Download - Interferon-gamma induces C/EBP beta expression and activity through MEK/ERK and p38 in T84 colon epithelial cells

Manuscript ID: #C-00293-2002.R1

INTERFERON-γ INDUCES C/EBPβ EXPRESSION AND ACTIVITY THROUGH

MEK/ERK AND p38 IN T84 COLON EPITHELIAL CELLS

Pertteli Salmenperä1, Sammy Hämäläinen1, Mika Hukkanen2 and Esko Kankuri1*

1 Institute of Biomedicine, Pharmacology, University of Helsinki, Finland

2 Institute of Biomedicine, Anatomy, University of Helsinki, Finland

Running head: INDUCTION OF C/EBPβ BY IFN-γ IN T84 CELLS

* Author for correspondence: Esko Kankuri

Institute of Biomedicine, Pharmacology

BIOMEDICUM Helsinki

PO BOX 63

FIN-00014 University of Helsinki

Finland

Tel. +358-9-191 25336

Fax. +358-9-191 25364

E-mail [email protected]

KEYWORDS: CCAAT-enhancer binding protein-beta, colon epithelial cells,

interferon-gamma, interleukin-6, mitogen-activated protein kinases

Copyright (c) 2002 by the American Physiological Society.

Articles in PresS. Am J Physiol Cell Physiol (December 27, 2002). 10.1152/ajpcell.00293.2002


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ABSTRACT

We investigated the role of IFN-γ and MAPKs on the expression and activity of the

transcription factor CCAAT/enhancer-binding protein-beta (C/EBPβ) in the T84 colon

epithelial cell line. IFN-γ induced the expression and activity of C/EBPβ, and

subsequently increased the secretion of IL-6 from these cells. Treatment with the p38

inhibitor SB203580, the MEK1 and MEK2 inhibitor U0126, or the translational inhibitor

cycloheximide inhibited the induction of C/EBPβ and IL-6 by IFN-γ, while the MEK1

inhibitor PD98059 or the tyrosine kinase inhibitor genistein had no effect. These results

suggest a role for MEK2 and p38 in IFN-γ mediated signal transduction, and induction

of C/EBPβ expression and activity associated with IL-6 secretion in colon epithelial

cells.


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INTRODUCTION

Persistent and uncontrolled activation of the gut mucosal immune system together with

a dysfunctional epithelial cell layer are major pathologic features of inflammatory bowel

diseases (IBD) (43). In IBD the mucosal cells produce increased amounts of pro-

inflammatory cytokines, which mediate cell-to-cell crosstalk and aggravate the

inflammatory process (38). In active inflammation, IFN-γ is produced in excess by cells

of the immune system in the lamina propria. In Crohn’s disease IFN-γ is produced

especially by T helper 1 (Th1) cells (29), which are considered important in the

pathogenesis of this disease (42). IFN-γ 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-γ signal is mediated via JAK1 and JAK2, which activate dimerisation of the

transcription factor STAT1 (22). It has been shown that IFN-γ may induce gene

expression also independently of STAT1 (34). Moreover, IFN-γ mediated transcriptional

activation can proceed through the transcription factor CCAAT/enhancer-binding

protein-beta (C/EBPβ, also know as NF-IL6) (40). IFN-γ was recently shown to

increase the expression and activity of C/EBPβ in the RAW264.7 murine macrophage

cell line through activation of MAPKK (MEK1), and subsequent phosphorylation and

activation of ERK (15). The authors further suggested that MEK1 activation in response


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to IFN-γ 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/EBPß has been less extensively studied.

C/EBPβ 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 set-up 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-γ on activation of C/EBPβ and on

secretion of IL-6 in colon epithelial cells. We also studied the role of the inflammation-

associated MAPK-pathways (25) in IFN-γ signaling.


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MATERIALS AND METHODS

Reagents. IFN-γ was purchased from Bender MedSystems (Vienna, Austria).

