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First published online 10 June 2008
doi: 10.1242/jcs.021782

Journal of Cell Science 121, 2197-2207 (2008)
Published by The Company of Biologists 2008
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Differential growth factor regulation of N-cadherin expression and motility in normal and malignant oral epithelium

Michelle E. Diamond1,*, Limin Sun1,2,*, Adam J. Ottaviano1, Mathew J. Joseph1 and Hidayatullah G. Munshi1,2,3,{ddagger}

1 Division of Hematology/Oncology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
2 The Jesse Brown VA Medical Center, Chicago, IL 60611, USA
3 The Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL 60611, USA


Figure 1
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  		Fig. 1. N-cadherin expression regulates migration of OSCC cells. (A) Tert-immortalized oral keratinocytes (OKF4 and OKF6) and malignant OSCC cells (SCC9, SCC25, SCC68 and UMSCC1) were serum-starved overnight, and E- and N-cadherin expression was then determined by western blot analysis. Using real-time PCR the relative mRNA expression of E-cadherin, N-cadherin and GAPDH was determined, and normalized to the level of E-cadherin in SCC9 cells and the level of N-cadherin in OKF4 cells. (B) SCC9 and SCC25 cells were transfected with 100 nM of control siRNA (siCtrl) or 1:1 mixture of two N-cadherin-specific siRNAs at 50 nM each (siNcad); E-cadherin, N-cadherin and ERK protein levels were determined 72 hours later by western blot analysis. Using real-time PCR the relative expression of E-cadherin, N-cadherin and GAPDH was determined and normalized to siCtrl-transfected cells. (C) SCC9 and SCC25 cells were transfected with siCtrl or siNcad, and 48 hours following transfection cells were added to porous polycarbonate filters that had been coated with 5 µg type I collagen. Cells were allowed to migrate for 24 hours, nonmigratory cells were removed from the upper chamber and migrating cells counted. *P<0.05, significantly different from siCtrl-transfected cells. Results are representative of at least three independent experiments.

 

Figure 2
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  		Fig. 2. TGFβ1 increases migration of oral keratinocytes by upregulating N-cadherin expression. (A) Equal numbers of cells of the different cell lines were plated, serum starved for 24 hours and then allowed to condition the medium for an additional 24 hours. The conditioned medium was analyzed for total TGFβ1 by ELISA, and cell lysates processed for TGFβ1 and GAPDH mRNA expression by using real-time PCR and normalized to the levels present in OKF4 cells. *P<0.05, significantly different from the OKF4 levels; **P<0.005, significantly different from the OKF4 levels. (B) Oral keratinocytes and OSCC cells were serum-starved overnight, treated with TGFβ1 for 24 hours and the effect on E- and N-cadherin expression was determined by western blot analysis. Using real-time PCR, expression levels of E-cadherin, N-cadherin and GAPDH mRNA were determined and normalized to untreated samples. (C) SCC25 cells were transfected with 100 nM of control siRNA (siCtrl) or siRNA targeting TβRI (siTβRI); then TβRI, ERK2, N-cadherin and E-cadherin protein levels were determined 72 hours later by western blot analysis. Using real-time PCR, the relative expression of TβRI, E-cadherin, N-cadherin and GAPDH was determined and normalized to siCtrl-transfected cells. (D) SCC25 cells were serum starved overnight and then treated with DMSO (vehicle control) or with the TβRI inhibitor (TbRi, 10 µM) for 72 hours. N-cadherin and E-cadherin protein expression were determined by western blot analysis. N-cadherin, E-cadherin and GAPDH mRNA expression were determined by real time PCR and relative expression normalized to DMSO samples. The results are representative of 3 independent experiments. (E) OKF6 cells were transfected with 100 nM of siCtrl or 50 nM of siNcad1 and siNcad2 (siNcad), serum starved and treated with TGFβ1 for 24 hours. The cells were added to porous polycarbonate filters coated with type I collagen (5 µg), and allowed to migrate for 24 hours in the presence or absence of TGFβ1. Nonmigratory cells were removed from upper chamber, and migrating cells were counted. *, significantly different from the siCtrl, TGF-b1+ samples with P<0.05. The results are representative of at least three independent experiments.

