Chemokine receptor CCR6 transduces signals that activate p130Cas and alter cAMP-stimulated ion transport in human intestinal epithelial cells

Charles C. Yang,* Hiroyuki Ogawa,* Michael B. Dwinell, Declan F. McCole, Lars Eckmann, and Martin F. Kagnoff

Laboratory of Mucosal Immunology, Departments of Medicine and Pediatrics, University of California, San Diego, La Jolla, California

Submitted 1 April 2004 ; accepted in final form 22 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human colon epithelial cells express the G protein-coupled receptor CCR6, the sole receptor for the chemokine CCL20 (also termed MIP-3{alpha}). CCL20 produced by intestinal epithelial cells is upregulated in response to proinflammatory stimuli and microbial infection, and it chemoattracts leukocytes, including CCR6-expressing immature myeloid dendritic cells, into sites of inflammation. The aim of this study was to determine whether CCR6 expressed by intestinal epithelial cells acts as a functional receptor for CCL20 and whether stimulation with CCL20 alters intestinal epithelial cell functions. The human colon epithelial cell lines T84, Caco-2, HT-29, and HCA-7 were used to model colonic epithelium. Polarized intestinal epithelial cells constitutively expressed CCR6, predominantly on the apical side. Consistent with this, apical stimulation of polarized intestinal epithelial cells resulted in tyrosine phosphorylation of the p130 Crk-associated substrate (Cas), an adaptor/scaffolding protein that localizes in focal adhesions and has a role in regulating cytoskeletal elements important for cell attachment and migration. In addition, CCL20 stimulation inhibited agonist-stimulated production of the second messenger cAMP and cAMP-mediated chloride secretory responses by intestinal epithelial cells. Inhibition was abrogated by pertussis toxin, consistent with signaling through G{alpha}i proteins that negatively regulate adenylyl cyclases and cAMP production. These data indicate that signaling events, occurring via the activation of the apically expressed chemokine receptor CCR6 on polarized intestinal epithelial cells, alter specialized intestinal epithelial cell functions, including electrogenic ion secretion and possibly epithelial cell adhesion and migration.

CCL20; macrophage inflammatory protein-3{alpha}; forskolin; G protein-coupled receptors; tyrosine phosphorylation


THE SINGLE LAYER of epithelial cells that lines the intestinal tract is a key component of a signaling network that is important for host defense against enteric microbes. Intestinal epithelial cells can generate mediators that chemoattract and activate immune and inflammatory cells after microbial infection (3, 12, 16, 23, 25, 34, 52). In addition, intestinal epithelial cells respond to cytokines that are produced by mucosal immune and inflammatory cells by altering gene expression and other functions such as electrolyte transport (13, 14, 25, 52). Intestinal epithelial cells are structurally and functionally polarized into apical and basolateral domains. Consistent with the subepithelial and intraepithelial production of proinflammatory mediators by mucosal immune and inflammatory cells, receptors for proinflammatory cytokines (e.g., IL-1, TNF-{alpha}, and IFN-{gamma}) on intestinal epithelial cells are expressed mainly on the basolateral cell surface.

We and others recently reported that human intestinal epithelial cells express receptors for several members of the chemokine family (e.g., CXCR4, CCR5, CCR6, and CX3CR1) (4, 10, 24). Chemokine receptors are seven-transmembrane G protein-coupled receptors, and signaling through those receptors on leukocytes generally, but not exclusively, occurs through pertussis-sensitive G{alpha}i subunits (38). This also appears to be the case for CXCR4 and CX3CR1 expressed by intestinal epithelial cells (4, 10, 11, 24).

CCR6 is the sole known receptor for the chemokine CCL20 [also termed macrophage inflammatory protein-3{alpha} (MIP-3{alpha}) (41), liver and activation-regulated chemokine (LARC) (18), or Exodus-1 (21)]. CCR6 is expressed by immature myeloid lineage dendritic cells, which can migrate to the subepithelial region of mucosal surfaces in response to CCL20 stimulation, and by circulating memory T cells in humans that express the {alpha}4{beta}7-integrin characteristic of mucosal homing lymphocytes (5, 9, 22, 30, 42, 51).

