Departments of 1 Pharmacology and 2 Anatomy, Institute of Biomedicine, FIN-00014 University of Helsinki, Finland
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ABSTRACT |
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We investigated the role of
IFN- and MAPKs on the expression and activity of the transcription
factor CCAAT/enhancer-binding protein-beta (C/EBP
) in the T84 colon
epithelial cell line. IFN-
induced the expression and activity of
C/EBP
and subsequently increased the secretion of IL-6 from these
cells. Treatment with the p38 inhibitor SB-203580, the MEK1 and MEK2
inhibitor U-0126, or the translational inhibitor cycloheximide
inhibited the induction of C/EBP
and IL-6 by IFN-
, whereas the
MEK1 inhibitor PD-98059 or the tyrosine kinase inhibitor genistein had
no effect. These results suggest a role for MEK2 and p38 in
IFN-
-mediated signal transduction and induction of C/EBP
expression and activity associated with interleukin-6 (IL-6) secretion
in colon epithelial cells.
CCAAT-enhancer binding protein-; colon epithelial cells,
interferon-
; interleukin-6; mitogen-activated protein kinases
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INTRODUCTION |
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PERSISTENT AND
UNCONTROLLED ACTIVATION of the gut mucosal immune system,
together with a dysfunctional epithelial cell layer, are major
pathological features of inflammatory bowel disease (IBD)
(43). In IBD, the mucosal cells produce increased amounts of proinflammatory cytokines, which mediate cell-to-cell crosstalk and
aggravate the inflammatory process (38). In active
inflammation, IFN- is produced in excess by cells of the immune
system in the lamina propria. In Crohn's disease, IFN-
is produced
especially by T helper 1 (Th1) cells (29), which are
considered important in the pathogenesis of this disease
(42). IFN-
is a pleiotropic cytokine, which enhances
immune functions (5), promotes activation of the
epithelium (8), increases epithelial permeability
(1), and induces the phenotype switch to
antigen-presenting cells (APC). This phenotype switch is linked to the
expression of ICAM-1 and major histocompatibility complex (MHC) class
II molecules on epithelial cells (31, 44, 45).
The IFN- signal is mediated via JAK1 and JAK2, which activate
dimerization of the transcription factor STAT1 (22). It
has been shown that IFN-
may induce gene expression also,
independently of STAT1 (34). Moreover, IFN-
-mediated
transcriptional activation can proceed through the transcription factor
CCAAT/enhancer-binding protein-
(C/EBP
, also know as NF-IL-6)
(40). IFN-
was recently shown to increase the
expression and activity of C/EBP
in the RAW264.7 murine macrophage
cell line through activation of MAPKK (MEK1) and subsequent
phosphorylation and activation of ERK (15). We further
suggested that MEK1 activation in response to IFN-
in these cells is
dependent of MEKK1 (39). Beneficial effect for MAPK
inhibition has been suggested in different experimental models of
inflammation (19, 26, 46); however, the role and regulation of C/EBP
has been less extensively studied.
C/EBP regulates the expression of genes related to the acute phase
reaction, inflammation, and cell differentiation (35). It
induces the production of IL-6 (2) but, depending on the experimental setup, may also mediate the effects of IL-6
(6). The production of IL-6 is induced in response to
inflammatory stimuli (30) and contributes to the
pathogenesis of chronic intestinal inflammation (36, 49).
In IBD, elevated production of IL-6 correlates with disease activity
(14, 33) and relapse frequency (47).
In this study, we investigated the effect of IFN- on activation of
C/EBP
and on secretion of IL-6 in colon epithelial cells. We also
studied the role of the inflammation-associated MAPK-pathways (25) in IFN-
signaling.
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MATERIALS AND METHODS |
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Reagents.
IFN- was purchased from Bender MedSystems (Vienna, Austria).
