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The Journal of Immunology, 2007, 178, 8013-8021
Copyright © 2007 by The American Association of Immunologists, Inc.
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Macrophage-Inflammatory Protein-3{alpha} Mediates Epidermal Growth Factor Receptor Transactivation and ERK1/2 MAPK Signaling in Caco-2 Colonic Epithelial Cells via Metalloproteinase-Dependent Release of Amphiregulin1

Sarah Keates, Xinbing Han, Ciarán P. Kelly and Andrew C. Keates2

Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previously, we reported that normal colonocytes produce the memory CD4+ T cell-directed chemokine MIP-3{alpha}, and that epithelial MIP-3{alpha} levels are elevated in inflammatory bowel disease. Interestingly, the unique receptor for MIP-3{alpha}, CCR6, is expressed by a variety of cell types including colonocytes, suggesting that MIP-3{alpha} may regulate additional biological activities in the intestine. The aim of this study was to determine whether MIP-3{alpha} can induce intestinal epithelial cell proliferation and to examine the signaling mechanisms that mediate this response. We show that nonstimulated Caco-2 and HT-29 colonic epithelial cells express CCR6, and that stimulation of Caco-2 cells by MIP-3{alpha} can dose dependently increase cell proliferation as well as activate the epidermal growth factor receptor (EGFR) and ERK1/2 MAPK. MIP-3{alpha}-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3{alpha}-mediated, but not EGF-mediated Caco-2 cell proliferation. Taken together, our findings indicate that MIP-3{alpha} can regulate mitogenic signaling in colonic epithelial cells and thus may serve an important homeostatic function in the intestine by regulating tissue turnover and maintenance of the epithelium, in addition to its role in regulating leukocyte recruitment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The chemokine superfamily of chemoattractant cytokines are small (8–10 kDa) proteins that specialize in directing homeostatic leukocyte movement as well as the recruitment of leukocytes to areas of immune challenge (1, 2). To date, over 40 chemokines have been identified which can be classified into one of four subfamilies according to the number and arrangement of conserved cysteine residues (C, CC, CXC, or CX3C) (1, 3, 4, 5). MIP-3{alpha} is a CC chemokine that is predominantly expressed by epithelial cells in the small intestine and colon, and is up-regulated by proinflammatory stimuli (6, 7, 8). Binding of MIP-3{alpha} to its unique receptor, CCR6, induces migratory responses in memory CD4+ T lymphocytes and immature dendritic cells (9, 10, 11, 12). Moreover, MIP-3{alpha} also induces T lymphocyte adhesion to the gastrointestinal-specific vascular addressin MAd-CAM-1 (10). In recent studies we, and others, have reported that MIP-3{alpha} mRNA and protein levels are markedly elevated in colonic tissues from inflammatory bowel disease (IBD)3 patients (7, 13, 14). Moreover, our group has also shown that enterocytes are a major site of MIP-3{alpha} production in normal, noninflamed human colon, and that epithelial MIP-3{alpha} protein levels are elevated in IBD (7). Increased colonic MIP-3{alpha} production may, therefore, play an important role in regulating the recruitment of {alpha}4beta7-integrin-bearing CD4+ T cells during intestinal inflammation.

In addition to their role in the regulation of leukocyte trafficking, it is now evident that chemokines are also involved in the control of a variety of other physiological functions including angiogenesis, metastasis, and wound healing (1, 15). In keeping with this evidence, previous studies have reported that the rat neutrophil-directed CXC chemokines MIP-2 and CINC-1 can exert mitogenic effects on alveolar epithelial cells and gastric epithelial cells, respectively (16, 17). The CC chemokine MCP-1 has also been reported to induce mitogenic responses in human bronchial epithelial cells (18). Interestingly, Dwinell et al. (19) have reported that CCR6, the specific receptor for MIP-3{alpha}, is constitutively expressed by a wide variety of colonic epithelial cells lines. Moreover, a subsequent study by the same group has demonstrated expression of CCR6 by epithelial cells in noninflamed human colon (14). Whether MIP-3{alpha} regulates homeostatic functions in the human colon, however, has not been extensively investigated.

The aim of this study was to determine whether MIP-3{alpha} can induce intestinal epithelial cell proliferation and, if so, to examine the signaling mechanisms that regulate this response. Our findings demonstrate that activation of Caco-2 human colonic epithelial cells by MIP-3{alpha} can dose dependently stimulate cell proliferation, as well as induce activation of the ERK1/2 MAPK signaling cascade. In addition, we also show that MIP-3{alpha}-mediated ERK1/2 activation and cell proliferation in Caco-2 cells requires metalloproteinase-dependent release of the endogenous epidermal growth factor (EGF) receptor (EGFR) ligand amphiregulin and transactivation of the EGFR.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cell culture

