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Differential Expression of Cannabinoid Receptors in the Human Colon: Cannabinoids Promote Epithelial Wound Healing

      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.

      Abbreviations used in this paper:

      AEA (anandamide), DNBS (dinitrobenzene sulphonic acid), ERK (extracellular-regulated kinase), FAAH (fatty acid amide hydrolase), GSK (glycogen synthase kinase), LPA (lysophosphatidic acid), MAPK (mitogen-activated protein kinase), NE (noladin ether), PI3K (phosphatidylinositol 3-kinase), PKB (protein kinase B), TBS (Tris-buffered saline), THC (tetrahydrocannabinol)
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      Materials and Methods

       Subjects

      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.

       Reagents and Drugs

      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).

       Immunohistochemical Analysis of Human Colon

      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.
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      • Daniels V.G.
      Further immunohistochemical validation was undertaken taken using CB1- and CB2-receptor antibodies from Santa Cruz.

       Cell Cultures

      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.

       Radioligand Binding Assays

      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).

       Protein Preparation and Immunoblotting

      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 blocking buffer was performed overnight. Primary antibodies were used at the following concentrations: anti-ERK, anti—phospho-ERK1/2, anti-PKB, anti—phospho-PKB, anti—phospho-GSK3α/β, and anti-GSK3β at a 1:1000 dilution and anti-CB1, anti-CB2, anti-FAAH, and anti-LPA1 at a dilution of 1:500. After three 10-minute washes with TBS plus .05% (vol/vol) NP40, secondary antibodies conjugated to horseradish peroxidase (DAKO) were used at a concentration of .05 μg/mL in TBS plus .05% (vol/vol) NP40 for 1 hour. After further washing, immunoblots were developed using the ECL system (Amersham, Cardiff, UK) and Kodak X-AR5 film (Rochester, NY). Immunoblots were stripped completely of antibodies by incubation at 60°C for 30 minutes with stripping solution (62.5 mmol/L Tris-HCl, pH 6.8, 2% [wt/vol] sodium dodecyl sulfate, and 100 mmol/L 2-mercaptoethanol). After extensive washing, blots were reblocked before reprobing.

       Primary Colonic Epithelial Cell Purification

      Surgical specimens were collected immediately after surgery from patients undergoing large bowel resection for colon cancer. Normal colonic tissue at least 10 cm from the tumor margins was placed in Hanks’ balanced salt solution, pH 7.3, kept on ice, and processed within 1 hour after surgery. After the specimen was washed 3 times with Ca2+-, Mg2+-free Hanks’ balanced salt solution, the mucosa was dissected from the submucosa and cut into strips. These were washed at room temperature in 10 mmol/L dithiothreitol for 30 minutes, followed by 2 washes (90 minutes each) in 1 mmol/L ethylenediaminetetraacetic acid. Cells liberated from both washes were harvested by centrifugation at 500g for 5 minutes at room temperature and washed twice with Ca2+-, Mg2+-free Hanks’ balanced salt solution, followed by further purification with a 50% (vol/vol) Percoll gradient (Pharmacia Biotech, Uppsala, Sweden). After centrifugation for 20 minutes at 300g, purified epithelial cells were harvested at the interphase and washed 3 times in Ca2+-, Mg2+-free Hanks’ balanced salt solution. Cells were counted and placed in serum-free medium at a concentration of 107/mL. After 1–4 hours at 37°C, cells were stimulated and lysed as described earlier. To determine purity, immunoblotting of whole-cell lysates of isolated epithelial cells, peripheral blood lymphocytes, and primary human colonic myofibroblasts revealed that the majority of these cells were of epithelial origin (α-human epithelial-specific antigen, clone VU-1D9; Sigma) and not of immune cell (α-protein tyrosine kinase, Lck, clone 3A5; Sigma) or fibroblastic lineage (α-smooth muscle actin, clone 1A4; Sigma).

       Wound-Healing Experiments

      Confluent monolayers of cells in 6-well tissue culture plates (Nunc, Fisher Scientific, Loughborough, UK) were wounded with a Gilson p2 pipette tip (Anachem, Bedfordshire, UK). Cells were washed 3 times with fresh serum-free medium and placed in low-serum medium (.1% fetal bovine serum) for experiments. Wound widths for each well were determined under microscopic vision with a graded eyepiece. Three measurements were performed for each wound before the addition of agonists and in a blinded fashion after 24 hours of stimulation. Each experiment was performed in triplicate and at least 3 times. Data are expressed as the percentage reduction in wound width and the data were analyzed by analysis of variance general linear model with a post hoc Dunnett’s test for multiple comparisons of means.

