Phosphoinositide-3 kinase-Rac1-c-Jun NH2-terminal kinase signaling mediates collagen I-induced cell scattering and up-regulation of N-cadherin expression in mouse mammary epithelial cells

Abstract

During epithelial-to-mesenchymal transitions (EMTs), cells must change their interactions with one another and with their extracellular matrix in a synchronized manner. To characterize signaling pathways cells use to coordinate these changes, we used NMuMG mammary epithelial cells. We showed that these cells become fibroblastic and scattered, with increased N-cadherin expression when cultured on collagen I. Rac1 and c-Jun NH2-terminal kinase (JNK) were activated when cells were plated on collagen I, and dominant inhibitory Rac1 (RacN17) or inhibition of JNK signaling prevented collagen I-induced morphological changes and N-cadherin up-regulation. Furthermore, inhibiting phosphoinositide-3 kinase (PI3K) activity prevented Rac1 and JNK activation as well as collagen I-induced N-cadherin up-regulation. These data implicate PI3K-Rac1-JNK signaling in collagen I-induced changes in NMuMG cells. To establish a role for N-cadherin in collagen I-induced cell scattering, we generated N-cadherin overexpressing and knockdown NMuMG cells and showed that knocking down N-cadherin expression prevented collagen I-induced morphological changes. Motility assays showed that cells overexpressing N-cadherin were significantly more motile than mock-transfected cells and that N-cadherin-mediated motility was collagen I dependent. In addition, we showed that cord formation and branching in three-dimensional culture (EMT-dependent events) required N-cadherin expression and PI3K-Rac1-JNK signaling.

Figure 1.

NMuMG/E10 cells undergo a morphological change in response to collagen I. NMuMG/E10 cells were cultured on noncoated (a and d), collagen I-coated (b and e), or fibronectin-coated (c and f) dishes (a–c) or coverslips (d–f). a–c, phase contrast pictures. d–f, immunofluorescence staining for E-cadherin (ECCD2). Photographs (a–c) were taken using a 10× objective. Photographs (d–f) were taken using a 40× oil objective.

Figure 2.

Collagen I–induced changes in NMuMG/E10 cells. NMuMG/E10 cells were cultured on noncoated (a and d), collagen I-coated (b and e), or fibronectin-coated (c and f) coverslips. a–c, phalloidin staining. d–f, paxillin staining. Photographs were taken using a 40× oil objective.

Figure 3.

Collagen I–induced N-cadherin up-regulation in NMuMG/E10 cells. (A) NMuMG/E10 cells were cultured on noncoated (non), collagen I-coated (col), or fibronectin-coated (fib) dishes. Two days after seeding cells were extracted and 30 μg of protein resolved by SDS-PAGE and immunoblotted for fibronectin, N-cadherin, and E-cadherin. (B) Immunoblots were quantified by densitometry using Adobe Photoshop and normalized to GAPDH. The columns represent mean values of three independent experiments and the bars represent the SD (∗p < 0.05; col versus non or fib). (C) RT-PCR was done for N-cadherin, E-cadherin, and GAPDH (as a control). (D) N-cadherin and E-cadherin mRNA levels were analyzed by quantitative real-time PCR. 18S rRNA was used as an endogenous control, and quantification of mRNA levels was performed in duplicate and repeated three times. The columns represent mean values and the bars represent the SD (∗p < 0.05; col versus non or fib). (E) N-cadherin mRNA was analyzed by quantitative real-time PCR from 3- to 48-h time points. Quantification was performed in duplicate and repeated two times.

Figure 4.

Collagen I–induced changes in cadherin expression in NMuMG/E10 cells. (A) NMuMG/E10 cells were cultured on noncoated (non) or collagen I-coated (col) dishes. One day to 4 d after seeding, cells were extracted, and 30 μg of protein was resolved by SDS-PAGE and immunoblotted for N-cadherin, E-cadherin, and tubulin (as a loading control). (B) Cell surface E-cadherin expression in NMuMG/E10 cells. NMuMG/E10 cells were cultured on noncoated (non) or collagen I-coated (col) dishes. One day to 4 d after seeding, cells were surface biotinylated, and 500 μg of protein was immunoprecipitated with anti-E-cadherin (4A2), resolved by SDS-PAGE, transferred to nitrocellulose, and probed with horseradish peroxidase-labeled streptavidin. (C) E-cadherin extractability. NMuMG/E10 cells were cultured on noncoated (non) or collagen I-coated (col) dishes for 1–4 d. The soluble fraction (sol-E-cad) was extracted with 1% Triton X-100 for 10 min at 4°C with gentle rocking. The insoluble fraction (insol-E-cad) was extracted using an equal volume of SDS sample buffer. An equivalent volume of each fraction was resolved by SDS-PAGE and immunoblotted for E-cadherin. The ratio of insoluble E-cadherin to soluble E-cadherin (insol/sol) was calculated.

Figure 5.

Involvement of integrin related molecules in response to collagen I. (A) NMuMG/E10 cells were extracted 3 h after plating on noncoated, collagen I-coated, or fibronectin-coated dishes. RIPA extracts were immunoblotted for phospho-FAK (Tyr 577) and total FAK. (B) Lysates (300 μg of protein) were immunoprecipitated with anti-paxillin mAb and resolved by SDS-PAGE. Phospho-tyrosine and total paxillin were detected by PY20 mAb and paxillin mAb immunoblots, respectively. (C) NMuMG/E10 cells infected with shEGFP (a and b) or shIntegrin β1 (c and d) were cultured on noncoated (a and c) or collagen I-coated (b and d) dishes. Two days after seeding, phase pictures were taken using a 10× objective. (D) Two days after seeding on noncoated (non) and collagen I-coated (col) dishes, cells were extracted, and 30 μg of protein was resolved by SDS-PAGE and immunoblotted for integrin β1, N-cadherin, and tubulin.

