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First published online 27 February 2007
doi: 10.1242/jcs.003335

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Metalloproteinase axes increase -catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

Ontario Cancer Institute, 610 University Avenue Toronto, Ontario M5G2M9, Canada

View larger version (93K): [in this window] [in a new window]   Fig. 1. Decreased cell aggregation and sensitivity to EGTA-induced detachment in primary Timp3–/– MEFs. (A) Representative examples of aggregation assay with independent MEF preparations. WT aggregates (arrows) were evident at 15 minutes and large aggregates seen at 45 minutes. Timp3–/– MEF aggregates (arrows) did not form until 45 minutes and were much smaller in size (fewer cells) than WT aggregates. Bars, 70 µm. (B) Representative images of fibroblast monolayers in a Ca2+ chelation assay. Timp3–/– MEFs detached before WT controls, as assessed by Hoechst staining and fluorescence microscopy of the cells remaining on the coverslips after the indicated time of EGTA treatment. Bars, 70 µm. (C) The number of viable cells in the supernatant following EGTA-induced detachment, assessed using Trypan Blue exclusion, showed higher numbers of Timp3–/– cells at all incubation times (*P<0.04; , WT; , Timp3–/–). Student's t-test was performed to assess statistical significance between WT and Timp3–/–. Values are expressed as mean ± s.e.m. (D) Western blot analyses of whole cell lysates from three of the five independent clones of WT and Timp3–/– MEFs, probed with the indicated antibodies. Amido Black staining was used to assess protein loading.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Primary Timp3–/– MECs are more resistant to EGTA-induced cell detachment and have higher -catenin levels. (A) Representative images from at least three different experiments. An increased number of Timp3–/– epithelial islands (outlined with dotted circles) remain following EGTA treatment as assessed by Hoechst staining and fluorescence microscopy. Bars, 2 mm. (B) Representative western blots of whole-cell lysates from WT and Timp3–/– (t3–/–) MECs probed with the indicated antibodies. Quantification by densitometry of (C) E-cadherin signal and (D) -catenin signal (, WT; , Timp3–/–; *P<0.05). (E) Western blot analyses of nuclear lysates of WT and Timp3–/– MECs using an anti--catenin antibody. (F) MEC total and nuclear lysates subjected to western blotting using an anti-dephosphorylated -catenin (de-P--cat). Panels B-F represent one of three pools of primary mammary epithelial cultures. Each MEC pool was harvested from all ten mammary glands of five WT and five Timp3–/– female mice. Amido Black and silver staining were used to assess protein loading. Student's t-test was performed to assess statistical significance between WT and Timp3–/–. Bar graphs were expressed as the mean ± s.e.m.