      Results

       Localization of Cannabinoid Receptors in Normal Colonic Tissue

      To investigate the expression of cannabinoid receptors in the large bowel, normal adult human tissue sections (n = 20) from the colon first were stained with H&E to identify the morphology and verify the origin of the tissue (Figure 1A). Immunostaining with the Cayman CB1-receptor antibody showed intense positive expression in the absorptive epithelial cells of the crypts with enhanced signal in the microvilli on the apical surface of the colonic epithelial border facing the lumen (Figure 1B, C, and D, red arrows). The goblet cells appear negative, although this may be a result of mucus-blocking antibody binding (Figure 1C and D, blue arrows). The lymphoid aggregates are negative (Figure 1E, blue arrow), whereas subepithelial plasma cells in the lamina propria are weakly positive (Figure 1C, D, and E, black arrows). Positive staining also was seen in the smooth muscle of the blood vessel walls (Figure 1F, red arrow) and the smooth muscle layers of the muscularis mucosae (Figure 1F, black arrow), including the submucosal nerve bundles (Figure 1G). Preabsorption and omission controls were performed for each set of immunostaining procedures (Figures 1H and 2E).
      Figure thumbnail gr1
      Figure 1Immunohistochemical analysis of CB1-receptor protein in normal human colon. Deep sections and colonic biopsy specimens were fixed and stained with a primary rabbit polyclonal anti-human CB1 antibody (Cayman). The magnitude of each image is shown and the intensity of signal is color coded such that red arrows indicate dense positive staining, black arrows depict moderate staining, and blue arrows show areas of negative staining. (A) The morphology of normal human colon was confirmed with the H&E stain. (B) CB1 is expressed specifically in the crypt epithelium. (C and D) At greater magnitude, dense immunoreactivity was noted on the microvilli present on the absorptive border facing the lumen (red arrows). Goblet cells were negative (blue arrows). (C, D, and E) Moderate staining of subepithelial plasma cells in the lamina propria was present (black arrows), whereas the germinal center was negative (E, blue arrow). (F) Intense CB1 immunoreactivity was found in the smooth muscle of the blood vessels (red arrow), whereas staining of the smooth muscle layers of the muscularis mucosae was more moderate (black arrow). (G) Submucosal myenteric nerve bundles (arrow), including the surrounding longitudinal and circular smooth muscle, were positive for CB1. Very weak nonspecific labeling persisted in the crypts of the preabsorption and omission controls (1H and 2E, respectively), but the microvillus border always was negative, as was the rest of the tissue. Results are representative of the staining pattern noted in the assessment of normal colon from 20 separate patient samples.
      Similar tissue sections stained with the Cayman CB2-receptor antibody showed a different expression profile. Subepithelial macrophages were strongly CB2 positive (Figure 2B and C, red arrows). The macrophage marker α-CD68 was used to verify the macrophage cell type (Figure 2D). Interestingly, similar to CB1, plasma cells, identified by the histopathologist as characteristically present within the lamina propria of normal bowel,
      • Wheater P.R.
      • Burkitt H.G.
      • Daniels V.G.
      were weakly CB2 positive, a consistent increase in signal when compared with controls (Figure 2C, black arrow). Crypt epithelium appears very slightly positive in the cytoplasm, although this pattern always was similar to the preabsorption and omission controls (Figure 2A and E). Unlike CB1, no CB2 staining was evident on the microvillus border at the apical surface (Figure 2B). The lymphoid aggregates were negative for CB2 expression.
      Figure thumbnail gr2
      Figure 2Immunohistochemical analysis of CB2-receptor protein in human colon. Sections were fixed and stained with a primary rabbit polyclonal anti-human CB2 antibody (Cayman). (A) The magnitude of each image is shown and the intensity of signal is color coded such that red arrows indicate dense positive staining, black arrows depict moderate staining, and blue arrows show areas of negative staining. (B) Dense CB2 immunoreactivity was found in the subepithelial macrophages present in the lamina propria (red arrow). (C) At higher magnification, the intense signal on macrophages can be seen (red arrow) alongside weaker staining of plasma cells (black arrow), the presence of which is characteristic of the lamina propria in normal bowel. (D) Immunoreactivity of the macrophage marker, CD68, was intense in the subepithelial lamina propria, again characteristic of normal bowel. (B) The weakly positive pattern of epithelial CB2 staining (A and E) matched the nonspecific immunoreactivity found in the crypts of the preabsorption and omission controls. Results are representative of the staining pattern noted in the assessment of normal colon from 20 separate patient samples. Acute-phase IBD sections stained with (F) CB1 and (G) CB2 (n = 6) (red arrow). (H) Epithelial CB1 and CB2 (red arrow) in Crohn’s disease (n = 4).
      Further immunostaining using antibodies for CB1 and CB2 receptors (obtained from Santa Cruz) were performed on colonic sections prepared for immunohistochemistry. A similar pattern of epithelial CB1-receptor immunoreactivity was obtained in that positive staining was observed in both the cytoplasm and the membrane, with some border enhancement (data not shown). Minor positive epithelial CB2 immunoreactivity was seen in some samples but not others (data not shown), possibly owing to normal variation in the inflammatory status of the gut, which is not evident microscopically. Therefore, we cannot discount formally that low levels of CB2 may be expressed in ostensibly normal colonic epithelium, although immunoblotting experiments using CB1- and CB2-receptor antibodies from Cayman Chemical, Santa Cruz, and Affinity BioReagents are shown in Figures 3A, and 4B, C, and D and support our immunohistochemical data.
      Figure thumbnail gr3
      Figure 3Cannabinoid receptors and signaling in human colonic epithelial cell lines. In all experiments, 107/mL resting cells were lysed immediately or after stimulation and 20 μg of protein was subjected to electrophoresis. For signaling experiments, immunoblotting with anti—phospho-ERK, anti—phospho-PKB, or anti—phospho-GSK3α/β was performed as shown. Each experiment depicted is representative of at least 3 experiments. (A) CB-receptor expression. Membranes were probed with anti—CB1- and anti—CB2-receptor antibodies as described in the Materials and Methods section. Molecular mass markers are shown in kilodaltons. (B) ERK phosphorylation induced by cannabinoids. The vehicle used was dimethyl sulfoxide and stimulations were for 5 minutes. NE (50 nmol/L), AEA (100 nmol/L), WIN (100 nmol/L), and JWH (20 nmol/L). (C) PKB phosphorylation induced by cannabinoids. Stimulations were for 5′ as described earlier. (D) GSK3α/β phosphorylation induced by cannabinoids. Ethanol (EtOH) was used as vehicle in this experiment and stimulations were for 5′. Membranes were stripped and reprobed with anti—pan-ERK, anti—pan-PKB, and anti—pan-GSK where shown.
      Figure thumbnail gr4
      Figure 4Characterization of primary human colonic epithelial cells. Freshly isolated cells were lysed (107 cells/mL) and 20 μg protein from each cell type was fractionated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (see Materials and Methods section). (A) Characterization of cell type. Loading order of whole-cell lysates was as follows: primary human colonic epithelial cells from 2 patients (lanes 1 and 2), primary human myofibroblasts (lane 3), and peripheral human blood lymphocytes (lane 4). Membranes were blotted with α-human epithelial—specific antigen, α-epithelial—specific antigen (clone VU-1D9), T- and B-cell—specific protein tyrosine kinase, Lck (clone 3A5) and α-smooth muscle actin, and α-smooth muscle actin (clone 1A4). Data shown are representative of 12 primary cell purification procedures. (B) CB1- and CB2-protein expression (Cayman). pbls, peripheral blood lymphocytes; molt-4, leukemic T-cell line. Protein (20 μg) from each cell type was fractionated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and identical membranes were probed with anti-human CB1 or anti-human CB2 antibody, as described in the Materials and Methods section. These blots are representative of cannabinoid-receptor expression performed for all purification procedures. (C) CB1- and CB2-protein expression (antibodies obtained from Santa Cruz and Affinity BioReagents). CB1 and CB2 expression in Jurkat or CEM T-cell lines and the DLD1 colonic epithelial cell line were compared with primary epithelial cells as shown. sc, Santa Cruz antibodies; abr, Affinity Bioreagents antibodies (see Materials and Methods section). Blots are representative of 2 separate experiments. (D) Fractionation experiments were performed on primary cells and the HT29 cell line as described in the Materials and Methods section. Membrane (m), cytoplasmic (c), and whole-cell extract (wce) fractions from each cell type were fractionated further by sodium dodecyl sulfate—polyacrylamide gel electrophoresis and identical membranes were probed with anti-FAAH, anti-human CB1, or anti-human CB2 antibody (Cayman), as described in the Materials and Methods section. These blots are representative of at least 2 separate experiments.
      Tissue sections from patients with IBD were stained similarly. These sections were derived from biopsy specimens, which do not incorporate the deeper layers of colon. Epithelial CB1 immunoreactivity was evident in acute-phase IBD and Crohn’s disease, as seen in normal tissue (Figure 2F and H). Although CB1 immunoreactivity appears more intense, this technique is not quantitative and the epithelial architecture differs in disease caused by goblet-cell depletion. In contrast, epithelial CB2 immunoreactivity always was evident in acute-phase IBD tissue and appears more intense in the cytoplasm and is expressed in the membrane on the microvillus border (Figure 2G, red arrow). Histopathologic observations of acute-phase IBD require further clinicopathologic correlation to make a final diagnosis, which, in 4 of 6 cases, was ulcerative colitis. In Crohn’s disease, intense CB2 expression was evident in the epithelium at the crypt fissure where ulceration had occurred (Figure 2H, red arrow).

