Department of Medicine, University of California, San Diego, La Jolla, California 92093-0623
Submitted 11 March 2003 ; accepted in final form 11 December 2003
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ABSTRACT |
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chemokine; chemokine receptors; G protein-coupled receptors; ion transport; adenosine 3',5'-cyclic monophosphate; stromal cell-derived factor-1; CXC chemokine ligand; CXC receptor
Biological activity of SDF-1/CXCL12 has concentrated largely on its role in the directed migration of hematopoietic precursor cells, embryogenesis, B lymphopoiesis, and, by occupancy of its cognate receptor, inhibition of HIV-1 infection of susceptible cells (26, 30, 34, 44). In addition, CXCR4 and SDF-1/CXCL12 have been reported to have a role in trafficking of metastatic breast carcinoma cells, the movement of maturing thymocytes out of the thymus, in the pathogenesis of rheumatoid arthritis, as well as in mitogenesis, cytotoxicity, and apoptosis of specific cell types (16, 18, 21, 24, 31, 42).
Cells of the intestinal epithelium function primarily in nutrient absorption and electrolyte transport and form a physical barrier between the external luminal environment and the host interior. Intestinal epithelial cells participate in innate mucosal defense through the constitutive expression and production of chemokines, cytokines, antimicrobial peptides, and growth factors (1214, 19, 22, 32, 36, 45, 48). Furthermore, receptors for neurotransmitters, growth factors, hormones, and cytokines that are expressed on the surface of polarized intestinal epithelial cells convey signals to epithelial cellular effectors and genes important in the constitutive and regulated maintenance of epithelial functions (8, 11, 25). We and others have recently reported that human intestinal epithelial cells express several chemokine receptors, including CXCR4, CCR5, CCR6, and CX3CR1 (8, 11, 23), thereby rendering these cells potential targets for chemokine signals. Our prior report indicated that CXCR4 was localized to both apical and basolateral cellular domains of normal human colonic epithelium in vivo (11).
The expression of CXCR4 by intestinal epithelial cells, and its cognate ligand SDF-1/CXCL12, by cells within the intestinal mucosa suggests that CXCR4 plays a role in intestinal mucosal function. Chemokine receptors, including CXCR4, are members of the G protein-coupled receptor superfamily and, on leukocytes, are primarily coupled to Gi protein subunits (38). In previous studies, ligand activation of CXCR4 on human colon epithelial cells was shown to upregulate the expression of neutrophil chemoattractants (e.g., IL-8/CXCL8 and growth-regulated oncogene-
/CXCL1) and intercellular adhesion molecule-1 (11, 23).
Epithelial cell secretory functions are known to be influenced by several G protein-coupled receptors, including those that regulate production of the second messenger cAMP (4). We show herein that activation of epithelial cell CXCR4 by SDF-1/CXCL12 regulates G protein-dependent cAMP production and epithelial cell Cl- secretion. These data support the notion that activation of epithelial cell CXCR4 by SDF-1/CXCL12 plays a role in the homeostatic regulation of epithelial cell electrolyte transport.
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MATERIALS AND METHODS |
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Cell culture. Human T84 colon carcinoma cells (10) were grown in 50% DMEM-50% Ham's F-12 medium supplemented with 5% newborn calf serum and 2 mM L-glutamine as described before (11). The human colon epithelial cell lines HT-29 (HTB 38) and Caco-2 (HTB 37) were obtained from the ATCC (Manassas, VA) and cultured in DMEM supplemented with 10% FBS and 2 mM L-glutamine. HT-2918.C1 cells (provided by M. H. Montrose, Indiana University) were cultured in DMEM high glucose (4.5 g/l) supplemented with 10% FBS, 2 mM L-glutamine, and 10 µg/ml apotransferrin. Polarized T84 and Caco-2 epithelial monolayers were grown on mixed cellulose ester filters (0.45 µm pore size, Millicell-HA; Millipore, Bedford, MA). Cells were maintained in 95% air-5% CO2 at 37°C.