We recently noted that CCR6 is expressed by human colon epithelial cell lines and by human colonic epithelium in vivo (23). In response to bacterial infection or stimulation with proinflammatory mediators (e.g., TNF-{alpha}, IL-1), intestinal epithelial cells upregulate mRNA expression and production of CCL20 protein as well as human {beta}-defensin 2 (hBD2), an antimicrobial peptide that also has been reported to signal dendritic cells through CCR6 (51). Notably, CCL20 was secreted predominantly from the basolateral and not the apical membrane of polarized monolayers of intestinal epithelial cells (23). These studies raised the question as to whether CCL20 potentially mediates its effects on the intestinal epithelium as a paracrine/autocrine factor acting through basolaterally expressed CCR6 or whether the expression of CCR6 by intestinal epithelial cells is apically polarized and not likely to have contact with CCL20 in the presence of a normal epithelial barrier.

In the present study, we investigated whether CCR6 acts as a functional signaling receptor in intestinal epithelial cells and examined its distribution on those cells. We report that CCR6 expressed by intestinal epithelial cells is apically polarized and acts as a functional G protein-coupled receptor in these cells. CCL20 stimulation of CCR6-expressing human intestinal epithelial cells results in tyrosine phosphorylation of an adaptor/scaffolding protein, p130Cas, a key component of the pathway by which focal adhesion kinase (FAK) promotes cell migration. In addition, CCR6 stimulation results in the modulation of cAMP-stimulated electrogenic chloride secretion.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Reagents. Recombinant human CCL20 and mouse monoclonal antibody (MAb) to human CCR6 (clone 53103) were obtained from R&D Systems (Minneapolis, MN). Rabbit antibodies to phospho-Akt (Ser473), Akt, and phospho-PKC (pan) and mouse MAb to phosphotyrosine (Tyr-100) were obtained from Cell Signaling (Beverly, MA). Biotin-conjugated mouse MAb to phosphotyrosine (IgG2b) (RC-20) and MAb to p130Cas (IgG1) were obtained from Transduction Laboratories (San Diego, CA). Somatostatin, forskolin, pertussis toxin, 3-isobutyl-1-methylxanthine (IBMX), protease inhibitor cocktail set III, phorbol 12-myristate 13-acetate (PMA), and protein G-agarose were obtained from Calbiochem (La Jolla, CA). Prostaglandin E2 (PGE2) and vasoactive intestinal peptide (VIP) were obtained from Sigma Chemical (St. Louis, MO). Cy3-conjugated goat anti-mouse IgG was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Alexa 488-conjugated phalloidin and Alexa 488-conjugated goat anti-rabbit IgG were obtained from Molecular Probes (Eugene, OR). Biotin-conjugated sheep anti-mouse IgG or anti-rabbit Ig horseradish peroxidase (HRP) and streptavidin-HRP were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Normal goat IgG and monoclonal mouse IgG1 were obtained from Sigma Chemical. R-phycoerythrin-labeled goat anti-mouse IgG was obtained from Southern Biotechnology (Birmingham, AL).

Human colon epithelial cell lines. The human colon adenocarcinoma cell lines HCA-7, Caco-2, and HT-29 were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 or 15% (Caco-2) heat-inactivated fetal bovine serum and 2 mM L-glutamine as described previously (10). T84 human colon carcinoma cells were grown in 50% DMEM-50% Ham's F-12 medium supplemented with 5% newborn calf serum and 2 mM L-glutamine (25). The cells were maintained in 95% air-5% CO2 at 37°C.

Flow cytometry. Confluent T84, Caco-2, HCA-7, and HT-29 cells (~1 x 106 cells) were detached with 20 mM EDTA in phosphate-buffered saline (PBS), fixed for 10 min at 4°C in 2% paraformaldehyde in PBS, and subsequently incubated for 90 min at 4°C with 10 µg/ml mouse anti-human CCR6 MAb or a mouse isotype-matched control antibody, followed by incubation for 1 h with R-phycoerythrin-labeled goat anti-mouse IgG. In other experiments, paraformaldehyde-fixed cells were permeabilized by inclusion of 0.1% saponin in the dilution and wash buffers. Cells were analyzed using flow cytometry (FACScan; Becton Dickinson, Sunnyvale, CA).

