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First published online 6 February 2003
doi: 10.1242/jcs.00321

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Adherens junctions and tight junctions are regulated via different pathways by progastrin in epithelial cells

¹ Laboratoire de Signalisation Cellulaire Normale et Tumorale, EA MNRT 2995, Faculté de Pharmacie, 15 Avenue C. Flahault, 34093 Montpellier, France
² University of Melbourne, Department of Surgery, Austin Hospital, Melbourne, VIC 3084, Australia
³ CNRS UPR 1086, Route de Mende, 34293 Montpellier, France

View larger version (51K): [in a new window]   Fig. 1. Progastrin dissociates AJs and TJs. (A) Treatment of the IMGE-5 cell line, which does not produce progastrin-derived peptides, for up to 4 hours with 5 nM recombinant human progastrin6-80 did not affect the expression of ß-catenin (ß-cat), E-cadherin (E-cad), ZO-1, occludin (Occl), claudin-1 (Cld-1), p125FAK or actin, as assessed by western blotting of cell lysates. (B) Immunostaining for AJ and TJ proteins was observed using a confocal microscope as described in Materials and Methods. Series of horizontal optical sections (0.328 µm each) were collected, and images represent a merging of sections spanning the apical region of IMGE-5 cells (ten sections, 3.28 µm). Claudin-1 (Cld-1), ZO-1 and the AJ proteins E-cadherin (E-cad) and ß-catenin (ß-cat) were located in the most apical region of the lateral membrane in untreated (U) cells. Treatment with 5 nM progastrin6-80 (PG) for 4 hours induced a partial delocalisation of all four proteins from the membrane to the cytoplasm. Bar, 7 µm. (C) Progastrin treatment also induced a time-dependent partial dissociation of the complex between occludin (white bars) and ZO-1 (black bars), as assessed by densitometric scanning of western blots of ZO-1 immunoprecipitates of cell lysates. Densitometric analysis represents the average of four experiments, and statistical significance was assessed by Student's t test. *P<0.05; **P<0.01.

View larger version (60K): [in a new window]   Fig. 2. Reduction of gastrin expression induces an epithelial-like morphology. (A) DLD-1 colorectal carcinoma cells stably transfected with an antisense gastrin construct (ASG, grey bars) produced (cell extracts) and secreted (conditioned media) significantly less progastrin (PG), Ggly and Gam than cells transfected with vector only (VO, black bars), as measured using the radioimmunoassays described previously (Hollande et al., 1997). The statistical significance of the differences between VO and ASG clones was assessed using ANOVA. *P<0.05; **P<0.01; n=3. The reduction of gastrin expression induced an epithelial-like morphology (B), as assessed by bright-field microscopy (bar, 20 µm, upper panels, or 10 µm, lower panels), and increased the expression of cortical actin (C), as assessed by immunofluorescent staining (bar, 7.5 µm). Both parameters were partially reversed by treatment of ASG clones with exogenous progastrin6-80 (5 nM) for 4 hours (B,C).

View larger version (48K): [in a new window]   Fig. 3. Reduction of gastrin expression strengthens TJs. (A) Three independent clones of DLD-1 colorectal carcinoma cells stably transfected with an antisense gastrin construct (ASG, columns 4-6) expressed, on average, more ZO-1, occludin (Occl), E-cadherin (E-cad), claudin-1 (Cld-1), claudin-2 (Cld-2) and p125FAK (FAK) than three clones of cells transfected with vector only (VO, columns 1-3), as assessed by western blots of cell lysates. No difference was observed in the amounts of ß-catenin and actin. (B) ZO-1, occludin (Occl), claudin-1 (Cld-1) and E-cadherin (E-cad) were localised in the cytoplasm and/or nucleus in VO clones. Membrane staining for these three AJ and TJ proteins significantly increased in ASG clones, and this increase was largely reversed by a 4 hour treatment with 5 nM progastrin6-80. Bar, 7.5 µm. (C) Treatment of ASG clones with 5 nM progastrin6-80 for up to 240 minutes induced a partial dissociation of the complex between E-cadherin (white bars) and ß-catenin (black bars), as assessed by densitometric scanning of western blots of ß-catenin immunoprecipitates of cell lysates. Little, if any, dissociation was observed in VO clones after progastrin treatment. Densitometric analysis represents the average of at least three experiments per clone, and statistical significance was assessed by Student's t test. *P<0.05; **P<0.01.