View larger version (75K): [in this window] [in a new window]   Fig. 3. Increased cell surface distribution of -catenin in Timp3–/– MECs following Wnt3A stimulation. (A) Representative confocal images from three different experiments. The colocalization of E-cadherin and -catenin was examined following treatment with W0 (control ligand) and Wnt3A in WT and Timp3–/– MECs. (B) Representative confocal images of WT and Timp3–/– mouse embryo fibroblasts treated with Wnt3A or control ligand. Arrows indicate nuclear localization of -catenin; arrowheads indicate increased membrane colocalization of E-cadherin and -catenin. Bars, 20 µm.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Increased transcriptional activity and selective gene response in Timp3–/– MECs. (A) -catenin transcriptional activity was measured using pTOPFLASH/pFOPFLASH luciferase reporter assay and was significantly higher in both Timp3–/– MEFs (*P=0.001; , WT; , Timp3–/–) and Timp3–/– MECs (**P=0.01) compared with their respective WT controls. Relative light unit values were normalized against a -galactosidase transfection control. (B,C) Expression of -catenin target genes was assessed by TaqMan real-time PCR and are normalized to 18S RNA values for each sample. (B) Ccnd1 mRNA level was significantly lower in both Timp3–/– MEFs (*P=0.05) and Timp3–/– MECs (**P=0.002). (C) Mmp7 mRNA level was significantly increased only in Timp3–/– MECs (*P=0.005). Two-tailed, Student's t-test was performed to assess statistical significance between WT and Timp3–/–. Values are expressed as mean ± s.e.m. of three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Metalloproteinase inhibitors decrease -catenin signaling in Timp3–/– cells. (A) Western blots of whole-cell lysates from WT and Timp3–/– MECs revealed that metalloproteinase inhibition with recombinant TIMP3 (25 µM), GM6001 (25 µM) and PD166793 (50 µM) for 24 hours returned dephosphorylated -catenin to levels comparable to the WT. (B) The reduction in Ccnd1 gene expression in Timp3–/– MECs was reversed by 24-hour treatment with recombinant TIMP3 (10 µM) (*P<0.001), GM6001 (10 µM) (**P<0.01) and PD166793 (25 µM) (***P<0.05) when compared with the untreated control. (C) The increase in Mmp7 gene expression in Timp3–/– MECs was significantly mitigated by treatment with recombinant TIMP3 (10 µM) (*P<0.001), GM6001 (10 µM) (*P<0.001) and PD166793 (25 µM) (**P<0.05) for 24 hours. ANOVA was performed to assess the statistical significance between WT and Timp3–/– cells. Values are expressed as mean ± s.e.m. of three independent experiments.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effects of GSK3 and E-cadherin inhibitors on Ccnd1 and Mmp7 gene expression. (A,B) WT () and Timp3–/– () MECs were treated with the GSK3 inhibitor, SB-216763 (20 mM for 24 hours) and the E-cadherin blocking antibodies, ECCD-1 (200 mg/ml for 6 hours) and DECMA-1 (0.5 mg/ml for 6 hours). Expression of -catenin target genes, Ccnd1 (A) and Mmp7 (B) was assessed by TaqMan real-time PCR and normalized to 18S RNA values. (A) Inhibition of GSK3 led to an overall increase in Ccnd1 mRNA levels in WT and Timp3–/– MECs, but still showed significantly lower levels in Timp3–/– MECs compared with treated WT MECs. Similarly, treatment with SB-216763 of WT and Timp3–/– MECs generally increased Mmp7 mRNA levels. Upon GSK3 inhibition WT and Timp3–/– expression of Mmp7 became comparable (B). E-cadherin inhibition using two different blocking antibodies decreased Ccnd1 and increased Mmp7 mRNA levels in treated WT MECs but had no effect on Timp3–/– MECs. (C) Treatment with SB-216763, ECCD-1 and DECMA-1 elevated WT MEC levels of dephosphorylated -catenin (de-P--cat) to those of untreated Timp3–/– MEC levels. The -catenin levels in Timp3–/– MECs may be maximal as treatment with the GSK3 inhibitor did not further increase dephosphorylated -catenin levels. ANOVA was performed to assess statistical significance between WT and Timp3–/– cells. Values are expressed as mean ± s.e.m. of three independent experiments.

View larger version (37K): [in this window] [in a new window]   Fig. 7. Accelerated mammary ductal elongation in Timp3–/– virgin glands. (A) Representative images of mammary gland whole mounts (n=4) during pubertal morphogenesis in WT and Timp3–/– mammary glands. Bars, 1 mm. A significant increase was seen in (B) ductal length at 20 (*P=0.016), 30 (**P=0.019) and 40 (***P=0.022) days of age. There were no statistically significant differences in (C) numbers of ducts and (D) branches per duct between WT and Timp3–/– mammary glands (, WT; , Timp3–/–). (E) Implantation of recombinant TIMP-containing pellets (blue) or control pellets (red) into contralateral WT glands revealed effects of individual TIMPs on mammary morphogenesis. (F) TIMP1, TIMP3 and TIMP4 retarded mammary ductal elongation by 12% (P=0.04), 25% (P=0.07) and 19% (P=0.002), relative to contralateral glands, respectively. TIMP2 promoted ductal elongation by 13% relative to contralateral gland (P<0.05). Student's t-test was performed to assess statistical significance between WT and Timp3–/– mammary glands. Values are expressed as mean ± s.e.m.

View larger version (22K): [in this window] [in a new window]   Fig. 8. A schematic outlining the relationships between metalloproteinases, metalloproteinase inhibitors and regulators of -catenin signaling. In Timp3+/+ setting, E-cadherin and GSK3 influence the cytoplasmic pool of -catenin, which can translocate into the nucleus and function as transcriptional co-activator for target genes, Ccnd1 (cyclin D1) and Mmp7. We propose that MMP and ADAM activity operates upstream of E-cadherin and GSK3 through yet unidentified mechanisms to influence the signaling pool of -catenin. In Timp3 deficient mammary epithelial cells, -catenin signaling activity is increased, leading to selective gene responses. These arise in a GSK3-dependent manner and are not influenced by E-cadherin blocking. (, increased expression or activity; , decreased expression or activity; , unchanged expression or activity.)