Differential Expression of Cannabinoid Receptors in the Human Colon: Cannabinoids Promote Epithelial Wound Healing
2005; Elsevier BV; Volume: 129; Issue: 2 Linguagem: Inglês
10.1016/j.gastro.2005.05.026
ISSN1528-0012
AutoresKaren L. Wright, Norio Ozaki, Mark Feeney, J J T Tate, Duncan A. Robertson, Melanie J. Welham, Stephen G. Ward,
Tópico(s)Dietary Effects on Health
ResumoBackground & Aims: Two G-protein—coupled cannabinoid receptors, termed CB1 and CB2, have been identified and several mammalian enteric nervous systems express CB1 receptors and produce endocannabinoids. An immunomodulatory role for the endocannabinoid system in gastrointestinal inflammatory disorders has been proposed and this study sought to determine the location of both cannabinoid receptors in human colon and to investigate epithelial receptor function. Methods: The location of CB1 and CB2 receptors in human colonic tissue was determined by immunohistochemistry. Primary colonic epithelial cells were treated with both synthetic and endogenous cannabinoids in vitro, and biochemical coupling of the receptors to known signaling events was determined by immunoblotting. Human colonic epithelial cell lines were used in cannabinoid-binding studies and as a model for in vitro wound-healing experiments. Results: CB1-receptor immunoreactivity was evident in normal colonic epithelium, smooth muscle, and the submucosal myenteric plexus. CB1- and CB2-receptor expression was present on plasma cells in the lamina propria, whereas only CB2 was present on macrophages. CB2 immunoreactivity was seen in the epithelium of colonic tissue characteristic of inflammatory bowel disease. Cannabinoids enhanced epithelial wound closure either alone or in combination with lysophosphatidic acid through a CB1—lysophosphatidic acid 1 heteromeric receptor complex. Conclusions: CB1 receptors are expressed in normal human colon and colonic epithelium is responsive biochemically and functionally to cannabinoids. Increased epithelial CB2-receptor expression in human inflammatory bowel disease tissue implies an immunomodulatory role that may impact on mucosal immunity. Background & Aims: Two G-protein—coupled cannabinoid receptors, termed CB1 and CB2, have been identified and several mammalian enteric nervous systems express CB1 receptors and produce endocannabinoids. An immunomodulatory role for the endocannabinoid system in gastrointestinal inflammatory disorders has been proposed and this study sought to determine the location of both cannabinoid receptors in human colon and to investigate epithelial receptor function. Methods: The location of CB1 and CB2 receptors in human colonic tissue was determined by immunohistochemistry. Primary colonic epithelial cells were treated with both synthetic and endogenous cannabinoids in vitro, and biochemical coupling of the receptors to known signaling events was determined by immunoblotting. Human colonic epithelial cell lines were used in cannabinoid-binding studies and as a model for in vitro wound-healing experiments. Results: CB1-receptor immunoreactivity was evident in normal colonic epithelium, smooth muscle, and the submucosal myenteric plexus. CB1- and CB2-receptor expression was present on plasma cells in the lamina propria, whereas only CB2 was present on macrophages. CB2 immunoreactivity was seen in the epithelium of colonic tissue characteristic of inflammatory bowel disease. Cannabinoids enhanced epithelial wound closure either alone or in combination with lysophosphatidic acid through a CB1—lysophosphatidic acid 1 heteromeric receptor complex. Conclusions: CB1 receptors are expressed in normal human colon and colonic epithelium is responsive biochemically and functionally to cannabinoids. Increased epithelial CB2-receptor expression in human inflammatory bowel disease tissue implies an immunomodulatory role that may impact on mucosal immunity. 