View larger version (44K): [in a new window]   Fig. 4. Functional consequences of the progastrin-induced reduction in cell-cell adhesion. (A) Paracellular permeability, as assessed by [3H]mannitol flux through confluent monolayers, was reduced by almost 30% over a 24 hour period in DLD-1 colorectal carcinoma cells expressing antisense gastrin () compared with control cells transfected with vector only (•). The reduction was reversed by treatment with 5 nM progastrin6-80 (), which had no effect on vector only cells (). (B) The permeability through a confluent monolayer of IMGE-5 cells was significantly increased after treatment with 5 nM progastrin6-80 () compared with media alone (). Significance was assessed by Student's t test. *P<0.05; n=4. (C) When a confluent cell monolayer was wounded using a pipette tip, the spontaneous motility of DLD-1/VO clones (VO, columns 1, 2) over a 12 or 24 hour period was greatly reduced in clones expressing antisense gastrin (ASG, columns 3, 4). Motility was partly restored when the latter clones were treated with 5 nM progastrin6-80 (ASG + PG, column 5). Bar, 60 µm. (D) When a confluent IMGE-5 monolayer was wounded using a pipette tip, treatment with 5 pM, 50 pM, 0.5 nM or 5 nM progastrin6-80 (PG) over 12 or 24 hours increased cell motility compared with cells left untreated in DMEM containing 0.1% FCS (U). Bar, 60 µm.

View larger version (35K): [in a new window]   Fig. 5. Involvement of Src kinase in the progastrin-induced delocalisation of junction proteins. (A) In DLD-1 colorectal carcinoma cells basal Src-kinase activity (black bars) in Src immunoprecipitates of cell lysates was higher in VO (a) than in ASG clones (c). Furthermore, 5 nM recombinant human progastrin6-80 (5 minutes) stimulated Src-kinase activity (white bars) in ASG clones (d) but not in VO clones (b). The specificity of this effect was shown by the lack of stimulation of Src kinase activity by 5 nM progastrin6-80 (f) in cells transfected with a dominant-negative mutant of Src (DLD-1/Src-/-) (e). (B) In IMGE-5 cells, which do not produce progastrin-derived peptides, basal Src kinase activity (black bars) (a,c) was significantly increased by 5 nM progastrin6-80 (5 minutes) (white bars) in VO cells (b) but not in cells transfected with dominant-negative Src (IMGE-5/Src-/-) (d). Bar, 20 µm. (C) Activation of Src was essential for the effect of progastrin on the delocalisation of TJ proteins, as in IMGE-5/Src-/- cells, progastrin6-80 (PG) caused little, if any, delocalisation of ZO-1 and symplekin compared with untreated cells (U). By contrast, in IMGE-5/Src-/- cells, 5 nM progastrin6-80 treatment still caused delocalisation of the AJ protein ß-catenin (ß-cat). (D) The partial dissociation of the complex between ZO-1 (black bars) and occludin (Occl, white bars) induced in VO clones (a) by 5 nM progastrin6-80 (b) was completely abolished in the IMGE-5/Src-/- cells (c,d), as assessed by densitometric scanning of western blots of ZO-1 immunoprecipitates of cell lysates. (E) By contrast, expression of the Src dominant-negative mutant in IMGE-5 cells (c) did not prevent the dissociation of the complex between ß-catenin (ß-cat, black bars) and E-cadherin (E-cad, white bars) induced in VO cells (a) by 5 nM progastrin6-80 (b,d), as assessed by densitometric scanning of western blots of ß-catenin immunoprecipitates of cell lysates. In A, B, D and E, densitometric analysis represents the average of at least three experiments, and statistical significance was assessed by Student's t test. *P<0.05; **P<0.01.

