected in either unstimulated or TL1-treated BPAEC (data not
shown).
Activation of SAPK/JNK and p38 MAPK--
The effects of TL1 on
SAPK/JNK activity in BPAEC are shown in Fig.
4. Exposure of endothelial cells to TL1
induced a rapid activation of SAPK/JNK. A significant increase in
SAPK/JNK activity was detected 20 min after stimulation, peaked at 40 min, and then returned to the basal levels after 60 min (Fig.
4A). As shown in Fig. 4B, TL1-induced activation
of SAPK/JNK in endothelial cells was a
concentration-dependent process. The SAPK/JNK activity was
increased by 5.6 ± 1.4-fold (p < 0.05, n = 4) and 9.1 ± 1.8-fold (p < 0.01, n = 6) over the basal level in the presence of 50 and 300 ng/ml TL1, respectively.
The effects of TL1 on p38 MAPK activity are shown in Fig.
5. TL1 activated p38 MAPK in BPAEC with a
similar time course as SAPK/JNK but a lesser extent. The peak of p38
MAKP activity was increased by 3.1 ± 0.5- and 3.8 ± 0.4-fold over the basal level in the presence of 100 and 300 ng/ml TL1,
respectively.
Effects on TL1-induced Apoptosis by Expression of
Dominant-interfering Mutant of c-Jun in BPAEC or by the p38 MAPK
Inhibitor, SB203580--
To investigate the role of SAPK/JNK in
TL1-induced apoptosis in BPAEC, we transfected BPAEC with a
dominant-interfering mutant of c-Jun, pCDNA1-Flag Activation of Caspases in BPAEC by TL-1--
As shown in Fig.
8, TL1-induced BPAEC apoptosis was
attenuated by ZVAD-fmk, an irreversible cell-permeable inhibitor of
caspase (16), added to the culture medium 1 h prior to TL1
treatment. Under the same conditions, the addition of Ac-YVAD-CHO, a
relatively specific inhibitor of caspase-1 (17), up to 100 µM showed no effect in enhancing BPAEC rescue (data not
shown). To further determine which of the caspase family members are
activated in the TL1-induced apoptotic process in the endothelial
cells, we examined cell extracts for proteolytic activity. The relative rates of AMC formation were measured with a series of defined peptide
sequence variants that are relatively specific for caspase 1, 3, 4, 7, or 8 under the optimal conditions as described previously (13) and
presented in Fig. 9A. Similar
results were observed from three repeated experiments. Cell extracts
from TL1-treated BPAEC were highly active on Ac-DEVD-AMC and to a
lesser extent on Ac-DQMD-AMC, but not active on the remaining three
substrates which are more specific for caspase 1, 4, and 8. The
proteolytic activity appeared at 6 h after the cells were treated
with TL1, peaked at 24 h, and gradually returned to basal levels
within 48 h. Fig. 9B shows a comparison of the relative
velocities of four substrate hydrolysis rates by the TL1-treated cell
extracts and the recombinant caspase-3. The relative velocities of the two enzyme sources on four substrates were very similar.
To further confirm the activation of caspase-3 in TL1-stimulated BPAEC,
immunocytochemical detection of its enzymatically active form, the
17-kDa subunit, was performed. The antibody used was raised against a
peptide derived from the C-terminal portion of the p17 subunit. The
neoepitope antibody only binds caspase-3 following specific cleavage
between the p10 and p20 subunits (13). As shown in Fig.
10, the 17-kDa subunit of caspase-3 was
detected in TL1-treated but not vehicle-treated BPAEC, and was
localized with fragmented nuclei within the cells.
The studies presented in this paper demonstrate that TL1, a novel
TNF-like cytokine and a type II transmembrane protein, induces intensive apoptosis in cultured endothelial cells as reflected by
morphological and biochemical criteria. Under our experimental conditions, spontaneous BPAEC death rate was approximately 2-4%, well
in accord with a previous observation (4). The effect of TL1 was
concentration-dependent with an EC50 value of
72 ng/ml (3.5 nM), and a significant number of apoptotic
cells were detected 6-8 h after treatment. Moreover, the expression of
pro-apoptotic gene, Fas, was demonstrated in TL1-treated BPAEC, which
is consistent with that observed in apoptotic endothelial cells
reported previously (12).
