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First published online 22 December 2004
doi: 10.1242/jcs.01601

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Both ERK and Wnt/ß-catenin pathways are involved in Wnt3a-induced proliferation

Mi-Sun Yun^1,*, Sung-Eun Kim^1,*, Soung Hoo Jeon¹, Jung-Soo Lee¹ and Kang-Yell Choi^1,2,

¹ Department of Biotechnology, Yonsei University, Seoul 120-752, Korea
² Protein Network Research Center, Yonsei University, Seoul 120-752, Korea

View larger version (14K): [in a new window]   Fig. 1. The effects of Wnt3a on activation of the Wnt/ß-catenin pathway. (A) NIH3T3 cells were grown to 70% confluence and either not treated or treated with 150 ng/ml of recombinant Wnt3a for 8 hours. Whole cell lysates were subjected to western blotting with ß-catenin, cyclin D1 or -tubulin primary antibodies. (B) NIH3T3 cells were grown to 50% confluence, then transfected with either 0.5 µg of pTOPFLASH or pFOPFLASH (Korinek et al., 1997). 48 hours after transfection, cells were harvested for a Luciferase assay. Where required, 150 ng/ml of recombinant Wnt3a was applied 8 hours before harvesting. The mean values and standard deviations of three independent experiments are shown.

View larger version (34K): [in a new window]   Fig. 2. Effects of ß-catenin siRNAs on Wnt3a-induced BrdU incorporation. NIH3T3 cells were grown on coverslips in DMEM and either not transfected or transfected with 1.68 µg of ß-catenin siRNA per 3.5 cm dish for 48 hours. Cells were labeled with 20 µM BrdU for 5 hours prior to cytochemical analysis. Where required, 150 ng/ml of purified Wnt3a was applied 24 hours before cytochemical analysis. Cell nuclei were stained with DAPI. Cells containing BrdU incorporated into the nucleus were scored as BrdU-positive. (A) Quantitative measurement of the percentage of BrdU-positive cells. Analyses were performed at least three times and 100 cells were counted in each case. Error bars indicate the standard deviations of three independent analyses. The right panel shows western blot analysis of ß-catenin after transfection of NIH3T3 cells with ß-catenin siRNA. (B) DAPI, BrdU and merged images of representative non-transfected and ß-catenin transfected cells after Wnt3a treatment as summarized in A. Bar, 50 µm.

View larger version (59K): [in a new window]   Fig. 3. Effects of ERK1 and ERK2 siRNAs on Wnt3a-induced BrdU incorporation. NIH3T3 cells were grown on coverslips in DMEM and either not transfected or transfected with ERK1 or ERK2 siRNAs, or ERK1/ERK2 siRNA for 48 hours. Cells were labeled with 20 µM BrdU 5 hours prior to cytochemical analysis. Where required, 150 ng/ml purified Wnt3a was applied 24 hours before cytochemical analysis. Cell nuclei were stained with DAPI. (A) Quantitative measurement of the percentage of BrdU-positive cells. Analyses were performed at least three times and 100 cells were counted in each case. Error bars indicate the standard deviations of three independent analyses. The right panel shows the western blot analysis of p-ERK1 and p-ERK2 after transfection of NIH3T3 cells with ERK1 and ERK2 siRNA, respectively (B) DAPI, BrdU and merged images of representative non-transfected and ERK1/2 transfected cells after Wnt3a treatment as summarized in A. Bar, 50 µm.

View larger version (56K): [in a new window]   Fig. 4. Activation of ERK, MEK and Raf-1 kinases by Wnt3a in NIH3T3 and L cells. (A) NIH3T3 cells were grown in DMEM and treated with 150 ng/ml recombinant Wnt3a. Cells were harvested at 0, 30 minutes, 12 hours and 24 hours. p-ERK, p-MEK, p-Raf (Ser338), -tubulin, ß-catenin and cyclin D1 proteins were detected by western blot analysis. The p-Raf-1 (Ser338) bands differentially modified were marked with arrows. (B) Left panels, NIH3T3 cells were grown in DMEM and treated with 100 µl Wnt3a-CM (upper panel) or Con-CM (lower panel). Cells were harvested at 0, 5 minutes, 15 minutes, 30 minutes, 1 hour, 2, 4, 8, 12 and 24 hours. The p-ERK, p-MEK, p-Raf (Ser338), ß-catenin and -tubulin proteins were detected by western blot analysis (see A) Right panels, NIH3T3 cells were grown in DMEM and treated with different amounts of Wnt3a-CM (0, 1, 10, 50, 100 or 200 µl; upper panel) or Con-CM (0, 10, 50, 100 and 200 µl; lower panel). Cells were harvested 30 minutes after either Con-CM or Wnt3a-CM treatment for western blot analysis (see A). (C) Upper panel, L cells were grown in DMEM and treated with 100 µl Wnt3a-CM. Cells were harvested at 0, 5 minutes, 30 minutes, 2 and 8 hours after Wnt3a-CM treatment. Lower panel, L cells were grown in DMEM and treated with different amounts of Con-CM (0, 10, 50 or 100 µl). Cells were harvested 30 minutes after Con-CM treatment and western blot analyses were then performed.

