Rac-dependent cyclin D1 gene expression regulated by cadherin- and integrin-mediated adhesion - PubMed (original) (raw)

Rac-dependent cyclin D1 gene expression regulated by cadherin- and integrin-mediated adhesion

Alaina K Fournier et al. J Cell Sci. 2008.

Abstract

Integrin-mediated adhesion to substratum is required for cyclin D1 induction in mesenchymal cells, but we show here that the induction of cyclin D1 persists despite blockade of ECM-integrin signaling in MCF10A mammary epithelial cells. E-cadherin-mediated cell-cell adhesion also supports cyclin D1 induction in these cells, and the combined inhibition of both E-cadherin and integrin adhesion is required to prevent the expression of cyclin D1 mRNA and protein. Our previous studies described a pro-proliferative effect of E-cadherin in MCF10A cells, mediated by Rac, and we now show that Rac is required for cyclin D1 mRNA induction by both E-cadherin and integrin engagement. The levels of p21Cip1 and p27Kip1, Cdk inhibitors that are also targets of integrin signaling, are not affected by E-cadherin-mediated cell-cell adhesion. Finally, we show that the increased expression of cyclin D1 mRNA associated with E-cadherin-dependent cell-cell adhesion is causally linked to an increased entry into S phase. Our results identify Rac signaling to cyclin D1 as a crucial pro-proliferative effect of E-cadherin-mediated cell-cell adhesion.

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Figures

Fig. 1

Blockade of integrin-mediated adhesion does not prevent cyclin D1 gene expression in MCF10A cells. (A) Serum-starved MCF10A cells were trypsinized, reseeded on dishes coated with collagen or agarose, and stimulated with 10% FBS and growth factor cocktail. Cell lysates were analyzed by western blotting for cyclin D1, Cdk6, phosphoY397-FAK, and FAK. (B) The experiment in A was repeated except that collected cells were analyzed for cyclin D1 mRNA by QPCR and western blotting using antibodies to dually phosphorylated ERK and total ERK. QPCR results show mean ± s.e.m. of two experiments after normalizing the levels of cyclin D1 mRNA to the level in serum-starved cells. (C) Quiescent MCF10A cells were preincubated in suspension with vehicle or AIIB2 β1-integrin-blocking antibody and RGD peptide. The cells were then plated on agarose-coated dishes and incubated for 3 and 9 hours with either 10% FBS and growth factor cocktail (GF), growth factor cocktail in serum-free medium, or growth factor cocktail in serum-free medium with AIIB2 and RGD. The suspended cells were collected by attachment to poly-L-lysine-coated coverslips and visualized by phase-contrast microscopy. Bar, 210 μm. (D) Attachment of MCF10A cells to collagen (COLL), vitronectin (VN), fibronectin (FN) or laminin-1 (LM) in the absence and presence of AIIB2 and/or RGD as described in the Materials and methods. The results are plotted as percentage maximal attachment relative to the positive control (no inhibitors) for each matrix protein.

Fig. 2

E-cadherin-mediated adhesion in suspended MCF10A cells. (A) Quiescent MCF10A cells were trypsinized, reseeded on dishes coated with agarose, stimulated with 10% FBS and growth factor cocktail for 9 hours, and analyzed by phase-contrast or epifluorescence microscopy for E-cadherin (E-cad) and β-catenin (β-cat). (B) MCF10A cells were transfected with control or E-cadherin siRNA, rendered quiescent by serum and growth factor starvation, trypsinized, reseeded on collagen-coated dishes, stimulated with 10% FBS and growth factor cocktail for 9 hours, and analyzed by epifluorescence microscopy for E-cadherin, β-catenin and DAPI-stained nuclei. (C) Cells prepared as in B were seeded on agarose-coated dishes with 10% FBS and growth factor cocktail for 9 hours, collected on poly-L-lysine-coated coverslips, and analyzed by phase-contrast microscopy. (D) Quiescent MCF10A cells were trypsinized and pre-incubated in suspension in the absence and presence of 2 mM EGTA (30 minutes at 37°C) in DMEM-F12 with 1 mg/ml heat-inactivated fatty acid-free BSA. The pre-treated cells were directly replated on agarose-coated dishes, and stimulated with 10% FBS and growth factor cocktail for 9 hours. The cells were then collected on poly-L-lysine-coated coverslips and analyzed by phase-contrast microscopy. Bars, 210 μm.

Fig. 3

E-cadherin-mediated cell-cell adhesion and cell-substratum signaling coordinately regulate cyclin D1 expression in MCF10A cells. MCF10A cells were transfected with control or E-cadherin (E-cad) siRNA, rendered quiescent by serum and growth factor starvation, trypsinized, and reseeded in a monolayer (Mono) or in suspension (Susp) on collagen- or agarose-coated dishes, respectively. The cells were stimulated with 10% FBS and growth factor cocktail. (A) Samples were collected at the indicated time points and analyzed by western blotting for cyclin D1, Cdk6, E-cadherin, β-catenin, phosphoY397-FAK and FAK. (B) The experiment in A was repeated with cells seeded in monolayer (on collagen-coated dishes containing glass coverslips) (Liu et al., 2006) or in suspension (on agarose-coated dishes) at 2×104 cells/cm2. Collected cells were analyzed by QPCR for cyclin D1 mRNA. (C) Cells seeded on collagen-coated dishes and stimulated with 10% FBS and growth factor cocktail were collected and analyzed by western blotting for E-cadherin, p21Cip1, p27Kip1, dually phosphorylated ERK, total ERK and GAPDH (loading control).