       Cannabinoid-Receptor Expression and Signaling in Colonic Epithelial Cell Lines

      CB1 receptors have been shown to be present in smooth muscle and enteric nerves of various animals,
      • Casu M.A.
      • Porcella A.
      • Ruiu S.
      • Saba P.
      • Marchese G.
      • Carai M.A.
      • Reali R.
      • Gessa G.L.
      • Pani L.
      Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.
      • Manara L.
      • Croci T.
      • Guagnini F.
      • Rinaldi-Carmona M.
      • Maffrand J.P.
      • Le Fur G.
      • Mukenge S.
      • Ferla G.
      Functional assessment of neuronal cannabinoid receptors in the muscular layers of human ileum and colon.
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • Cannich A.
      • Monory K.
      • Cravatt B.F.
      • Ferri G.L.
      • Sibaev A.
      • Storr M.
      • Lutz B.
      The endogenous cannabinoid system protects against colonic inflammation.
      but never on normal gastrointestinal epithelium. A colonic epithelial cell line, Caco2, has been reported to express CB1 but not CB2 receptors.
      • Ligresti A.
      • Bisogno T.
      • Matias I.
      • De Petrocellis L.
      • Cascio M.G.
      • Cosenza V.
      • D’Argenio G.
      • Scaglione G.
      • Bifulco M.
      • Sorrentini I.
      • Di Marzo V.
      Possible endocannabinoid control of colorectal cancer growth.
      Therefore, we initially examined 4 human colonic epithelial cell lines to verify this finding. CB1- and CB2-receptor expression was determined in whole-cell extracts of human colonic epithelial cell lines, HT29, Caco2, T84, and DLD1 (Figure 3A). A 52-kilodalton protein corresponding to the predicted weight of CB1 was seen in all cell extracts (Figure 3A, top panel). In addition, an 83-kilodalton band also was detected in all cell lines, described in the literature as resulting from posttranslational modifications such as glycosylation and phosphorylation.
      • Pettit D.A.
      • Harrison M.P.
      • Olson J.M.
      • Spencer R.F.
      • Cabral G.A.
      Immunohistochemical localization of the neural cannabinoid receptor in rat brain.
      • Song C.
      • Howlett A.C.
      Rat brain cannabinoid receptors are N-linked glycosylated proteins.
      The predicted molecular weight for the CB2 receptor is 39 kilodaltons and immunoblotting of the same cell lines showed expression of a 47-kilodalton protein (Figure 3A, bottom panel), previously described as a modified form of CB2
      • Carayon P.
      • Marchand J.
      • Dussossoy D.
      • Derocq J.M.
      • Jbilo O.
      • Bord A.
      • Bouaboula M.
      • Galiegue S.
      • Mondiere P.
      • Penarier G.
      • Fur G.L.
      • Defrance T.
      • Casellas P.
      Modulation and functional involvement of CB2 peripheral cannabinoid receptors during B-cell differentiation.
      and also seen previously in DLD1 cells.
      • Ligresti A.
      • Bisogno T.
      • Matias I.
      • De Petrocellis L.
      • Cascio M.G.
      • Cosenza V.
      • D’Argenio G.
      • Scaglione G.
      • Bifulco M.
      • Sorrentini I.
      • Di Marzo V.
      Possible endocannabinoid control of colorectal cancer growth.
      Overexposure of the same membrane did not show a 39-kilodalton protein (data not shown).
      The stimulation of cannabinoid receptors initiates several downstream signal transduction pathways and we focused on phosphorylation of ERK, PKB, and GSK3α/β. Synthetic and endogenous cannabinoids, with varying specificities to CB1 and/or CB2, were used to determine whether the receptors expressed on these cell lines could elicit any of these biochemical responses as an indicator of their functionality. It should be noted that because of the instability of 2-arachidonoyl glycerol in cell culture media,
      • Rouzer C.A.
      • Ghebreselasie K.
      • Marnett L.J.
      Chemical stability of 2-arachidonylglycerol under biological conditions.
      we did not attempt to use this endocannabinoid in this study. The data shown are the results from experiments with HT29 cells, although it should be noted that similar experiments were performed with Caco2 cells with comparable responses to cannabinoids (data not shown). Cells exhibited detectable basal levels of ERK phosphorylation, which was enhanced rapidly on stimulation by cannabinoids (Figure 3B). The endogenous CB1/2 agonist, AEA (100 nmol/L) and NE, a CB1-selective agonist at 50 nmol/L, induced notable increases in ERK phosphorylation, whereas the synthetic CB1/2 agonist, WIN 55,212-2 (WIN, 100 nmol/L) and the CB2-selective synthetic agonist, JWH133 (JWH, 20 nmol/L) induced more robust signals (Figure 3B). Phosphorylation above basal levels of PKB was achieved by all cannabinoid receptor agonists used (Figure 3C). Interestingly, the increase in phosphorylation of PKB induced by the CB1-selective agonist, NE, was not as robust as the synthetic CB2-selective agonist, JWH, or the CB1/2 agonists, AEA and WIN. In addition, phosphorylation of both the α and β isoforms of GSK3 was induced by cannabinoids, although at this time point and dose, NE induced a negligible increase in phosphorylation (Figure 3D). Vehicle controls were either ethanol or dimethyl sulfoxide, as indicated.

