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. 2008 Aug 29;283(35):23514-23.
doi: 10.1074/jbc.M801125200. Epub 2008 Jun 30.

Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins

Misako Haraguchi  1 , Tadashi Okubo, Yayoi Miyashita, Yasunori Miyamoto, Masao Hayashi, Tania N Crotti, Kevin P McHugh, Masayuki Ozawa
Affiliations

Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins

Misako Haraguchi et al. J Biol Chem. .

Abstract

Snail, a transcriptional repressor of E-cadherin expression, plays a role in the process of epithelial-mesenchymal transition. However, the molecular basis of the role of snail in epithelial-mesenchymal transition has not been fully clarified. Here we show that the expression of snail in epithelial Madin-Darby canine kidney (MDCK) and A431 cells enhances both cell detachment and attachment. Snail did not confer resistance to anoikis induced by loss of contact but instead enhanced cell attachment to extracellular matrices such as fibronectin. This attachment was inhibited by Arg-Gly-Asp (RGD) peptides. Up-regulation of the promoter activity of integrin alphaV was observed in snail-expressing MDCK (MDCK/snail) cells. Snail also enhanced MDCK cell migration toward osteopontin that is a ligand for integrin alphaVbeta3. We confirmed the reduction of basement membrane proteins such as laminin (LN) alpha3, beta3, and gamma2 (laminin-5/LN-5) and of receptors for LN-5 such as integrins alpha3, alpha6, or beta4 in MDCK/snail or in snail-expressing A431 (A431/snail) cells. Nevertheless, suppression of LN-alpha3 chain by transient transfection of small interference RNAs resulted in no enhancement of cell detachment. We also found an induction of matrix metalloproteinase-3 in MDCK/snail and A431/snail cells. However, the inhibition of matrix metalloproteinase-3 showed no significant effect on the detachment of MDCK/snail cells. These results suggest that snail enhances cell detachment by multiple mechanism and leads to cell migration and reattachment at a second site, at least in part, by changing the expression of integrins in the cells.