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The purpose of the present study was to determine the location of both CB1 and CB2 receptors in both normal and inflammatory bowel disease (IBD) human colonic tissue and to confirm the functionality of these receptors by establishing whether both endogenous and synthetic cannabinoids couple to known biochemical effector pathways in colonic epithelial cells. In addition, evidence for a cannabinoid role in epithelial wound healing is presented. Human colonic biopsy specimens from routine colonoscopy, histopathologically assessed to exclude microscopic inflammation, were retrieved from files at the Royal United Hospital, Bath, United Kingdom. In addition, colonic tissue, which included normal colon at least 20–25 cm from the tumor, was removed from patients undergoing colonic resections. This procedure had the approval of the Bath local research ethics committee, Royal United Hospital Bath National Health Service Trust, United Kingdom. Tissue blocks were fixed in 4% (wt/vol) formaldehyde and embedded in paraffin. Cell culture media and plastic ware were purchased from Invitrogen (Paisley, UK). Synthetic (arachidonylcyclopropylamide, JWH 133, and WIN 55,212-2,) and endogenous (AEA, methanadamide, and NE) cannabinoids and the cannabinoid receptor antagonist (AM251) were from Tocris (Bristol, UK). Enzyme inhibitors LY294002 and PD98059 were from Merck Biosciences (Nottingham, UK) and all other chemicals came from Sigma-Aldrich (Dorset, UK). Antibodies to components of the endogenous cannabinoid system were purchased for immunohistochemistry and immunoblotting as follows: anti-CB1 (PA1-743) and anti-CB2 (PA1-744) were from Affinity BioReagents (Cambridge BioScience, Cambridge, UK); anti-CB1 (sc20754) and anti-CB2 (sc25494) were from Santa Cruz (Autogen Bioclear, Wiltshire, UK); anti-CB1 (101500) and anti-CB2 (101550) were from Cayman Chemical (IDS Ltd, Tyne and Wear, UK), and anti-FAAH (11-A) was from Alpha Diagnostic (San Antonio, TX). Other antibodies used in this study included anti—lysophosphatidic acid (LPA)1 (Edg2) from Upstate (Dundee, UK), and anti-ERK, anti—phospho-ERK1/2, anti-PKB, anti—phospho-PKB, anti—phospho-GSK3α/β, and anti-GSK3β from Cell Signaling Technology, New England Biolabs (Hertfordshire, UK). The DAKO ChemMate System kit (Cambridgeshire, UK) was used for the immunohistochemical staining of the sections. Briefly, tissue sections (3–5 μm) were mounted on slides. After the sections were deparaffinized with xylene and rehydrated through a series of graded alcohol, antigen retrieval was achieved through boiling in .01 mol/L sodium citrate buffer, pH 6.0, at high pressure for 2 minutes. Sections were blocked in 5% bovine serum albumin in Tris-buffered saline (TBS), pH 9.0, for 1 hour before application of primary antibodies. CB1 and CB2 antibodies (Cayman Chemical) at a 1:1000 dilution in TBS, pH 9.0, were incubated overnight at 4°C. For control slides, primary antibodies were omitted or a 10-fold excess of blocking peptide was used as suggested by the manufacturer (Cayman Chemical). Sections then were incubated in rabbit-specific secondary antibody for 25 minutes and then in 3,3′-diaminobenzidine tetrahydrochloride (DAKO) for 5 minutes. Sections were counterstained with a progressive hematoxylin. To identify macrophages, anti-human CD68 staining was performed as described earlier, except the TBS was at a pH of 7.6. Plasma cells were identified by the consultant gastrointestinal histopathologist, who confirmed that these cells are characteristic of normal bowel and always are located in the lamina propria.48Wheater P.R. Burkitt H.G. Daniels V.G. Functional histology. Churchill Livingstone, London, UK1979Google Scholar Further immunohistochemical validation was undertaken taken using CB1- and CB2-receptor antibodies from Santa Cruz. Human colonic epithelial cell lines HT-29, Caco2, and DLD1 were cultured routinely in 80-cm2 tissue culture flasks in McCoy's 