       Analysis of Freshly Isolated Primary Normal Colonic Epithelial Cells

      The expression of the CB1 receptor on the colonic epithelium initially was surprising, but having shown that human colonic epithelial cell lines expressed the receptors and responded to a range of cannabinoids we sought to validate these findings and our immunohistochemical data further by purifying normal human colonic epithelial cells and probing for cannabinoid receptors at the protein level. After every isolation procedure, we verified that the cells were indeed of epithelial origin based on the expression and absence of appropriate cell markers. Immunoblotting of whole-cell lysates of isolated epithelial cells, peripheral blood lymphocytes, and primary human colonic myofibroblasts showed that the isolated colonic epithelial cells expressed human epithelial-specific antigen, but not immune cell—specific protein tyrosine kinase, Lck, or myofibroblast-specific smooth muscle actin (Figure 4A). CB1- and CB2-receptor expression was determined in whole-cell extracts of purified epithelial cells and compared with primary peripheral blood lymphocytes, the leukemic T-cell line, MOLT-4, and primary colonic myofibroblasts. A 52-kilodalton protein corresponding to the predicted weight of CB1 was seen in all primary epithelial cell extracts from different patients (n = 12) (Figure 4B). In addition, an 83-kilodalton band also was detected in all cell types, although it was not the predominant band in primary epithelial cells. The 64-kilodalton band detected in primary colonic myofibroblasts also is thought to be a differentially modified form of the CB1 receptor.
      • Pettit D.A.
      • Harrison M.P.
      • Olson J.M.
      • Spencer R.F.
      • Cabral G.A.
      Immunohistochemical localization of the neural cannabinoid receptor in rat brain.
      • Song C.
      • Howlett A.C.
      Rat brain cannabinoid receptors are N-linked glycosylated proteins.
      The 47-kilodalton CB2-receptor protein, previously described as a modified form of CB2,
      • Carayon P.
      • Marchand J.
      • Dussossoy D.
      • Derocq J.M.
      • Jbilo O.
      • Bord A.
      • Bouaboula M.
      • Galiegue S.
      • Mondiere P.
      • Penarier G.
      • Fur G.L.
      • Defrance T.
      • Casellas P.
      Modulation and functional involvement of CB2 peripheral cannabinoid receptors during B-cell differentiation.
      is expressed in peripheral blood lymphocytes, MOLT-4, and primary colonic myofibroblasts, but not in primary epithelial cells (Figure 4B). Only the leukemic T-cell line exhibited the 39-kilodalton CB2 protein. For comparison, we used CB-receptor antibodies from Affinity BioReagents and Santa Cruz for immunoblotting of primary epithelial cells. CB1-receptor protein was expressed similarly and CB2-receptor protein was absent (Figure 4C).
      To characterize further the presence of components of the endogenous cannabinoid system in primary colonic epithelial cells, fractionation experiments were performed. The endocannabinoid degrading enzyme, FAAH, was found to be membrane-bound exclusively (n = 6) (Figure 4D, left top panel). In parallel, immunoblotting showed both membrane and cytoplasmic localization of the 52-kilodalton CB1 receptor (n = 6) (Figure 4D, left middle panel), whereas no membrane-bound CB2 was detected (n = 6) (Figure 4D, left bottom panel). There was some variation in the expression of cytoplasmic CB2 in primary cells. In most cases there was no expression (n = 4) (Figure 4D, bottom panel), but in 2 cases there was a low level of expression after overexposure, which nonetheless barely was detectable (n = 2, data not shown). HT29 fractionation analysis showed differential localization of the components of the endogenous cannabinoid system. FAAH again was localized to the membrane (Figure 4C, right top panel). CB1-receptor localization remained similarly detected in both membrane and cytoplasmic fractions (Figure 4C, right middle panel). Interestingly, CB2 receptor (47 kilodaltons) was expressed differentially in HT29 (Figure 4C, right bottom panel). Membrane localization was enhanced substantially when compared with cytoplasmic localization.

       Cannabinoid Binding Experiments

      Ligand binding experiments with the nonselective CB1/CB2 agonist, [3H]-CP-55,940, were performed to verify epithelial CB1 expression. We were unable to purify sufficient primary epithelial cells to perform these experiments and thus used the colonic epithelial cell line, DLD1. These cells express both CB1 and CB2 receptors and data from competition studies showed a median inhibitory concentration (IC50) of 1.3 nmol/L for ACPA (a CB1-specific synthetic agonist) and 2.7 nmol/L for JWH (the CB2-specific agonist) (Figure 5A and B). We therefore used 10 nmol/L for both agonists in subsequent experiments. Displacement curves for methanandamide (a stable analogue for anandamide) showed a lower median inhibitory concentration (14.4 nmol/L) than for anandamide (82.8 nmol/L) (Figure 5C). The displacement curve for AM251, the CB1 antagonist/inverse agonist, defined 2 median inhibitory concentration values of 6.1 nmol/L and 1.4 μmol/L for CB1 and CB2, respectively (Figure 5D).
      Figure thumbnail gr5
      Figure 5Displacement of [3H]-CP-55,940 binding to human colonic epithelial cells. Binding assays were performed as described in the Materials and Methods section using DLD1 cell membranes, .2 nmol/L [3H]-CP-55,940 and increasing concentrations of unlabeled cannabinoid, (A) ACPA, (B) JWH, (C) AEA and mAEA, and (D) AM251. Data are from 2 separate experiments performed in duplicate and are expressed as the percentage of specific binding in the absence of unlabeled cannabinoid.

