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First published online 25 August 2004
doi: 10.1242/jcs.01322

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Critical role of N-cadherin in myofibroblast invasion and migration in vitro stimulated by colon-cancer-cell-derived TGF-ß or wounding

¹ Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital, De Pintelaan 185, 9000 Gent, Belgium
² Department of Dermatology, Ghent University Hospital, De Pintelaan 185, 9000 Gent, Belgium
³ INSERM U482, Hôpital Saint-Antoine, 184 rue du Faubourg, Saint-Antoine, 75571 Paris CEDEX 12, France

View larger version (57K): [in a new window]   Fig. 1. Colon-cancer-cell-derived TGF-ß is necessary and sufficient to stimulate invasion of myofibroblasts into collagen type I and Matrigel. (A) Myofibroblast spheroids were embedded in a collagen type I gel on top of which serum-free DMEM was added without (untreated), with 106 HCT-8/E11 cells or with CMHCT-8/E11 cells, in the presence of control IgG1 or of neutralizing TGF-ß mAb. (B) Myofibroblast spheroids in collagen type I were cultured for 7 days with the indicated treatments. The medium was refreshed on days 3 and 5. A representative phase-contrast micrograph is shown for each condition, taken after 48 hours (panel A) or after 7 days (panel B). Experiments in A and B were repeated at least three times and had similar results. Scale bars, 100 µm. (C) Single-cell myofibroblasts at a density of 4x105 were seeded upon a Matrigel coated filter. HCT-8/E11 cells at a density of106 were seeded or rTGF-ß1 was placed in the lower compartment in the presence of control IgG1 or neutralizing TGF-ß mAb. Bars indicate means of three results ± s.d. Asterisks show statistically significant difference from untreated control.

View larger version (81K): [in a new window]   Fig. 2. Role of N-cadherin in rTGF-ß1-stimulated invasion of myofibroblasts. (A) 4x105 single myofibroblasts were seeded upon a Matrigel coated filter with rTGF-ß1 or HCT-8/E11 cells in the lower compartment to stimulate invasion. N-cadherin was neutralized by the GC-4 mAb which was added together with the myofibroblasts. Bars indicate means of three results ± s.d. Asterisks show statistically significant inhibition of invasion. (B) Myofibroblast spheroids cultured in collagen type I for 48 hours in the presence of HCT-8/E11 cells, and cultured in collagen type I for 7 days untreated or treated with rTGF-ß1, were challenged with control IgG1 (80 µg/ml) or GC-4 mAb (80 µg/ml). In the 7-day experiment, medium was exchanged on day 3 and 5. A representative phase-contrast micrograph is shown for each condition. Scale bar, 100 µm. (C) Western blot, showing the effect of combinations of siN-CAD oligonucleotides on N-cadherin expression and cadherin-11 expression in myofibroblasts. Tubulin was used as loading control. (D) Spheroid myofibroblasts of cells that had been electroporated with siN-CAD oligonucleotides and cultured in collagen type I for 2 or 4 days in the presence of rTGF-ß1. con, control oligonucleotide. Scale bar, 100 µm.

View larger version (31K): [in a new window]   Fig. 3. Effect of rTGF-ß1 on N-cadherin expression and the organization of N-cadherin-catenin--SMA complex in myofibroblasts. (A) Treatment of myofibroblasts with rTGF-ß1 for 7 days stimulates N-cadherin protein expression in a dose-dependent manner. Expression levels of ß-ctn and -SMA proteins are not significantly changed. Tubulin protein expression is used as loading control. (B) Immunoprecipitation (IP) on lysates of myofibroblasts treated or not for 7 days with rTGF-ß1 (1 ng/ml) were performed to investigate the association of N-cadherin with ß-catenin and -SMA. Total lysates were stained for N-cadherin and tubulin (loading control).

