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

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

Departments of 1 Pharmacology and 2 Anatomy, Institute of Biomedicine, FIN-00014 University of Helsinki, Finland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the role of IFN-gamma and MAPKs on the expression and activity of the transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta ) in the T84 colon epithelial cell line. IFN-gamma induced the expression and activity of C/EBPbeta 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/EBPbeta and IL-6 by IFN-gamma , 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-gamma -mediated signal transduction and induction of C/EBPbeta expression and activity associated with interleukin-6 (IL-6) secretion in colon epithelial cells.

CCAAT-enhancer binding protein-beta ; colon epithelial cells, interferon-gamma ; interleukin-6; mitogen-activated protein kinases


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma is produced in excess by cells of the immune system in the lamina propria. In Crohn's disease, IFN-gamma is produced especially by T helper 1 (Th1) cells (29), which are considered important in the pathogenesis of this disease (42). IFN-gamma 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-gamma signal is mediated via JAK1 and JAK2, which activate dimerization of the transcription factor STAT1 (22). It has been shown that IFN-gamma may induce gene expression also, independently of STAT1 (34). Moreover, IFN-gamma -mediated transcriptional activation can proceed through the transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta , also know as NF-IL-6) (40). IFN-gamma was recently shown to increase the expression and activity of C/EBPbeta 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-gamma 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/EBPbeta has been less extensively studied.

C/EBPbeta 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-gamma on activation of C/EBPbeta and on secretion of IL-6 in colon epithelial cells. We also studied the role of the inflammation-associated MAPK-pathways (25) in IFN- gamma  signaling.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Reagents. IFN-gamma 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/EBPbeta , 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-gamma (500 ng/ml) was studied at 0, 4, 8, 12, and 24 h. Selection of this relatively high dose of IFN-gamma 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-gamma . 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-gamma . 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/EBPbeta , p38, p-p38, ERK, p-ERK1/2, and ICAM-1. Expression of C/EBPbeta , 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/EBPbeta 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/EBPbeta was determined in T84 cells cultured on microscope slides (Nalge Nunc International, Naperville, IL) using confocal microscope. The cells were stimulated with IFN-gamma or vehicle for 24 h, subsequently fixed in -20°C methanol, washed with PBS, and incubated with the C/EBPbeta (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.


    RESULTS
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In T84 cells, the expression of the transcription factor C/EBPbeta increased after 8 h of stimulation with IFN-gamma . At 24 h, it reached a 3.3-fold induction compared with unstimulated cells (Fig. 1A). Induction of C/EBPbeta expression by IFN-gamma was associated with increased C/EBPbeta DNA-binding activity, which peaked at 4 h after stimulation with IFN-gamma and remained slightly elevated compared with controls, as shown by EMSA (Fig. 1B). Demonstration of nuclear localization of IFN-gamma -induced C/EBPbeta expression (Fig. 2) by immunocytochemistry further supported this transcriptional activation. Although the activation of C/EBPbeta diminished after 4 h, the protein expression increased and the induced protein localized to the nucleus.


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Fig. 1.   Epithelial cell responses to the interferon-gamma (IFN-gamma ) stimulation at 0, 4, 8, 12, and 24 h. A: time dependency of CCAAT/enhancer-binding protein-beta (C/EBPbeta ) expression in IFN-gamma -stimulated () and unstimulated () T84 epithelial cells (n = 4 for each treatment and time point). B: DNA-binding activity of C/EBPbeta with or without IFN-gamma stimulation in T84 colon epithelial cells. A representative figure of 2 separate experiments. C: time dependency of interleukin-6 (IL-6) production in IFN-gamma -stimulated () and unstimulated () T84 epithelial cells (n = 6 for each treatment and time point). D: immunoblot analysis of intercellular adhesion molecule-1 (ICAM-1) expression in T84 epithelial cells after stimulation with IFN-gamma . A representative figure of 2 separate experiments. Data are expressed as means ± SE. *P < 0.05; **P < 0.01; *** P < 0.001 compared with the unstimulated controls.



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Fig. 2.   Nuclear localization of C/EBPbeta expression after 24 h of immunological staining of C/EBPbeta in untreated T84 cells (A), propidium iodide staining of the nucleus in untreated T84 cells (B), immunological staining of C/EBPbeta in IFN-gamma -stimulated T84 cells (C), and propidium iodide staining of the nucleus in IFN-gamma -stimulated T84 cells (D).

In response to IFN-gamma , 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/EBPbeta preceded the increased IL-6 secretion, thereby suggesting activation of IL-6 production by C/EBPbeta , or at least a sequential correlation between these responses.

When stimulated with IFN-gamma , 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-gamma , 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/EBPbeta and IL-6 by IFN-gamma , 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-gamma -stimulated C/EBPbeta expression by 46 and 34%, respectively. The MEK1 inhibitor PD-98059 and the tyrosine kinase inhibitor genistein had no effect. Cycloheximide inhibited the IFN-gamma -induced C/EBPbeta expression by 63% (Fig. 3A). These results suggest that MEK2 and p38, as well as novel protein synthesis, regulate the induction of C/EBPbeta expression in response to IFN-gamma in T84 colon epithelial cells.