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Figures

FIGURE 1.
FIGURE 1.
Cell detachment is enhanced in snail-expressing cells. A, detachment assays of MDCK/neo and MDCK/snail cells in 24-well plates. Cells were sparsely seeded (1.2 × 104 cells/well) on 24-well plates to and then incubated for 24 h at 37 °C. Cell monolayers were then treated with a solution of 0.125% trypsin/0.1 mm EDTA for the indicated times. The remaining cells adhered to the plates were counted by staining with crystal violet and the data are presented as the percentage of the adhered cells to total (untreated) cells. B, detachment assays of A431/neo and A431/snail cells in 96-well plates. Cells were seeded (2 × 104 cells/well) on 96-well plates and then incubated for 24 h at 37 °C in DMEM without FCS. Detachment assay was performed as described above. C, detachment assays of MDCK/neo and MDCK/snail cells in Transwell plates. Cells were seeded (1 × 105 cells/well) in the upper chamber of the Transwell plates, incubated for 24 h at 37 °C, and then treated with 0.125% trypsin/0.1 mm EDTA that was added to the lower chamber. At the indicated times, the remaining cells in the upper chamber were counted by a MTT assay as previously described (26). Data are the mean ± the S.E. for triplicate determinations. *, p < 0.05 versus MDCK/neo (A and C) or A431/neo (B) cells.
FIGURE 2.
FIGURE 2.
The ECM produced by MDCK/snail cells is less adhesive for MDCK/neo cells than that produced by MDCK/neo cells. To obtain the deposited ECM for this assay, MDCK/neo and MDCK/snail cells were seeded (5 × 104 cells/well) on 24-well plates and incubated for 48 h. The cells were then removed from the matrix protein by treatment with 0.1% deoxycholic acid (DC) or 0.125% trypsin/0.1 mm EDTA (trypsin). The ECM deposited by MDCK/neo (neo DC and neo trypsin) or by MDCK/snail (snail DC and snail trypsin) were washed with DMEM with 10% FCS and PBS. Blocking of nonspecific interactions was achieved by incubation with 0.2% BSA. MDCK/neo and MDCK/snail cells were then seeded (5 × 104 cells/well) onto the ECM-covered plates and incubated for 40 min at 37 °C in DMEM without FCS. After removing the non-adherent cells with PBS, adherent cells were quantified by staining with crystal violet. Data are the mean ± the S.E. for triplicate determinations. *, p < 0.05 versus ECM deposited by MDCK/neo cells.
FIGURE 3.
FIGURE 3.
Snail regulates the expression of integrins and ECM proteins. A and B, effect of snail on the protein expression of integrins and ECM proteins. MDCK/neo and MDCK/snail cells (A) and A431/neo and A431/snail cells (B) were lysed in SDS sample buffer and subjected to Western blot analysis with the indicated antibodies. Vinculin was served as an internal control for protein loading. Measurement of protein expression level was achieved by using ImageJ (National Institutes of Health). The data are presented as the relative intensity of the bands of from MDCK/snail cells to that in MDCK/neo cells (C) and the bands from A431/snail to that in A431/neo cells (D). *, p < 0.05 versus MDCK/neo (C) or A431/neo (D) cells. Effect of snail on mRNA expression of integrins and ECM proteins in MDCK cells (E) and in A431 cells (F). RT-PCR of glyceraldehyde-3-phosphate dehydrogenase was used as an internal control. The sequences for PCR primers are listed in Tables 1 and 2.
FIGURE 4.
FIGURE 4.
Suppression of LN-α3 expression by siRNA does not affect cell detachment. A, RT-PCR analysis of LN-α3 transcripts. MDCK/neo cells were transfected with control siRNA (Con si) or with siRNA targeted against LN-α3 (LN-α3 si). The expression of LN-α3 mRNA or of control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was assayed by RT-PCR using specific primers listed in Table 1. B, effect of LN-α3 siRNA on detachment of MDCK/neo cells. MDCK/neo cells, MDCK/neo cells that were transfected with control siRNA (Con si) or with LN-α3 siRNA (LN-α3 si) and MDCK/snail cells were seeded (1 × 105 cells/well) on 24-well plates and incubated for 24 h at 37 °C. Detachment assays were performed as described in the legend to Fig. 1A. The data are presented as the percentage of the cells that remained adhered to the plate. Data are the mean ± the S.E. for triplicate determinations. *, p < 0.05 versus MDCK/neo cells.
FIGURE 5.
FIGURE 5.