View larger version (57K): [in a new window]   Fig. 6. PI3-kinase is involved in the progastrin-induced cytoplasmic shift of ß-catenin. (A) Basal Src kinase activity (black bars), assessed as described in the legend to Fig. 5, in IMGE-5 cells expressing a SH2 dominant-negative mutant of the p85 regulatory subunit of PI3-kinase (IMGE-5/PI3-k-/-) (c) was similar to cells transfected with vector only (VO) (a). Furthermore, activation of Src kinase activity by 5 nM progastrin6-80 (b,d, white bars) was not PI3-kinase dependent. (B) Treatment of IMGE-5/PI3-k-/- cells with 5 nM progastrin6-80 induced a partial or complete disappearance from the plasma membrane of ZO-1 and symplekin (SYM), respectively, compared with untreated (U) controls. The disappearance was prevented by preincubation with the Src kinase inhibitor PP2. Conversely, the cytoplasmic shift of ß-catenin (ß-cat) induced by progastrin6-80 in wild-type or IMGE-5/Src-/- cells was prevented in IMGE-5/PI3-k-/- clones, as well as in IMGE-5/Src-/- cells preincubated with the PI3-kinase inhibitor LY294002. In DLD-1/Src-/- cells, ZO-1 was mostly located at the membrane, whereas DLD-1/PI3-k-/- cells showed a largely cytoplasmic expression of E-cadherin (E-cad). Bar, 20 µm. (C) When compared with untreated cells (U, black bars), 5 nM progastrin6-80 (PG, white bars) induced a partial dissociation of the occludin (Occl)/ZO-1 complex, assessed as described in the legend to Fig. 1, in IMGE-5/PI3-k-/- cells (left panel), of similar amplitude to that observed in wild-type or IMGE-5/VO cells. However, dissociation of the E-cadherin (E-cad)/ß-catenin (ß-cat) complex after progastrin6-80 treatment, assessed as described in the legend to Fig. 3, did not occur in IMGE-5/PI3-k-/- clones (right panel). In A and C, densitometric analysis represents the average of at least three experiments, and statistical significance was assessed by Student's t test. *P<0.05.

View larger version (35K): [in a new window]   Fig. 7. Involvement of PKC in the progastrin-induced cytoplasmic shift of ß-catenin. (A) Of an EGFP-tagged PKC isoform was transiently overexpressed in wild-type IMGE-5 cells (a). Treatment of EGFP-PKC transfected cells with 5nM progastrin6-80 for 15 minutes (b) or 30 minutes (c), or with phorbol dibutyrate for 30 minutes (d), induced a delocalisation of EGFP-PKC to a perinuclear area and to the plasma membrane. Preincubation with the PI3-kinase inhibitor LY 294002 (f), for 30 minutes before, and during, progastrin stimulation prevented the delocalisation of EGFP-PKC to the membrane. Preincubation with the Src kinase inhibitor PP2 (e), or the PKC inhibitor calphostin C (g) had no effect on delocalisation. Control cells were transfected with an empty pEGFP vector similar to the vector used for PKC overexpression (h) and stimulated with 5 nM progastrin6-80 for up to 45 minutes (i). Bars, 10 µm for a and c, 5 µm for all other panels. (B) In wild-type IMGE-5 cells, treatment with 5 nM progastrin6-80 was found to increase transiently the association between ß-catenin (top line), the p85 subunit of PI3-kinase (filled bars) and PKC (open bars), as detected by densitometric scanning of western blots of anti-ß-catenin immunoprecipitates of cell lysates. (C) In IMGE-5/Src-/- cells, preincubation with calphostin C reversed the progastrin6-80-induced cytoplasmic shift of ß-catenin (ß-cat) shown in Fig. 5C. By contrast, the changes in ZO-1 and symplekin (SYM) localisation induced by progastrin6-80 were barely affected by preincubation with calphostin C in wild-type (F.H., unpublished) and IMGE-5/PI3-k-/- clones (Fig. 6B). Bar, 20 µm. (D) Preincubation with calphostin C (Calph) partially inhibited the increase in paracellular permeability for [3H]mannitol induced by 5 nM progastrin6-80 (PG). The increase in permeability was almost completely blocked by the Src kinase inhibitor PP2, but was unaffected by preincubation with the PI3-kinase inhibitor LY294002. In B and D, densitometric analysis represents the average of at least three experiments, and statistical significance was assessed by Student's t test. *P<0.05, **P<0.01.