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First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02594

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TGFß-induced downregulation of E-cadherin-based cell-cell adhesion depends on PI3-kinase and PTEN

Roger Vogelmann^1,*, Marc-Daniel Nguyen-tat¹, Klaudia Giehl², Guido Adler¹, Doris Wedlich³ and Andre Menke^1,

¹ Department of Internal Medicine I, University of Ulm, Robert-Koch-Strasse 8, 89070 Ulm, Germany
² Department of Pharmacology and Toxicology, University of Ulm, Robert-Koch-Strasse 8, 89070 Ulm, Germany
³ Institute of Zoology II, University of Karlsruhe, 76131 Karlsruhe, Germany

View larger version (92K): [in a new window]   Fig. 1. PANC-1 and BxPC-3 cells were serum-starved for 24 hours and treated with solvent (–TGFß) or 10 ng/ml TGFß1 (+TGFß1) for 2 days. (A) The left panel shows phase contrast pictures. (B) The right panel shows immunofluorescence staining of E-cadherin. Bars, 20 µm. (C) Western blot analyses of E-cadherin, - and ß-catenin in total lysates of PANC-1 and BxPC-3 cells treated with 10 ng/ml TGFß1 for the indicated periods of time. Equal loading was demonstrated by staining of ß-actin. Molecular mass standards are given in Mr x103. Representative blots out of four independent experiments are shown.

View larger version (20K): [in a new window]   Fig. 2. (A) Cell aggregation assays were performed by incubation of pancreatic carcinoma cells PANC-1 and BxPC-3 under constant agitation in HEPES buffer plus CaCl2 supplemented with solvent, TGFß1 (10 ng/ml), EDTA/EGTA (5 mM each) or TGFß1 (10 ng/ml) + E-cadherin neutralising antibody (nAB=DECMA1, 4 µg/ml). The aggregation index was determined by A=(No–Ne)/No, No represents the total particle number before and Ne the particle number after 30 minutes of incubation with constant rotation at 70 rpm. Mean values ±s.e.m. are shown of three independent experiments. (B) Cell migration of PANC-1 and BxPC-3 was analysed using uncoated or collagen type I-coated transwell cell culture inserts with 8 µm pores. After inhibition of cell proliferation by treatment with 10 µg/ml mitomycin C, 20 ng/ml TGFß1 or solvent were added to the lower compartment. After 48 hours of incubation the number of cells, which had migrated through the pores, was estimated by counting 5 independent visual fields. Three independent assays were performed in triplicate. Mean values ±s.e.m. are shown.

View larger version (31K): [in a new window]   Fig. 3. Triton-soluble and -insoluble protein fractions were analysed regarding their amounts of E-cadherin, - and ß-catenin in PANC-1 and BxPC-3 cells after treatment with TGFß1 (10 ng/ml) or solvent for the time points indicated (A). Equal amounts of each fraction were separated by SDS-PAGE and blotted onto nitrocellulose. E-cadherin, - and ß-catenin were detected by immunostaining. The ß-actin concentration served as control to prove equal loading. Representative blots out of four independent experiments are shown. (B) E-cadherin was precipitated from lysate of TGFß1-stimulated or unstimulated PANC-1 cells and coprecipitated - and ß-catenin was analysed by western blotting. After serum starvation for 24 hours, cells were incubated for 90 minutes either with 100 µM PP1 to inhibit Src-kinase, 25 µM PD98059 to inhibit MEK-1 or with 25 µM LY294002 to inhibit PI3-kinase and stimulated with 10 ng/ml TGFß1 or solvent for additional 6 hours. E-cadherin was precipitated from 1 mg of NOP lysate. The amount of co-immunoprecipitated - and ß-catenin was examined by immunoblotting. The blots were restained for E-cadherin to document equal amounts of precipitated protein. Three independent experiments were performed.

View larger version (37K): [in a new window]   Fig. 4. (A) Transwell migration assays were performed as described before. The inhibitors LY294002 (25 µM) or PD98059 (25 µM) were added to the upper compartment. TGFß1 (20 ng/ml) or solvent was added to the lower chamber. The number of migrated cells was estimated after 48 hours of incubation. Mean values ±s.d. of one representative assay are shown out of three independent experiments. (B) The amount of PI3-kinase associated with the E-cadherin complex was analysed by co-immunoprecipitation. Beta catenin was precipitated from lysates of PANC-1 or BxPC-3 cells treated for 30 minutes, 6 hours or 48 hours with TGFß1. The amount of co-precipitated p85 was analysed by western blotting. (C) The amount of p110 co-precipitated with ß-catenin was analysed in PANC-1 or BxPC-3 lysates. Beta-catenin was precipitated from 2 mg of PANC-1 or 1 mg of BxPC-3 NOP lysate treated with 10 ng/ml TGFß1 or solvent. One representative blot out of three is shown. (D) Immunolocalisation of p85 was performed in PANC-1 cells treated with 10 ng/ml TGFß1 or solvent (–TGFß1) for 3 hours. P85 localisation was analysed with confocal laser microscopy. Bar, 20 µm.