Caco-2 cells and HT-29 cells colonic epithelial cells (American Type Culture Collection) were maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% FCS, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and nonessential amino acids (Caco-2 only) at 37°C in an atmosphere of 5% CO2 and 95% air. For most experiments, epithelial cell monolayers were grown on 12-well plates and maintained in serum-free medium for 24 h before the experiment. The cells were then incubated with MIP-3{alpha} (0–100 ng/ml; R&D Systems) or EGF (0–100 ng/ml; R&D Systems) in serum-free medium for varying lengths of time. In some experiments, Caco-2 cells were pretreated with the EGFR kinase inhibitor AG1478 (1 µM; Calbiochem), goat neutralizing polyclonal Abs directed against various EGFR ligands (EGF, heparin-binding EGF, TGF-{alpha}, amphiregulin, 20 µg/ml; R&D Systems), the broad spectrum metalloproteinase inhibitor GM6001 (20 µM; Calbiochem) or the TNF-{alpha} protease inhibitor (TAPI)-1 (10 µM; Calbiochem) for 1 h before stimulation.

Analysis of CCR6 mRNA levels by RT-PCR

Total RNA was isolated from nonstimulated Caco-2 cells or HT-29 cells using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. RNA (1 µg) was reversed transcribed using random hexamer primers and Moloney murine leukemia virus reverse transcriptase as previously described (7), and the resulting cDNA was stored at –20°C. PCR amplification of CCR6 cDNA was performed as described by Dwinell et al. (19). PCR amplification of GAPDH cDNA was performed using our previously published method (7). Amplification products were then analyzed by electrophoresis through 1% agarose gels containing 500 ng/ml ethidium bromide and visualized using an UV transilluminator at 312 nm (Fisher Biotech). Expected sizes for CCR6 and GAPDH PCR products were 1021 and 600 bp, respectively.

Analysis of CCR6 protein expression by immunofluorescence

Caco-2 or HT-29 cells (grown on glass coverslips) were washed with PBS and fixed with Cytofix (BD Biosciences) for 20 min at room temperature. After washing three times with PBS, the cells were permeabilized using 0.2% Triton X-100 in PBS for 2 min at room temperature, then washed three times with PBS. The cells were then blocked for 1 h at room temperature with PBS containing 3% donkey serum and 1% BSA, and incubated with an anti-CCR6 mAb (R&D Systems) or an isotype control IgG2b mAb (DakoCytomation) at a concentration of 25 µg/ml overnight at 4°C in a humid chamber. The next day, the cells were washed three times with PBS and then incubated with a rhodamine-labeled donkey anti-mouse IgG Ab (1/100 dilution; Jackson ImmunoResearch Laboratories) for 1 h at room temperature in the dark. The cells were then washed four times with PBS and mounted using VectorShield (Vector Laboratories). Cellular fluorescence was then assessed using a Bio-Rad MRC 1024 laser scanning confocal microscope.

Cell proliferation assays

Caco-2 and HT-29 cell proliferation was determined using a CellTiter 96 Non-Radioactive Cell Proliferation Assay kit (Promega) according to the manufacturer’s instructions with minor modifications. Briefly, 50-µl aliquots of serum-free medium containing various concentrations of MIP-3{alpha} or EGF were added to the wells of a 96-well plate. In some experiments, goat neutralizing polyclonal Abs directed against EGF and/or amphiregulin or normal goat IgG were also added. The plate was then placed in a tissue culture incubator to equilibrate for 1 h. Following the equilibration period, 50 µl of Caco-2 cell suspension (1 x 104 cells) was added to each well and the plate was incubated for 72 h. Dye solution (15 µl) was then added to each well and the plate was incubated for 4 h, after which 100 µl of solubilization/stop solution was added. To allow for complete solubilization of the formazan product, the plate was incubated at 37°C overnight and the absorbance at 570 nm (formazan) and 750 nm (reference) was determined for each well.

For some experiments, Caco-2 and HT-29 cell proliferation was also assessed using [3H]thymidine uptake. Briefly, 250-µl aliquots of serum-free medium containing various concentrations of MIP-3{alpha} or EGF were added to the wells of a 12-well plate. Following equilibration for 1 h, 250-µl aliquots of Caco-2 or HT-29 cell suspension (1 x 105 cells) were added to each well and the plate was incubated for a further 72 h. For the final 6 h of this incubation, 2 µCi of [methyl-3H]thymidine (PerkinElmer) was added to each well. At the end of the labeling period, the cells were washed twice with PBS then fixed with 500 µl of ice-cold methanol for 15 min. After fixation, the cells were treated with 500 µl of 15% TCA, washed three times with 1 ml of distilled water, and dried overnight at room temperature. The cells were then dissolved for 15 min in 200 µl of 0.2 M NaOH, and a 50-µl aliquot was subjected to scintillation counting.