       Cannabinoid Signaling in Primary Colonic Epithelial Cells

      To assess whether the CB receptors that were detected by immunostaining and immunoblotting were functional, we investigated whether any biochemical responses could be elicited from primary epithelial cells by a range of cannabinoid ligands. An important caveat to using these particular primary cells is, first, the paucity of cells isolated from each specimen (1–2 × 107) and, second, the resultant apoptosis of these cells over 24-hour postpurification. Thus, the diversity of experiments and the ability to perform exact experimental replicates is limited. Nevertheless, we were able to perform short-term transient signaling experiments with freshly isolated cells. We used synthetic and endogenous cannabinoids with specificity for either CB1 or that could bind both receptors and, once again, measured their ability to phosphorylate ERK, PKB, and GSK3α/β by immunoblotting. Primary colonic epithelial cells exhibit moderate to high basal levels of ERK phosphorylation, which can be enhanced rapidly on stimulation by cannabinoids (Figure 6A). The synthetic CB1/2 agonist, WIN 55,212-2 (100 nmol/L), and the endogenous CB1/2 agonist, anandamide (100 nmol/L), induced small transient changes in ERK phosphorylation, which were sometimes difficult to assess when basal levels of phosphorylation were high. However, a robust signal was achieved with the CB1-selective agonist NE (50 nmol/L), which was reproducible in 6 different patient samples. Although the presence of this endocannabinoid has not been measured in gut, it proved a useful tool in subsequent experiments. Phosphorylation of PKB was undetectable in these cells using immunoblotting techniques, although PKB was expressed (data not shown). Interestingly, GSK3α/β was phosphorylated rapidly in response to NE (Figure 6B). Pretreatment with the CB1 antagonist, AM251, at the CB1-selective concentration of 10 nmol/L showed that activation of ERK was CB1-receptor dependent (Figure 6C, top panel). Another approach for assessing the involvement of the PI3K—PKB signaling axis is to measure phosphorylation of ERK and GSK3α/β after inhibition of PI3K. However, pretreatment of cells with the PI3K inhibitor LY294002 (10 μmol/L) had no effect on baseline or induced phosphorylation of ERK, indicating that this signal was not PI3K-dependent (Figure 6C, top panel). PD 098059 is a cell-permeable, specific inhibitor of the kinases that phosphorylate and activate ERK (MAPK/ERK kinase [MEK1/2]). It is used widely to show mitogen-activated protein kinase—dependent processes in intact cells, and pretreatment of cells with PD098059 completely blocked the NE-induced ERK phosphorylation (Figure 6C, bottom panel). Phosphorylation of GSK3α/β was CB1-dependent because this signal was blocked by AM251 (Figure 6D, top left panel). Interestingly, phosphorylation of the α isoform of GSK3 was inhibited partially by the MEK inhibitor, whereas phosphorylation of the β isoform was inhibited completely by the MEK inhibitor (Figure 6D, top right and bottom left panels). Similar to ERK phosphorylation, the inactivation of GSK3α/β was unaffected by PI3K inhibition (Figure 6D, bottom panels).
      Figure thumbnail gr6
      Figure 6Cannabinoid-induced signaling in primary human colonic epithelial cells. Each experiment is from one patient and experimental design progressed through different patients (P1—P7). Repeat stimulations in different formats occurred at least twice. In all cases, freshly isolated primary cells (107/mL) were lysed after stimulation and 20 μg of protein was subjected to electrophoresis and immunoblotting with either anti—phospho-ERK or anti—phospho-GSK3α/β as shown. Membranes were stripped and reprobed with anti—pan-ERK, anti—pan-PKB, and anti—pan-GSK where shown. (A) ERK phosphorylation induced by cannabinoids. P1, the vehicle used was dimethyl sulfoxide and stimulations were for 5 minutes. NE (50 nmol/L), AEA (100 nmol/L), and WIN (100 nmol/L). P2, time course for NE (50 nmol/L), vehicle control was ethanol for 5 minutes. P3, Repeat time course for NE (50 nmol/L) to include 5-minute stimulation, vehicle was ethanol for 2 minutes. P4, time course for AEA (100 nmol/L), ethanol vehicle for 2 minutes. (B) GSK3α/β phosphorylation induced by NE. By using remaining cell lysates from P2 and P3 as described earlier, a new membrane was generated as shown. NE (50 nmol/L) stimulations were 2 minutes. After probing for phospho-GSK3α/β, the membrane was stripped twice and reprobed as shown. (C) Upstream regulators of NE-induced ERK phosphorylation in primary cells. P5 and P6, CB1 receptor antagonist, AM251 (10 nmol/L) was added 5′ before NE (50 nmol/L) stimulations for 5 minutes. Similarly, the PI3K inhibitor, LY294002 (10 μmol/L), and the MEK inhibitor, PD98059 (10 μmol/L), were added 30′ before stimulation with NE (50 nmol/L). Both dimethyl sulfoxide and ethanol were used as vehicles in this experiment and stimulations were for 5′. (D) Upstream regulators of NE-induced GSK3α/β phosphorylation in primary cells. Remaining cell lysates from P5 and P6 as described earlier were used as indicated to generate new membranes. P7, cells were treated in the presence or absence of either vehicle or inhibitors at the concentrations and times described in (C). Cells were stimulated further with either vehicle or NE (50 nmol/L) for 5 minutes.