SB-203580, PD-98059, and U-0126 were obtained from Tocris Cookson (Bristol, UK), and cycloheximide and genistein were from Sigma Chemicals (St. Louis, MO). C/EBP
, p38, ERK1, phosphorylated-ERK1/2 (p-ERK1/2), intercellular adhesion molecule-1 (ICAM-1), and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibodies were from Santa
Cruz Biotechnology (Santa Cruz, CA). Phosphorylated-p38 (p-p38) was
from Cell Signaling Technology (Beverly, MA), HRP-conjugated anti-mouse
IgG2a was from Zymed Laboratories (San Francisco, CA), and
Alexa Fluor 488 anti-mouse IgG was from Molecular probes (Eugene, OR).
EMSA supplies were purchased from Pierce (Rockford, IL). All other
materials were from Sigma Chemicals unless otherwise specified.
Cell culture.
The human intestinal epithelial cell line T84 (CCL-248, American Type
Culture Collection, Manassas, VA) was cultured in Dulbecco's modified
Eagle's medium (DMEM):F12 medium (GIBCO BRL, Grand Island, NY)
containing antibiotics (penicillin G 100 U/ml, amphotericin B 250 ng/ml, and streptomycin 100 µg/ml) (GIBCO) and 5% fetal calf serum
(Biological Industries, Kibbutz Beit Haemek, Israel). The cells tested
negative against mycoplasma contamination (Roche Diagnostics, Mannheim,
Germany). They were grown as subconfluent monolayers and were
serum-starved 24 h before the experiments. The effect of IFN-
(500 ng/ml) was studied at 0, 4, 8, 12, and 24 h. Selection of
this relatively high dose of IFN-
was based on preliminary
experimentation at our laboratory (Kankuri E, personal communication)
and on previous in vivo results (7, 12). For studying the
drug effects, the cells were preincubated for 90 min with drugs before
stimulation with IFN-
. The effects of a tyrosine kinase inhibitor
(genistein, 100 µM), a p38 inhibitor (SB-203580, 30 µM), a MEK1
inhibitor (PD-98059, 30 µM), a MEK1 and MEK2 inhibitor (U-0126, 30 µM), or a translation inhibitor (cycloheximide, 20 nM) were studied
at 24 h after treatment with IFN-
. After each experiment, the
culture medium was collected and the cells were harvested by scraping.
Samples were stored at
70°C until analyzed.
Immunoblotting of C/EBP, p38, p-p38, ERK, p-ERK1/2, and
ICAM-1.
Expression of C/EBP
, p38, p-p38, ERK, p-ERK1/2, and ICAM-1 was
determined by immunoblotting. Cells were homogenized by freezing and
thawing three times in boiling lysis buffer (1% SDS, 1.0 mM Na3VO4 in PBS, pH 7.4), followed by
brief sonication. The protein content of supernatants was measured
according to the method of Lowry et. al. (28).
Immunoblotting was carried out as previously described
(13). Equal protein loading and transfer to the membranes were confirmed using Ponceau S dye.
Cytokine ELISA. The medium contents of IL-6 were measured using commercial ELISA kits and reagents (CLB, Amsterdam, The Netherlands). The sensitivity of the assay was 0.2 pg/ml, and it had no crossreactivity with other cytokines or chemokines.
EMSA.
T84 cell nuclear extracts were prepared using NU-PER nuclear protein
extraction kit (Pierce). The 3'-ends of oligonucleotides containing the
binding sequence of C/EBP (5'-TGC AGA TTG CGC AAT CTG CA-3' and its
consensus sequence) were labeled using a biotin 3'-end labeling kit
(Pierce). In the binding reaction, nuclear proteins (8 µg) were
allowed to bind with labeled oligonucleotides in binding buffer.
Mutated oligonucleotides (5'- TGC AGA GAC TAG TCT CTG CA-3'
and its consensus sequence) were used to confirm binding specificity.
The same C/EBP antibody that was used for the immunoblots was also
used for supershift assays. Bound oligonucleotides were separated from
free oligonucleotides in a 5% PAGE containing 0.5% Tris-borate EDTA
buffer (Bio-Rad Laboratories, Hercules, CA).
Immunocytochemistry.