Fig. 4

Cyclin D1 half-life is not regulated by E-cadherin. MCF10A cells were transfected with control or E-cadherin siRNA during serum and growth factor starvation. (A) The starved cells were trypsinized, reseeded in suspension on agarose-coated dishes and stimulated with 10% FBS and growth factor cocktail for 6 hours to induce cyclin D1. Cycloheximide (CHX) was then added, and the cells were collected every 15 minutes for 1 hour. Collected cells were analyzed by western blotting using anti-cyclin D1 and anti-GAPDH (loading control). The cyclin D1 blot was exposed for 10 and 30 seconds (s) so that all bands could be readily detected and easily compared between samples (e.g. compare 10 s ‘control siRNA’ with 30 s ‘E-cadherin siRNA’). (B) MCF10A cells were transfected with control or E-cadherin siRNA and then infected with β-galactosidase (LacZ) (20 MOI) or cyclin D1 adenoviruses (2.5-20 MOI) during serum and growth factor starvation. The cells were trypsinized, reseeded in monolayer [M; β-galactosidase control only] or suspension (Susp) on dishes coated with collagen or agarose, respectively. The cells were stimulated with 10% FBS and growth factor cocktail for 9 hours. Cell lysates were analyzed by western blotting for cyclin D1, E-cadherin, and actin (loading control).

Fig. 5

Cell-cell- and cell-substratum-dependent expression of cyclin D1 mRNA is mediated by Rac. MCF10A cells were infected with adenoviruses encoding GFP, β-galactosidase (LacZ), N17Rac or β2-chimerin and serum starved. Control viruses were used at the highest MOI of the test virus. The infected, serum-starved cells were plated on collagen-coated dishes and stimulated with 10% FBS and growth factor cocktail. (A) Cells were mitogen stimulated for 9 hours, and the effect of N17-Rac on cyclin D1 mRNA was determined by QPCR. (B) Cells were mitogen stimulated for 9 hours, and the effect of β2-chimerin on cyclin D1 mRNA was determined by QPCR. (C) MCF10A cells were transfected with control or E-cadherin siRNA and then infected with β-galactosidase (LacZ) or N17-Rac adenovirus during serum- and growth factor-starvation. Quiescent, transfected/infected cells were trypsinized, reseeded in monolayer (Mono) or suspension (Susp) on collagen- or agarose-coated dishes, respectively. The cells were stimulated with 10% FBS and growth factor cocktail for 12 hours. Total RNA was isolated and analyzed for cyclin D1 mRNA by QPCR.

Fig. 6

Rac inhibition blocks cyclin D1 expression. (A,B) MCF10A cells were infected with adenoviruses encoding GFP, β-galactosidase (LacZ), N17-Rac, or β2-chimerin and serum starved. Control viruses were used at the highest MOI of the test virus. The infected, serum-starved cells were plated on collagen-coated dishes, stimulated with 10% FBS and growth factors for 9 hours. Collected cells were analyzed by western blotting with the antibodies shown. (C) MCF10A cells were transfected with irrelevant control or Rac1 siRNAs and serum starved. The cells were plated on collagen-coated dishes, stimulated with 10% FBS and growth factor cocktail for the times shown, and analyzed by western blotting using antibodies to cyclin D1, dually phosphorylated ERK, total ERK, Rac and GAPDH (loading control).

Fig. 7

Causal relationship between the stimulatory effects of E-cadherin-mediated cell-cell adhesion on cyclin D1 gene expression and S-phase entry. (A) Serum-starved control and E-cadherin (E-cad) siRNA-transfected MCF10A cells were trypsinized, reseeded in full maintenance medium (see Materials and Methods) on collagen-coated, 25-mm glass coverslips at 2×104 cells/cm2 (with cell-cell adhesion) or 2×103 cells/cm2 (without cell-cell adhesion) as described by Liu et al. (Liu et al., 2006). BrdU was added when the cells were seeded and stimulated; the number of BrdU-labeled nuclei (relative to DAPI-stained nuclei) was determined after 24 hours. (B) Duplicate samples were collected at 12 hours and analyzed for cyclin D1 mRNA by QPCR. Results were calculated relative to the level of cyclin D1 mRNA in serum-starved cells expressing control siRNA. (C) Serum-starved MCF10A cells infected with Ad-GFP or Ad-cyclin D1 (100 MOI) were treated and analyzed as in A. Results for each panel show means ± s.e.m. of two experiments.

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Rac-dependent cyclin D1 gene expression regulated by cadherin- and integrin-mediated adhesion - PubMed (original) (raw)