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First published online April 23, 2007
doi: 10.1242/10.1242/jcs.003129

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TGF3 inhibits E-cadherin gene expression in palate medial-edge epithelial cells through a Smad2-Smad4-LEF1 transcription complex

Ali Nawshad^1,*,, Damian Medici^2,*, Chang-Chih Liu¹ and Elizabeth D. Hay²

¹ Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, Lincoln, NE 68583, USA
² Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

View larger version (56K): [in this window] [in a new window]   Fig. 1. EMT is associated with nuclear localization of Smad2-P, Smad4 and LEF1, but not -catenin. (A) Immunoblotting demonstrated that palatal adherence increases TGF3 expression. Unfused cells do not express TGF3, whereas fused cells show moderate protein expression. (B) Immunocytochemistry showed nuclear localization of Smad2-P, Smad4, and LEF1 in fused MEE cells and in fused cells treated with exogenous TGF3. Acquisition of the mesenchymal phenotype was observed when cells were treated with TGF3. -catenin remained in the cytoplasm during EMT. Bar, 10 µm. (C) Smad2-P and total Smad2 expression was observed by immunoblotting. No expression was detected in unfused MEE cells for Smad2-P, whereas moderate levels were found in fused cells. Protein levels were heavily increased upon treatment with TGF3. Increased levels of total Smad2 were observed upon palatal adherence. (D) LEF1 protein expression was not detected in unfused cells, but showed steady increases in fused cells and in fused cells treated with TGF3. Addition of dominant-negative Smad4 prevented these increases. (E) Immunoblotting from cytoplasmic and/or membrane (Cyt/Memb) and nuclear (Nucl) protein fractions showed no evidence of nuclear -catenin under any condition. LEF1 was observed in the nuclear fraction of fused cells, with increased levels in cells treated with TGF3. GAPDH and histone H3 were used as cytoplasmic and nuclear controls respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Confirmation of Smad and LEF1 transcriptional activities. (A) p3TP-Lux reporter gene assay demonstrated that Smad transcriptional activity increased from unfused to fused MEE cells, with a sharp increase in activity upon stimulation with TGF3. A dominant-negative Smad4 (DN Smad4) construct inhibited luciferase activity (mean ± s.d.; n=3; *P<0.05 compared with Unfused; **P<0.05 compared with p3TP-Lux). (B) pTOPFLASH-Lux (TF) reporter gene assay showed that LEF1 transcriptional activity continually increases as TGF3 signaling progresses. Mutated LEF1 binding sites of the pFOPFLASH-Lux reporter, as well as treatment with DN Smad4 (Smad4 being necessary for LEF1 gene expression) or dominant-negative LEF1 (DN LEF1) inhibited transcriptional activity whereas antisense -catenin/-catenin oligodeoxynucleotide (AS B-catenin ODN) did not (mean ± s.d.; n=3; *P<0.001 compared with Unfused; **P<0.01 compared with TF).

View larger version (23K): [in this window] [in a new window]   Fig. 3. TGF3 signaling promotes formation of a Smad2-P–Smad4–LEF1 transcription complex. (A) Co-immunoprecipitation (IP) of cell extracts from MEE cells was performed using antibodies against Smad2-P, Smad4 or LEF1. Immunoblot (IB) analysis showed that Smad4, Smad2-P and LEF1 are bound together in the extracts of fused MEE cells; increased levels were observed when cells were exposed to TGF3. Smad3-P and -catenin were not found in this complex. Unfused MEE cells showed no traces of interaction because LEF1 is not expressed in these cells. (B) Immunoprecipitation and immunoblotting for -actin was used as an internal control.