Preparation of cell lysates, membrane, and cytosolic proteins. T84 cells were solubilized for 30 min in ice-cold lysis buffer [50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors (Protease Inhibitor Cocktail III; CalBiochem)], and samples were cleared by centrifugation. To separate membrane and cytosolic protein fractions, T84 cells were solubilized in hypotonic lysis buffer (5 mM Tris·HCl, pH 7.4, 5 mM EDTA, and protease inhibitors) and centrifuged for 5 min at 4°C at 2,500 g to pellet the nuclei. This postnuclear supernatant fraction was, in turn, ultracentrifuged (30 min at 40,000 g, rotor TLA100.3; Beckman). The cytosolic fraction was then removed, and the pellet containing cell membranes was diluted in PBS, concentrated using ammonium sulfate precipitation, and resuspended in PBS supplemented with protease inhibitors. Protein concentrations were determined by a modified Lowry assay (DC Protein Assay; Bio-Rad, Hercules, CA).
Immunoblotting. Equal amounts of total cell lysates, membrane, or cytosolic fractions were suspended in sample buffer (50 mM Tris·HCl, pH 6.8, 2% SDS, 0.01% bromphenol blue, 10% glycerol, and 2% -mercaptoethanol), size-separated using 10% SDS-PAGE, and electrotransferred to polyvinylidene difluoride membranes (Immobilon-P; Amersham Pharmacia). Blots were fixed in methanol and blocked in Tris-buffered saline (TBS; 50 mM Tris base, pH 7.2, 150 mM NaCl, and 2.6 mM KCl) containing 5% dry milk and 0.1% Tween 20, followed by overnight incubation at 4°C with rabbit anti-CXCR4 in TBS, 5% dry milk, and 0.1% Tween 20. Blots were washed in TBS-Tween, incubated with HRP-conjugated donkey anti-rabbit Ig, and visualized using enzyme chemiluminescence (SuperSignal West-Pico; Pierce, Rockford, IL). Equal protein loading was verified by Coomassie blue staining.
Analysis of mRNA expression by RT-PCR. Isolated total cellular RNA (1 µg) was reverse transcribed as described before (11). CXCR4 and SDF-1/CXCL12 mRNA were amplified from cDNA by PCR using specific primers. Primers to SDF-1/CXCL12 were sense primer 5'-TGA GCT ACA GAT GCC CAT GC-3' and antisense primer 5'-TTC TCC AGG TAC TCC TGA ATC C-3', designed from published cDNA sequences (GenBank accession no. U16752
[GenBank]
). The primers used do not distinguish between the two SDF-1/CXCL12 isoforms (43). The amplification profile for SDF-1/CXCL12 was 35 cycles of 1 min denaturation at 94°C and 2.5 min of annealing and extension at 62°C and yielded a PCR product of 177 bp. CXCR4 and -actin primers and amplification profiles were as described before (11). As negative controls, RNA was omitted from the reverse transcription reaction (no RNA), and cDNA was omitted from the PCR amplification reaction. To confirm that RNA preparations were free of contaminating genomic DNA, specific primers were used to amplify a region of the mannose-binding lectin promoter (27). PCR products were size separated on 1% agarose gels containing ethidium bromide and photographed. RNA isolated from human intestinal microvascular endothelial cells (gift of D. Binion; see Refs. 6 and 20) was used as a positive control for CXCR4 and SDF-1/CXCL12 expression.
Confocal microscopy. Caco-2 cells were plated on 30-mm cell culture filter inserts in a six-well tissue culture dish. Cell monolayers cultured for 14 days expressed increased levels of alkaline phosphatase as a marker of differentiation, had a transepithelial resistance 400
·cm2, and were judged to be polarized. These Caco-2 cell monolayers on cell insert membranes were removed from the surrounding plastic frame, embedded in optimum cutting temperature compound, and flash-frozen in isopentane/dry ice. Cryostat sections (5 µm) were prepared, fixed with 2% paraformaldehyde (pH 7.0), and incubated overnight at 4°C with monoclonal antibody to CXCR4 (clone 12G5) or an IgG2a isotype control antibody. Slides were washed and incubated for 60 min at 22°C with Cy3-conjugated goat anti-mouse antibody concomitant with phalloidin-Alexa 488 to visualize epithelial actin. Slides were analyzed using a Bio-Rad MRC1024 confocal microscope.