Confocal microscopy. Polarized HCA-7 cells were cultured on microporous collagen-coated filter inserts (0.4-µm pore size, Transwell; Costar, Cambridge, MA) and allowed to grow for approximately 7 days as described previously (31), at which time they manifested a resistance of 500–700 {Omega}·cm2. Cells were fixed in 4% paraformaldehyde in PBS for 10 min at 4°C, followed by incubation in PBS/1% bovine serum albumin (BSA) with 10% goat serum for 1 h at room temperature. Cells were then incubated at 37°C for 4 h with mouse anti-human CCR6 MAb (10 µg/ml) or a mouse monoclonal IgG2b isotype control applied from both the apical and basolateral sides. Cells were subsequently washed and incubated with Cy3-labeled goat anti-mouse IgG as secondary antibody and Alexa 488-coupled phalloidin at 4°C overnight. Filters were removed, mounted on glass slides, and examined using laser scanning confocal microscopy (MRC Bio-Rad 500; Bio-Rad Laboratories, Hercules, CA).

Immunoprecipitation and immunoblotting. HCA-7, Caco-2, and T84 cells were washed in DMEM or 50% DMEM-50% Ham's F-12 (T84) medium supplemented with 1% BSA and 2 mM L-glutamine, allowed to equilibrate for 1 h at 37°C, and then stimulated with CCL20 or PMA. Cells were washed in ice-cold PBS and lysed in ice-cold lysis buffer [10 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 0.5% protease inhibitor cocktail set III] for 30 min, sonicated, and spun at 14,000 rpm for 10 min, after which supernatants were assayed for protein content (Bio-Rad protein assay kit). Protein (20 µg/lane) was electrophoresed on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Hybond ECL; Amersham Pharmacia). Membranes were blocked with Tris-buffered saline (TBS) containing 10% nonfat dry milk, 1% donkey serum, and 0.1% Tween 20 and then incubated overnight at 4°C in a 1:1,000 dilution of rabbit antibodies against the different signaling molecules in dilution buffer (1% nonfat dry milk, 1% donkey serum, 0.1% Tween 20 in TBS). Blots were washed and incubated for 1 h with donkey anti-rabbit Ig-HRP. Alternatively, membranes were developed using biotin-conjugated sheep anti-rabbit IgG followed by streptavidin-HRP. Western blots were developed using enhanced chemiluminescence agents according to the manufacturer's instructions (Amersham) and were exposed to imaging film (XAR; Kodak).

For immunoprecipitation, equal concentrations of cell lysates were incubated overnight with anti-phosphotyrosine MAb (Tyr-100; 5 µg/ml), after which protein G-agarose was added. After centrifugation, pellets were extensively washed in lysis buffer, resuspended in 2x gel loading buffer [50 mM Tris (pH 6.8), 2% SDS, 200 mM dithiothreitol, 20% glycerol, and 0.2% bromphenol blue], and boiled before separation by SDS-polyacrylamide gel electrophoresis. Resolved proteins were transferred to a nitrocellulose membrane and probed with anti-p130Cas MAb followed by biotin-conjugated sheep anti-mouse IgG and streptavidin-HRP and then developed as described above. Total cell lysates of polarized HCA-7 cells stimulated either apically or basolaterally with CCL20 or PMA were incubated with anti-phosphotyrosine MAb, precipitated, transferred to nitrocellulose membranes, and probed with mouse anti-p130Cas followed by HRP-conjugated sheep anti-mouse IgG as described above.

cAMP assays. Confluent T84 cells in 12-well plates were washed with HEPES-buffered Ringer solution and incubated with 1 mM IBMX. After 10 min, CCL20 or somatostatin was added. After an additional 10 min, 1 µM forskolin, 0.1 µM PGE2, or 1 nM VIP was added, and cells were incubated for 10 min. In some cases, cells were pretreated with pertussis toxin (100 ng/ml) for 18 h before CCL20 stimulation, as indicated. cAMP levels in cell extracts were assayed using a cAMP enzyme immunoassay system (Amersham Pharmacia Biotech). In other experiments, confluent T84 cells were stimulated with CCL20 (100 ng/ml) for 90, 60, 30, 15, or 10 min before the addition of forskolin. IBMX (1 mM) was added 10 min before forskolin stimulation. Cells were incubated for 10 min after the addition of forskolin (1 µM), and cAMP levels were measured as described above. In time course studies, confluent T84 cells were treated for 10 min with CCL20, and cAMP levels were measured at 0, 5, 10, and 30 min after forskolin stimulation. To parallel conditions in the electrophysiological studies described below, the latter experiments were done in the absence of IBMX.