Localization of C/EBP was determined in T84 cells cultured on
microscope slides (Nalge Nunc International, Naperville, IL) using
confocal microscope. The cells were stimulated with IFN-
or vehicle
for 24 h, subsequently fixed in
20°C methanol, washed with
PBS, and incubated with the C/EBP
(1:200) primary antibody for
1 h. They were then washed three times with PBS and incubated with
Alexa Fluor 488 anti-mouse IgG for 30 min. Before mounting, the cells
were incubated with the nuclear dye propidium iodide and washed in water.
Statistical analysis. Results are presented as means ± SE. Statistical analysis was carried out using ANOVA, followed by Bonferroni multiple comparisons test. Differences at P values of <0.05 were considered significant.
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RESULTS |
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In T84 cells, the expression of the transcription factor C/EBP
increased after 8 h of stimulation with IFN-
. At 24 h, it reached a 3.3-fold induction compared with unstimulated cells (Fig.
1A). Induction of C/EBP
expression by IFN-
was associated with increased C/EBP
DNA-binding activity, which peaked at 4 h after stimulation with
IFN-
and remained slightly elevated compared with controls, as shown
by EMSA (Fig. 1B). Demonstration of nuclear localization of
IFN-
-induced C/EBP
expression (Fig. 2) by immunocytochemistry further
supported this transcriptional activation. Although the activation of
C/EBP
diminished after 4 h, the protein expression increased
and the induced protein localized to the nucleus.
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In response to IFN-, the T84 cells produced increased amounts of
IL-6. Its secretion was significantly increased at 12-24 h after
stimulation (1.9-fold at 24 h) (Fig. 1C). Induction of C/EBP
preceded the increased IL-6 secretion, thereby suggesting activation of IL-6 production by C/EBP
, or at least a sequential correlation between these responses.
When stimulated with IFN-, the T84 colon epithelial cell line has
been shown to undergo a switch into an immunologically active
phenotype. As a marker of this event in our setup, we used ICAM-1
(20). Its expression was undetectable in unstimulated cells. ICAM-1 was first detected at 8 h after stimulation with IFN-
, and its expression increased constantly throughout the 24-h
experiment, as shown in Fig. 1D.
To study the role of MAPK pathways and tyrosine kinases in the
induction of C/EBP and IL-6 by IFN-
, the T84 cells were
treated with U-0126 (MEK1 and MEK2 inhibitor), PD-98059
(MEK1 inhibitor), SB-203580 (p38 inhibitor), or genistein (tyrosine
kinase inhibitor). The role of de novo protein synthesis was assessed
by treatment with the translational inhibitor, cycloheximide.
Inhibition of MEK1 and MEK2 by U-0126 and inhibition of p38 by
SB-203580 decreased IFN--stimulated C/EBP
expression by 46 and
34%, respectively. The MEK1 inhibitor PD-98059 and the tyrosine kinase
inhibitor genistein had no effect. Cycloheximide inhibited the
IFN-
-induced C/EBP
expression by 63% (Fig.
3A). These results suggest
that MEK2 and p38, as well as novel protein synthesis, regulate the
induction of C/EBP
expression in response to IFN-
in T84 colon
epithelial cells.
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Because phosphorylation has been shown to affect the transcriptional
activity of C/EBP (35), the effects of kinase
inhibitors on C/EBP
activity were assayed using EMSA. The
IFN-
-induced DNA binding activity of C/EBP
was decreased by
U-0126 and SB-203580, whereas PD-98059 and genistein had no effect.
Also, cycloheximide inhibited the IFN-
-stimulated C/EBP
activity
(Fig. 1B).
Increased secretion of IL-6 by IFN- was inhibited by U-0126 (by
43%) and by SB-203580 (by 46%), whereas genistein and PD-98059 had no
effect. Cycloheximide inhibited the IFN-
-stimulated IL-6 secretion
by 36% (Fig. 1C). Thus the effects of drugs on IL-6 secretion were similar to their effects on the expression and activity
of C/EBP
.