 

Figure 3
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  		Fig. 3. Smad signaling regulates TGFβ1-mediated N-cadherin expression to promote migration. (A) OKF6 cells were transfected with 100 nM of control siRNA (siCtrl) or S4Si, allowed to recover overnight, serum starved and then treated with TGFβ1 for 24 hours. Smad4, ERK2, E- and N-cadherin protein levels were determined by western blotting. Using real-time PCR, the mRNA levels of Smad4, N-cadherin and GAPDH mRNA expression were determined and normalized to siCtrl TGF-b1 (–) samples. (B) OKF6 cells were transfected with 100 nM of siCtrl, S2Si or S3Si, and then treated with TGFβ1 for 24 hours. Smad2, Smad3, ERK2, E-cadherin and N-cadherin protein expression was determined by western blotting. Using real-time PCR the mRNA levels of Smad2, Smad3, N-cadherin and GAPDH were determined and normalized to siCtrl TGFβ1 (–) samples. (C) OKF6 cells were transfected with siCtrl or S4Si, serum starved and treated with TGFβ1 for 24 hours. The cells were then added to porous polycarbonate filters that had been coated with 5 µg type I collagen, and were allowed to migrate for an additional 24 hours in the presence or absence of TGFβ1. Nonmigratory cells were removed from upper chamber, and migrating cells were counted to determine migration relative to untreated siCtrl-transfected cells. *P<0.05, significantly different from the siCtrl, TGF-b1+ samples. The results are representative of at least three independent experiments. (D) OKF6 cells were transfected with siCtrl or S4Si, and 16 hours later the cells were infected as detailed in the Materials and Methods with viral particles containing the pQCXIN empty vector or pQCXIN-Ncad vector at two different concentrations for 8 hours, serum starved for 16 hours, and then treated with TGFβ1 for an additional 24 hours. N-cadherin, E-cadherin, Smad4 and ERK2 (loading control) were examined by western blotting. siRNA-transfected cells were infected with pQCXIN empty vector or pQCXIN-Ncad vector at the lower concentration and then added to porous polycarbonate filters that had been coated with 5 µg type I collagen, and allowed to migrate for 24 hours in the presence or absence of TGFβ1. Nonmigratory cells were removed from the upper chamber, and migrating cells were counted. *P<0.05, significantly different from the pQCXIN,siCtrl,TGF-b1(+) samples. **P<0.05, significantly different from pQCXIN,S4Si,TGF-b1(+) samples. The results are representative of at least two independent experiments.

 

Figure 4
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  		Fig. 4. TGFβ1-mediated N-cadherin expression does not involve ERK1/2, p38 MAPK, Src, PI3-kinase, Rho-associated kinase or Jun, but is blocked by EGF in oral keratinocytes. (A) OKF6 cells were serum starved overnight, pre-treated with DMSO (vehicle control), MEK1/2 inhibitor U0126 (10 µM), p38 MAPK inhibitor SB202190 (SB, 10 µM) or the Src inhibitor PP2 (5 µM) for 30 minutes, and then treated with TGFβ1 for 24 hours. N-cadherin and E-cadherin protein levels were determined by western blot analysis. N-cadherin and GAPDH mRNA expression were determined by real-time PCR and relative expression normalized to DMSO-treated TGF-b1 (–) samples. (B) OKF6 cells were serum starved overnight, pre-treated with DMSO (vehicle control), PI3-kinase inhibitor LY294002 (LY, 10 µM), Rho-associated kinase inhibitor Y27632 (10 µM) or the JNK inhibitor SP600125 (SP, 10 µM) for 30 minutes and then treated with TGFβ1 for 24 hours. N-cadherin and E-cadherin protein levels were determined by western blot analysis. N-cadherin and GAPDH mRNA expression were determined by real time PCR and relative expression normalized to DMSO-treated TGF-b1 (–) samples. The results are representative of three independent experiments. (C) OKF4, OKF6, SCC9, SCC25, SCC68 and UMSCC1 were serum starved overnight and then treated with 20 ng/ml EGF 30 minutes prior to treatment with TGFβ1 (10 ng/ml) for 24 hours. E- and N-cadherin protein expression was determined by western blot analysis. The relative levels of N-cadherin and GAPDH mRNA expression in OKF6 and UMSCC1 cells were determined by real-time PCR, and normalized to untreated samples. The results are representative of at least four independent experiments.

 