Electrophysiological studies. Vectoral ion transport was examined in modified Ussing chambers as described previously (26). The mucosal and serosal baths contained Ringer buffered salt solution supplemented with glucose (115 mM NaCl, 25 mM NaHCO3, 2.4 mM K2HPO4, 0.4 mM KH2PO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose; adjusted to pH 7.4), which was gassed with 95% O2-5% CO2 at 37°C. The transepithelial voltage clamp connected to the Ussing chambers provided continuous monitoring of short-circuit current (Isc). Open-circuit potential difference was measured every 1–5 min, and transepithelial resistance was calculated using Ohm's law. After initial stabilization, baseline measurements of Isc, potential difference, and conductance were recorded for 40 min. Subsequently, agonists were added to the apical and basolateral baths to stimulate chloride secretion, and changes in electrophysiological parameters were recorded at regular intervals. These studies were done in the absence of IBMX.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Expression of CCR6 by intestinal epithelial cell lines. CCR6 is expressed by normal human intestinal epithelium in vivo (23). To study the functional significance of CCR6 expression, we used cultured lines of human colon epithelial cells as model epithelia. As shown in Fig. 1 for T84 cells, human colon epithelial cells constitutively expressed cell surface CCR6, and this also was the case for three other human colon epithelial cell lines: HCA-7, Caco-2, and HT-29 cells (not shown). Stimulation of those cell lines with IFN-{gamma} (40 ng/ml), granulocyte-macrophage colony-stimulating factor (20 ng/ml), TNF-{alpha} (20 ng/ml), or IL-1 (20 ng/ml) for 6 or 24 h did not significantly affect CCR6 mRNA levels or surface or intracellular expression levels of CCR6, even though in other studies (25, 52) and in data not shown these cell lines respond to the above cytokines at those concentrations by altering the expression of several other cellular mediators.



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Fig. 1. Constitutive chemokine receptor CCR6 expression by intestinal epithelial cells. T84 cells were immunostained for CCR6 and analyzed using flow cytometry. Open area depicts CCR6 staining, and closed area shows staining with an isotype control. Similar results were obtained in 4 repeated experiments using T84, HCA-7, HT-29, and Caco-2 cells.

 
CCL20 inhibits forskolin-stimulated cAMP production. G{alpha}i/G{alpha}o subunits play a key role in chemokine receptor signaling in leukocytes (40). Signaling through G{alpha}i inhibits the activity of adenylyl cyclases and the formation of the second messenger cAMP, which plays a pivotal role in stimulating intestinal epithelial cell chloride secretion. CCL20 stimulation did not affect baseline cAMP levels in T84 (Fig. 2), Caco-2, and HCA-7 cells (data not shown). We therefore examined whether CCL20 modulates epithelial cAMP levels induced by other agonists. Forskolin is a potent stimulator of adenylyl cyclases and cAMP production in intestinal epithelial cells, and, as shown in Fig. 2A, stimulation of T84 cells with CCL20 inhibited forskolin-stimulated cAMP production in a concentration-dependent manner. Moreover, maximal inhibition of cAMP with CCL20 approximated the inhibition of forskolin-stimulated secretion that occurred with somatostatin, which was used as a positive control for activating a G protein-coupled receptor that signals intestinal epithelial cells via G{alpha}i subunits and inhibits adenylyl cyclases and cAMP production (47, 48). Pertussis toxin reversed the inhibitory effect of CCL20 on forskolin-induced cAMP production with a magnitude similar to its effects on somatostatin-mediated inhibition of cAMP production (Fig. 2B), suggesting that CCL20 signaling involved G{alpha}i subunits. Inhibition of cAMP occurred when cells were stimulated with CCL20 up to 15 min before forskolin stimulation (Fig 2C). Moreover, CCL20 decreased cAMP production in response to forskolin over a 30-min period after stimulation (Fig. 3A).