Western blotting

ERK1/2 MAPK and EGFR activation were evaluated by Western blot analysis using phospho-specific Abs for ERK1/2 (Cell Signaling Technology) and EGFR (Santa Cruz Biotechnology). At the end of each experiment, Caco-2 cell monolayers were washed three times with PBS and lysed with 1x SDS sample buffer. Samples were then heated to 100°C for 5 min, and ~10 µl of each lysate was separated by SDS-PAGE using a 6% or 8% gel and blotted onto nitrocellulose membranes. Blots were then blocked by incubation in TBS containing 5% milk for 1 h at room temperature and then incubated overnight at 4°C with phospho-specific MAPK (Cell Signaling Technology) or EGFR Abs (Santa Cruz Biotechnology) diluted 1/1000 in blocking buffer. Membranes were then washed three times with TBS, and incubated at room temperature for 1 h with peroxidase-conjugated goat anti-rabbit IgG (1/3000 dilution; Santa Cruz Biotechnology). SuperSignal chemiluminescent substrate (Pierce) was used for detection. To demonstrate equal loading, blots were stripped and reprobed with either Abs directed against total ERK2 or total EGFR (1/000 dilution; Santa Cruz Biotechnology).

Amphiregulin ELISA

Human amphiregulin was assayed using a commercially available ELISA (R&D Systems) according to the manufacturer’s instructions. This assay showed no cross-reactivity with human EGF, EGFR, heparin-binding EGF, or TGF-{alpha} and had a lower limit of detection of 15.6 pg/ml.

Data analysis

Statistical analyses were performed using SigmaStat 3.1 software (Jandel Scientific). ANOVA followed by protected t tests was used for intergroup comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Caco-2 and HT-29 colonic epithelial cells express CCR6

Recent studies indicate that, in addition to their well-documented role in leukocyte recruitment, chemokines also participate in a variety of other important biological activities such as wound healing (1, 15). Interestingly, CCR6, the unique receptor for MIP-3{alpha}, is highly expressed by colonic epithelial cells in normal, noninflamed human colon (14), suggesting MIP-3{alpha}/CCR6 may have important homeostatic functions in the intestine. To begin to address this important question, we first examined whether cultured colonic epithelial cells also express CCR6. As shown in Fig. 1A, we found that CCR6 mRNA was clearly evident in nonstimulated Caco-2 cells, as well as nonstimulated HT-29 cells. To determine whether these cells also expressed CCR6 protein we next performed an immunofluorescence study. As shown in Fig. 1B, we found that CCR6 protein could be readily detected in Caco-2 and HT-29 cells stained with a mAb directed against CCR6. In contrast, little or no staining was observed when Caco-2 or HT-29 cells were incubated with an isotype control IgG2b mAb.


Figure 1
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  		FIGURE 1. Caco-2 and HT-29 colonic epithelial cells express CCR6. A, Total RNA was isolated from confluent Caco-2 or HT-29 cell monolayers and reversed transcribed as described in Materials and Methods. Reversed transcribed (RT) or nonreversed transcribed (no RT) RNA was then subjected to PCR, and CCR6 and GAPDH mRNA levels were assessed by 1% agarose gel electrophoresis. B, Fixed and permeabilized Caco-2 and HT-29 cells were treated with either an anti-CCR6 mAb (left panels) or an isotype control IgG2b mAb (right panels), and then incubated with a rhodamine-labeled donkey anti-mouse IgG Ab. Cellular fluorescence was assessed using a Bio-Rad MRC 1024 laser scanning confocal microscope. Original magnification, x200.

 
MIP-3{alpha} induces mitogenic responses in Caco-2 and HT-29 colonic epithelial cells

To determine whether CCR6 expression by colonocytes may be functionally important we next examined whether Caco-2 and HT-29 cells proliferate in response to stimulation by MIP-3{alpha}. As expected, stimulation of Caco-2 or HT-29 colonic epithelial cells by EGF, a well-known mitogen, dose dependently increased cell proliferation as measured by MTT assay (Fig. 2A). Compared with nonstimulated controls, Caco-2 and HT-29 cell numbers were increased ~30–40% following treatment with 10 ng/ml EGF for 72 h. Similar results were obtained when EGF-mediated epithelial cell proliferation was assessed by [3H]thymidine incorporation (Fig. 2C).


Figure 2
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  		FIGURE 2. MIP-3{alpha} induces mitogenic responses in Caco-2 and HT-29 cells. Subconfluent Caco-2 or HT-29 monolayers were serum starved for 24 h then stimulated by 0–100 ng/ml EGF (A and C) or 0–100 ng/ml MIP-3{alpha} (B and D) for 72 h. At the end of the experiment, cell proliferation was assessed by MTT assay or [3H]thymidine incorporation as described in Materials and Methods. Absorbance (A) at 570 and 750 nm was determined. Data are expressed as mean ± SEM (n = 3–6 experiments).

 
Enhanced Caco-2 and HT-29 cell proliferation (measured by MTT assay) was also seen following stimulation with varying doses of MIP-3{alpha}. As shown in Fig. 2B, maximal Caco-2 cell proliferation (~20% compared with untreated cells) was observed following stimulation by 10 ng/ml MIP-3{alpha}. In contrast, 100 ng/ml MIP-3{alpha} was required to stimulate maximal HT-29 cell proliferation (Fig. 2B). Again, results comparable to those obtained using the MTT assay were observed when MIP-3{alpha}-stimulated epithelial cell proliferation was assessed using [3H]thymidine incorporation (Fig. 2D). Taken together, these findings indicate that activation of CCR6 can induce mitogenic responses in human colonic epithelial cells.

