1 Inflammatory Bowel Disease
Center, Fas is expressed
constitutively by colonic epithelial cells, and its ligand is expressed
by intraepithelial and lamina propria lymphocytes. Fas ligation induces
apoptosis in colonic epithelial cells and is implicated in the
epithelial damage seen in ulcerative colitis. To understand the
pleiotropic effects of Fas in the intestinal mucosa, we have examined
signaling pathways activated by Fas in HT-29 colonic epithelial cells.
HT-29 cells were stimulated with anti-Fas in the presence or absence of
interferon-
c-Jun NH2-terminal kinase; activator protein-1; extracellular signal-regulated kinase activation
FAS IS A transmembrane receptor in the tumor necrosis
factor (TNF) receptor family (11, 19). Several lines of evidence suggest that Fas-Fas ligand (FasL) interactions are important in
immune-epithelial communication in the intestine. Fas is expressed constitutively by the basolateral membrane of normal colon and small
intestinal epithelium (17, 20). The ligand for Fas is expressed by
intraepithelial lymphocytes and lamina propria lymphocytes, and its
expression is increased in the lamina propria of ulcerative colitis
patients (5, 25, 26). Cross-linking of Fas on freshly isolated colonic
crypts leads to accelerated apoptosis of these cells (25). There are no
data, however, about the molecular events associated with ligation of
Fas in colonic epithelial cells.
Previously, we described a model of Fas-mediated signals in intestinal
epithelial cells using the human colon cancer line HT-29 (1). HT-29
cells display a dual response to Fas ligation: cross-linking Fas
stimulates secretion of interleukin (IL)-8 and, in combination with
interferon- In the present study, we examine the effect of Fas on activation of the
mitogen-activated protein kinase (MAPK) family of signal transduction
molecules and the regulation of transcription factors downstream of
MAPK activation in HT-29. We chose to study the MAPK family of signal
transduction molecules because they are central regulators of cellular
responses to growth factors, cytokines, and stress-induced signals
(14). Recent evidence suggests that oligomerization of Fas recruits a
death complex that results in activation of the stress-activated branch
of the MAPK family, c-Jun
NH2-terminal kinase (JNK), as well
as cleavage and activation of caspases (10, 31). Fas-mediated
activation of JNK has been causally linked to apoptosis in T cells (8, 27), 293 cells, HeLa cells (31), and neuroblastoma cells (10). Importantly, Fas-mediated apoptosis of T cells (18) and HeLa cells (28)
can occur through a caspase-dependent, JNK-independent pathway as well.
The present study demonstrates that ligation of Fas activates JNK,
activator protein-1 (AP-1) binding, and AP-1 transcriptional activity
in colonic epithelial cells. IFN- Cell culture. HT-29 cells obtained
from the American Type Culture Collection (Rockville, MD) were
maintained in McCoy's medium with L-glutamine (Mediatech,
Washington, DC), supplemented with 10% FCS. Cells were kept at
subconfluence in a humidified incubator at 37°C with 5%
CO2.
Reagents and antibodies. The
monoclonal antibody (Ab) to Fas, CH-11, was purchased from Kamiya
Biomedical (Thousand Oaks, CA), and subsequently CH-11 was purchased
from Upstate Biotechnology (Lake Placid, NY). The level of endotoxin
contamination for CH-11 is 0.048 EU/µg as determined by
the limulus amebocyte lysate test. IFN- Flow cytometry. Cells were prepared
for flow cytometry by incubating with primary anti-Fas Ab or irrelevant
monoclonal IgG at a 1:1,000 dilution followed by PE-conjugated anti-IgG
in PBS with 2% FCS. Cells were fixed in PBS with 2% paraformaldehyde and were analyzed with a Becton-Dickinson flow cytometer.