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Fig. 3.   The effects of genistein, SB-203580, PD-98059, U-0126, and cycloheximide on IFN-gamma -stimulated T84 epithelial cells. A: drug effects on the C/EBPbeta expression and its densitometric analysis (n = 4 for each treatment). B: drug effects on the IFN-gamma -induced C/EBPbeta DNA-binding activity in T84 epithelial cells as determined by EMSA. A representative figure of 2 separate experiments. C: drug effect on the IFN-gamma -stimulated secretion of IL-6 in T84 epithelial cells (n = 6 for each treatment). D: drug effects on the IFN-gamma -induced ICAM-1 expression in T84 epithelial cells (n = 4 for each treatment). E: drug effects on the IFN-gamma -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/EBPbeta antibody was used to determine specificity of the C/EBPbeta band. Data are expressed as means ± SE. *P < 0.05, **P < 0.01; ***P < 0.001 compared with the IFN-gamma -stimulated control cells.

Because phosphorylation has been shown to affect the transcriptional activity of C/EBPbeta (35), the effects of kinase inhibitors on C/EBPbeta activity were assayed using EMSA. The IFN-gamma -induced DNA binding activity of C/EBPbeta was decreased by U-0126 and SB-203580, whereas PD-98059 and genistein had no effect. Also, cycloheximide inhibited the IFN-gamma -stimulated C/EBPbeta activity (Fig. 1B).

Increased secretion of IL-6 by IFN-gamma 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-gamma -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/EBPbeta .

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-gamma induces the expression of C/EBPbeta 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-gamma 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-gamma 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-gamma -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/EBPbeta expression and activity. We found a small constitutive phosphorylation of p38, which was not regulated by IFN-gamma stimulation or any of the used kinase inhibitors (data not shown), which may contribute to the induction of C/EBPbeta .


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we examined the effect of IFN-gamma on the activation of C/EBPbeta and on the production of IL-6 in the T84 colon epithelial cell line. Treatment with IFN-gamma increased the expression and DNA-binding activity of C/EBPbeta and also the production of IL-6. Both of these IFN-gamma -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/EBPbeta in T84 epithelial cells. Our study is apparently the first to report the induction of C/EBPbeta and concomitant production of IL-6 in response to IFN-gamma 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-gamma (29) and IL-6 (14). Activated lymphocytes, especially Th1 cells, are active producers of IFN-gamma (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/EBPbeta is a member of the C/EBP-transcription factor family that consists of six (alpha -zeta ) 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/EBPbeta 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-gamma -stimulated activation of C/EBPbeta and production of IL-6 in colon epithelial cells. Our results suggest that both the activation of C/EBPbeta and the increased production of IL-6 by IFN-gamma are MEK dependent and emphasize on the role of MEK2. It was recently shown that IFN-gamma induces C/EBPbeta 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-gamma , on the activation of transcription factor C/EBPbeta in T84 colon epithelial cells. IFN-gamma 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-alpha (23, 31). The mechanisms of this synergy are incompletely understood, but our present data suggest that the IFN-gamma -induced activation of C/EBPbeta may have a role in this response. Further studies are warranted to elucidate this effect.

In the present study, the induction of C/EBPbeta and subsequent production of IL-6, in response to IFN-gamma , 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-gamma (10, 41).

Activation of C/EBPbeta and production of IL-6 were independent of tyrosine phosphorylation, suggesting JAK1 and JAK2-unrelated signal transduction from IFN-gamma receptor. This result is supported by similar findings in JAK1-deficient fibroblasts, which were able to activate C/EBPbeta in response to IFN-gamma (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), which has been shown to be C/EBPbeta dependent (40). Furthermore, in agreement with the GATE-mediated transcription in response to IFN-gamma (48), our results also suggest dependency of IFN-gamma -induced C/EBPbeta 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-gamma . 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-gamma -induced ICAM-1 expression have not been reported earlier. Our results suggest that induction of ICAM-1 in response to IFN-gamma 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-gamma is not prevented by tyrosine kinase inhibitors genistein and herbimycin A (18).

The inhibition of p38 activity with SB-203580 upregulated the IFN-gamma -induced ICAM-1 expression. A similar effect was noticed by induction with TNF-alpha (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/EBPbeta activity may enhance the inflammatory process by increasing the secretion of cytokines, such as IL-6. Because epithelial C/EBPbeta was associated with increased ICAM-1 expression, it may also regulate recruitment of neutrophils in mucosal inflammation. In conclusion, our results suggest that IFN-gamma -stimulated expression and activity of C/EBPbeta , 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.


    ACKNOWLEDGEMENTS

We thank Lahja Eurajoki for expert technical assistance.


    FOOTNOTES

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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ABSTRACT
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RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 284(5):C1133-C1139
0363-6143/03 $5.00 Copyright © 2003 the American Physiological Society




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