The receptor(s) mediating TL1 activity has not been identified as yet.
To examine whether TL1 acts via distinct receptor(s) or shares the
known TNFR1 or TNFR2, we tested the effects of sTNFR1 and sTNFR2 on
TL-1-induced apoptosis in BPAEC. These two TNFRs have been shown
previously to block the cell surface TNFR1 and TNFR2 mediated TNF
bioactivities on responsive cell lines (data from R&D Systems).
However, neither sTNFR1 nor sTNFR2 inhibited the effect of TL1 on
BPAEC. In contrast, TNF Recent research efforts on TNF family members have demonstrated that
TNF Recent work has supported a central role for the caspase family
members, as effectors of apoptosis (24). However, the role of caspases
in endothelial cell apoptosis has not been sufficiently explored. Among
the caspase family, caspase-3 (CPP32) has been considered as a central
component of the proteolytic cascade during apoptosis and plays a key
role in this family (25, 26). TL1-induced BPAEC apoptosis was inhibited
by ZVAD-fmk, indicating a potential role for the caspase family in this
effector pathway for apoptosis. To determine which of the caspase
family members are involved, we examined the substrate specificity of
proteolytic activity in the extracts from TL1-activated BPAEC by
measuring the relative rate of AMC formation from six different
substrates, which are relatively specific for caspases 1, 3, 4, 7, and
8 (27). Treatment of BPAEC with TL1 resulted in a significant increase
in proteolytic activity toward DEVD-AMC mainly and DQMD-AMC to some
extent, both of which show the relative specificity for caspase-3 (13,
27). There was no induction in proteolytic activity in TL1-activated cell extracts when Ac-YVAD-AMC, LEED-AMC, or VETD-AMC were used as the
substrate, indicating that caspases 1, 4, and 8 might not be involved.
Moreover, comparison of the substrate specificity of the extracts from
TL1-treated BPAEC with the recombinant caspase-3 showed a similar
pattern, further suggesting that caspase-3 may be the predominant
member in the caspase family activated by TL1. Furthermore,
immunocytochemical studies detected the active form of caspase-3 in
TL1-treated BPAEC. It was reported that multiple caspase homologues
were found in both the cytoplasm and nucleus in etoposide-induced
apoptosis in HL-60 cells (28). Interestingly, in TL1-induced apoptotic
BPAEC, the immunoreactive 17-kDa subunit of caspase-3 was only
localized with fragmented nuclei, further indicating a role of
caspase-3 in TL1-induced apoptosis. Whether this active caspase-3 was
transported into the neucleus or the inactive caspase-3 is already in
the nucleus awaiting activation promoted by TL1 requires further
investigation. Taken together, these results suggest that caspase-3 was
activated by TL1 in BPAEC and may mediate TL1-induced cell apoptosis.
However, our results cannot exclude other members of this family,
especially those closely related to caspase-3, such as caspase-7, in
mediating TL1-induced apoptosis.