GTP35S binding assay. The membrane fraction of T84 cells was incubated with SDF-1/CXCL12 in the presence of the nonhydrolyzable GTP analog thio-GTP
35S. The reaction was stopped with 0.9 M NaCl. Membrane fractions passed through a glass-fiber filter (25 mm, type APF; Millipore), after which radiolabel incorporation was assessed using a
-scintillation counter (Packard).
Cell stimulation and cAMP enzyme immunoassay. For cell stimulation and cAMP assays, T84 cells were grown as confluent monolayers in 12-well tissue culture dishes, or as polarized epithelia on 12-mm cellulose filters. Before stimulation, cells were incubated with IBMX (10 mM, 30 min) to inhibit phosphodiesterase activity. Cells were stimulated with SDF-1/CXCL12 diluted in HBSS/IBMX for 10 min, after which they were incubated for an additional 10 min with forskolin (1 µM) to stimulate cAMP production. To further test the ability of SDF-1/CXCL12 to alter receptor-mediated cAMP production, T84 cells were incubated with SDF-1/CXCL12 followed by the addition of 1 µM PGE2 to stimulate cAMP production. Controls included unstimulated cells and cells stimulated with SDF-1/CXCL12 or forskolin alone. Cells were washed two times in ice-cold HBSS/IBMX and solubilized, and cAMP was assayed using a competitive enzyme immunoassay (Biotrak; AmershamPharmacia Biotech).
ELISA. SDF-1/CXCL12 is produced as - and
-isoforms, resulting from alternatively spliced mRNA transcripts (43), although different biological activities have not yet been ascribed to these isoforms. We assayed for each isoform using a sandwich ELISA with commercially available antibodies (R&D Systems). SDF-1/CXCL12 assays were sensitive to 75 pg/ml.
Electrophysiological studies. Confluent T84 epithelial cell monolayers grown on 12-mm Millicell-HA cellulose filters for 1215 days were mounted in Ussing chambers (area = 0.6 cm2) and bathed in oxygenated (95% O2-5% CO2) Ringer solution at 37°C. Epithelial cell monolayers were voltage clamped to zero potential difference by continuous application of short-circuit current. Changes in current reflect electrogenic Cl- secretion (28). Polarized T84 cells were incubated with either forskolin (0.3, 1.0, or 10 µM) alone to stimulate Cl- transport or pretreated 2 or 15 min with 20 or 100 ng/ml SDF-1/CXCL12 to inhibit cAMP production before the addition of varying doses of forskolin. Additional polarized T84 monolayers were pretreated 5 min with SDF-1/CXCL12 (100 ng/ml) to assess effects of SDF-1/CXCL12 on ion transport stimulated by DB-cAMP (200 µM).
Statistical analysis. Statistical analysis used Student's t-test or ANOVA for comparisons between groups.
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RESULTS |
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SDF-1/CXCL12 inhibits cAMP production and stimulates T84 cells via Gi. CXCR4 is known to transduce signals via the Gi
-subunit of the heterotrimeric G protein complex in leukocytes after stimulation of those cells with SDF-1/CXCL12 (38). To determine if G proteins were activated in intestinal epithelial cells in response to SDF-1/CXCL12 stimulation, T84 cells were stimulated with that chemokine (10 ng/ml) for 5 min, after which the transfer of radiolabeled thio-GTP
35S to the cell membrane was assayed. T84 cell membrane fractions exhibited a 40% increase in radiolabel incorporation compared with unstimulated controls (n = 3, P < 0.05).