ICAM-1 expression was inhibited by the combined MEK1 and MEK2 inhibitor U-0126 (by 34%) and by cycloheximide (by 57%), whereas genistein, SB-203580, and PD-98059 failed to inhibit it (Fig. 1D).
The present results suggest that IFN- induces the expression of
C/EBP
through the MEK-ERK and p38 pathways. To confirm the effects
of the kinase inhibitors on ERK1, ERK2, and p38 phosphorylation, expression of p-ERKs and p-p38 was studied using immunoblotting. IFN-
stimulation produced a 2.5-fold increase in the phosphorylation of ERK1 and ERK2 (the substrates for MEK1 and MEK2), supporting the
role of MEK-ERK pathway in IFN-
signaling (Fig. 1E).
Phosphorylation of ERK1 and ERK2 was inhibited by U-0126 (by 72%),
whereas SB-203580 and PD-98059 had no effect. This result confirms the
inhibitory action of U-0126 on MEK in our experimental setup.
Interestingly, genistein and cycloheximide enhanced the
IFN-
-stimulated ERK phosphorylation by 2-fold and 3.8-fold,
respectively (Fig. 1E). Similar effects of genistein and
cycloheximide on ERK phosphorylation have also been reported by others
(21, 27). Inhibition of tyrosine kinases by genistein may
have unspecific effects on the phosphorylation of inactive ERK, because
it did not affect C/EBP
expression and activity. We found a small
constitutive phosphorylation of p38, which was not regulated by IFN-
stimulation or any of the used kinase inhibitors (data not shown),
which may contribute to the induction of C/EBP
.
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DISCUSSION |
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In the present study, we examined the effect of IFN- on the
activation of C/EBP
and on the production of IL-6 in the T84 colon
epithelial cell line. Treatment with IFN-
increased the expression
and DNA-binding activity of C/EBP
and also the production of IL-6.
Both of these IFN-
-induced events were inhibited by treatment with
the MEK1 and MEK2 inhibitor U-0126, the p38 inhibitor SB-203580, and
the translational inhibitor cycloheximide but were unaffected by
treatment with the MEK1 inhibitor PD-98059 or the tyrosine kinase
inhibitor genistein. These results suggest a role for MEK2 and p38
protein kinases, as well as novel protein synthesis in activation of
C/EBP
in T84 epithelial cells. Our study is apparently the first to
report the induction of C/EBP
and concomitant production of IL-6 in
response to IFN-
in colon epithelial cells.
The functions of intestinal epithelial cells, which have an essential
role in both physiological and pathophysiological processes in the gut,
are regulated and modified by signals derived from other cells in the
mucosa. In gut inflammation, the mucosal cells produce increased
amounts of proinflammatory cytokines such as IFN- (29)
and IL-6 (14). Activated lymphocytes, especially Th1
cells, are active producers of IFN-
(9), whereas a
major part of IL-6 production under inflammatory conditions is derived from epithelial cells (24). IL-6 induces the proliferation
and differentiation of cytotoxic T cells and antibody production from B
cells at the site of inflammation (30). It also inhibits
Th1 cell apoptosis (4), thereby affecting
polarization of T helper cells to favor a type 1 cytokine response, as
seen in Crohn's disease (42). Inhibition of IL-6
signaling has been shown to inhibit disease progression in experimental
models of colitis and to induce apoptosis in T cells of IBD
patients (4). However, the regulation of IL-6 production
in colon epithelial cells is not completely understood.
The transcription factor C/EBP is a member of the
C/EBP-transcription factor family that consists of six (
-
)
subspecies. It is induced in various cell types in response to
different inflammatory stimuli (35) and has recently been
shown to increase the production of IL-6 in intestinal epithelial cells
(11, 17, 37). The activity of C/EBP
is regulated
through phosphorylation and novel protein synthesis (35).