MIP-3{alpha} activates ERK1/2 MAPK in Caco-2 cells

To begin to examine the signaling mechanisms that regulate MIP-3{alpha}-induced Caco-2 cell proliferation, we next examined whether ERK MAPK signaling cascade was activated in MIP-3{alpha}-treated cells because this pathway is known to regulate cellular mitogenic responses (20, 21). As shown in Fig. 3, control cells contained undetectable levels of active (i.e., phosphorylated) ERK1/2. Stimulation of Caco-2 cells with MIP-3{alpha} resulted in a dose-dependent increase in the level of phosphorylated ERK1/2 (Fig. 3A). Moreover, MIP-3{alpha}-induced ERK1/2 activation in Caco-2 cells was rapid with maximal activation observed after 10 min (Fig. 3B and see Fig. 5B). As expected, levels of phosphorylated ERK1/2 were markedly elevated when Caco-2 cells were treated with 10 ng/ml EGF (Fig. 3 and see Fig. 5A). Increased levels of phosphorylated p38 MAPK were also observed in Caco-2 cells treated with MIP-3{alpha} or IL-1beta, however, JNK MAPK activation was not observed in MIP-3{alpha}-stimulated Caco-2 cells (data not shown).


Figure 3
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  		FIGURE 3. MIP-3{alpha} activates ERK1/2 MAPK in Caco-2 cells. A, Dose response of MIP-3{alpha}-mediated ERK1/2 activation. Caco-2 monolayers were serum starved for 24 h then stimulated by MIP-3{alpha} (0–10 ng/ml) or EGF (10 ng/ml) for 10 min. Whole cell lysates were then separated on 8% SDS-PAGE gels, and phospho-ERK1/2 levels were assessed by Western blotting as described in Materials and Methods. To demonstrate equal loading, stripped blots were reprobed with a control ERK2 Ab. B, Time course of MIP-3{alpha}-mediated ERK1/2 activation. Caco-2 monolayers were serum starved for 24 h then stimulated by MIP-3{alpha} or EGF (both 10 ng/ml) for 0–10 min. Whole cell lysates were then separated on 8% SDS-PAGE gels and phospho-ERK1/2 levels were assessed by Western blotting as described. To demonstrate equal loading, stripped blots were reprobed with a control ERK2 Ab.

 

Figure 5
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  		FIGURE 5. Inhibition of EGFR kinase activity markedly reduces MIP-3{alpha}-mediated ERK1/2 MAPK phosphorylation in Caco-2 cells. Confluent, serum starved Caco-2 monolayers were pretreated with or without AG1478 (1 µM) for 1 h and then stimulated by 10 ng/ml EGF (A) or 10 ng/ml MIP-3{alpha} (B) for 0–30 min. Whole cell lysates were then separated on 8% SDS-PAGE gels, and phospho-ERK1/2 levels were assessed by Western blotting as described in Materials and Methods. To demonstrate equal loading, stripped blots were reprobed with a control ERK2 Ab.

 
EGFR activation is required for MIP-3{alpha}-mediated ERK1/2 MAPK phosphorylation in Caco-2 cells

Previous studies from our group, and others, have shown that ligands for G protein-coupled receptors can activate the ERK MAPK signaling pathway via transactivation of the EGFR complex in epithelial cells (22, 23, 24, 25). To test whether MIP-3{alpha} induces EGFR phosphorylation confluent Caco-2 monolayers were pretreated with or without the EGFR kinase inhibitor AG1478 (1 µM) for 1 h before stimulation with MIP-3{alpha} or EGF (both at 10 ng/ml). As shown in Fig. 4, little or no phosphorylated EGFR was present in nonstimulated Caco-2 cells. As expected, activation of Caco-2 cells with EGF led to a rapid and sustained increase in the level of phosphorylated EGFR (Fig. 4A). Moreover, pretreatment of Caco-2 cells with AG1478 before EGF stimulation completely abolished EGFR phosphorylation. Increased levels of phosphorylated EGFR were also seen in Caco-2 cell stimulated by MIP-3{alpha}. However, in contrast to the sustained increase in phosphorylated EGFR levels seen following EGF stimulation, MIP-3{alpha} induced activation of the EGFR appeared to be maximal after 2.5 min after which it gradually returned toward baseline (Fig. 4B). Pretreatment of Caco-2 cells with the EGFR kinase inhibitor AG1478, however, completely abolished MIP-3{alpha}-mediated EGFR phosphorylation.