SB203580, PD98059 and U0126 were obtained from Tocris Cookson (Bristol, UK) and

cycloheximide and genistein from Sigma Chemicals (St. Louis, MO). C/EBPβ, p38,

ERK1, phosphorylated-ERK1/2 (p-ERK1/2), ICAM-1 and horseradish peroxidase

(HRP)-conjugated anti-rabbit IgG antibodies were from Santa Cruz Biotechnology

(Santa Cruz, CA), phosphorylated-p38 (p-p38) from Cell Signaling Technology

(Beverly, MA), HRP-conjugated anti-mouse IgG2a from Zymed Laboratories (San

Francisco, CA), and Alexa Fluor® 488 anti-mouse IgG 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 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 BRL) and 5 % foetal calf serum (Biological

Industries, Kibbutz Beit Haemek, Israel). The cells were tested negative against

mycoplasma contamination (Roche Diagnostics, Mannheim, Germany). They were

grown as sub-confluent monolayers and were serum-starved 24 hours prior to the

experiments. The effect of IFN-γ (500 ng/ml) was studied at 0, 4, 8, 12 and 24 hours.

Selection of this relatively high dose of IFN-γ was based on preliminary

experimentation at our laboratory (Kankuri E, personal communication), and on


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previous in vivo results (7, 12). For studying the drug effects, the cells were

preincubated for 90 min with drugs prior to stimulation with IFN-γ. The effects of a

tyrosine kinase inhibitor (genistein, 100 µM), a p38 inhibitor (SB203580, 30 µM), a

MEK1 inhibitor (PD98059, 30 µM), a MEK1 and MEK2 inhibitor (U0126, 30 µM) or a

translation inhibitor (cycloheximide, 20 nM) were studied at 24 hours following

treatment with IFN-γ. After each experiment the culture medium was collected, and the

cells were harvested by scraping. Samples were stored at –70 oC until analyzed.

Immunoblotting of C/EBPβ, p38, p-p38, ERK, p-ERK1/2 and ICAM-1. Expression of

C/EBPβ, p38, p-p38, ERK, p-ERK1/2 and ICAM-1 were determined by immunoblotting.

Cells were homogenized by freezing and thawing three times in boiling lysis buffer (1

% SDS, 1.0 mM Na3VO4, 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 was 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 chemoknes.

EMSA. T84 cell nuclear extracts were prepared using NU-PER nuclear protein

extraction kit (Pierce). The 3’-ends of oligonucleotides containing the binding sequence


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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 let 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/EBPβ antibody as

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/EBPβ was determined in T84 cells cultured on

microscope slides (Nalge Nunc International, Naperville, IL) using confocal

microscope. The cells were stimulated with IFN-γ or vehicle for 24 hours, were

subsequently fixed in –20 °C methanol, washed with PBS, and incubated with the

C/EBPβ (1:200) primary antibody for 1 hour. They were then washed three times with

PBS and incubated with Alexa Fluor® 488 anti-mouse IgG for 30 minutes. Before

mounting, the cells were incubated with the nuclear dye propidium iodide, and washed

in water.

Statistical analysis. Results are presented as mean ±SEM. 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

In T84 cells, the expression of the transcription factor C/EBPβ increased after 8 hours

of stimulation with IFN-γ. At 24 hours it reached a 3.3-fold induction as compared to

unstimulated cells (fig. 1A). Induction of C/EBPβ expression by IFN-γ was associated

with increased C/EBPß DNA-binding activity, which peaked at 4 hours after stimulation

with IFN-γ and remained slightly elevated as compared to controls, as shown by EMSA

(fig. 1B). Demostration of nuclear localization of IFN-γ -induced C/EBPβ expression

(fig. 2) by immunocytochemistry further supported this transcriptional activation.

Although the activation of C/EBPß diminished after four hours, the protein expression

increased and the induced protein localized to the nucleus.

In response to IFN-γ, the T84 cells produced increased amounts of IL-6. Its secretion

was significantly increased at 12-24 hours after stimulation (1.9-fold at 24 h) (fig 1C).

Induction of C/EBPβ preceded the increased IL-6 secretion, thereby suggesting

activation of IL-6 production by C/EBPβ, or at least a sequential correlation between

these responses.

When stimulated with IFN-γ, 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 hours after stimulation with IFN-γ, and its


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expression increased constantly throughout the 24-hour experiment as shown in figure

1D.

