SMAD4 Regulates Cell Motility through Transcription of N-Cadherin in Human Pancreatic Ductal Epithelium

<div><p>Expression of the cellular adhesion protein N-cadherin is a critical event during epithelial-mesenchymal transition (EMT). The SMAD4 protein has been identified as a mediator of transforming growth factor-β (TGF-β) superfamily signaling, which regulates EMT, but the mechanisms linking TGF-β signaling to N-cadherin expression remain unclear. When the TGF-β pathway is activated, SMAD proteins, including the common mediator SMAD4, are subsequently translocated into the nucleus, where they influence gene transcription via SMAD binding elements (SBEs). Here we describe a mechanism for control of <i>CDH2</i>, the gene encoding N-cadherin, through the canonical TGFβ–SMAD4 pathway. We first identified four previously undescribed SBEs within the <i>CDH2</i> promoter. Using telomerase immortalized human pancreatic ductal epithelium, we found that TGF-β stimulation prompted specific SMAD4 binding to all four SBEs. Luciferase reporter and SMAD4-knockdown experiments demonstrated that specific SMAD4 binding to the SBE located at −3790 bp to −3795 bp within the promoter region of <i>CDH2</i> was necessary for TGF-β-stimulated transcription. Expression of N-cadherin on the surface of epithelial cells facilitates motility and invasion, and we demonstrated that knockdown of SMAD4 causes decreased N-cadherin expression, which results in diminished migration and invasion of human pancreatic ductal epithelial cells. Similar reduction of cell motility was produced after <i>CDH2</i> knockdown. Together, these findings suggest that SMAD4 is critical for the TGF-β-driven upregulation of N-cadherin and the resultant invasive phenotype of human pancreatic ductal epithelial cells during EMT.</p></div

N-cadherin alteration after TGF-β treatment in HPNE, HPNE/shScr, and shSMAD4 cells.

<p>(a) Western blot analysis of cellular expression levels of N-cadherin and SMAD4 in cells treated with TGF-β (5 ng/ml) for 2 hours, 8 hours, 24 hours, or 5 days. Actin was used as the loading control. (b) <i>CDH2</i> mRNA level was measured by RT-PCR after TGF-β treatment (5 ng/ml) in 2 hours, 8 hours, and 24 hours' time points. <i>GAPDH</i> was used as the housekeeping gene control.</p

Invasion and migration assay after <i>CDH2</i> knockdown in HPNE, HPNE/shScr, and HPNE/shCDH2 cells.

<p>(a) N-cadherin was suppressed though shCDH2. Actin was used as the loading control. (b) RT-PCR (left) and real-time PCR (right) confirmed that <i>CDH2</i> mRNA was significantly decreased in HPNE shCDH2 cells. (c) Modified Boyden chamber assay was performed with transfected cells. Cells were treated with or without 10 ng/ml TGF-β in serum-free medium in the top inserts, and 20% FBS medium was used in the bottom chamber as a chemoattractant. Invasive cells were counted in 3 fields at 10× magnification in duplicated inserts. (d) Wound-healing assay was performed with transfected cells treated with or without 10 ng/ml TGF-β. Three random images (4× magnification) were taken at the time of the scratch (0 hours) and at 20 hours. Migration rate was determined as the ratio of the distance traveled at 20 hours versus 0 hours in the wound's gap using Adobe Photoshop software. **P<0.01, ***P<0.001.</p

Primers used in this study for RT-PCR or real time PCR.

<p>Primers used in this study for RT-PCR or real time PCR.</p

TGF-β Stimulated CDH2 promoter Dual-luciferase Activity.

<p>(a) Schematic of five pGL2-<i>CDH2</i> promoter constructs, including part of the <i>CDH</i> first exon (white rectangles), and potential SBE sites (downward arrows). (b,c) Luciferase activity ratios of <i>CDH2</i> clone #5 and F1/R1 construct treated with or without 5 ng of TGF-β at 0, 2, 8, and 24 hours in HPNE and 293T cells. **P<0.01, ***P<0.001. (d) The second SBE sequence in the <i>CDH2</i> promoter and the <i>Mut2</i> mutant sequence (G→C). (e) Luciferase activity ratio of <i>Mut2</i> with or without 5 ng TGF-β at 0, 2, 8, and 24 hours in HPNE and 293T cells.</p

Primers used for CDH2 ChiP assay.

<p>Primers used for CDH2 ChiP assay.</p

Knocked down SMAD4 reduces N-cadherin protein level and inhibits invasion and migration in HPNE cells.

<p>(a) Western blot analysis of cell expression of SMAD4 and N-cadherin and CK19. Actin was used as the loading control. (b) RT-PCR results showing cell mRNA levels of <i>CDH1, CDH2, CK19, VIM, BCAT</i>, and <i>FN1</i>, and (c) <i>TWIST1, TWIST2, SNAIL2</i>, and <i>ZEB1</i>. <i>GAPDH</i> was used as the housekeeping control. (d) Immunofluorescence staining of CK19 and N-cadherin in HPNE/shScr or HPNE/shSMAD4 cells. CK19 and N-cadherin were labeled with red fluorescent Alexa Fluor 594 goat anti-rabbit IgG (A11012, Invitrogen). GFP-positive cells represent cells transfected with shScr or shSMAD4. Nuclei were counterstained with blue fluorescent 4,6-diamidino-2-phenylindole. Images were merged using Olympus CellSens software. (e) Modified Boyden chamber assay. HPNE/shScr and HPNE/shSMAD4 Cells were added with or without 10 ng/ml TGF-β to serum-free media inserts in the top chamber, and 20% FBS was placed in the bottom chamber as a chemoattractant. Invasive cells were counted in 3 fields at 10× magnification in duplicated inserts. (f) Wound-healing assay. HPNE/shScr and HPNE/shSMAD4 cells were treated with or without10 ng/ml TGF-β. The y-axis represents cell migration distance at the time of the scratch and after 20 hours. Three random images (4×) were taken at these time points, and migration rate was determined as the ratio of distance at 20 hours versus 0 hours in the wound's gap using Adobe Photoshop software. Results are the mean ± s.d. of 3 independent experiments.</p

SMAD4 and N-cadherin expression levels in patient tumors and matched xenograft samples.

<p>(a) Representative immunostained images of patients' primary tumors and (b) and matched patient-direct xenograft tumors (PATX). Images were captured with an Olympus DP72 camera (10× magnification). (c) Western blotting results for corresponding patient-direct xenograft tumor lysates. Actin was used as the loading control.</p

Map of multiple SBEs in <i>CDH2</i> promoter.

<p>(a) Three SBEs with a CAGACA sequence (blue circles 1, 2, and 3) and one SBE with a GTCTAGAC sequence (red circle 4). Sections A, B, and C represent 3 primers and an amplifying region for ChIP assay in the promoter. (b) Electrophoretic mobility shift assay results showed that 4 SBE oligos had strong DNA and nuclear protein interaction bands (lane 1), binding was quenched by wild-type (WT) oligos (lane 2) and not by mutant (M) oligos (lane 3), and anti-SMAD4 antibody (Ab) inhibited binding activity (lane 4). (c) SBE binding activity was regulated by TGF-β treatment at 0, 2, 8, and 24 hours. Oct-1 DNA binding activities were determined as loading controls in (b) and (c). (d-f) ChIP assays and real-time PCR of primers A, B, and C comparing the ratio of IgG to anti-SMAD4 antibody with or without 5 ng of TGF-β at 0 2, 8, and 24 hours. *P<0.05, **P<0.01, ***P<0.001.</p