Electromobility shift assay. HT-29
cells were grown to confluency in 100-mm plates. IFN- For collection of nuclear protein, cells were rinsed two times with
ice-cold PBS with 0.1% BSA. Cells were removed from the plate with a
cell scraper, transferred to a Microfuge tube, and centrifuged at 3,000 rpm for 5 min at 4°C (12). Cell pellets were resuspended in 900 µl of cold 1× RSB buffer [10 mM Tris (pH 7.4), 10 mM
NaCl, 3 mM MgCl2, 0.5 mM
dithiothreitol (DTT), 2 µM leupeptin, 1 µg/ml aprotinin, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM EGTA]. After
resuspension, 100 µl of 1× RSB containing 5% Nonidet P-40 was
added to each tube and was incubated for 5 min on ice. Lysates were
centrifuged at 5,400 rpm for 5 min, and supernatant was completely
removed. Pellets were washed one time with 1× RSB and centrifuged
at 8,100 rpm for 5 min. Pellets were resuspended in 60 µl of cold
buffer containing 20 mM HEPES (pH 7.4), 0.42 mM NaCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 25% glycerol, 0.01% NaN3, 0.5 mM DTT, 1 mM
PMSF, 20 µM leupeptin, and 10 µg/ml aprotinin and were placed on
ice for 40 min. Samples were spun at 13,000 rpm for 10 min,
supernatants were transferred to a fresh tube, and an equal volume of
buffer with protease inhibitors was added [20 mM HEPES (pH 7.4),
50 mM KCl, 0.2 mM EDTA, 20% glycerol, and 0.01%
NaN3].
Protein concentration was determined using the Coomassie protein assay
reagent (Pierce) as per the manufacturer's directions. AP-1 consensus
oligomer (3.5 pmol; Promega, Madison, WI) was labeled with
[ Plasmids, transfections, and retroviral
infections. The cDNA MEKK-dominant active (MEKK-DA) and
MEKK-dominant negative (MEKK JNK assay. HT-29 cells (2 × 106 cells/sample) were cultured in
1% FCS overnight after IFN- Immunoblots. Nuclear protein was
extracted as described above. Nuclear protein (4 µg) was analyzed by
10% SDS-PAGE, and proteins were transferred to nitrocellulose
membranes (Schleicher & Schuell, Keene, NH). Membranes were blocked in
8% milk in PBS-Tween for 2 h and then were incubated with primary Ab
(c-Jun, JunD, JunB, Fos) at a dilution of 1:1,500 for 1 h. Membranes
were washed three times with PBS-Tween and incubated with horseradish
peroxidase-linked secondary Ab (Amersham) at a 1:5,000 dilution for 45 min. After washing, membranes were reacted with the enhanced
chemiluminescent solutions as per the manufacturer's protocol
(Amersham) and were exposed to film. For ERK Western blots, 10 µg of
total cellular protein were analyzed on 10% SDS-PAGE, and transferred
proteins were blotted with anti-ERK1 and ERK2 Ab (Zymed) at a 1:5,000
dilution. The remainder of the protocol was performed as above. MEKK1
Western blots were performed with anti-MEKK1 polyclonal Ab (Santa Cruz) at 1 µg/ml dilution.
Fas is constitutively expressed by HT-29 cells, and
its expression is increased after IFN-
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(IFN-
). Activation of mitogen-activated protein
kinase pathways was assessed by kinase assay, Western blots, and
promoter-reporter assays. Electromobility shift assays were used to
assess activator protein-1 (AP-1) binding activity. IFN-
increases
expression of Fas on HT-29 cells. Signaling via Fas receptor, as
determined by induction of c-Jun
NH2-terminal kinase (JNK) activity
and transcriptional activation of AP-1, is enhanced in IFN-
-primed
cells. Dominant-interfering mutants of the JNK pathway do not block
Fas-mediated apoptosis. Signaling through Fas results in activation of
JNK and AP-1 binding activity that is increased in the presence of
IFN-
. Inhibition of JNK does not block Fas-mediated apoptosis in
these cells. Fas-Fas ligand interactions in the intestinal mucosa may
lead to complex signal transduction cascades and gene regulation that
culminate in apoptosis, cytokine secretion, or other novel functions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(IFN-
), rapidly undergo apoptosis. Because of the
diverse effects of Fas in the intestinal mucosa and its modulation by
IFN-
, we hypothesized that Fas activates critical signal
transduction pathways involved in apoptosis and proinflammatory
cytokine secretion and that activation of these pathways is modified by
the presence of IFN-
.