Figure 5
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  		Fig. 5. EGF attenuates TGFβ1-mediated Smad2 C-terminal phosphorylation and Smad-driven promoter activity, but does not attenuate TGFβ1-mediated expression of TGIF, SnoN or cSki. (A,B) OKF6 cells were serum starved overnight and then pre-treated with EGF (20 ng/ml) for 30 minutes prior to the addition of TGFβ1 (10 ng/ml) for the indicated time points. The cell lysates were then probed for phosphorylated Smad2, and then stripped and re-probed for total Smad2. OKF6 cells were plated onto glass coverslips, serum starved overnight and then pre-treated with EGF (20 ng/ml) for 30 minutes, and then treated with TGFβ1 for 1-8 hours. The cells were then fixed in glutaraldehyde, permeabilized with Triton X-100, incubated with anti-Smad2 antibody followed by Alexa-Fluor-488-labeled secondary antibody. The immunofluorescence signal was detected using Zeiss microscope. (C) OKF6 cells were transfected with 1 µg of SBE-4 reporter luciferase construct, and then equal numbers of cells were plated overnight in 12-well tissue culture plates, serum starved for 8 hours and then pre-treated with EGF (20 ng/ml) for 30 minutes prior to treatment with TGFβ1 (10 ng/ml) for 24 hours. The cells were lysed, luciferase activity was quantified, normalized to untreated samples arbitrarily set at 1.0. **P<0.005, significantly different from EGF-,TGF-b1+ samples with. The results are representative of at least four independent experiments. (D) OKF6 cells were serum starved overnight and then treated with 20 ng/ml EGF 30 minutes prior to treatment with TGFβ1 (10 ng/ml) for 24 hours. The relative levels of TGIF, SnoN, cSki and GAPDH mRNA expression were determined by real time PCR, and normalized to untreated samples. The results are representative of at least two independent experiments.

 

Figure 6
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  		Fig. 6. EGF promotes phosphorylation of the linker region of Smad2 by ERK1/2 in oral keratinocytes. (A) OKF6 cells were serum starved overnight and then treated with EGF (20 ng/ml), TGFβ1 (10 ng/ml), or with EGF (20 ng/ml) for 30 minutes prior to the addition of TGFβ1 (10 ng/ml) for the indicated time points. The cell lysates were probed for phosphorylated ERK1/2, and then stripped and re-probed for total ERK1/2. (B) OKF6 or UMSCC1 cells were serum starved overnight and then pre-treated with DMSO (as vehicle control) or MEK1/2 inhibitor U0126 (10 µM) for 30 minutes prior to the addition of EGF (20 ng/ml) for the indicated time points. The cell lysates were probed for phosphorylated ERK1/2, and then stripped and re-probed for total ERK1/2. (C) OKF6 and UMSCC1 cells were serum starved overnight and then pre-treated with DMSO (vehicle control) or U0126 (10 µM) for 30 minutes prior to the addition of EGF (20 ng/ml) for the indicated time points. Cell lysates were probed for Smad2 phosphorylation in the linker region (p-Smad2L), and then stripped and re-probed for total Smad2. The results are representative of at least two independent experiments.

 

Figure 7
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  		Fig. 7. ERK1/2 mediates the effect of EGF on TGFβ1-mediated Smad-driven promoter activity. OKF6 (A) and UMSCC1 (B) cells were transfected with 1 µg of SBE-4 luciferase construct, and equal numbers of cells were plated overnight, serum starved for 8 hours, and then treated with either DMSO or U0126 for 30 minutes prior to treatment with EGF (20 ng/ml). The samples were then treated with TGFβ1 (10 ng/ml) 30 minutes later for additional 24 hours. The samples were lysed, luciferase activity was quantified, and values normalized to DMSO-treated untreated samples arbitrarily set at 1.0. *P<0.05, significantly different from the DMSO,EGF(–),TGF-b1(+) samples. The results are representative of at least three independent experiments.

 

Figure 8
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  		Fig. 8. MAPK/ERK1/2 inhibition reverses the effect of EGF on TGFβ1-mediated N-cadherin expression. Serum-starved OKF4 and OKF6 cells were pre-treated with DMSO (as vehicle control), or MEK1/2 inhibitor U0126 (10 µM) for 30 minutes prior to treatment with EGF (20 ng/ml). The samples were then treated 30 minutes later with TGFβ1 (10 ng/ml) for an additional 24 hours. (A) E- and N-cadherin protein levels were determined by western blot analysis. (B) N-cadherin and GAPDH mRNA levels were determined in OKF6 cells using real-time PCR, and normalized to DMSO,EGF(–),TGF-b1(–) samples arbitrarily set at 1.0. The results are representative of four independent experiments.

 

Figure 9
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  		Fig. 9. EGF attenuates TGFβ1-mediated migration of oral keratinocytes, but increases TGFβ1-mediated migration of OSCC cells. Serum-starved OKF6 and UMSCC1 cells were untreated, treated with EGF (20 ng/ml) or TGFβ1 (10 ng/ml), or pre-treated with EGF (20 ng/ml) for 30 minutes and then treated with TGFβ1 for 24 hours. The cells were then added to transwell chambers that had been coated with type I collagen, for additional 24 hours in the presence or absence of TGFβ1 and/or EGF. Nonmigratory cells were removed from upper chamber, and migrating cells were counted. *P<0.05, significantly different from the EGF(–),TGF-b1(+) OKF6 cells. The results are representative of at least two independent experiments.

 

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© The Company of Biologists Ltd 2008