View larger version (57K): [in a new window]   Fig. 4. Effect of MAPK inhibitors on rTGF-ß1-stimulated N-cadherin expression and N-cadherin dependent myofibroblast invasion. (A) 4x105 single myofibroblasts were seeded on a Matrigel coated filter, with rTGF-ß1 in the lower compartment to stimulate invasion. MAPK inhibitors were added in the upper compartment together with the myofibroblasts. Incubation was for 72 hours. Bars indicate the mean number of cells at the lower side of the filter (means of three results ± s.d.). The asterisk shows statistically significant inhibition of invasion. (B) Myofibroblast spheroids were cultured for 7 days in collagen gels in presence or not of rTGF-ß1 and MAPK inhibitors. Medium was exchanged on day 3 and 5. A representative micrograph for each condition is shown. Scale bars, 100 µm. (C) Western blot analysis of lysates from myofibroblasts treated for 7 days with rTGF-ß1 in presence or not of MAPK inhibitors. The upper part of the blot was probed for N-cadherin, the lower part for tubulin.

View larger version (36K): [in a new window]   Fig. 5. Kinetics of rTGF-ß1 activation of p38, ERK1/2 and JNK MAPK in myofibroblasts. Western analysis of lysates from myofibroblasts that had been treated with rTGF-ß1 for different times (h=hours) and probed for the phosphorylated forms of p38MAPK (p38MAPK-P), ERK1/2 (ERK-P), and the JNK substrate Jun, mainly phosphorylated ser63 or ser73 (Jun-P63 and Jun-P73). Blots were stripped and re-probed for total amounts of p38MAPK, ERK1/2 and Jun and tubulin. The relative intensity of total ERK1/2, Jun and p38MAPK was normalized to tubulin levels. Consequently, the relative intensity of phosphorylated forms of ERK1/2, Jun and p38MAPK was normalized to total values of ERK, Jun and p38MAPK.

View larger version (32K): [in a new window]   Fig. 6. Role of N-cadherin in wound-healing migration of myofibroblasts. (A-B) Confluent monolayers of serum-starved myofibroblasts were wounded with a razor blade. Migration of myofibroblasts was quantified as number of intersections/microscopic field at different distances after a 24-hour treatment with (A) N-cadherin neutralizing mAb or peptide and (B) electroporation in the presence of N-cadherin siRNA. Of the different conditions tested in A, representative pictures were taken. The arrowhead indicates the scratch (time 0) made by the razor blade. Bar, 100 µm.

View larger version (30K): [in a new window]   Fig. 7. Role of N-cadherin in filopodia formation of myofibroblasts. (A) Bars show mean numbers of filopodia revealed by F-actin staining and counted on 50 myofibroblasts at the migration front (means of three results ± s.d.); asterisks indicated statistically significant difference. (B) Representative pictures of F-actin-staining of myofibroblasts at the migration front of cultures treated with control IgG1 or the GC-4 mAb. Bar, 25 µm (C) Representative pictures of F-actin and N-cadherin-staining at the migration front of myofibroblasts electroporated with control (con), or siN-CAD3 and siN-CAD4 oligonucleotides. Bar, 25 µm.

View larger version (50K): [in a new window]   Fig. 8. Role of N-cadherin in the polarity of myofibroblasts. (A) Representative phase-contrast micrographs of DHD-Fib myofibroblasts, taken 8 hours after wounding. Cells were treated with huN-CAD10scr or huN-CAD10 peptide. Loss of polarity is indicated by loss of unidirectional migration. Bars, 50 µm. (B) (upper panel) Golgi-complex immunostaining of DHD-Fib myofibroblasts in confluent conditions or 8 hours after wounding. Direction of movement is shown by arrows; (lower panel) bars show percentage of cells with their Golgi complex facing the wound for 50 cells of the first 2 front rows or at random in wounded or confluent monolayers. Bar, 25 µm.

View larger version (27K): [in a new window]   Fig. 9. Effect of MAPK inhibitors on wound healing-induced migration of myofibroblasts. Serum-starved confluent myofibroblast monolayers were wounded with a razor blade. Migration of myofibroblasts was quantified as number of intersections/microscopic field at different distances after 24 hours in cultures and treated as indicated.

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