In summary, the present studies have demonstrated that TL1, a novel
member of TNF cytokine family, causes endothelial cell apoptosis. TL1
appears to act through a receptor that is distinct from TNF receptors 1 or 2. The effect of TL1 is via activation of the stress protein
kinases, SAPK/JNK and p38 MAPK, and the caspases, mainly caspase-3-like
protease. Apoptotic programmed cell death has been suggested to be a
cause of endothelial cell damage contributing to various inflammatory
disorders and cardiovascular injury (29). Moreover, endothelial cell
apoptosis may be an important mechanism involved in a balance between
antiangiogenic and proangiogenic processes, and loss of this balance
will lead to a variety of diseases such as solid tumor metastasis and
retinopathy (30, 31). Therefore, the biological significance and the
potential roles of TL1 in these pathophysiological conditions, and the
mechanism for regulation of TL1 production in endothelial cells require further studies.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(TNF
)1 (1). This newly
identified member of the TNF family has been demonstrated to be
abundantly expressed in endothelial cells as well as in kidney, lung,
and prostate. TL1 expression in HL-60 and THP1 cells was induced by
phorbol 12-myristate 13-acetate treatment. Radiation hybrid mapping
localized TL1 gene on chromosome 9q32, near CD30L. Because of its
robust expression in endothelial cells, TL1 has been suggested to
possibly play a role in vascular functions (1). However, no functional
study with this novel protein has been reported so far. The present
study was undertaken to explore whether TL1 induces endothelial cell
apoptosis, a phenomenon suggested to be one cause of endothelial cell
damage contributing to various inflammatory disorders and
cardiovascular dysfunction (2, 3). To examine this possibility, we used
bovine pulmonary artery endothelial cells (BPAEC) in which
TNF
-induced apoptosis has been demonstrated (4). Apoptosis was
detected on the basis of morphological (including ultrastructural) and
biochemical characteristics including DNA fragmentation. In addition,
we studied the effects of TL1 on the activity of stress kinases, p38
mitogen-activated protein kinase (p38 MAPK) and stress-activated
protein kinase (SAPK/JNK), and certain caspases. Both signaling
pathways are believed to be implicated in programmed cell death (5-8).
The expression of Fas and Bcl-2 in TL-1-stimulated BPAEC was also determined in view of the death-promoting effect of Fas and the anti-apoptotic effect of Bcl-2 (9, 10).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-D-galactopyranoside, and protein was
purified from the inclusion bodies into 8 M urea. The
solubilized protein was passed over a PD-10 column in 2×
phosphate-buffered saline (PBS), thereby removing the urea, and
refolding the protein. To remove endotoxin, the protein was passed over
a polymyxin column. The resulting TL1 preparation was found to be more
than 90% pure by SDS-PAGE and N-terminal sequencing. Automated
N-terminal sequencing was carried out using a model ABI-494 sequencer
(Perkin-Elmer/Applied Biosystems, Inc.) and the Gas-phase Blot cycles.
Endotoxin levels were assayed using the Amebocyte Lysate Test
(Bio-Whittaker) and were <10 EU of endotoxin/mg of protein (11).
-32P]ATP (20 Ci/mmol). The
phosphorylated products were resolved by SDS-PAGE and visualized by PhosphorImager.
169 (5) using the Calphos Maximizer transfection kit (CLONTECH)
according to the manufacturer's recommendation. Following
transfection, the cells were allowed to recover in complete medium for
24 h. The cells were treated with TL1, and the number of apoptotic
cells was assessed by nuclear staining after fixation as described
under Morphological Assessment and Quantification of
Apoptosis.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 1.
TL1-induced-apoptosis in BPAEC.
A, fluorescence photomicrographs showing nuclear morphology
of BPAEC treated with vehicle (A) or TL1 (50 ng/ml)
(B) for 24 h and stained with Hoechst 33324 (original
magnification, ×400). B, transmission electron micrographs
of BPAEC cultures treated with vehicle (a) and TL1 (50 ng/ml) (b) for 8 h (Bar = 2.5 µm).
C, electrophoretic analysis of internucleosomal DNA
fragmentation in TL1-treated BPAEC. Cells were treated with vehicle
(lane 5) or TL1 at 30 (lane
4), 100 (lane 3), and 300 (lane 2) ng/ml for 24 h. Lane
1, DNA size markers. D, in situ
detection of DNA fragments. BPAEC were cultured in two-chamber slides
and treated with vehicle (A) or TL1 (50 ng/ml)
(B) for 24 h. Cells were washed in PBS, fixed,
permeabilized, and then labeled with fluorescent dUTP and analyzed by
fluorescent microscopy.
, at 10 ng/ml, induced apoptosis in BEAPC by 16.7 ± 3.2%
(n = 4).
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Fig. 2.
Time- and concentration-dependent
induction of apoptosis in BPAEC by TL1. A, cells
were cultured with vehicle (blank bars) or TL1
(100 ng/ml) (black bars) for the indicated time
intervals. B, cells were cultured with the indicated
concentration of TL1 for 24 h. The production of cells undergoing
apoptosis was quantified by fluorescence microscopy after Hoechst 33324 staining. Values are mean ± S.E.(n = 4). *,
p < 0.05; **, p < 0.01 versus time 0 (A) or basal (B).