Figure 4
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  		FIGURE 4. MIP-3{alpha} stimulates phosphorylation of the EGFR in Caco-2 cells. Confluent, serum starved Caco-2 monolayers were pretreated with or without AG1478 (1 µM) for 1 h and stimulated by 10 ng/ml EGF (A) or 10 ng/ml MIP-3{alpha} (B) for 0–30 min. Whole cell lysates were then separated on 8% SDS-PAGE gels, and phospho-EGFR levels were assessed by Western blotting as described in Materials and Methods. To demonstrate equal loading, stripped blots were reprobed with a control EGFR Ab.

 
To confirm that MIP-3{alpha}-mediated ERK MAPK activation required EGFR activation we also examined levels of phosphorylated ERK1/2 in Caco-2 cells pretreated with the EGFR kinase inhibitor AG1478. As shown in Fig. 5, stimulation of Caco-2 cells by MIP-3{alpha} or EGF resulted in a marked increase in the level of phosphorylated ERK1/2 that was completely inhibited when the cells were pretreated with AG1478. These findings, taken together, strongly suggest that MIP-3{alpha}-induced activation of ERK1/2 in Caco-2 cells requires transactivation of the EGFR.

Neutralization of amphiregulin bioactivity reduces MIP-3{alpha}-mediated, but not EGF-mediated, EGFR and ERK1/2 phosphorylation in Caco-2 cells

Release of endogenous membrane-bound EGFR ligands (e.g., TGF-{alpha}, heparin-binding EGF, amphiregulin) has been previously identified as an important mechanism leading to EGFR transactivation in many cell types (26, 27). To determine whether MIP-3{alpha}-mediated transactivation of EGFR required the participation of one or more endogenous EGF ligands, confluent Caco-2 monolayers were pretreated for 1 h with neutralizing polyclonal Abs directed against EGF, TGF-{alpha}, heparin-binding EGF, or amphiregulin before stimulation with MIP-3{alpha}. Caco-2 cell levels of phosphorylated EGFR and phosphorylated ERK1/2 were then analyzed by Western blotting.

As shown in Fig. 6A, pretreatment of Caco-2 cells with an EGF neutralizing polyclonal Ab completely blocked EGF-mediated increases in EGFR phosphorylation and ERK1/2 phosphorylation. Moreover, pretreatment with normal goat IgG or neutralizing polyclonal Abs directed against the endogenous EGF ligands TGF-{alpha}, heparin-binding EGF, or amphiregulin had no effect on EGFR or ERK1/2 activation in Caco-2 cells following EGF stimulation. In contrast to these findings, we found that MIP-3{alpha}-mediated increases in EGFR phosphorylation and ERK1/2 phosphorylation could only be attenuated when Caco-2 cells were preincubated with a neutralizing polyclonal Ab directed against the endogenous EGF ligand amphiregulin (Fig. 6B). Again, pretreatment with normal goat IgG had no effect on MIP-3{alpha}-induced EGFR or ERK1/2 activation. To further confirm the data presented in Fig. 6B, we also performed additional control experiments to demonstrate the specificity of anti-HB-EGF, anti-TGF-{alpha}, and anti-amphiregulin neutralizing Abs. As shown in Fig. 6, C–E, we found that each Ab was only able to block ERK1/2 phosphorylation mediated by its corresponding ligand.


Figure 6
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  		FIGURE 6. Neutralization of amphiregulin bioactivity reduces MIP-3{alpha}-mediated, but not EGF-mediated, EGFR and ERK1/2 phosphorylation in Caco-2 intestinal cells. Confluent, serum-starved Caco-2 cell monolayers were pretreated with neutralizing polyclonal Abs directed against EGF, heparin-binding EGF, TGF-{alpha}, and amphiregulin or normal goat IgG for 1 h and then stimulated by 10 ng/ml EGF (A) or 10 ng/ml MIP-3{alpha} (B) for 10 min. Whole cell lysates were then separated on 8% SDS-PAGE gels and phospho-EGFR levels were assessed by Western blotting as described in Materials and Methods. Blots were then stripped and reprobed to determine phospho-ERK1/2 levels. To demonstrate equal loading, the blots were stripped once again and reprobed with a control ERK2 Ab. To assess neutralizing Ab specificity, Caco-2 cell monolayers were pretreated with anti-EGF, anti-heparin-binding EGF, anti-TGF-{alpha}, and anti-amphiregulin Abs or normal goat IgG for 1 h and then stimulated with 10 ng/ml heparin-binding EGF (C), 10 ng/ml TGF-{alpha} (D), or 10 ng/ml amphiregulin (E) for 10 min. Whole cell lysates were then separated on 8% SDS-PAGE gels and phospho-ERK1/2 levels were assessed by Western blotting. To demonstrate equal loading, the blots were stripped once again and reprobed with a control ERK2 Ab.

 
MIP-3{alpha} stimulates the release of amphiregulin from Caco-2 cells

The results of the Ab neutralization experiment suggested that MIP-3{alpha} stimulates the release of endogenous amphiregulin from Caco-2 cells that then binds to and activates the EGFR. To confirm that MIP-3{alpha} does indeed promote the release of amphiregulin, we next measured amphiregulin levels in conditioned medium from MIP-3{alpha}-stimulated Caco-2 cells by ELISA.