       Cannabinoids Modulate Epithelial Wound Healing

      IBD is characterized by ulcerative symptoms and delayed wound healing.
      • Podolsky D.K.
      Inflammatory bowel disease.
      Continuity of the epithelial barrier after injury is rapid (minutes to hours), and restitution is achieved by 3 processes that probably overlap and may not be distinct.
      • Sturm A.
      • Dignass A.U.
      Modulation of gastrointestinal wound repair and inflammation by phospholipids.
      Initially, restitution is achieved by epithelial dedifferentiation and migration, followed by proliferation, and, finally, differentiation and maturation. LPA has been shown to enhance intestinal epithelial wound healing through increased epithelial cell migration
      • Sturm A.
      • Sudermann T.
      • Schulte K.M.
      • Goebell H.
      • Dignass A.U.
      Modulation of intestinal epithelial wound healing in vitro and in vivo by lysophosphatidic acid.
      and, in a rat model of colitis, LPA treatment showed a reduction in weight loss, mucosal inflammation, and necrosis.
      • Sturm A.
      • Sudermann T.
      • Schulte K.M.
      • Goebell H.
      • Dignass A.U.
      Modulation of intestinal epithelial wound healing in vitro and in vivo by lysophosphatidic acid.
      LPA also has been shown to induce DLD1 migration at low concentrations (10–100 nmol/L).
      • Shida D.
      • Kitayama J.
      • Yamaguchi H.
      • Okaji Y.
      • Tsuno N.H.
      • Watanabe T.
      • Takuwa Y.
      • Nagawa H.
      Lysophosphatidic acid (LPA) enhances the metastatic potential of human colon carcinoma DLD1 cells through LPA1.
      We therefore used LPA as a positive control for wound-healing experiments with 2 colonic epithelial cell lines, HT29 and DLD1, and investigated whether cannabinoids could modulate wound closure in vitro (Figure 7 and Table 1). HT29 cells do not spontaneously migrate after wounding, whereas DLD1 cells migrate after wounding even without serum (data not shown). As expected, LPA stimulated a modest wound closure in HT29, although this was only at the highest concentration used (5 μmol/L) and not at 100 nmol/L. Similarly, the endogenous cannabinoids AEA and NE, at physiologically relevant concentrations, also were able to stimulate modest wound closure. The CB1-selective agonist ACPA also induced wound closure, but the CB2-selective agonist (JWH133) had no effect. Interestingly, AEA, NE, and ACPA significantly (P ≤ .05) enhanced wound closure when added together with LPA, whereas JWH133 had no further effect on LPA-induced wound closure. This implies that colonic epithelial wound healing is modulated by the CB1 receptor. Similar effects were observed in DLD1 cells where LPA, NE, and ACPA at low nanomolar concentrations were able to further induce wound closure over spontaneous closure. However, notably, AEA and JWH133 were unable to enhance the spontaneous migration of these cells whereas AEA and ACPA were able to enhance wound closure further when added together with LPA (10 nmol/L). However, neither NE nor JWH133 had additive effects with LPA at either 10 nmol/L or 100 nmol/L. At the concentrations used for wound healing experiments, neither LPA nor the cannabinoids induced changes in proliferation (data not shown).
      Figure thumbnail gr7
      Figure 7Wound healing in DLD1 cells. Confluent monolayers of epithelial cells were wounded as described in the Materials and Methods section. Wound widths and images (40×) were taken before and after 24-hour stimulation with compounds at the concentrations shown.
      Table 1Percentage Reduction in Wound Width 24 Hours After Stimulation
      TreatmentHT29DLD1
      Vehicle control ethanol (.001%)0 ± 019.91 ± .9
      LPA 10 nmol/L29.00 ± 1.3
      P ≤ .05 compared with vehicle control.
      LPA 100 nmol/L0 ± 030.74 ± 3.6
      P ≤ .05 compared with vehicle control.
      LPA 5 μmol/L6.76 ± 1.2
      P ≤ .05 compared with vehicle control.
      22.72 ± 1.1
      AEA 100 nmol/L9.09 ± 3.1
      P ≤ .05 compared with vehicle control.
      25.07 ± 5.2
      NE 50 nmol/L16.66 ± 4.4
      P ≤ .05 compared with vehicle control.
      28.85 ± 3.7
      P ≤ .05 compared with vehicle control.
      ACPA 10 nmol/L11.11 ± 4.8
      P ≤ .05 compared with vehicle control.
      27.95 ± 5.6
      P ≤ .05 compared with vehicle control.
      JWH 10 nmol/L0 ± 020 ± 4.4
      LPA (10 nmol/L) + AEA32.29 ± 1.7
      P ≤ .05 compared with LPA 5 μmol/L (HT29) and LPA 10 nmol/L or 100 nmol/L (DLD1).
      LPA (100 nmol/L) + AEA27.28 ± 10.6
      LPA (5 μmol/L) + AEA22.22 ± 4.9
      P ≤ .05 compared with LPA 5 μmol/L (HT29) and LPA 10 nmol/L or 100 nmol/L (DLD1).
      LPA (10 nmol/L) + NE28.78 ± 8.8
      LPA (100 nmol/L) + NE27.67 ± 6.0
      LPA (5 μmol/L) + NE26.52 ± 5.3
      P ≤ .05 compared with LPA 5 μmol/L (HT29) and LPA 10 nmol/L or 100 nmol/L (DLD1).
      LPA (10 nmol/L) + ACPA41.86 ± 7.7
      P ≤ .05 compared with LPA 5 μmol/L (HT29) and LPA 10 nmol/L or 100 nmol/L (DLD1).
      LPA (100 nmol/L) + ACPA23.08 ± 10.7
      LPA (5 μmol/L) + ACPA18.3 ± 2.7
      P ≤ .05 compared with LPA 5 μmol/L (HT29) and LPA 10 nmol/L or 100 nmol/L (DLD1).
      LPA (10 nmol/L) + JWH25.1 ± 2.6
      LPA (100 nmol/L) + JWH33.33 ± 3.5
      LPA (5 μmol/L) + JWH4.54 ± 1.1
      NOTE. Confluent monolayers of epithelial cells were wounded as described in the Materials and Methods section. Wound widths were measured before and after 24 hours of stimulation with compounds at the concentrations shown. Changes in wound widths are expressed as a percentage and compared with the relevant control (vehicle or LPA). Each experiment was performed in triplicate and at least 3 times. All results from all experiments were pooled and statistically analyzed (see Materials and Methods section for more detail).
      a P ≤ .05 compared with vehicle control.
      b P ≤ .05 compared with LPA 5 μmol/L (HT29) and LPA 10 nmol/L or 100 nmol/L (DLD1).
      By using this information, we performed more detailed studies of the cannabinoid-induced wound healing in DLD1 cells. The stable analogue of anandamide, methanandamide, alone induced wound closure and enhanced LPA-induced wound closure in a classic bell-shaped response, maximal at 10 nmol/L (Figure 8A). Pertussis toxin (PT), LY294002, and PD98059 were able to inhibit spontaneous and ACPA-induced wound closure (Figure 8B). AM251 at 10 nmol/L blocked ACPA-induced wound closure, whereas at 100 nmol/L spontaneous wound closure also was inhibited partially. CB1 immunoprecipitation studies were performed on wounded monolayers, stimulated with either ACPA (10 nmol/L) alone or in combination with LPA (10 nmol/L). Immunoblotting of CB1 with the LPA1 receptor antibody showed that increasing concentrations of ACPA in combination with LPA (10 nmol/L) resulted in an increased association between the CB1 and LPA1 receptors (Figure 8C). These results support a role for CB1 ligation in wound closure and propose limited cross-talk between LPA and CB receptors.
      Figure thumbnail gr8
      Figure 8Modulation of wound healing in DLD1 cells. Confluent monolayers of epithelial cells were wounded as described in the Materials and Methods section. Wound widths were measured before and after 24-hour stimulation with compounds at the concentrations shown. Changes in wound widths are expressed as a percentage and compared with the relevant control (vehicle or LPA). Each experiment was performed in duplicate and at least 3 times. (A) mAEA (.1 nmol/L—1 μmol/L) alone or concurrently with LPA (10 nmol/L). (B) Inhibitors were applied to wounded monolayers at the following concentrations and times before addition of ACPA (10 nmol/L) for 24 hours: PT (pertussis toxin) 100 ng/mL (16 h), PD (PD98059) 10 μmol/L (1 h), LY (LY294002) 10 μmol/L (30′), and AM (AM251) 10 nmol/L and 100 nmol/L (10′). (C) Wounded monolayers were stimulated with either vehicle (EtOH) or LPA (10 nmol/L) alone or in combination with ACPA at the concentrations shown. Cells were lysed after 5′. Anti-CB1 (Santa Cruz) immunoprecipitates were separated on 12% gels and membranes were probed with anti-LPA1. Blot is representative of 2 separate experiments.