priming of these cells leads to an
increase in Fas expression and enhanced signaling through these
pathways. Expression of a dominant negative mutant of MAPK kinase
kinase-1 (MEKK1), the upstream activator of JNK, does not
block Fas-mediated apoptosis. In addition, expression of a
constitutively active MEKK1 mutant leads to increased JNK activation
but does not sensitize cells to Fas-mediated apoptosis. These data
taken together suggest that Fas-mediated apoptosis of intestinal
epithelial cells is JNK independent.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was purchased from R&D
Systems (Minneapolis, MN). For Fas surface staining, biotin-conjugated
anti-human CD95 (Fas/Apo-1), biotin-conjugated anti-human Fas ligand,
and streptavidin-phycoerythrin (PE) were purchased from
Pharmingen. The ApoBrDU apoptosis detection kit was purchased from
Pharmingen and was used as per the manufacturer's directions. For Jun,
Fos, and nuclear factor-
B (NF-
B) Western blots and supershift
assays, all Ab were purchased from Santa Cruz (Santa Cruz, CA).
Anti-extracellular signal-regulated kinase (ERK) 1 and ERK2 Ab were
purchased from Zymed (San Francisco, CA).
was added at a
concentration of 40 ng/ml for 6 h, and cells were washed three times
and kept in fresh medium overnight containing 5% bovine serum albumin,
5 µg/ml transferrin, and 5 µg/ml insulin (Sigma, St. Louis, MO).
Anti-Fas (CH-11; 100 ng/ml) was added for the times indicated.
-32P]ATP using T4
polynucleotide kinase to a specific activity of ~1 × 106
counts · min
1 · µl
1.
Nuclear protein (4 µg) was reacted with 1 µl of labeled oligomer in
5× binding buffer containing 20% glycerol, 5 mM
MgCl2, 2.5 mM EDTA, 25 mM DTT, 250 mM NaCl, 50 mM Tris (pH 7.5), and 0.05 mg/ml
poly(dI-dC) · poly(dI-dC) at room
temperature for 20 min and was analyzed on a 6% acrylamide gel (80:1
ratio of acrylamide to bis-acrylamide). Gels were dried and exposed to
film (Amersham, Arlington Heights, IL). For cold competition, 100-fold
excess of unlabeled oligomer was added. A Helena densitometer
was used to semiquantify nuclear protein binding activity.
K432M; MEKK-DN; cDNA gift of Dr. G. Johnson, National Jewish Center for Immunology and Research, Denver,
CO) were subcloned into the retroviral vector pSR
MSVtkneo (16, 21).
Amphotropic packaging plasmid and pSR
MSVtkneo containing MEKK-DA or
MEKK-DN were used to transfect 293T cells, and supernatant containing
retrovirus was collected. Retroviral vector alone or containing MEKK
mutants was used to infect HT-29 cells, and cells were subsequently
selected in G418 at 500 µg/ml.
incubation as above. Cells were stimulated with 250 ng/ml anti-Fas for indicated times, rinsed with
ice-cold PBS, and lysed directly on the plate [25 mM HEPES (pH
7.4), 50 mM
-glycerophosphate, 1 mM EGTA, 2.5 mM
MgCl2, 1% Triton, 10 mM
p-nitrophenyl phosphate, 1 mM sodium
vanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM
PMSF]. After centrifugation, 100 µg of sample protein were
added to 15 µg of c-Jun-(1
79)-GST fusion protein (kind gift of
Roger Davis; see Ref. 6) bound to glutathione-Sepharose beads
(Pharmacia) and rocked for 1 h at 4°C. Beads were washed three
times in wash buffer (same as lysis buffer but with 50 mM NaCl) and
resuspended in kinase buffer [25 mM HEPES (pH 7.4), 50 mM
-glycerophosphate, 1 mM EGTA, 2.5 mM
MgCl2, and 1 mM DTT] in the presence
of 10 µCi [
-32P]ATP for
30 min. Samples were boiled in 3× sample buffer containing 2-mercaptoethanol and analyzed on 12% SDS-PAGE. Equal loading of
samples was determined by Coomassie staining. Gels were dried and
exposed to film.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
incubation.