The prototypic function of Gi proteins is the binding to and inhibition of adenylyl cyclase-mediated cAMP production (33). To determine if this is also the case in human colon epithelial cells, we assessed if activation of CXCR4 with SDF-1/CXCL12 blocks adenylyl cyclase-mediated cAMP production in T84 cells. T84 cells were incubated with titrated concentrations of SDF-1/CXCL12, followed by stimulation with forskolin, to upregulate cAMP production. As shown in Fig. 3, SDF-1/CXCL12 significantly inhibited forskolin-stimulated cAMP responses in T84 cells in a dose-dependent manner. We also assessed the ability of SDF-1/CXCL12 to inhibit receptor-mediated activation of adenylyl cyclase and in turn production of cAMP. PGE2 was selected as an appropriate receptor agonist because it is known to increase cAMP production in T84 cells (3, 37) and plays an important role in mucosal immune responses (5, 15). SDF-1/CXCL12 (100 ng/ml) significantly inhibited cAMP production evoked by 1 µM PGE2 in T84 cells (64.8 ± 8.3% of PGE2 control cAMP production, P = 0.001, n = 3 experiments). Similar inhibition was observed at 10 ng/ml SDF-1/CXCL12 (n = 3 experiments, data not shown).
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To determine if SDF-1/CXCL12 inhibition of cAMP production, in fact, involves the activation of Gi subunits, T84 cells were incubated for 12 h with pertussis toxin, which specifically targets and prevents the activation of Gi
and Go
subunit proteins (33). As shown in Fig. 4A, the inhibition of forskolin-stimulated cAMP production by SDF-1 was abrogated by pertussis toxin. Pertussis toxin alone had no effect on background cAMP production or on cAMP production in cells stimulated with forskolin alone (data not shown). Taken together, these results suggest that CXCR4 is coupled to Gi
subunits in T84 cells.
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To demonstrate that SDF-1/CXCL12-mediated inhibition of cAMP production occurred via the activation of CXCR4, cells were treated with a CXCR4 neutralizing antibody (12G5) before stimulation with SDF-1/CXCL12 and forskolin. As shown in Fig. 4B, the inhibitory effect of SDF-1/CXCL12 on forskolin-stimulated cAMP production was significantly decreased by pretreatment of cells with 12G5.
Activation of G protein-coupled receptors, including chemokine receptors on leukocytes, leads to receptor desensitization. To determine if this was also the case for CXCR4 expressed by T84 cells, cells were first treated with SDF-1/CXCL12 and subsequently restimulated with the same dose of SDF-1/CXCL12 followed by forskolin. As shown in Table 1, there was little, if any, inhibition of forskolin-stimulated cAMP production in SDF-1/CXCL12-pretreated cells. These findings are consistent with the activation and desensitization of epithelial CXCR4 by its ligand SDF-1/CXCL12.
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SDF-1/CXCL12 attenuates ion transport in model intestinal epithelia. We next determined if SDF-1/CXCL12-activated CXCR4 signaling alters specialized functions of intestinal epithelial cells that are cAMP regulated and that differ from those characteristic of leukocytes. Because cAMP is the major second messenger known to regulate ion secretion in intestinal epithelia, and in T84 cells (4), we hypothesized that SDF-1/CXCL12 alters electrogenic ion transport. Consistent with measurements of increased cAMP levels, and as shown in Fig. 5, SDF-1/CXCL12 significantly inhibited forskolin-stimulated ion transport. Of note, SDF-1/CXCL12 inhibition of electrogenic ion transport was comparable for 10 µM (Fig. 5) and 1 µM (Fig. 5, inset) doses of forskolin. In separate experiments, cells pretreated with 20 ng/ml SDF-1/CXCL12 had no significant change in short-circuit current compared with control cells receiving a markedly submaximal 0.3 µM dose of forskolin alone (22.7 ± 3.8 compared with 20.8 ± 0.9 µA/cm2, respectively, values are mean ± SE, n = 6). As a control, voltage-clamped T84 cells did not manifest significant changes in current, conductance, or resistance when incubated with 20 or 100 ng/ml SDF-1/CXCL12 alone. These data suggest that SDF-1/CXCL12 modulates ion transport via binding to and inhibiting activity of adenylyl cyclase enzymes. Nonetheless, additional mechanisms may also be involved, since we found that SDF-1/CXCL12 also could inhibit ion transport stimulated by 200 µM cell-permeant DB-cAMP (range 910%, n = 2), although the extent of inhibition was consistently less and delayed compared with that seen after forskolin stimulation.