View larger version (52K): [in this window] [in a new window]   Fig. 4. Smad2-P–Smad4–LEF1 directly bind to the promoter of the E-cadherin gene. (A) Promoter analysis of the murine E-cadherin gene revealed one unique LEF1-binding site (independent of the standard TCF and/or LEF site located in the E-pal) and a Smad-binding element (SBE), both within close proximity to the E-pal LEF1-binding region. (B) Binding of the Smad2-P–Smad4–LEF1 protein complex to the endogenous loci was confirmed by chromatin immunoprecipitation. Chromatin was immunoprecipitated with antibodies against IgG (negative control), LEF1, Smad4, Smad2-P or -catenin. PCR analysis using precipitated DNA showed binding of LEF1, Smad4 and Smad2-P to LEF1 (non-E-pal), LEF1 (E-pal) and SBE regions of the E-cadherin promoter. Whereas LEF1 and Smad4 interact with their respective binding regions, all three proteins are observed because they form a transcription complex. No signal was observed from unfused MEE cells whereas a moderate signal was detected from fused MEE cells that was heavily increased upon treatment with exogenous TGF3. -catenin was not detected for interaction with these binding sites.

View larger version (26K): [in this window] [in a new window]   Fig. 5. Smad2-P–Smad4–LEF1 directly inhibits E-cadherin gene expression. (A) Reporter gene analysis of E-cadherin promoter activity (pGL3-E-cad-Lux) demonstrated decreased expression under the influence of TGF3. Treatment of cells with dominant-negative Smad4 or LEF1 (DN Smad4 or DN LEF1, respectively) prevented the repression of E-cadherin, but antisense -catenin/-catenin oligodeoxynucleotide (AS B-catenin ODN) did not (mean ± s.d.; n=3; *P<0.001 compared with Unfused; **P<0.05 compared with E-cad). (B) Site directed mutagenesis of the Smad4-binding (SBE) and LEF1-binding (E-pal and non E-pal) regions also prevented loss of E-cadherin promoter activity (mean ± s.d.; n=3; *P<0.001 compared with Unfused; **P<0.01 compared with E-cad). (C) Real-time quantitative PCR for E-cadherin gene expression relative to the unfused MEE negative control showed a steady decrease in fused MEE cells and fused cells treated with TGF3. DN-LEF1 and DN Smad4 prevented this suppression, whereas AS B-catenin ODN had no effect (mean ± s.d.; n=3; *P<0.001 compared with Unfused; **P<0.01 compared with + TGF3).

View larger version (46K): [in this window] [in a new window]   Fig. 6. Loss of E-cadherin is associated with increased expression of mesenchymal markers. (A) Immunocytochemistry demonstrated progressive repression of E-cadherin from fused MEE cells to those exposed to exogenous TGF3. Bar, 10 µm. (B) E-cadherin expression was also assessed by immunoblotting, showing a moderate loss in fused cells, followed by a complete loss in fused cells treated with TGF3. Dominant-negative Smad4 or LEF1 (DN Smad4 or DN LEF1, respectively) prevented this repression, whereas antisense -catenin/-catenin oligodeoxynucleotide (AS -catenin ODN) had no effect. Contrarily, we found that expression of the mesenchymal markers vimentin and Fibronectin was steadily increased; DN Smad4 and DN LEF1 prevented these increases. AS -catenin ODN had no effect on these proteins.

View larger version (58K): [in this window] [in a new window]   Fig. 7. TGF3 signaling promotes post-EMT cell migration of MEE cells. (A) Scratch wound assays demonstrated lack of cell migration in unfused or fused MEE cells. Addition of exogenous TGF3 stimulated cell migration of fused cells, whereas addition of dominant-negative LEF1 or Smad4 (DN LEF-1 or DN Smad4, respectively) prevented migration under these conditions. Antisense -catenin/-catenin oligodeoxynucleotide (AS -catenin ODN) did not prevent TGF3-induced migration. Bar, 60 µm. (B) Transwell cell migration assays showed little migration of unfused or fused MEE cells, whereas fused cells exposed to TGF3 were highly migratory. DN Smad4 and DN LEF1 prevented migration, but AS -catenin ODN did not (mean ± s.d.; n=3; *P<0.001 compared with Unfused; **P<0.05 compared with + TGF3).

View larger version (13K): [in this window] [in a new window]   Fig. 8. Diagram of the proposed interaction between the Smad2-P–Smad4–LEF1 transcription complex and the E-cadherin gene promoter is shown to demonstrate our hypothesis of transcriptional repression.