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Fig. 2. CCL20 inhibition of forskolin (FSK)-stimulated cAMP production. A: T84 cells were left untreated or incubated with increasing concentrations of CCL20 for 10 min, after which cells were stimulated with FSK (1 µM) for 10 min and cellular cAMP production was assayed. Values are means ± SE of 3–6 experiments. *P < 0.05 compared with FSK alone. B: CCL20 (20 ng/ml) inhibited FSK-stimulated (1 µM) cAMP production to an extent similar to that of somatostatin (SST; 1 µM) used as a positive control. In both cases, inhibition was reversed by pertussis toxin (PTX; 100 ng/ml). Values are means ± SE of 4–6 repeated experiments. C: T84 cells were preincubated with CCL20 (100 ng/ml) or SST (1 µM) for the indicated times before stimulation with FSK for 10 min, after which cAMP production was assayed. Values are means ± SE of 2 experiments.

 


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Fig. 3. Time course of CCL20 inhibition of FSK-stimulated chloride secretion and inhibition of cAMP production. A: time course of cAMP production by T84 cells treated ({square}) or not ({bullet}) with CCL20 (200 ng/ml) for 10 min before FSK stimulation at time 0. B: CCL20 (200 ng/ml) or SST (1 µM) was added bilaterally to confluent monolayers of polarized T84 epithelial cells mounted in Ussing chambers 10 min before stimulation with FSK (0.5 µM) at time 0 (indicated by arrow). CCL20 ({square}) and SST ({triangleup}) significantly attenuated the secretory response compared with FSK alone ({bullet}) as assessed by changes in short-circuit current (Isc) (CCL20 + FSK vs. FSK alone, P < 0.05, n = 3; SST + FSK vs. FSK alone, P < 0.05, n = 3). Data are expressed as means ± SE and represent increases in Isc ({Delta}Isc) measured in µA/cm2.

 
PGE2 is released during microbial infection and mucosal inflammation and, acting through cell surface prostaglandin receptors, stimulates adenylyl cyclases and cAMP production (50). VIP is also a potent receptor-mediated stimulator of adenylyl cyclase and cAMP in T84 cells (8). CCL20 pretreatment of T84 cells inhibited both PGE2 (0.1 µM)- and VIP (1 nM)-stimulated cAMP production [%inhibition (means ± SE): PGE2, 25.3 ± 3.0, P < 0.05, n = 4; VIP, 32.0 ± 5.7%, P < 0.05, n = 3].

CCL20 inhibits forskolin-stimulated chloride secretion by intestinal epithelial cells. Increased electrogenic chloride secretion is an important functional consequence of increased cAMP levels in intestinal epithelial cells and is primarily responsible for promoting intestinal fluid secretion and secretory diarrhea. Therefore, we investigated whether CCL20 inhibition of forskolin-stimulated increases in cAMP were accompanied by decreased forskolin-stimulated epithelial cell chloride secretion, as assessed by alterations in Isc in Ussing chambers. Pretreatment of polarized T84 cells with CCL20 significantly reduced the peak Isc response to forskolin from 39 ± 5 µA/cm2 to 25 ± 6 µA/cm2, a decrease of 39 ± 7% (P < 0.05, n = 3 experiments) (Fig. 3B). The magnitude of this decrease was comparable to that seen after pretreatment of T84 monolayers with somatostatin (1 µM) before forskolin stimulation of electrogenic chloride secretion (39 ± 5%, P < 0.05, n = 3 experiments). These data indicate that CCL20 can function as a negative regulator of intestinal epithelial cell chloride secretion and are consistent with the observed decrease in cAMP production by CCL20-treated cells over the same time frame (Fig. 3A).

Because chemokines activate calcium fluxes in leukocytes (38) and calcium fluxes in intestinal epithelial cells also stimulate epithelial cell chloride secretion (45), in further studies we examined the ability of CCL20 to stimulate calcium fluxes in colonic epithelial cells. CCL20 stimulation did not affect baseline intracellular Ca2+ levels or basal Isc in T84 cells, which is consistent with the absence of calcium fluxes noted after stimulation with other chemokines (11) Although stimulation with a calcium ionophore, ionomycin, induced significant calcium fluxes in T84, Caco-2, and HCA-7 cells, stimulation with CCL20, as in T84 cells, induced little or no change in intracellular Ca2+ levels in Caco-2 or HCA-7 cells (data not shown), indicating that the inhibitory effects of CCL20 effects on epithelial chloride secretion are likely to be specific for the cAMP-mediated chloride secretory pathway.

