doi: 10.1242/10.1242/jcs.00091
Transforming growth factor-ß and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (EMT) through a MEK-dependent mechanism in primary cultured pig thyrocytes
Mats Grände1,*,
Åsa Franzen2,
Jan-Olof Karlsson1,
Lars E. Ericson1,
Nils-Erik Heldin2 and
Mikael Nilsson1
1 Institute of Anatomy and Cell Biology, Göteborg University, Göteborg, Sweden
2 Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University Hospital, Uppsala, Sweden
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Fig. 1. Effect of TGF-ß1 and EGF on cell proliferation and apoptosis in filter-cultured pig thyroid epithelial cells. (A) [3H]Thymidine incorporation. Confluent cells stimulated with EGF (10 ng/ml) or TGF-ß1 (10 ng/ml) alone or in combination were simultaneously exposed to 1 µCi/ml [3H]thymidine, added to the basal medium of the bicameral chamber, for 0-24 or 24-48 hours, after which incorporated radioactivity was counted. Mean±s.d. (n=3). (B) Caspase-3 activity. Filter cultures were lysed after growth factor stimulation at the indicated times and the degradation rate of a caspase-3 specific fluorogenic substrate (DEVD-AMC) was measured. Mean±s.d. (n=3).
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Fig. 2. Electron micrographs of filter-cultured pig thyrocytes after TGF-ß1 and EGF stimulations. (A) Portion of an untreated cell monolayer. The cells are polarised with numerous microvilli (arrows) at the apical plasma membrane. (B) Cells exposed to TGF-ß1 (10 ng/ml) for 48 hours. The monolayer organisation is similar to that of control cultures. Apical microvilli are lost and the appearance of submembranous microfilament condensations is evident (arrowheads). (C) Cells treated with EGF (10 ng/ml) for 48 hours. The cells are tall and crowded due to increased proliferation, but the monolayered epithelium is largely maintained. The apical plasma membrane displays microvilli. (D) Cells co-stimulated with TGF-ß1 and EGF (both 10 ng/ml) for 48 hours. The cells are flat and elongated and extend on top of each other in multiple layers. Distinct junctional complexes are not present.
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Fig. 3. Effects of TGF-ß1 and EGF on epithelial barrier function in filter-cultured thyrocytes. (A) Transepithelial resistance (TER) measured consecutively in the same cultures at the indicated times. Mean±s.d. (n=3). (B) Apical-to-basal transepithelial flux of [3H]inulin (0.1 µCi/ml) recorded after growth factor stimulation for 24 hours. Mean±s.d. (n=3). E, EGF (10 ng/ml); T, TGF-ß1 (10 ng/ml).
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Fig. 4. Expression of occludin and claudin-1 proteins in growth factor-stimulated thyrocytes. Cultures were exposed to TGF-ß1 (10 ng/ml) and/or EGF (10 ng/ml) for the indicated times, after which cell lysates at equivalent protein concentrations were analysed with SDS-PAGE and western blotting. ß-actin was used as an internal control for equal protein loading. E, EGF; T, TGF-ß1.
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Fig. 7. Effects of MEK inhibitor U0126 and PI3K inhibitor LY294002. (A) Filter-grown cells were stimulated with TGF-ß1 (10 ng/ml) or EGF (10 ng/ml) or both in the presence or absence of 25 µM U0126 for 24 hours, and then processed for western blot analysis of N-cadherin, occludin and claudin-1. ß-actin (ß-actin) was used as an internal control for equal protein loading. (B) Cells were exposed to EGF with or without TGF-ß1 in the presence or absence of U0126 for the indicated times and then analysed for the presence of phosphorylated Erk (P-Erk 1/2). Total Erk (Erk 1/2) indicates equal protein loading. (C) Cells were exposed to EGF and TGF-ß1 in the presence or absence of 5 µM LY294002. Cell lysates at equivalent protein concentrations were analysed for N-cadherin with SDS-PAGE and western blotting. ß-actin was used as an internal control for equal protein loading.
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Fig. 5. Expression of E-cadherin and N-cadherin in growth factor-stimulated thyrocytes. (A) Immunofluorescent staining of filter-cultured cells treated with TGF-ß1 (10 ng/ml) and EGF (10 ng/ml) for 24 hours. (Upper panel) E-cadherin (E-cad); (lower panel) N-cadherin (N-cad). Specimens were counterstained with DAPI (red) to visualise the cells' nuclei. N-cad was immunolocalised with clone CH-19, which is a pan-cadherin antibody raised against a sequence of the highly conserved cytoplasmic domain of N-cadherin. CH-19 gives results identical to that of the 3B9 monoclonal N-cadherin antibody in western blot analysis, but was used for immunofluorescence due to superior staining intensity. Bar, 15 µm. (B) Western blot analysis of E-cadherin and N-cadherin (using clone 3B9) after stimulation with TGF-ß1 (T; 10 ng/ml) and/or EGF (E; 10 ng/ml) for the indicated times. ß-actin indicates equivalent protein loading. (C) Appearance of distinct small size bands with E- or N-cadherin immunoreactivity after co-stimulation for 48 hours.
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Fig. 6. MAPK signalling in pig thyrocytes treated with EGF and TGF-ß1. (A) Phosphorylation of Erk by TGF-ß1 or EGF. Cells were stimulated with TGF-ß1 (10 ng/ml) or EGF (10 ng/ml) for 15-45 minutes and then solubilised. Proteins were detected by antibodies against phosphorylated Erk (P-Erk 1/2) and total Erk (Erk 1/2). Note diminutive amounts of Erk phosphorylation in TGF-ß1-treated cells only after 15 minutes. (B) Effect of TGF-ß1 on EGF-induced Erk phosphorylation. Cells were exposed to EGF in the presence or absence of TGF-ß1 for the indicated times and then analysed for the presence of phosphorylated Erk and total Erk as outlined in A.
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