As shown in Fig. 7, conditioned medium from nonstimulated control Caco-2 cells contained very little, if any, amphiregulin. In contrast, significantly increased levels of amphiregulin were detected in Caco-2 cell conditioned medium following stimulation by MIP-3{alpha} for 2.5 min. Moreover, amphiregulin levels in conditioned medium continued to increase throughout the course of the experiment reaching a maximum concentration of ~140 pg/ml after 30 min.


Figure 7
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  		FIGURE 7. MIP-3{alpha} stimulates the release of soluble amphiregulin from Caco-2 cells. Confluent Caco-2 monolayers were serum starved for 24 h then stimulated by MIP-3{alpha} (10 ng/ml) for 0–30 min. Conditioned medium was then collected, and amphiregulin levels determined by ELISA as described in Materials and Methods. Data are expressed as mean ± SEM (n = 6 experiments).

 
Neutralization of amphiregulin bioactivity reduces MIP-3{alpha}-induced Caco-2 cell proliferation

To further elucidate the role of amphiregulin in the regulation of MIP-3{alpha}-mediated mitogenic responses we next examined the effect of neutralizing amphiregulin bioactivity on MIP-3{alpha}-induced Caco-2 cell proliferation. For these studies, subconfluent Caco-2 monolayers were stimulated with MIP-3{alpha} or EGF in the presence or absence of neutralizing polyclonal Abs directed against EGF or amphiregulin for 72 h after which cell proliferation was assessed.

As shown in Fig. 8A, EGF-mediated cell proliferation was markedly attenuated when Caco-2 cells were incubated in the presence of EGF neutralizing polyclonal Ab. No inhibitory effect was observed, however, when Caco-2 cells were incubated with an amphiregulin neutralizing polyclonal Ab or normal goat IgG. Consistent with our previous findings, we found that MIP-3{alpha}-mediated Caco-2 cell proliferation was almost completely abolished in the presence of an amphiregulin neutralizing polyclonal Ab, but was unaffected when Caco-2 cells were incubated with the EGF neutralizing polyclonal Ab or normal goat IgG (Fig. 8B). Taken together, these data demonstrate that release of amphiregulin by Caco-2 cells is required for cell proliferation in response to stimulation by MIP-3{alpha}.


Figure 8
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  		FIGURE 8. Neutralization of amphiregulin bioactivity reduces MIP-3{alpha}-induced Caco-2 cell proliferation. Subconfluent, serum-starved Caco-2 cell monolayers were pretreated with neutralizing polyclonal Abs directed against EGF and amphiregulin (AR) as well as normal goat IgG for 1 h and then stimulated by 10 ng/ml EGF (A) or 10 ng/ml MIP-3{alpha} (B) for 72 h. At the end of the experiment, cell proliferation was assessed as described in Materials and Methods. Absorbance (A) at 570 and 750 nm was determined. Data are expressed as mean ± SEM (n = 4 experiments).

 
Blockade of matrix metalloproteinase activity reduces MIP-3{alpha}-mediated, but not EGF-mediated, ERK1/2 MAPK phosphorylation in Caco-2 cells

Previous studies have shown that the release of endogenous EGFR ligands from various cell types is dependent on the action of cell surface metalloproteinases, in particular, members of the ADAM (a disintegrin and metalloprotease) family of zinc-dependent proteases (26, 27). To determine whether MIP-3{alpha}-induced mitogenic signaling also required the activation of metalloproteinases, we next investigated the effect of pretreating the Caco-2 cells with various metalloproteinase inhibitors before stimulation by MIP-3{alpha}.

As expected, we found that pretreatment of Caco-2 cells with a broad-spectrum metalloproteinase inhibitor, GM6001, or with a specific inhibitor of TNF-{alpha} converting enzyme (TACE)/ADAM-17, TAPI-1, had no effect on EGF-mediated ERK1/2 activation (Fig. 9, A and C). In contrast, MIP-3{alpha}-induced phosphorylation of ERK1/2 was markedly inhibited when Caco-2 cells were pretreated with GM6001 or TAPI-1 (Fig. 9, B and D). Pretreatment of Caco-2 cells with the GM6001 control, however, had no effect on MIP-3{alpha}-induced ERK1/2 activation. Taken together, these findings indicate that activation of cell surface metalloproteinases is required for MIP-3{alpha}-mediated mitogenic signaling in Caco-2 cells.