In order to study the role of MAPK pathways and tyrosine kinases in the induction of

C/EBPβ and IL-6 by IFN-γ, the T84 cells were treated with U0126 (MEK1 and MEK2

inhibitor), PD98059 (MEK1 inhibitor), SB203580 (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 U0126 and inhibition of p38 by SB203580 decreased

IFN-γ stimulated C/EBPβ expression by 46 % and 34 %, respectively. The MEK1

inhibitor PD98059 and the tyrosine kinase inhibitor genistein had no effect.

Cycloheximide inhibited the IFN-γ -induced C/EBPβ expression by 63 % (fig. 3A).

These results suggest that MEK2 and p38 as well as novel protein synthesis regulate

the induction of C/EBPβ expression in response to IFN-γ in T84 colon epithelial cells.

Since phosphorylation has been shown to affect the transcriptional activity of C/EBPβ

(35), the effects of kinase inhibitors on C/EBPβ activity were assayed using EMSA. The

IFN-γ -induced DNA binding activity of C/EBPβ was decreased by U0126 and

SB203580, whereas PD98059 and genistein had no effect. Also cycloheximide

inhibited the IFN-γ -stimulated C/EBPβ activity (fig. 1B).


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Increased secretion of IL-6 by IFN-γ was inhibited by U0126 (by 43 %) and by

SB203580 (by 46 %), whereas genistein and PD98059 had no effect. Cycloheximide

inhibited the IFN-γ 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/EBPβ.

ICAM-1 expression was inhibited by the combined MEK1 and MEK2 inhibitor, U0126,

(by 34 %) and by cycloheximide (by 57 %), whereas genistein, SB203580 and

PD98059 failed to inhibit it (fig. 1D).

The present results suggest that IFN-γ induces the expression of C/EBPβ through the

MEK-ERK and p38 pathways. In order 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-γ -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-γ signaling (fig. 1E). Phosphorylation of ERK1

and ERK2 was inhibited by U0126 (by 72 %), whereas SB203580 and PD98059 had

no effect. This result confirms the inhibitory action of U0126 on MEK in our

experimental set-up. Interestingly, genistein and cycloheximide enhanced the IFN-γ

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, since it did not affect


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C/EBPβ expression and activity. We found a small constitutive phosphrylation of p38,

which was not regulated by IFN-γ stimulation or any of the used kinase inhibitors (data

not shown), which may contribute to the induction of C/EBPβ.


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DISCUSSION

In the present study we examined the effect of IFN-γ on the activation of C/EBPβ, and

on the production of IL-6 in the T84 colon epithelial cell line. Treatment with IFN-γ

increased the expression and DNA-binding activity of C/EBPβ, and also the production

of IL-6. Both of these IFN-γ -induced events were inhibited by treatment with the MEK1

and MEK2 inhibitor U0126, the p38 inhibitor SB203580, and the translational inhibitor

cycloheximide, but were unaffected by treatment with the MEK1 inhibitor PD98059 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/EBPβ in T84

epithelial cells. Our study is apparently the first to report the induction of C/EBPβ and

concomitant production of IL-6 in response to IFN-γ 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 pro-inflammatory cytokines such as IFN-γ (29) and

IL-6 (14). Activated lymphocytes, especially Th1-cells, are active producers of IFN-γ

(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 polarisation of T helper cells to favour

a type 1 cytokine response as seen in Crohn’s disease (42). Inhibition of IL-6 signaling


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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/EBPβ is a member of the C/EBP-transcription factor family

that consists of six (α-?) 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/EBPβ 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-γ stimulated activation of C/EBPβ and production of IL-6 in colon

epithelial cells. Our results suggest that both the activation of C/EBPβ and the

increased production of IL-6 by IFN-γ are MEK-dependent, and emphasize on the role

of MEK2. It was recently shown that IFN-γ induces C/EBPβ 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-γ, on the activation of transcription

factor C/EBPß in T84 colon epithelial cells. IFN-γ has been shown to enhance the

expression of many inflammatory events in these cells when stimulated in conjunction

with other pro-inflammatory cytokines, e.g., IL-1 and TNF-a (23, 31). The mechanisms

of this synergy are incompletely understood, but our present data suggest that the IFN-


13

γ –induced activation of C/EBPß may have a role in this response. Further studies are

warranted to elucidate this effect.

In our present study the induction of C/EBPβ and subsequent production of IL-6, in

response to IFN-γ, also showed p38 dependency (as suggested by the effect of

SB203580). This finding suggests a similar signal transduction pathway, as shown in

earlier reports, by inductors other than IFN-γ (10, 41).