CCL20 stimulates tyrosine phosphorylation of p130Cas. To further investigate pathways by which CCR6 transduces signals into human colon epithelial cells, we stimulated HCA-7, T84, and Caco-2 cells with CCL20 and assessed the phosphorylation of Akt as an important downstream effector of signaling through phosphoinositide 3-kinase and the conventional PKC{alpha}/{beta} and novel PKC{delta} isoforms as important signal transduction molecules downstream of the lipid second messenger diacylglycerol. CCL20 stimulation did not result in phosphorylation of Akt or the PKC isoforms, although Akt was phosphorylated by signaling through the EGF receptor and the PKC isoforms were activated by stimulation with phorbol esters (data not shown). We next assessed whether CCL20 increases tyrosine phosphorylation of cellular proteins. Interestingly, these studies revealed a marked increase in tyrosine phosphorylation of a protein with a molecular mass of ~130 kDa within 1 min of CCL20 stimulation, which peaked by 5–10 min and began to decrease by 15 min (Fig. 4A).



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Fig. 4. CCL20 stimulation increases phosphorylation of p130Cas protein. A: total cell lysates from HCA-7, T84, and Caco-2 cells stimulated with CCL20 (200 ng/ml) for 1, 5, 10, and 15 min were transferred to nitrocellulose membranes and probed with biotin-conjugated anti-phosphotyrosine antibody. Data are from a representative experiment. Similar data were obtained in 3 repeated experiments. B: HCA-7 cells were stimulated for the indicated times with CCL20 (200 ng/ml) or PMA (1 µg/ml), after which phosphotyrosine immunoprecipitates prepared from total cell lysates (top) or nonprecipitated total cell lysates as controls (bottom) were transferred to nitrocellulose membranes and probed with p130Cas MAb. Data are from a representative experiment. Similar data were obtained in 4 repeated experiments. Relative increases compared with controls were determined by densitometry and are indicated by numbers beneath each sample.

 
Chemoattractant cytokine signaling in leukocytes results in the phosphorylation of adaptor molecules that are important in the formation of focal adhesions (38). p130Cas is an adaptor/scaffolding protein in a signaling cascade that leads to the formation of focal adhesions (6, 27, 29, 44). Therefore, we tested the possibility that the prominent phosphotyrosine-containing band at ~130 kDa after CCL20 stimulation might reflect the tyrosine phosphorylation of p130Cas. As shown in Fig. 4B, this was, in fact, the case. Tyrosine phosphorylation of p130Cas occurred within 1 min of CCL20 stimulation of HCA-7 cells, whereas total levels of p130Cas remained unchanged.

CCR6 expression is predominately apical in polarized colon epithelial cells. Intestinal epithelial cells are morphologically and functionally polarized in vivo. To assess the distribution of CCR6 on polarized intestinal epithelial cells, HCA-7 cells, grown as polarized monolayers on microporous filter supports, were immunostained for CCR6 and examined using confocal microscopy. As shown in Fig. 5, the most intense staining for CCR6 was in the apical region. A similar apical distribution of CCR6 was found in polarized T84 and Caco-2 cells (not shown).



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Fig. 5. Distribution of CCR6 on polarized HCA-7 cells. A–D: confocal images of polarized HCA-7 monolayers stained with anti-CCR6 MAb (red fluorescence). E–H: polarized HCA-7 cells costained with anti-CCR6 and Alexa 488-phalloidin for F-actin (green fluorescence). I–L: polarized HCA-7 monolayers costained with isotype control antibody and Alexa 488-phalloidin. Apical sections (A, E, and I), midsections (B, F, and J), and basolateral sections (C, G, and K) are shown. Images in D, H, and L are the respective X-Z reconstructions. Original magnification, x400. Data are from a single experiment and are representative of those obtained in 3 repeated experiments.