Figure 9
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  		FIGURE 9. Blockade of metalloproteinase activity reduces MIP-3{alpha}-mediated, but not EGF-mediated, ERK1/2 MAPK phosphorylation in Caco-2 cells. Confluent, serum-starved Caco-2 cell monolayers were pretreated with the broad-spectrum metalloproteinase inhibitor GM6001 or its control (both 20 µM) or the TACE/ADAM-17 inhibitor TAPI-1 (10 µM) for 1 h, then stimulated by 10 ng/ml EGF (A and C) or 10 ng/ml MIP-3{alpha} (B and D) for 10 min. Whole cell lysates were then separated on 8% SDS-PAGE gels and phospho-ERK1/2 levels were assessed by Western blotting as described in Materials and Methods. To demonstrate equal loading, stripped blots were reprobed with a control ERK2 Ab.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In recent studies we have shown that enterocytes are a major site of MIP-3{alpha} production in human colon and that enterocyte MIP-3{alpha} protein levels are elevated in IBD (7). Whereas CCR6, the specific receptor for MIP-3{alpha}, is mainly found on memory T cells, B cells, and immature dendritic cells, it is also expressed by a variety of nonimmune cell types including intestinal epithelial cells (12, 14, 28, 29). The ability of epithelial cells to produce both MIP-3{alpha} and CCR6 led us to investigate whether these factors regulate other enterocyte processes. In this study, we have examined whether MIP-3{alpha} induces intestinal epithelial cell proliferation and the signaling mechanisms that regulate this response. Our findings show that stimulation of Caco-2 human colonic epithelial cells by MIP-3{alpha} dose dependently increased cell proliferation as well as activated the EGFR and ERK1/2 MAPK signaling pathways. Our findings also indicate that MIP-3{alpha}-mediated ERK1/2 activation in Caco-2 cells appeared to require metalloproteinase-dependent release of the endogenous EGFR ligand amphiregulin and transactivation of the EGFR. Moreover, blockade of amphiregulin bioactivity using a neutralizing polyclonal Ab significantly reduced MIP-3{alpha}-mediated, but not EGF-mediated, Caco-2 cell proliferation. A model summarizing our experimental findings is presented in Fig. 10.


Figure 10
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  		FIGURE 10. A model for MIP-3{alpha}-mediated proliferation of Caco-2 human intestinal epithelial cells. Stimulation of Caco-2 cells by MIP-3{alpha} leads to the activation of the cell surface metalloproteinase TACE/ADAM-17 and the release of the endogenous EGFR ligand amphiregulin. Subsequent binding to, and activation of, the EGFR by amphiregulin leads to activation of the ERK MAPK signaling cascade and increased cell proliferation.

 
Although chemokines primarily control the migration of immune cells, it is becoming increasingly evident that these molecules likely play an important role in regulating epithelial cell proliferation. Studies by Driscoll et al. (16) have demonstrated increased proliferation of rat alveolar epithelial cells in response to stimulation by the CXC chemokines MIP-2 or CINC. Increased proliferation and ERK1/2 signaling has also been observed for the rat gastric epithelial cell line RGM-1 following stimulation by CINC (17). Consistent with these studies, epithelial resurfacing of excisional wounds in CXCR2 knockout mice is significantly delayed compared with wild-type animals (30). In another study Ottaiano et al. (31) reported that stromal cell-derived growth factor-1{alpha} was capable of inducing ERK1/2 signaling and proliferative responses in SW480 human colonic epithelial cells. Moreover, these effects could be blocked by pretreating SW480 cells with a neutralizing Ab directed against CXCR4, the receptor for stromal cell-derived growth factor-1{alpha}. Stimulation of normal human bronchial epithelial cells by the CXC chemokine I-TAC has also been shown to enhance cell proliferation as well as increase p38 and ERK1/2 MAPK phosphorylation (32). MCP-1-stimulated BEAS-2B bronchial epithelial cells exhibit similar proliferative responses that can be ameliorated via MCP-1 neutralizing Abs (18).

In this study, we demonstrate that stimulation of Caco-2 colonic epithelial cells by MIP-3{alpha} can activate the ERK1/2 signaling cascade resulting in increased cellular proliferation. In keeping with these findings, Brand et al. (33) have recently reported similar data using MIP-3{alpha}-stimulated SW480 or HT-29 human intestinal epithelial cells. Interestingly, these authors also showed a marked increase in the migration of HT-29 cells following stimulation by MIP-3{alpha}. This finding is consistent with a previous report by Yang et al. (34) showing that apical stimulation of polarized HCA-7 intestinal epithelial cell monolayers by MIP-3{alpha} can lead to the phosphorylation of p130Cas, an adaptor protein that interacts with focal adhesion proteins regulating cell migration. Taken together, these findings indicate that, in addition to directing leukocyte recruitment, MIP-3{alpha} may also play an important role in regulating mucosal homeostasis in the intestine.