Activation of C/EBPβ and production of IL-6 were independent of tyrosine

phosphorylation, suggesting JAK1 and JAK2 -unrelated signal transduction from IFN-γ

receptor. This result is supported by similar findings in JAK1-deficient fibroblasts, which

were able to activate C/EBPß in response to IFN-γ (15), and in murine macrophages, in

which tyrosine kinase inhibitors (genistein and herbimycin) were ineffective in

preventing gamma-activated transcriptional element (GATE) mediated transcription

(48), that has been shown to be C/EBPß dependent (40). Furthermore, in agreement

with the GATE mediated transcription in response to IFN-γ (48), our results also

suggest dependency of IFN-γ -induced C/EBPβ 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-γ.

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-γ -induced ICAM-1


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expression have not been reported earlier. Our results suggest that induction of ICAM-

1 in response to IFN-γ 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-γ is not prevented by tyrosine kinase inhibitors genistein and

herbimycin A (18).

The inhibition of p38 activity with SB203580 upregulated the IFN-γ -induced ICAM-1

expression. A similar effect was noticed by induction with TNF-a (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 SB203580 may have

contributed to upregulation of ICAM-1. The exact mechanisms remain to be clarified.

C/EBPβ activity may enhance the inflammatory process by increasing the secretion of

cytokines, such as IL-6. Since epithelial C/EBPβ was associated with increased ICAM-

1 expression, it may also regulate recruitment of neutrophils in mucosal inflammation.

In conclusion, our results suggest that IFN-γ stimulated expression and activity of

C/EBPβ, 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

We thank Mrs. Lahja Eurajoki for expert technical assistance.


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FIGURE LEGENDS

FIGURE 1. The epithelial cell responses to the IFN-γ stimulation at 0, 4, 8, 12 and 24

h. A) Time dependency of C/EBPβ expression in IFN-γ -stimulated (filled squares) and

unstimulated (open squares) T84 epithelial cells (n=4 for each treatment and time

point). B) DNA-binding activity of C/EBPβ with or without IFN-γ stimulation in T84 colon

epithelial cells. A representative figure of two separate experiments. C) Time

dependency of IL-6 production in IFN-γ -stimulated (filled squares) and unstimulated

(open squares) T84 epithelial cells (n=6 for each treatment and time point). D)

Immunoblot analysis of ICAM-1 expression in T84 epithelial cells after stimulation with

IFN-γ. A representative figure of two separate experiments. Data are expressed as

mean ± SEM, * p<0.05, ** p<0.01 and *** p<0.001 as compared to the unstimulated

controls

FIGURE 2. Nuclear localization of C/EBPβ expression after 24 hours of A)

immunological staining of C/EBPβ in untreated T84 cells, B) propidium iodide staining

of the nucleus in untreated T84 cells, C) immunological staining of C/EBPβ in IFN-γ

stimulated T84 cells and D) propidium iodide staining of the nucleus in IFN-γ stimulated

T84 cells.


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FIGURE 3. The effects of genistein, SB203580, PD98059, U0126 and cycloheximide

on IFN-γ -stimulated T84 epithelial cells. A) The drug effects on the C/EBPβ expression

and its densitometric analysis (n=4 for each treatment). B) The drug effects on the IFN-

γ -induced C/EBPβ DNA-binding activity in T84 epithelial cells as determined by EMSA.

A representative figure of two separate experiments. C) The drug effect on the IFN-γ -

stimulated secretion of IL-6 in T84 epithelial cells (n=6 for each treatment). D) The drug

effects on the IFN-γ -induced ICAM-1 expression in T84 epithelial cells (n=4 for each

treatment). E) The drug effects on the IFN-γ -stimulated ERK1 and ERK2

phosphorylation in T84 epithelial cells and its densitometric analysis (n=4 for each

treatment). F) Addition of excess cold, unlabeled wild-type or mutant C/EBP

oligonucleotides, or C/EBPβ antibody were used to determine specificity of the C/EBPβ

band. Data are expressed as mean ± SEM, * p<0.05, ** p<0.01 and *** p<0.001 as

compared to the IFN-γ -stimulated control cells.

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