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DISCUSSION |
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The second messenger cAMP modulates diverse cellular functions (2). cAMP is synthesized from ATP by several isoforms of adenylyl cyclase that are activated upon ligand binding to Gs protein-coupled receptors and negatively regulated by activated Gi
protein-coupled receptors. Once produced, cAMP is rapidly degraded by cytosolic phosphodiesterases. As shown herein, SDF-1/CXCL12 signals through CXCR4 expressed by intestinal epithelial cells and inhibits adenylyl cyclase-mediated production of cAMP. As in other cell types (e.g., leukocytes or cells transfected with CXCR4; see Ref. 38), our data suggest that epithelial-expressed CXCR4 is coupled to a Gi
protein, as evidenced by the sensitivity of its effects to pertussis toxin (33).
Signaling through CXCR4 in intestinal epithelial cells regulates adenylyl cyclase-directed cAMP production and cAMP-mediated Cl- secretion in epithelial cells. cAMP is the major second messenger that regulates ion transport in intestinal epithelial cells in vitro and by the intestinal epithelium in vivo (4, 9). Nonetheless, except for somatostatin (47), which regulates both cAMP and calcium-dependent ion secretion in colonic epithelium, there has been little prior characterization of physiological extracellular stimuli that can downregulate cAMP-dependent epithelial cell secretion. Three of the known adenylyl cyclase isoforms possess binding sites for the Gi protein (2). In contrast, forskolin and PGE2 each activate all nine known adenylyl cyclase isoforms. Given this disparity in adenylyl cyclase stimulation by varying ligands, the lack of complete inhibition of forskolin- or PGE2-stimulated, cAMP-dependent, Cl- secretion by SDF-1/CXCL12 was expected. Consistent with electrogenic ion transport being a multifactorial process (4), our data suggest that SDF-1/CXCL12-CXCR4 signaling likely impacts additional mechanisms, as evidenced by the inhibition of electrogenic ion transport evoked by cell-permeant cAMP.
Inflammatory responses in the intestine often are accompanied by increased epithelial cell secretion, associated with increased epithelial cAMP production (4). Of note, we have shown that SDF-1/CXCL12 can inhibit ion transport evoked by PGE2, an important arachidonic acid metabolite with a known role in modulating ion transport (5, 15). The data herein indicate that SDF-1/CXCL12, a chemokine that is constitutively produced by cells of the innate mucosal immune system, can negatively regulate PGE2 receptor-mediated cAMP-mediated electrogenic ion transport by epithelial cells. As such, this chemokine may play a role in regulating intestinal secretion in the intestinal mucosa. Although the role of the intestinal epithelium in the pathogenesis of mucosal inflammation that occurs in Gi knockout mice is unknown, the failure to normally downregulate cAMP-mediated intestinal epithelial secretion may play a pathogenic role (40).
These studies focused on the functional role of a chemokine receptor expressed by intestinal epithelial cells. Although not addressed in this study, changes in cAMP production in intestinal epithelial cells mediated via CXCR4 may also modulate other intestinal epithelial cell functions. For example, within various cell types, cAMP is known to act in proliferative or apoptotic signaling pathways (17, 31, 35) and SDF-1/CXCL12 can signal the chemotaxis of metastatic tumor cells (29). Given the unique characteristics of epithelial cell proliferation, migration, and apoptosis within the crypt-villus unit of the intestinal epithelium, it is possible that activation of epithelial Gi-coupled CXCR4, via regulation of cAMP levels, also plays a role in regulating epithelial survival and/or migratory mechanisms. In summary, our data suggest a paradigm wherein chemokine receptors that are broadly expressed on intestinal epithelial cells can transduce signals that regulate distinct epithelial cell type-specific functions.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-35108 and DK-58960 (M. F. Kagnoff) and DK-28305 (K. E. Barrett). M. B. Dwinell was supported by NIDDK training Grant T32 DK-07202 and by Career Development Award K01 DK-02808.
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FOOTNOTES |
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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.
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REFERENCES |
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