Collagen IV suppresses the detachment of MDCK/snail and A431/snail cells. A, effects of various ECM proteins on cell detachment of MDCK cells. The wells of 24-well plates were coated with 0.25 ml of 1 μg/ml purified LN-5, 10 μg/ml collagen I, collagen IV, FN, or 10% FCS. Twenty-four hours later, MDCK/neo and MDCK/snail cells were seeded (3.5 × 104 cells/well) onto these wells and incubated for 2.5 or 24 h in DMEM without FCS. These cells were then treated with 0.025% trypsin/0.02 mm EDTA for 90 s. The detached cells were removed, and the remaining cells on the plate were fixed with 70% ethanol and stained with crystal violet and quantified. *, p < 0.05 versus trypsin-treated cells on BSA-coated wells. B, effects of various ECM proteins on cell detachment of A431 cells. A431/neo and A431/snail cells were seeded (8 × 104 cell/well). Detachment assay was performed as described above. *, p < 0.05 versus trypsin-treated cells on BSA-coated wells. C, effect of MMP-3 inhibitor on detachment of MDCK/snail cells. Cells were incubated without or with MMP-3 inhibitor at 1 μm for 24 h and subjected to detachment assay using enzyme free cell dissociation buffer.
FIGURE 6.
FIGURE 6.
Snail does not confer resistance to anoikis. A, analysis of anoikis of MDCK/neo and MDCK/snail cells by PI staining and subsequent FACS analysis. MDCK/neo and MDCK/snail cells (5 × 105 cells/well) were cultured either on non-coated, or on poly-HEMA-coated 6-well plates for 24 h at 37 °C. After incubation, detached and suspended cells from poly-HEMA-coated plates were harvested (24 h). Cells from non-coated plastic tissue were trypsinized and harvested (0 h). Cells were stained with PI solution, and the percentage of dead cells was quantified by flow cytometry. B, analysis of anoikis with the LDH assay. MDCK/neo and MDCK/snail cells (1 × 105 cells/well) were cultured either on non-coated or poly-HEMA-coated 24-well tissue culture plates for 24 h at 37 °C. The medium was recovered, and LDH that released from dying cells was measured. Data for released LDH were normalized relative to total LDH and expressed as a percentage. All measurements were performed in triplicate.
FIGURE 7.
FIGURE 7.
Snail enhances cell adhesion to FCS- and FN-coated plates. A, attachment of MDCK/neo and MDCK/snail cells to tissue culture plates. Representative micrographs of the cells, photographed at the indicated time after seeding, are shown. B, attachment assays of MDCK/neo and MDCK/snail cells to various ECM substrates. The wells of 96-well plates were coated with FCS or 10 μg/ml of FN or LN-1. Twenty-four hours later, cells were seeded (2.5 × 104 cells/well) onto these wells and incubated for 30 min at 37 °C in DMEM without FCS. After removing non-adherent cells, the adhered cells were quantified by staining with methylene blue. *, p < 0.05 versus MDCK/neo cells. C, inhibition of cell attachment by RGD peptides. MDCK/snail cells were incubated with or without 1 mm RGD or RGE peptides for 10 min before seeding on 96-well plates. An attachment assay was performed as described above. *, p < 0.05 versus RGE-treated cells. D, attachment assays of A431/neo and A431/snail cells to various ECM substrates. *, p < 0.05 versus A431/neo cells.
FIGURE 8.
FIGURE 8.
The expression of snail enhances the promoter activities of integrin αV. MDCK/neo and MDCK/snail cells were seeded (1 × 105 cells/well) onto 24-well and transfected with 1 μg of reporter vector and 20 ng of pRL-CMV vector. After 48-h incubation, both firefly (FL) and Renilla luciferase (RL) activities were measured using the Dual luciferase reporter assay kit (Promega). FL activities were normalized to the RL activities. Data are the mean ± the S.E. for triplicate determinations. *, p < 0.05 versus MDCK/neo cells.
FIGURE 9.
FIGURE 9.
Snail enhances cell migration toward FN, osteopontin, and the conditioned medium of osteoblasts. A, migration assay of MDCK/neo and MDCK/snail cells to various attractant. Cells (1 × 106 cells/0.1 ml) were seeded into the upper wells. FN or osteopontin was added to the lower wells at a final concentration of 10 μg/ml. BSA was used at 0.1%. Conditioned medium of MC3T3 cells (CM MC3T3) or fresh medium containing 10% FCS was added to lower wells at 25% v/v. After 4 h of incubation at 37 °C, cells that had migrated to the lower surface of the filter were counted using the MTT assay. Data are the mean ± the S.E. for triplicate determinations. *, p < 0.05 versus MDCK/neo cells. B, inhibition of migration of MDCK/snail cell to osteopontin by function blocking integrin αVβ3 antibody. MDCK/snail cells were incubated with 25 μg/ml function blocking integrin αVβ3 antibody or control antibody in DMEM supplemented with 0.1% BSA for 20 min. Cells (8 × 104 cells/0.1 ml) were then seeded into the upper wells. Five μg/ml osteopontin, 1 μg/ml FN, and 1% FCS in DMEM supplemented with 0.1% BSA were added to the lower wells. Migration assay was performed as described above. *, p < 0.05 versus control antibody-treated cells.
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