The main finding of the present study is that MIP-3{alpha} can induce phosphorylation of the EGFR in Caco-2 colonic epithelial cells, and that this appears to result from ectodomain shedding of the endogenous EGFR ligand amphiregulin. MIP-3{alpha}-induced EGFR phosphorylation then leads to activation of ERK1/2 signaling and increased proliferation (Fig. 10). Transactivation of the EGFR is thought be an important mechanism regulating proinflammatory and mitogenic signaling pathways in gastrointestinal epithelial cells. Indeed, previous studies from our group have shown that substance P, neurotensin and Clostridium difficile toxin B can induce EGFR transactivation and ERK1/2 activation in nontransformed NCM460 colonic epithelial cells via metalloproteinase-mediated release of TGF-{alpha} (24, 25, 35). Moreover, we have also shown that cag+ Helicobacter pylori, via shedding of heparin-binding EGF, can transactivate the EGFR in AGS gastric epithelial cells (36). Despite the wide variety of stimuli that can induce EGFR transactivation, there have been few studies demonstrating a role for chemokines in this process. Stimulation of NCI-H292 human bronchial epithelial cells by the CC chemokine eotaxin has been shown to induce EGFR transactivation as well as ERK1/2 MAPK activation (37). IL-8-mediated EGFR transactivation has also been previously reported in SK-OV-3 human ovarian cancer cells and human microvascular endothelial cells following stimulation by IL-8 (38). Studies by Tanida et al. (39) have demonstrated IL-8-mediated EGFR transactivation also occurs in KATO III gastric epithelial cells. Moreover, these investigators found that this activity was dependent on the metalloproteinase ADAM-10. Finally, the same group has recently reported that IL-8 can induce EGFR-dependent cell proliferation and migration of Caco-2 colonic epithelial cells (40). In contrast to the findings of this study, however, IL-8-mediated EGFR transactivation appeared to be mediated by ectodomain shedding of heparin-binding EGF rather than amphiregulin, suggesting that chemokine-dependent mitogenic signaling can be regulated by multiple pathways in colonic epithelial cells.

In this study, we show that pretreatment of Caco-2 colonic epithelial cells with TAPI-1, a specific inhibitor of TACE/ADAM-17, can significantly attenuate MIP-3{alpha}-mediated ERK1/2 activation. This finding indicates that EGFR-dependent mitogenic signaling in MIP-3{alpha}-stimulated Caco-2 cells is mediated, at least in part, by TACE/ADAM-17. These data are in keeping with a previous studies by Gschwind et al. (41, 42) demonstrating that processing of cell surface proamphiregulin by TACE/ADAM-17 mediates EGFR transactivation and MAPK activation in head and neck squamous carcinoma cells stimulated by LPA or carbachol. Another study has shown that TACE/ADAM-17 can induce ectodomain shedding of amphiregulin in NCI-H292 lung epithelial cells stimulated by cigarette smoke (43). Although TACE/ADAM-17 has emerged as a key mediator of amphiregulin ectodomain shedding in various cell types exactly, how this protein is regulated remains to be established. Previous studies have shown that the cytoplasmic domains of most ADAM family proteins contain multiple phosphorylation sites as well as proline rich regions capable of binding Src homology 3 domains (44). In keeping with this research, recent reports have suggested that the ERK and p38 MAPK signaling pathways can regulate TACE/ADAM-17-mediated EGFR ligand shedding (45). Several investigators have also demonstrated that TACE/ADAM-17 can be phosphorylated following stimulation by the phorbol ester PMA or growth factors (46, 47). Moreover, direct interactions between TACE/ADAM-17 and several intracellular proteins have also been reported (48, 49, 50). Whether any of these potential mechanisms participate in MIP-3{alpha}-mediated activation of TACE/ADAM-17 is the subject of ongoing investigations in our laboratory.

In summary, we show that MIP-3{alpha}, a CC chemokine primarily expressed by epithelial cells in the intestine, can activate mitogenic signaling cascades in colonocytes leading to increased cell proliferation. We also demonstrate that MIP-3{alpha} mitogenic signaling in colonic epithelial cells occurs via a mechanism involving metalloproteinase-dependent transactivation of the EGFR by amphiregulin. To our knowledge, this is the first demonstration that MIP-3{alpha} can mediate transactivation of the EGFR or that MIP-3{alpha} causes ectodomain shedding of amphiregulin. These findings indicate that in noninflamed colon MIP-3{alpha} may serve a homeostatic function by regulating tissue turnover and maintenance of the epithelium. Moreover during intestinal inflammation, increased MIP-3{alpha} production may facilitate enterocyte proliferation and tissue repair, in addition to its role in regulating leukocyte recruitment.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants DK075942, DK58858, AIO53069, and P01 DK033506 from the National Institutes of Health, by Grant DK43551 from the Massachusetts General Hospital/New England Regional Primate Research Center for the Study of Inflammatory Bowel Diseases, by a First Award from the Crohn’s and Colitis Foundation of America and the William and Shelby Modell Family Foundation (to A.C.K.), and by an American Gastroenterological Association/TAP Endowed Research Award in Acid Related Diseases (to S.K.). Back

2 Address correspondence and reprint requests to Dr. Andrew C. Keates, Division of Gastroenterology, Dana 501, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail address: akeates{at}bidmc.harvard.edu Back

3 Abbreviations used in this paper: IBD, inflammatory bowel disease; EGF, epidermal growth factor; EGFR, EGF receptor; TACE, TNF-{alpha} converting enzyme; TAPI, TNF-{alpha} protease inhibitor. Back

Received for publication July 13, 2006. Accepted for publication April 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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