      Discussion

      The present study provides evidence for the distribution of both CB1 and CB2 cannabinoid receptors in the human colon. Recently, the effects of cannabinoids on the gastrointestinal tract have been given some attention. Although these studies have been related to homeostatic functions such as gut motility,
      • Izzo A.A.
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      pathophysiologic roles in paralytic ileus,
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      intestinal secretion,
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      An endogenous cannabinoid tone attenuates cholera toxin-induced fluid accumulation in mice.
      and gastrointestinal inflammation
      • Izzo A.A.
      • Fezza F.
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      • Esposito G.
      • Mascolo N.
      • Di Marzo V.
      • Capasso F.
      Cannabinoid CB1-receptor mediated regulation of gastrointestinal motility in mice in a model of intestinal inflammation.
      • McVey D.C.
      • Schmid P.C.
      • Schmid H.H.
      • Vigna S.R.
      Endocannabinoids induce ileitis in rats via the capsaicin receptor (VR1).
      • Massa F.
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      • Cannich A.
      • Monory K.
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      • Ferri G.L.
      • Sibaev A.
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      The endogenous cannabinoid system protects against colonic inflammation.
      • Croci T.
      • Landi M.
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      • Marini P.
      Role of cannabinoid CB1 receptors and tumor necrosis factor-alpha in the gut and systemic anti-inflammatory activity of SR 141716 (Rimonabant) in rodents.
      also have been attributed to AEA and the CB1 receptor. In human beings, CB1 receptors functionally are present in ileum and colon, resulting in inhibition of excitatory cholinergic pathways subserving smooth muscle contraction
      • Croci T.
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      In vitro functional evidence of neuronal cannabinoid CB1 receptors in human ileum.
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      Functional assessment of neuronal cannabinoid receptors in the muscular layers of human ileum and colon.
      and normal human mucosa contains AEA and 2-arachidonoyl glycerol and transcript for both CB receptors, although the cellular origin was not investigated.
      • Ligresti A.
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      Possible endocannabinoid control of colorectal cancer growth.
      In our study, CB1 expression was particularly high on the microvillus border, which faces the lumen, and we believe this to be a novel finding. The presence and distribution of CB1 on smooth muscle and nerve cells is comparable with a recent murine study
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      Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.
      and a previous study in human beings.
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      • Ferla G.
      Functional assessment of neuronal cannabinoid receptors in the muscular layers of human ileum and colon.
      Interestingly, the murine study did report a dense pattern of CB1 expression on murine colonic epithelial villi,
      • Casu M.A.
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      Differential distribution of functional cannabinoid CB1 receptors in the mouse gastroenteric tract.
      but disregarded it because it persisted in preabsorption and omission controls. We found the CB1 antibody to be particularly pH sensitive and the CB2 antibody to a lesser extent and therefore performed our immunohistochemical studies at a pH of 9.0, rather than a pH of 7.4, which is the standard protocol. In addition, our preabsorption controls were negative for CB1 in the microvillus border. Further support for the epithelial CB1-receptor expression came from the immunoblotting experiments. Purified primary colonic epithelial cells from all 12 tissue samples expressed CB1 receptors. Membrane fractions of both primary cells and HT29 cells showed CB1 in the cytoplasm and membrane.
      Normal bowel is regarded as continuously being activated immunologically, driven by the presence of normal luminal flora.
      • Swidsinski A.
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      Mucosal flora in inflammatory bowel disease.
      • Panja A.
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      Synthesis and regulation of accessory/proinflammatory cytokines by intestinal epithelial cells.
      For example, the presence of macrophages and plasma cells in the lamina propria is characteristic of normal bowel.
      • Wheater P.R.
      • Burkitt H.G.
      • Daniels V.G.
      In response to bacterial products and antigens from dietary sources, surface epithelium can produce cytokines and chemokines that recruit and activate mucosal immune cells.
      • Podolsky D.K.
      Inflammatory bowel disease.
      Because the CB2 receptor is believed to have immune function, it is interesting to note that in our study macrophages in the lamina propria express CB2 and the antibody-secreting B cells express both receptors. Immune cells in the germinal centers lack CB1 and CB2 receptors, which initially was unexpected. However, this distribution of CB2 correlates with a previous study that showed a role for CB2 in B-cell differentiation
      • Carayon P.
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      Modulation and functional involvement of CB2 peripheral cannabinoid receptors during B-cell differentiation.
      such that CB2 expression was weak in tonsillar virgin B cells in the germinal centers, absent during differentiation, and marked on memory B cells. Interestingly, the lack of CB2 receptors in Caco2 cells seen by Ligresti et al
      • Ligresti A.
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      Possible endocannabinoid control of colorectal cancer growth.
      is proposed to relate to differentiation status. It is conceivable that our primary epithelial cells were at a different stage of differentiation. On the other hand, gut epithelium is pivotal to host defense
      • Podolsky D.K.
      Inflammatory bowel disease.
      and the increase of CB2 receptor in the epithelium of IBD tissue implies an additional role for this receptor in inflammation. Indeed, an anti-inflammatory role for CB2 in the colonic epithelial cell line HT29 has been reported.
      • Ihenetu K.
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      • Parsons M.E.
      • Whelan C.J.
      Inhibition of interleukin-8 release in the human colonic epithelial cell line HT-29 by cannabinoids.
      Increased levels of interleukin-8 are thought to contribute to the pathogenesis of IBD
      • Mahida Y.R.
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      Enhanced synthesis of neutrophil-activating peptide-1/interleukin-8 in active ulcerative colitis.
      and CB2-mediated inhibition of interleukin-8 secretion from HT29 cells supports an anti-inflammatory role for CB2 in IBD. We were able to replicate this result using JWH133 at 10 nmol/L (data not shown). In addition, the activation of rat intestinal CB2 receptors in response to lipopolysaccharide, to inhibit intestinal transit, highlights the possibility that CB2 mediates this mechanism for the re-establishment of normal gastrointestinal transit after an inflammatory stimulus.
      • Mathison R.
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      • Sharkey K.A.
      Effects of cannabinoid receptor-2 activation on accelerated gastrointestinal transit in lipopolysaccharide-treated rats.
      An antiproliferative role for the CB2 receptor in tumor cells (DLD1) also has been proposed
      • Ligresti A.
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      • Di Marzo V.
      Possible endocannabinoid control of colorectal cancer growth.
      and one could speculate that CB2 expression can be induced during inflammation as a modulator of the immune response, whereby CB2-mediated PI3K activation has anti-inflammatory potential and, in chronic inflammation, contributes to malignant transformation.
      Further analysis of the CB1 receptors found in the epithelium was undertaken to confirm that they were functional through their ability to couple to previously characterized signal transduction pathways induced by synthetic and endogenous cannabinoids. Our data indicate that the colonic epithelial cell lines can respond to synthetic and endogenous cannabinoids through the phosphorylation of ERK, PKB, and GSK3α/β. Coupling of the PI3K pathway to both CB1 and CB2 has been observed in a prostate epithelial cell line, PC-3,
      • Sanchez M.G.
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      Activation of phosphoinositide 3-kinase/PKB pathway by CB(1) and CB(2) cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction.
      and this correlates with our data in colonic epithelial cell lines.
      In freshly isolated primary cells, cannabinoids induced phosphorylation of both the ERK and GSK3α/β pathways. Three lines of evidence suggest that these events are CB1-mediated. First, no detectable CB2-receptor expression was evident in the epithelium of primary tissue or in isolated primary epithelial cells. Second, NE exhibits selectivity toward CB1 at the concentration used in this study (50 nmol/L). Third, the CB1-selective antagonist, AM251 (10 nmol/L), inhibited both signals. In addition, cannabinoid-induced ERK phosphorylation clearly was MEK-dependent, as evidenced by complete inhibition by PD 098059. MEK inhibition partially reduced GSK3α/β phosphorylation to varying extents, indicating a limited dependence on ERK activation for this signal, as has been described elsewhere.
      • Sutherland C.
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      • Cohen P.
      Inactivation of glycogen synthase kinase-3 beta by phosphorylation new kinase connections in insulin and growth-factor signalling.
      • Sutherland C.
      • Cohen P.
      The alpha-isoform of glycogen synthase kinase-3 from rabbit skeletal muscle is inactivated by p70 S6 kinase or MAP kinase-activated protein kinase-1 in vitro.
      • Cross D.A.
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      • Cohen P.
      • Andjelkovich M.
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      Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B.