 
Apical CCL20 stimulation of polarized colon epithelial cells CCL20 activates p130Cas phosphorylation and inhibits forskolin-stimulated cAMP production. Given the predominant apical distribution of CCR6 on polarized HCA-7 cells, we next determined whether phosphorylation of p130Cas and inhibition of forskolin-stimulated cAMP production occur after apical stimulation with CCL20. Polarized monolayers of HCA-7 cells were stimulated with CCL20 added to either the apical or basolateral chamber of Transwell cultures, after which total cell lysates were immunoprecipitated with anti-phosphotyrosine antibody and immunoblotted with an antibody to p130Cas. Phosphorylation of p130Cas was most marked after apical stimulation of polarized HCA-7 cells with CCL20 (Fig. 6A). Similarly, as shown for polarized monolayers of T84 cells cultured in Transwells, apical stimulation with CCL20 significantly inhibited forskolin-stimulated cAMP production, whereas basolateral CCL20 stimulation did not (Fig. 6B). Together, these finding indicate that signaling through CCR6 on polarized intestinal epithelial cells is more marked after apical rather than basolateral stimulation with CCL20.



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Fig. 6. Apical CCL20 stimulation of polarized intestinal epithelial cells phosphorylates p130Cas and inhibits FSK-stimulated cAMP production. A: p130Cas was phosphorylated after apical stimulation of HCA-7 cells with CCL20. CCL20 [also termed macrophage inflammatory protein-3{alpha} (MIP-3{alpha}); 200 ng/ml] was added to the apical or basal chamber of polarized HCA-7 monolayers in Transwell cultures for 10 min, after which total cell lysates were isolated, immunoprecipitated with anti-phosphotyrosine antibody, transferred to nitrocellulose membranes, and probed with anti-p130Cas. Cultures were stimulated with PMA (1 µg/ml) as a control. Data are from a representative experiment. Relative increases compared with controls were determined by densitometry and are indicated by numbers beneath each sample. Similar data were obtained in 3 repeated experiments. B: polarized T84 cells were incubated for 10 min with CCL20 (200 ng/ml), added to either the apical (CCL20 A) or basolateral (CCL20 B) chamber of T84 monolayers in Transwell cultures as indicated, after which FSK (1 µM) was added to both chambers and cAMP levels were determined. SST (1 µM) was added to both chambers for 10 min before FSK stimulation. Data are from a representative experiment. *P < 0.05 compared with FSK alone. **P < 0.05 compared with basolateral stimulation. Similar data were obtained in 2 repeated experiments. NS, not significant.

 

    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Stimulation of intestinal epithelial cells with CCL20, which is the only known chemokine ligand for the chemokine receptor CCR6, generated cellular signals that resulted in the phosphorylation of p130Cas, inhibition of agonist-stimulated cAMP production, and, as a functional correlate of the latter, inhibition of agonist-stimulated electrogenic chloride secretion. Prior work on CCR6 had mostly focused on leukocytes (1, 30), in which signaling through G protein-coupled chemokine receptors characteristically activates leukocyte chemotaxis. Consistent with this, CCR6 has a role in the population of the intestinal mucosa with myeloid dendritic cells and CCR6-expressing lymphoid cells and in the chemoattraction of dendritic cells into the proximity of the intestinal epithelium, as shown by in vitro studies and studies in mice genetically deficient in CCR6 (7, 28). Our studies emphasize that CCR6 is a functional receptor on intestinal epithelial cells and is likely to have functions beyond regulating leukocyte chemotaxis.

Electrogenic chloride secretion, induced by the second messenger cAMP, is the major driving force for intestinal epithelial cell ion secretion (2). Chemokine receptors are G protein-coupled receptors that transduce signals through pertussis toxin-sensitive G{alpha}i subunits (11, 35), which results in inhibition of adenylyl cyclases and cAMP production. Stimulation with CCL20, at concentrations that are produced by human intestinal epithelial cells (23), attenuated agonist-stimulated cAMP production and electrogenic chloride secretion by intestinal epithelial cells, and, consistent with signaling through G{alpha}i subunits, this inhibition was abrogated by pertussis toxin. Moreover, inhibition of agonist-stimulated cAMP production and its reversal by pertussis toxin was similar in CCL20- and somatostatin-treated cells, with the latter being a prototypic agonist that activates G{alpha}i subunits and inhibits adenylyl cyclases and cAMP (47, 48).