View larger version (38K): [in a new window]   Fig. 5. (A) Phosphorylation of - and ß-catenin after TGFß1 treatment (10 ng/ml) for 30 minutes, 6 hours and 48 hours for BxPC-3 and additionally after 12 hours for PANC-1 cells was determined by immunoprecipitation of the individual proteins from isolated membrane fractions. Phosphorylated tyrosine was detected using a phosphotyrosine-specific antibody. Blots were restained with antibodies used for immunoprecipitation to document equal amounts of precipitated proteins. Representative blots out of three independent experiments are shown. (B) Phosphorylation of ß-catenin or (C) -catenin was examined after treatment of PANC-1 cells with TGFß1, the PI3-kinase inhibitor LY294002 or the Src inhibitor PP1 (only for ß-catenin phosphorylation). Beta-catenin or -catenin was precipitated from 1 mg of PANC-1 RIPA-lysates treated with TGFß1, TGFß1 plus PP1 or TGFß1 plus LY294002. Blots were restained for ß- or -catenin to document equal amounts of precipitated protein. (D) TGFß1-induced ß-catenin phosphorylation was analysed in the presence of the farnesyltransferase inhibitor FTI 277 (2 µM) to inhibit Ras activity. In addition PANC-1 cells, which stably expressed EGFP/H-Ras N17, were analysed for ß-catenin phosphorylation in response to 10 ng/ml TGFß1 or solvent. Beta-catenin immunoprecipitated from 1 mg of PANC-1 RIPA lysate was analysed regarding its phosphorylation by western blotting with a phospho-specific antibody. Equal amounts of ß-catenin were documented by restaining the blots with ß-catenin antibody. Representative blots are shown (n=3). (E) In addition to the pictures shown in Fig. 4D, immunolocalisation of p85 was performed in PANC-1 cells stably expressing EGFP/H-Ras N17 treated with TGFß1 or solvent (–TGFß1) for 3 hours and analysed with confocal laser microscopy. Bar, 20 µm.

View larger version (46K): [in a new window]   Fig. 6. (A) Immunoprecipitation of E-cadherin, - or ß-catenin was performed from 500 µg of PANC-1 RIPA lysate and PTEN was detected by immunoblotting. Equal amounts of immunoprecipitated proteins were documented by restaining the blots with the appropriated antibody. (B) Beta-catenin was precipitated from 2 mg of BxPC-3 or PANC-1 lysates treated with 10 ng/ml TGFß1 or solvent. Co-precipitated PTEN was detected by western blotting. The blots were restained with anti-ß-catenin antibody to demonstrate equal amounts of protein. (C) E-cadherin was precipitated from 0.5 mg of total lysate from PANC-1 cells transiently transfected with PTEN, PTENC or vector alone (mock). Co-precipitated - and ß-catenin was determined by western blotting. Restaining of the blot for E-cadherin confirmed equal amounts of precipitated proteins. In all experiments representative blots out of three independent studies are shown.

View larger version (43K): [in a new window]   Fig. 7. (A) Beta-catenin was immunoprecipitated from 1 mg of PANC-1 lysate of cells transfected with pEGFP, pEGFP-PTEN or pEGFP-PTENC, treated with 10 ng/ml TGFß1 or solvent for 6 hours and analysed for its tyrosine phosphorylation. The blot was restained for ß-catenin to demonstrate equal amounts of protein. (B) For an in vitro phosphatase assay, tyrosine phosphorylated ß-catenin was incubated for 30 minutes at 30°C with EGFP-PTEN-constructs, which were immunoprecipitated with anti-EGFP antibody. The amount of phosphorylated ß-catenin was analysed by western blotting. Restaining of the blots with anti-ß-catenin antibody revealed equal amounts of phosphatase substrate and restaining with anti-PTEN documents the presence of both EGFP-tagged PTEN proteins. Representative assays out of four independent experiments are shown. (C) Beta-catenin was immunoprecipitated from 1 mg of lysate from PANC-1 cells transfected with siRNA for PTEN and treated with TGFß1 (10 ng/ml) or solvent (two independent experiments each) or an unrelated control siRNA and analysed for its tyrosine phosphorylation. Restaining for ß-catenin is shown to document equal loading. PTEN protein expression was analysed by western blots stained for PTEN. Representative assays are shown (n=3). (D) Phosphorylation of Ser380 of PTEN, which was co-immunoprecipitated with ß-catenin, was analysed after TGFß1 treatment. Beta-catenin was immunoprecipitated from 2 mg of PANC-1 lysate treated with TGFß1 for 30 minutes or 6 hours. In addition PANC-1 cells were examined, which were pretreated with the farnesyltransferase inhibitor FTI 277 (2 µM) for 2 hours prior to addition of TGFß1 for 6 hours. Co-precipitated PTEN was analysed regarding the phosphorylation at Ser380 with a phospho-specific antibody. Restaining for ß-catenin documented equal amounts of precipitated protein. A representative blot out of three independent experiments is shown.

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