-induced apoptosis in BPAEC was reduced by sTNFR1 significantly (data not shown).
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Fig. 3.
Immunocytochemical detection of Fas receptor
protein in BPAEC. The cells were treated with vehicle
(A) or TL1 (50 ng/ml) for 8 h (B) or 24 h (C).
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Fig. 4.
Time- and concentration-dependent
activation of SAPK by TL1. BPAEC were treated with TL1 (100 ng/ml)
for the indicated periods of time (A) or at the
concentrations indicated for 40 min (B) and lysed. The
activity of SAPK was assayed using GST-c-Jun-(1-81) as the substrate.
Upper panels in A and B are
the quantitative results of three to five independent experiments and
the lower panels in A and B
are representative autoradiograms. *, p < 0.5; **,
p < 0.01 versus time 0 (A) or
vehicle (B).
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Fig. 5.
Time and concentration dependence of
TL1-induced activation of p38 MAPK in BPAEC. BPAEC were treated
with TL1 (100 ng/ml) for the indicated period of time (A)
(n = 3) or at the concentrations indicated for 40 min
(B) (n = 3-4). The activity of p38 MAPK was
assayed using GST-ATF2 as substrate as described under "Experimental
Procedures." *, p < 0.05; **, p < 0.01 versus basal.
169, in which a
deletion in the N-terminal transactivation domain that includes the
binding site for JNK (5). As shown in Fig.
6, expression of dominant-interfering c-Jun construct in BPAEC reduced TL1-induced apoptosis by 62.8% (p < 0.05). TL1-induced apoptosis in BPAEC was also
attenuated by a specific p38 MAPK inhibitor, SB203580 (23), in a
concentration-dependent manner as shown in Fig.
7. In the presence of 3 and 10 µM SB203580, TL1-induced BPAEC apoptosis was reduced by
33% (p < 0.05) and 51% (p < 0.01),
respectively. No further inhibition was observed when the concentration
of SB203580 was increased.
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Fig. 6.
Inhibition of TL1-induced apoptosis in BPAEC
by expression of dominant-interfering mutant of c-Jun. Cells were
transfected with 1 µg of the empty cloning vector (pCDNA1;
control) or the dominant-interfering c-Jun mutant pCDNA1-Flag
169. Transfected cells were detected by cotransfection with
Pegfp-c-1 as a fluorescent marker. Cells were treated with vehicle or
TL1 (50 ng/ml) for 16 h, and the cells undergoing apoptosis were
quantified by nuclear staining. The data are mean ± S.E.
(n = 4). The respective total number of transfected
cells that were counted is given within each column. **,
p < 0.01 versus column
1; *, p < 0.05 versus
column 2; #, p < 0.05 versus column 3.
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Fig. 7.
Effect of SB203580 on TL1-induced apoptosis
in BPAEC. The BPAEC were incubated with vehicle or SB203580 at the
concentrations indicated for 30 min, and TL1 (100 ng/ml) was added, and
the incubation continued for 20 h. The cells were fixed and
stained with Hoechst, and the number of apoptotic cells was counted.
The data are mean ± S.E. of three to four independent experiments
performed in duplicate. *, p < 0.05; **,
p < 0.01 versus TL1 alone.
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Fig. 8.
Inhibition of TL1-induced apoptosis in BPAEC
by ZVAD-fmk. Cells were treated with vehicle or ZVAD-fmk (100 µM) for 1 h prior to addition of TL-1 (50 ng/ml),
and incubation was continued for the indicated time period. Apoptotic
cells were quantified by fluorescence microscopy after staining with
Hoechst 33345. Values are mean ± S.E. of three independent
experiments. **, p < 0.01 versus TL1 alone
at the same time point; #, p < 0.05 versus
basal.
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Fig. 9.