      The partial sensitivity of GSK3 phosphorylation to MEK inhibition is interesting because GSK3 is also a known substrate for the PI3K effector, PKB.
      • Cross D.A.
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      Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B.
      However, we were unable to detect phosphorylation of PKB in the primary epithelial cells and PI3K inhibition with LY 294002 had no effect on either ERK or GSK3 phosphorylation. The activation of PI3K-PKB by cannabinoids in epithelial cell lines, but not primary cells, could be accounted for by (1) dysregulated PI3K—PKB signal transduction in tumor cells or (2) phosphorylation of PKB in primary cells occurs below the detection limit of the assay. Alternatively, there is evidence that CB1 and CB2 are coupled differentially to the PI3K—PKB pathway. A recent study in mast cells showed that cannabinoid-induced PKB activation is CB2-dependent and not mediated by CB1
      • Samson M.T.
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      Differential roles of CB1 and CB2 cannabinoid receptors in mast cells.
      and our normal primary cells have no CB2 receptor. GSK3 phosphorylation may occur through cannabinoid-induced inhibition of GSK-directed phosphatases, independently of PKB.
      Biochemical coupling of the CB1 receptor to signaling pathways in primary cells then was related to a functional outcome in vitro. Cannabinoids have been shown to have an antiproliferative role in colonic epithelial cell lines, Caco2 and DLD1,
      • Ligresti A.
      • Bisogno T.
      • Matias I.
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      • Cosenza V.
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      • Scaglione G.
      • Bifulco M.
      • Sorrentini I.
      • Di Marzo V.
      Possible endocannabinoid control of colorectal cancer growth.
      although at the concentrations used in our study (low nmol/L), we found no effect on proliferation in all 3 cell lines. Cannabinoids induce chloride secretion in ocular epithelium
      • Shi C.
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      • Hung O.
      • Kelly M.E.
      A3 adenosine and CB1 receptors activate a PKC-sensitive Cl- current in human nonpigmented ciliary epithelial cells via a G beta gamma-coupled MAPK signaling pathway.
      and inhibit gap junctions in liver epithelium,
      • Upham B.L.
      • Rummel A.M.
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      • Trosko J.E.
      • Ouyang Y.
      • Crawford R.B.
      • Kaminski N.E.
      Cannabinoids inhibit gap junctional intercellular communication and activate ERK in a rat liver epithelial cell line.
      which may be relevant to the regulation of barrier permeability in colonic epithelium. However, a recent publication showed that CB1 mediates intrinsic protective signals against dinitrobenzene sulphonic acid (DNBS)-induced colitis through an early control of inflammation-induced irritation of smooth muscle cells.
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • Cannich A.
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      • Cravatt B.F.
      • Ferri G.L.
      • Sibaev A.
      • Storr M.
      • Lutz B.
      The endogenous cannabinoid system protects against colonic inflammation.
      DNBS treatment increased the percentage of myenteric neurons expressing CB1 receptors, suggesting an enhancement of cannabinoid signaling during colitis. In addition, CB1-receptor knock-out mice and CB1R blockade increased the severity of induced colitis.
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • Cannich A.
      • Monory K.
      • Cravatt B.F.
      • Ferri G.L.
      • Sibaev A.
      • Storr M.
      • Lutz B.
      The endogenous cannabinoid system protects against colonic inflammation.
      The macroscopic score of colonic inflammation, which assesses colonic damage, was reduced by cannabinoid administration in wild-type mice. FAAH−/− mice, in which an increased presence of anandamide is inferred, also had improved macroscopic scores. Massa et al
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • Cannich A.
      • Monory K.
      • Cravatt B.F.
      • Ferri G.L.
      • Sibaev A.
      • Storr M.
      • Lutz B.
      The endogenous cannabinoid system protects against colonic inflammation.
      noted particular colonic epithelial damage in the CB1−/− mice after DNBS-induced colitis.
      • Massa F.
      • Marsicano G.
      • Hermann H.
      • Cannich A.
      • Monory K.
      • Cravatt B.F.
      • Ferri G.L.
      • Sibaev A.
      • Storr M.
      • Lutz B.
      The endogenous cannabinoid system protects against colonic inflammation.
      Our in vitro wound-healing data show that (1) wound widths in HT29 cells closed marginally to LPA and the cannabinoids, which was enhanced significantly when LPA and cannabinoids were added together, and (2) nanomolar concentrations of LPA induced wound closure in DLD1 cells, which AEA, mAEA, and ACPA were able to enhance this wound closure significantly. Interestingly, the maximal wound closure induced by anandamide was 100 nmol/L and this 10-fold shift in concentration when compared with mAEA may relate to the metabolic stability of mAEA over the 24-hour period of the assay. The CB1 antagonist, AM251, at 10 nmol/L blocked the ACPA-induced wound-healing response. In addition, AM251 at 100 nmol/L not only blocked the ACPA response but also partially inhibited the spontaneous wound closure. The mechanism of this additional inhibition is unknown, but a possible explanation could be related to the processes involved in spontaneous wound closure. Wounding may induce an increase in the levels of endogenous cannabinoids or LPA (or both) to facilitate wound closure, and AM251 could influence this process in either or both of 2 ways: (1) antagonism of endogenously released cannabinoids at CB1 receptors and (2) increased inverse agonist activity.
      • Pertwee R.G.
      Inverse agonism and neutral antagonism at cannabinoid CB(1) receptors.
      Cannabinoid-induced small wound closure is likely to be mediated by the CB1 receptor for 3 reasons: (1) the CB1-specific agonist ACPA was able to induce wound closure at concentrations shown to bind CB1 and not CB2, (2) the CB1-specific antagonist AM251 was able to reverse this response, and (3) the CB2-specific agonist had no effect. Cross-talk between LPA and CB receptors has been observed
      • Molderings G.J.
      • Bonisch H.
      • Hammermann R.
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      Noradrenaline release-inhibiting receptors on PC12 cells devoid of alpha(2(-)) and CB(1) receptors similarities to presynaptic imidazoline and edg receptors.
      • Bouaboula M.
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      • Carayon P.
      • Combes T.
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      Gi protein modulation induced by a selective inverse agonist for the peripheral cannabinoid receptor CB2 implication for intracellular signalization cross-regulation.
      • Hiley C.R.
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      Cannabinoid pharmacology in the cardiovascular system potential protective mechanisms through lipid signalling.
      and our biochemical data provide evidence for a heteromeric-receptor complex that produces an additive functional effect in colonic epithelium. Thus, exposure of colonic epithelium to LPA and cannabinoids facilitated closure of in vitro small wounds through heteromeric-receptor association and enhanced cell migration instead of proliferation. The involvement of ERK1/2 and GSK3 in cell spreading recently has been proposed
      • Matsubayashi Y.
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      ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing.
      • Roberts M.S.
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      • Roberts M.S.
      • Woods A.J.
      • Dale T.C.
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      Protein kinase B/Akt acts via glycogen synthase kinase 3 to regulate recycling of alpha v beta 3 and alpha 5 beta 1 integrins.
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      Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity.
      and with the use of inhibitors, our data provide evidence for a G-protein—coupled and an ERK1/2-dependent mechanism by which lipid mediators may influence intestinal wound healing in vitro. A partial dependence on PI3K also is evident, although the role of class II PI3Ks cannot be discounted. In vivo, disruptions to these signaling pathways observed in IBD might account for the ulcerative symptoms and delayed wound healing.
      • Podolsky D.K.
      Inflammatory bowel disease.
      These studies strengthen the hypothesis of a physiologic role for endocannabinoids in the human gastrointestinal tract. Consideration should be given that CB1 receptors are expressed preferentially in the apical zone of normal colonic epithelium where CB2 receptors largely are absent. The physiologic relevance of the cannabinoid-induced phosphorylation of ERK and GSK3α/β pathways within minutes may be related to a response to epithelial injury although it cannot be discounted that the epithelial CB1 receptor has an immunomodulatory role, where bacterial products from the lumen and locally produced endocannabinoids serve to ensure an appropriate immune response to normal gut flora. Increased epithelial CB2 expression in the acute phase of IBD, through which interleukin-8 secretion is reduced, implies a potentially anti-inflammatory role for this receptor.
      The immunohistochemical, biochemical, pharmacologic, and functional evidence shown here strengthens the proposal that cannabinoids may have a direct influence on the human large intestine. In addition, having identified the regions where functional cannabinoid receptors are expressed, further investigations into their differential physiology and potential as modulators of gastrointestinal pathophysiology should be performed.
      The authors thank Priscilla Russell (Royal United Hospital, Bath, United Kingdom), who provided excellent technical expertise for the immunohistochemical studies; and Darran Cronshaw and Andreas Kouroumalis (University of Bath, Bath, United Kingdom), who generously provided extracts of peripheral blood lymphocytes, CEM, and MOLT-4 T-cells (to D.C.) and primary colonic myofibroblasts (to A.K.).

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