Figure 6.

Rac1 is activated when cells are plated on collagen I. (A) NMuMG/E10 cells were extracted 3 to 24 h after plating on noncoated or collagen I-coated dishes. Lysates (1 mg of protein) were incubated with GST-CRIB–coupled beads and resolved by SDS-PAGE. Rac1-GTP and total Rac1 were detected by Rac1 immunoblots. (B) Rac1-GTP was quantified and normalized to total Rac1. Each pull-down experiment was repeated two times. (C) NMuMG/E10 cells were infected with retrovirus encoding the neomycin-resistance gene (MOCK), RacN17-GFP, or RacV12-GFP. Three hours after seeding on noncoated or collagen I-coated dishes, protein was extracted and pull-down assays performed as in A. (D) RIPA extracts of NMuMG/E10 cells expressing the neomycin-resistance gene (MOCK), RacN17-GFP, or RacV12-GFP were immunoblotted for phospho-JNK (p-JNK; Thr183/Thy185) and total JNK1. Cells treated with anisomycin (Aniso; 1 μg/ml) to activate JNK were used as a control to indicate the phosphorylated forms of JNK (lane 7).

Figure 7.

RacN17 inhibits collagen I–induced morphological changes and N-cadherin up-regulation. (A) MOCK, RacN17, and RacV12 infected NMuMG/E10 cells were cultured for 2 d on noncoated (a, c, and e) or collagen I-coated (b, d, and f) dishes and photographed using a 10× objective. (B) Extracts (30 μg of protein) from cells cultured on noncoated or collagen I-coated dishes for 2 d were resolved by SDS-PAGE and immunoblotted for N-cadherin, E-cadherin, and tubulin (loading control). (C) N-cadherin mRNA levels were analyzed by quantitative real-time PCR as in Figure 3D (∗p < 0.05, col RacN17 versus col MOCK or col RacV12).

Figure 8.

PI3K and JNK activities are necessary for collagen I–induced changes. (A) NMuMG/E10 cells were cultured on noncoated or collagen I-coated dishes for 2 d in the presence of the vehicle DMSO (a and b), the PI3K inhibitor LY294002 (10 μM; c and d), or the JNK inhibitor SP600125 (10 μM; e and f). Photographs were taken using a 10× objective. (B and C) RIPA extracts were made from cells cultured on noncoated or collagen I-coated dishes for 3 h in the presence of DMSO, LY294002 (10 μM; B and C), or SP600125 (10 μM; C). Pull-down assays for Rac-GTP and total Rac were performed as in Figure 6A. Immunoblots were done for phospho-Akt (Ser479), total Akt1, phospho-JNK (Thr183/Thy185) and total JNK1 (B), and with N-cadherin and GAPDH (C). (D) N-cadherin mRNA levels were analyzed by quantitative real-time PCR as in Figure 3D (∗p < 0.05, DMSO col versus LY294002 col or SP600125 col).

Figure 9.

N-cadherin knockdown prevents collagen I–induced changes. (A) Mock-infected, N-cadherin knockdown, and N-cadherin–overexpressing NMuMG/E10 cells were cultured on noncoated (a–c) or collagen I-coated (d–f) dishes. Photographs were taken of living cells using a 10× objective. (B) RIPA extracts (30 μg of protein) from mock-infected, N-cadherin knockdown, and N-cadherin–overexpressing NMuMG/E10 were resolved by SDS-PAGE and immunoblotted for N-cadherin, E-cadherin, and GAPDH. (C) Extracts (1 mg of protein) from cells cultured on noncoated or collagen I-coated dishes for 3 h were used for pull-down assay as in Figure 6A.

Figure 10.

N-cadherin knockdown limits cell motility. (A) Mock infected, N-cadherin knockdown, and N-cadherin–overexpressing NMuMG/E10 cells were plated for 4 h on transwell filters coated in both the top and bottom sides with collagen I or fibronectin. Cells traversing the filter were photographed using a 10× objective (A) and quantified (B). The columns represent mean values and the bars represent the SD (∗p < 0.05, 1 versus 2; ∗∗p < 0.05, 5 versus 1 or 3). (C) NMuMG/E10 cells were plated on coated filters, and DMSO (vehicle), LY294002 (10 μM), or SP600125 (10 μM) was added to both the top and bottom chambers. NMuMG/E10 cells expressing Rac1N17 were plated on coated filters. The columns represent mean values and the bars represent the SD (∗p < 0.05, 1 versus 3, 5, or 7).

Figure 11.

N-cadherin knockdown limits branching in 3D collagen gel culture. (A) 3D collagen gel cultures were performed using mock transfected, N-cadherin knockdown, and N-cadherin–overexpressing NMuMG/E10 cells. (B) 3D collagen gel cultures were performed using NMuMG/E10 cells in the presence of LY294002 (10 μM) or SP600125 (10 μM), or using NMuMG/E10 cells expressing Rac1N17. (C) Quantification of branching was performed by counting all identifiable branch points in each colony. For the experiments using inhibitors, LY294002 (10 μM) or SP600125 (10 μM) was added to the culture medium above the collagen gel. The columns represent mean values, and the bars represent the SD (p < 0.05; 1 versus 2 or 3, 1 versus 4, p < 0.05; 1 versus 5, p = 0.11; 1 versus 6, p = 0.06).