We have previously reported that cross-linking of Fas on HT-29 cells
leads to IL-8 secretion and, in the presence of IFN-
, results in
apoptosis (1). An example of the synergism between IFN-
and anti-Fas in causing HT-29 apoptosis can be seen in Fig.
1A.
Although neither cross-linking Fas alone nor IFN-
treatment alone
leads to significant apoptosis compared with untreated cells, the
combination of the two results in apoptosis, as measured by terminal
deoxy-nucleotidyl transferase end labeling (TUNEL) assay
(Fig. 1A). In spite of the
requirement for IFN-
with respect to Fas-mediated apoptosis of
HT-29, cross-linking of Fas alone results in IL-8 secretion, and the
amount of IL-8 secreted does not change with IFN-
preexposure, even
after correction for cell loss (1). This model system, therefore,
permits us to study function-specific Fas signaling.
View larger version (38K):
[in a new window]
Fig. 1.
A: TUNEL staining of HT-29 cells after
cross-linking Fas in the presence or absence of interferon (IFN)- .
TUNEL staining of HT-29 cells exposed to conditions as indicated:
unstimulated cells (No Treatment), 40 ng/ml IFN-
for 6 h on day
before collection (IFN-
), 250 ng/ml anti-Fas stimulation for 4 h
(anti-Fas), and IFN-
preincubation after anti-Fas stimulation for 4 h (IFN-
+ anti-Fas). Percent of cells staining positive for
apoptosis is indicated. This is 1 representative experiment of 3. B: IFN-
increases expression of Fas
on HT-29 cells. HT-29 and Caco-2 cells were stained with irrelevant
antibody (Ab), anti-Fas Ab, or anti-Fas Ab after 40 ng/ml IFN-
for 6 h on day before staining. Jurkat cells were stained with irrelevant Ab
or anti-Fas Ab but were not pretreated with IFN-
.
One potential role for IFN- in this system is to upregulate the
expression of Fas. Upregulation of Fas may lead to a critical mass of
Fas receptors required for apoptosis. To determine the level of Fas
expression on the surface of HT-29, flow cytometry was performed with a
Fas-specific monoclonal Ab. Fas receptors are present on resting HT-29
cells (Fig. 1B,
left). After a 6-h incubation with
IFN-
, the level of Fas expression is increased and approaches that
of Jurkat cells, which are sensitive to Fas-mediated killing. Caco-2
cells, which are not sensitive to Fas-mediated apoptosis, do not
express Fas receptors at baseline or after incubation with IFN-
.
These data suggest a correlation between the level of Fas expression
and the ability of cells to undergo Fas-mediated apoptosis in colonic
epithelial cells.
Ligation
of Fas in HT-29 results in JNK activation, which is enhanced by IFN-
priming. The MAPK signal transduction family is
involved in cellular responses to diverse extracellular stimuli. Ligation of Fas has been shown to cause activation of the JNK branch of
the MAPK family and has been causally linked to apoptosis in different
cell types (10, 31). Based on these observations, we hypothesized that
Fas activates the JNK branch of the MAPK pathway in HT-29 cells.
Because our HT-29 model requires IFN-
for Fas-mediated apoptosis but
not for IL-8 secretion, we further hypothesized that the magnitude of
JNK activation in response to Fas would be altered by the presence of
IFN-
. Ligation of Fas leads to a fourfold activation of JNK at 60 min compared with control or IFN-
-treated cells (Fig.