In the present study, we investigated the roles of MAPKs, tyrosine
phosphorylation, and protein synthesis in IFN-
-stimulated activation
of C/EBP
and production of IL-6 in colon epithelial cells. Our
results suggest that both the activation of C/EBP
and the increased
production of IL-6 by IFN-
are MEK dependent and emphasize on the
role of MEK2. It was recently shown that IFN-
induces C/EBP
expression and activity through the MEK-ERK pathway in RAW264.7 murine
macrophages and mouse fibroblasts (15). The present study
was carried out to investigate the effect of the inflammatory
"response modifying" cytokine, IFN-
, on the activation of
transcription factor C/EBP
in T84 colon epithelial cells. IFN-
has been shown to enhance the expression of many inflammatory events in
these cells when stimulated in conjunction with other proinflammatory
cytokines, e.g., IL-1 and TNF-
(23, 31). The mechanisms
of this synergy are incompletely understood, but our present data
suggest that the IFN-
-induced activation of C/EBP
may have a role
in this response. Further studies are warranted to elucidate this effect.
In the present study, the induction of C/EBP and subsequent
production of IL-6, in response to IFN-
, also showed p38 dependency (as suggested by the effect of SB-203580). This finding suggests a
similar signal transduction pathway, as shown in earlier reports, by
inductors other than IFN-
(10, 41).
Activation of C/EBP and production of IL-6 were independent of
tyrosine phosphorylation, suggesting JAK1 and JAK2-unrelated signal
transduction from IFN-
receptor. This result is supported by similar
findings in JAK1-deficient fibroblasts, which were able to activate
C/EBP
in response to IFN-
(15), and in murine macrophages, in which tyrosine kinase inhibitors (genistein and herbimycin) were ineffective in preventing
-activated
transcriptional element (GATE)-mediated transcription
(48), which has been shown to be C/EBP
dependent
(40). Furthermore, in agreement with the GATE-mediated
transcription in response to IFN-
(48), our results
also suggest dependency of IFN-
-induced C/EBP
activity on novel
protein synthesis.
IL-6 was co-induced with ICAM-1 molecule, suggesting that in epithelial
cells expression of these molecules is linked to the phenotype switch
induced by IFN-. Increased expression of ICAM-1 in intestinal
epithelial cells has been shown in several studies (16, 20,
32); however, the roles of MEK1 and MEK2 on IFN-
-induced ICAM-1 expression have not been reported earlier. Our results suggest
that induction of ICAM-1 in response to IFN-
is MEK2 dependent, but
independent of MEK1 and tyrosine phosphorylation. This result clarifies
the earlier observations that induction of ICAM-1 in response to
IFN-
is not prevented by tyrosine kinase inhibitors genistein and
herbimycin A (18).
The inhibition of p38 activity with SB-203580 upregulated the
IFN--induced ICAM-1 expression. A similar effect was noticed by
induction with TNF-
(3). These authors hypothesized
that the induced p38 activity may act as a negative regulator of
cytokine induced ICAM-1 expression. In our study, p38 phosphorylation
was not enhanced; however, inhibition of low basal level of p38
activity by SB-203580 may have contributed to upregulation of ICAM-1.
The exact mechanisms remain to be clarified.
C/EBP activity may enhance the inflammatory process by increasing
the secretion of cytokines, such as IL-6. Because epithelial C/EBP
was associated with increased ICAM-1 expression, it may also regulate
recruitment of neutrophils in mucosal inflammation. In conclusion, our
results suggest that IFN-
-stimulated expression and activity of
C/EBP
, and associated production of IL-6, are mediated via induction
of MEK2. The basal activity of p38 may modulate this effect in T84
colon epithelial cells.
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ACKNOWLEDGEMENTS |
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We thank Lahja Eurajoki for expert technical assistance.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Kankuri, Institute of Biomedicine, Pharmacology, BIOMEDICUM Helsinki, PO BOX 63, FIN-00014 Univ. of Helsinki, Finland (E-mail: esko.kankuri{at}helsinki.fi).
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.
First published December 27, 2002;10.1152/ajpcell.00293.2002
Received 24 June 2002; accepted in final form 18 December 2002.
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