5A, Dulbecco's modified Eagle, and RPMI media, respectively. Media were supplemented with penicillin (10 U/mL), streptomycin (10 μg/mL), fungizone (.5 μg/mL), and 5% (vol/vol) fetal bovine serum. In addition, Dulbecco's modified Eagle medium was supplemented with 1× nonessential amino acids (Sigma) (referred to as complete medium). Cultures were maintained at 37°C in an atmosphere of 5% CO2. The medium was changed every 2–3 days and cells were passaged weekly into 80-cm2 tissue culture flasks for further culture, or into 6-, 24-, or 96-well plates or Petri dishes for experimental protocols. Unless otherwise stated, cells were grown until confluent. Before experiments, monolayers were washed and cultured in medium without fetal bovine serum for 24 hours. Growth-arrested cultures were treated with fresh fetal bovine serum—free medium and stimulated with the appropriate doses of either drugs, cannabinoids, or vehicle controls (ethanol or dimethyl sulfoxide, as appropriate) for the times described in the Results section. Total cellular proteins were extracted as described later. Cells grown to confluence were collected by scraping and spun at 200 × g for 10 minutes at 4°C. Crude membranes were prepared by homogenization of cells in 5 mmol/L Tris-HCl, pH 7.5, and centrifugation at 1000g for 5 minutes. The supernatant was centrifuged at 40,000g for 40 minutes at 4°C, and the pellet was resuspended in a buffer consisting of 50 mmol/L Tris-HCl, pH 7.5, 5 mmol/L MgCl2, 1 mmol/L ethylenediaminetetraacetic acid, and stored at −80°C until use. To determine the effect of cannabinoids on displacement of [3H]-CP-55,940 (a nonselective cannabinoid agonist), membranes (50 μg of protein) were incubated at 30°C for 1 hour in 1 mL (final volume) binding buffer (50 mmol/L Tris-HCl, pH 7.7, 5 mmol/L MgCl2, 1 mmol/L ethylenediaminetetraacetic acid, and .5% [wt/vol] bovine serum albumin) with .2 nmol/L [3H]-CP-55,940 and increasing concentrations of unlabeled cannabinoid. A rapid filtration technique using Whatman GF/B filters (soaked in cold wash buffer containing .05 mol/L Tris-HCl, pH 7.7, and .25% bovine serum albumin buffer; Fisher Scientific, Loughborough, UK) and a 12-well filtration apparatus (Brandel; SEMAT Technical, St. Albans, UK) was used to harvest and rinse labeled membranes with cold wash buffer. Filter-bound radioactivity was counted with 4 mL of biofluor liquid scintillator (Perkin Elmer, Beaconsfield, UK). Nonspecific binding was determined in the presence of 1 μmol/L CP-55,940. Data from competition studies were analyzed by nonlinear regression for 2-site competition using GraphPad Prism software (San Diego, CA). Cells were lysed in ice-cold solubilization buffer (50 mmol/L Tris-HCl, pH 7.5, 10% [vol/vol] glycerol, 1% [vol/vol] Nonidet P-40, 150 mmol/L NaCl, 5 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L sodium vanadate, 1 mmol/L sodium molybdate, 10 mmol/L NaF, 40 μg/mL phenymethylsulphonyl fluoride, 10 μg/mL aprotinin, 10 μg/mL soybean trypsin inhibitor, 10 μg/mL leupeptin, and .7 μg/mL pepstatin). Insoluble cell debris was removed by centrifugation at full speed for 1 minute and the supernatant was transferred to a clean tube. For fractionation studies, the Calbiochem (Merck Biosciences) Subcellular Proteome Extraction Kit (539790) was used per the manufacturer's instructions. Protein concentrations were determined with the Bio-Rad Protein Assay reagent (Bio-Rad, Hertfordshire, UK). For immunoprecipitation studies, 5 μL of anti-CB1 (sc20754) was added to lysates (1 mg/mL) and immunocomplexes subsequently were coupled to protein G-sepharose beads (1 h at 4°C). Protein (20 μg) was resolved on a 7.5% polyacrylamide gel (12%, for immunoprecipitates) and transferred to nitrocellulose membranes. Membranes were blocked in 5% (wt/vol) nonfat milk in TBS for 2 hours. Immunoblotting with primary antibody diluted 1:5 in bl
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