2A). Pretreatment
with IFN-
followed by cross-linking of Fas leads to a rapid
induction of JNK activity at 15 min that is ~10-fold higher than
control cells and is sustained at 60 min.
|
The specificity of JNK activation by Fas ligation was demonstrated by
parallel study of ERK activation. There was no significant activation
of ERK based on Western blot analysis of phosphorylated ERK in response
to ligation of Fas in the presence or absence of IFN- (Fig.
2B). The demonstration of increased
JNK activity within 15 min after cross-linking of Fas is
consistent with a temporal relationship, albeit not a causal
relationship, between JNK activation and apoptosis.
Activation of the JNK pathway in HT-29 cells results
in increased AP-1 nuclear protein binding.
Cross-linking Fas leads to activation of JNK, resulting in the
phosphorylation of the AP-1 transcription factor c-Jun (6). To further
understand the molecular pathways involved in Fas signal transduction
in colonic epithelial cells, we studied the ability of Fas to induce
nuclear protein binding to an AP-1 consensus sequence. We hypothesized
that IFN- and cross-linking Fas would result in increased AP-1
binding with kinetics similar to JNK activation. To test this
hypothesis, nuclear extracts from cells treated with anti-Fas alone or
after IFN-
preincubation were tested for their ability to bind an
AP-1 consensus sequence (Fig. 3). Compared with
untreated cells, nuclear protein from cells stimulated with anti-Fas
had gradually increasing AP-1 activity that peaked at 60 min (Fig. 3).
In contrast, nuclear extracts from cells treated with anti-Fas in the
presence of IFN-
had much greater AP-1 binding compared with
untreated cells. In the presence of IFN-
, peak AP-1 binding occurred
15 min after ligation of Fas. These data are consistent with the JNK
data with regard to the temporal relationship of AP-1 binding and JNK
activation. An excess of unlabeled AP-1 oligomer eliminates the
DNA-protein complexes, but unlabeled irrelevant oligomers such as
NF-
B and Sp1 do not compete with the DNA-protein
complexes, verifying specificity of nuclear protein binding to AP-1
(data not shown). Western blots of nuclear protein showed that
the absolute amounts of c-Jun, JunD, or Fos nuclear protein do not
change with IFN-
preincubation or in response to cross-linking Fas
(data not shown). Thus ligation of Fas in the presence of IFN-
causes an early increase in AP-1 binding activity followed by
downregulation coincident with the onset of apoptosis.
|
Expression of MEKK1-DN does not inhibit
apoptosis. Because the apoptotic combination of
anti-Fas and IFN- induces JNK activation, we asked whether JNK
activation was required for HT-29 apoptosis. To address this question,
HT-29 cells were stably infected with retrovirus containing a dominant
negative mutant of MEKK1, MEKK-DN. This construct has been shown to
inhibit downstream JNK activation in response to several stimuli (3,
9). Expression of the truncated MEKK-DN protein was demonstrated by
Western blot analysis (Fig.
4A,
bottom). HT-29 expressing MEKK-DN
have a 50% reduction in JNK activity after IFN-
and cross-linking
of Fas compared with cells infected with vector control (Fig.
4B). HT-29 cells expressing MEKK-DN
or the vector control do not show any differences in Fas-mediated
apoptosis (Fig. 4C). These results
suggest that a non-JNK-dependent pathway is present in HT-29 cells that
signals apoptosis in cells exposed to anti-Fas and IFN-
or that
partial inhibition of JNK is insufficient to block Fas-mediated
apoptosis.
|
Although JNK activation does not seem to be necessary for Fas-mediated
apoptosis, we asked whether JNK activation was sufficient to induce
apoptosis in HT-29 cells or bypass the requirement for IFN-
pretreatment. HT-29 cells were derived that expressed a constitutively
active form of MEKK1, MEKK-DA. This mutant form of MEKK1 induces
apoptosis in fibroblasts and T cells (8, 13). HT-29 cells expressed
MEKK-DA protein as demonstrated by Western blot analysis and had
fourfold JNK activation at baseline compared with vector control cells
(Fig. 4A). Activation of JNK was not sufficient to induce apoptosis and did not sensitize cells to Fas-mediated apoptosis as demonstrated by TUNEL staining (Fig. 4C). These data suggest that JNK
activity is neither necessary nor sufficient to induce apoptosis in
HT-29 cells.