cAMP-dependent chloride secretion in colonic epithelial cells is dependent on cytoskeletal remodeling (17, 32, 43). Stabilization of F-actin with phalloidin, which prevents actin depolymerization, markedly inhibits forskolin-stimulated activity of the Na+-K+-2Cl cotransporter (NKCCl), a key component of the chloride secretory pathway (32, 33). Although we have shown that CCL20 partially inhibits forskolin-stimulated chloride secretion, doubtless due at least in part to a reduction in cAMP production, a possible influence of CCL20 on the cytoskeletal component of forskolin-stimulated ion transport should not be ruled out. Indeed, CCL20 stimulation of human colon epithelial cells resulted in tyrosine phosphorylation of p130Cas, an adaptor/scaffolding protein that associates with cytoskeletal and other focal adhesion proteins involved in cell adhesion and chemotaxis (6, 15, 37, 46). Cell migration requires the regulation of kinases and phosphatases, which modulate the phosphorylation and dephosphorylation of adaptor/scaffolding molecules in adhesions. Although little information is available regarding the activity of p130Cas in intestinal epithelial cells, in other cell types (e.g., fibroblasts) the FAK-Src complex mediates p130Cas tyrosine phosphorylation (49). Moreover, actin filament assembly and actin stress fiber organization is abnormal in p130Cas-deficient fibroblasts (19, 20). Our findings suggest the possibility that CCL20, in addition to its inhibitory effects on cAMP-dependent chloride secretion, has a role in epithelial cell adhesion/migration. If this is the case, it is tempting to speculate that alterations in intestinal epithelial cell adhesion/migration in response to CCL20 might also facilitate the passage of dendritic cell processes between epithelial cells (39).

Intestinal epithelial cells produce CCL20 and constitutively express its cognate receptor, CCR6 (23), which suggests that CCL20 can mediate autocrine/paracrine effects on intestinal epithelial cells. The gene encoding CCL20 is transcriptionally regulated by NF-{kappa}B in human intestinal epithelial cells, and CCL20 is produced and secreted by those cells in response to proinflammatory mediators, microbial, infection and microbial products (e.g., bacterial flagellin) (23). The question then arises as to how, and under what conditions, CCL20 produced by intestinal epithelial cells alters intestinal epithelial cell functions. Our findings suggest that the answer to this question lies in the selective apical distribution of CCR6 on polarized intestinal epithelial cells. Stimulation of polarized intestinal epithelial cells with proinflammatory cytokines such as TNF-{alpha} or IL-1, which are produced by cells in the lamina propria and therefore stimulate the basolateral domains of epithelial cells, results in the basolateral secretion of CCL20 (23). Under those conditions, CCL20 would act mainly as a chemoattractant for target leukocyte populations in the lamina propria (e.g., dendritic cells, B cells) (7, 28). However, CCL20 access to the apical membrane could occur during inflammatory reactions or microbial infections sufficient to alter barrier integrity of the intestinal epithelium. In this case, CCL20 could signal epithelial cells through apical CCR6 receptors, leading to downregulation of epithelial secretory processes and changes in epithelial cell adhesion/migration that might favor restoration of the epithelial barrier. Alternatively, it should be considered that apically expressed CCR6 might serve as a target for ligands other than CCL20. For example, the antimicrobial peptide hBD2 is an NF-{kappa}B target gene that is upregulated in intestinal epithelium in response to proinflammatory signals and has been reported to signal leukocytes through CCR6 (36, 51). Consistent with the present data, we also have found CCR6 to be predominately apically expressed in human intestinal epithelium in vivo.


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 ABSTRACT
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-35108 and DK-58960 (to M. F. Kagnoff). H. Ogawa was supported in part by a research fellowship from the Yamada Science Foundation. C. C. Yang and M. B. Dwinell were supported by National Institutes of Health Training Grant DK-07202.


    ACKNOWLEDGMENTS
 
Present addresses: M. B. Dwinell, Dept. of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI; H. Ogawa, Dept. of Molecular Medicine, Osaka University Medical School, Osaka, Japan.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. Kagnoff, Laboratory of Mucosal Immunology, Univ. of California, San Diego, Mail Code 0623, 9500 Gilman Drive, La Jolla, CA 92093-0623 (E-mail: mkagnoff{at}ucsd.edu)

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.

* C. C. Yang and H. Ogawa contributed equally to this work. Back


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