A, activation of caspases in BPAEC by
TL1. BPAEC were treated with TL1 (100 ng/ml) for the indicated time
period. Cell extracts were assayed at each time point with five
different substrates under the condition optimal for each caspase as
reported in Ref. 13. AMC formation was measured, and the specific
activities were calculated from rates of product formation from each
tetrapeptide-AMC substrate. B, comparison of BPAEC extract
with recombinant caspase-3 for substrate specificity.
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Fig. 10.
Immunocytochemical detection of the 17-kDa
caspase-3 subunit in BPAEC treated with vehicle (A) or TL1
(50 ng/ml) for 18 h (B).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-induced apoptosis in BPAEC was significantly
reduced by sTNF1. The results suggest clearly that TL1-induced cell
death is independent of TNFR1 or TNFR2.
and Fas activate stress protein kinases, SAPK/JNK and p38 MAPK,
in a variety of cell types (18, 19); however, the effects of other
members of this family on SAPK and p38 MAPK have not been well studied.
Moreover, controversies regarding the role of SAPK/JNK and p38 MAPK in
TNF
- or Fas-mediated cell death have been reported. For example,
TNF
-induced apoptosis is dependent on JNK activity in U937 cells (6,
20) but not in fibroblasts (21), indicating that the consequences of
JNK activation vary considerably among cell types. Fas-mediated JNK activation occurs with a different kinetics from that of TNF
, suggesting that TNF
and Fas most likely activate JNK through a
different mechanism (22). Moreover, Juo et al. (19) reported recently that blockade of p38 MAPK by a specific p38 MAPK inhibitor did
not affect Fas-mediated apoptosis in Jurkat cells. Therefore, we were
interested in finding whether TL1 activates JNK and p38 MAPK, and what
is the role of this activation in TL1-mediated apoptosis in BPAEC. The
present investigation clearly demonstrates that both JNK and p38 MAPK
were rapidly activated by TL1 in a similar fashion to that observed in
TNF
-activated U937 cells. Moreover, expression of
dominant-interfering mutant of c-Jun in BPAEC reduced TL1-induced cell
death indicating that TL1-induced apoptosis in BPAEC was dependent on
JNK activity. To address the potential involvement of p38 MAPK in
TL1-mediated apoptosis in BPAEC, a specific p38 MAPK inhibitor SB203580
was tested. This inhibitor has been shown to specifically inhibit p38
MAPK activity in vitro, with no effect on a variety of
kinases tested, including JNK-1 and ERK-1 (23). As shown in Fig. 7,
TL1-induced apoptosis in BPAEC was also reduced by SB203580 in a
concentration-dependent manner, indicating that p38 MAPK
signaling pathway is involved in TL1-mediated BPAEC apoptosis. This
effect is different from that observed in Fas-mediated apoptosis in
Jurkat cells, in which SB203580 had no protective effect (19).
Moreover, TL1-induced p38 MAPK activation occurs with much faster
kinetics in BPAEC than that observed in Jurkat cells, in which the peak
of p38 MAPK activation was at 2-4 h after stimulation by Fas,
indicating TL1 and Fas most likely activate p38 MAPK through a
different mechanism with a different outcome. Our data further suggest
that different members of the TNF family may have different signaling
pathways to mediate cell death or have different effects in different
cell types.
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ACKNOWLEDGEMENTS |
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We thank Dr. D. Lee for kindly providing Ac-DQMD-AMC, Ac-LEED-AMC, and Ac-VETD-AMC and B. Maleeff and K. Pillarisetti for excellent technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF039390.
§ To whom correspondence should be addressed: Dept. of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406-0939. Tel.: 610-270-5313; Fax: 610-270-5080; E-mail: tian-li_yue{at}sbphrd.com.
The abbreviations used are: TNF, tumor necrosis factor; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; SAPK, stress-activated protein kinase; MAPK, mitogen-activated protein kinase; SB, SmithKline Beecham; sTNFR, soluble TNF receptor; GST, glutathione S-transferase; BPAEC, bovine pulmonary artery endothelial cells; AMC, 7-amino-4-methylcoumarin; CHO, aldehyde; fmk, fluoromethyl-ketone, JNK, c-Jun N-terminal protein kinase; TUNEL, in situ end labeling.
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REFERENCES |
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