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DISCUSSION |
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The important role of FasL interactions in the intestinal mucosa is
beginning to emerge. Known functions mediated by Fas in colonic
epithelial cells include proinflammatory cytokine secretion and
apoptosis, but the full range of Fas effects in the intestinal mucosa
is not known (25). This study examines the subcellular signaling
pathways triggered by cross-linking Fas on colonic epithelial cells. We
chose HT-29 as a model system because cross-linking Fas stimulates
secretion of IL-8 but, in the presence of IFN-, cells rapidly
undergo apoptosis (1). This model system, therefore, permits us to
study function-specific signaling in response to ligation of Fas.
In this paper, we have demonstrated that cross-linking Fas in colonic
epithelial cells stimulates the stress-activated branch of the MAPK
pathway, resulting in JNK activation, phosphorylation of c-Jun, and
increased AP-1 binding activity. The parallel ERK branch of the MAPK
pathway is not activated by ligation of Fas. In other systems,
activation of JNK and downregulation of ERK are sufficient to cause
apoptosis (30). Importantly, however, inhibition of the
MEKK1JNK pathway in HT-29 does not inhibit apoptosis. These
data are consistent with the existence of multiple pathways resulting
in Fas-mediated apoptosis of colonic epithelial cells. Recent work by
Yang et al. (31) demonstrates that Fas can activate parallel pathways
resulting in JNK activation or caspase activation and that both can
result in apoptosis (31). We have also expressed a constitutively
active form of MEKK1 that induces apoptosis in diverse cell types (8,
13). HT-29 cells expressing dominant-active MEKK1 did not have a higher
spontaneous rate of apoptosis and were not sensitized to Fas-mediated
apoptosis. These data suggest that JNK is not causally involved
in apoptosis in HT-29 cells.
Our results suggest a model in which Fas-expressing intestinal
epithelial cells are not competent to undergo apoptosis without costimulation by IFN-. At least one effect of IFN-
in this system is to increase the level of surface expression of Fas. Although the
level of Fas expression does not correlate directly with the ability of
cells to undergo apoptosis (24, 29), in HT-29 cells and Caco-2 cells
Fas expression parallels sensitivity to apoptosis. Our findings of
increased second signaling in IFN-
-treated cells can be explained by
an increase in Fas expression. In addition, IFN-
may be exerting its
effect on any of the components of the Fas death machinery. Binding of
FasL leads to aggregation of Fas and recruitment of a death complex
that results in JNK activation and sequential cleavage of caspases (2,
4, 7, 22). Cells deficient in the IFN-
-regulated STAT-1
transcription factor are defective in TNF-
-mediated apoptosis as a
result of a deficiency in caspases (15). It is plausible, therefore,
that the individual components of the Fas death machinery such as
FADD or caspases exist in rate-limiting quantities and are
increased by the presence of IFN-
. An understanding of the different
pathways utilized by intestinal epithelial cells to signal cytokine
secretion and apoptosis may eventually permit selective targeting of
these pathways as a treatment for chronic inflammation or cancer (23).
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ACKNOWLEDGEMENTS |
---|
This work was supported by a Crohn's and Colitis Foundation of America Career Development Award (M. T. Abreu-Martin) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43211 (S. R. Targan).
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FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. T. Abreu-Martin, Inflammatory Bowel Disease Center, 8700 Beverly Blvd., D4063, Los Angeles, CA 90048 (E-mail: abreu{at}csmc.edu).
Received 6 April 1998; accepted in final form 23 November 1998.
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