View larger version (63K): [in a new window]   Fig. 5. Effects of Wnt3a on nuclear accumulation of the p-ERK and ß-catenin proteins in NIH3T3 cells. NIH3T3 cells were grown in DMEM and treated with 150 ng/ml recombinant Wnt3a. Cells were subjected to immunocytochemical analysis at 0, 30 minutes and 8 hours after Wnt3a treatment. Cells were then treated with anti-p-ERK and anti-ß-catenin antibodies. p-ERK proteins were detected as a green color using CyTM2-conjugated goat anti-mouse IgG, ß-catenin proteins were detected as a red color using anti-rabbit-Rhodamine Red TM-X-conjugated secondary antibody. Cell nuclei were stained with DAPI. Bar, 10 µm.

View larger version (44K): [in a new window]   Fig. 6. The effects of ERK pathway inhibitors on Wnt3a-induced Raf-1, MEK, and ERK kinase activation. (A) NIH3T3 cells were grown in DMEM and either not treated or treated with 150 ng/ml recombinant Wnt3a (left panel) or with 100 µl Con-CM or Wnt3a-CM (right panel) for 30 minutes before harvesting. Where required, 20 µM U0126 was added 1 hour before the Con-CM or Wnt3a-CM treatment. (B) NIH3T3 cells were grown in DMEM and transfected with 0.5 µg pCMV vector, 0.5 µg pSVSport1-dn-Raf-1, 0.5 µg pCMV-MEK2A vector, 0.5 µg pMTSM-MycMKP-3 (left panel) and 0.5 µg pcDNA3.0 vector or pcDNA3.1 H-Ras N17 (right panel). 48 hours after transfection, the cells were treated for 30 minutes with 100 µl Con-CM or Wnt3a-CM. Cells were harvested and subjected to western blot analysis.

View larger version (32K): [in a new window]   Fig. 7. The effects of ß-catenin siRNA on Wnt3a-induced ERK activation. NIH3T3 cells were either not transfected or transfected with ß-catenin siRNA for 48 hours. (A) Cells were either treated or not treated with 150 ng/ml recombinant Wnt3a for 30 minutes before harvesting. (B) Cells were treated with 100 µl Con-CM or Wnt3a-CM for 30 minutes before harvesting. Cell extracts were then prepared, and western blot analyses were performed.

View larger version (35K): [in a new window]   Fig. 8. Activation of the ERK pathway by ß-catenin overexpression, and effects of dominant-negative Tcf-4 on ß-catenin-induced ERK pathway activation. (A) NIH3T3 cells were grown in DMEM and transfected with either 0.5 µg pcDNA3.0 vector or 0.5 µg Flag-ß-catenin-pcDNA3.0. Cells were harvested 48 hours after transfection. (B) NIH3T3 cells were grown in DMEM and transfected with either 0.5 µg pcDNA3.0 vector or 0.5 µg Flag-ß-catenin-pcDNA3.0. Where required, cells were treated with 20 µM U0126 1 hour before harvesting. Anti-Flag antibody was used for detection of Flag-ß-catenin. (C) NIH3T3 cells were grown in DMEM and transfected with either 0.5 µg pcDNA3.0 vector or 0.5 µg Flag-ß-catenin-pcDNA3.0. Cells were co-transfected with 0.5 µg of vector or N-Tcf-4E together with Flag-ß-catenin-pcDNA3.0. The cells were harvested after 48 hours and western blot analyses were performed with ß-catenin, Flag, ERK, p-ERK, p-MEK, Raf-1 or -tubulin antibodies.

View larger version (13K): [in a new window]   Fig. 9. The effect of Wnt3a on cell cycle progression and the effect of U0126 on NIH3T3 cells. (A) NIH3T3 cells were grown in DMEM and synchronized with double thymidine blocking (Park et al., 2002). Cells were either not treated or treated with 150 ng/ml recombinant Wnt3a for 24 hours before harvest for FACS analysis. Where required, 20 µM U0126 was added 1 hour before treatment with Wnt3a. Error bars indicate the standard deviations of three independent experiments. (B) The effect of U0126 on Wnt3a-induced BrdU incorporation in NIH3T3 cells. NIH3T3 cells were grown in DMEM and either not treated or treated for 24 hours with 150 ng/ml of recombinant Wnt3a in DMEM containing 1% FBS. Where required, 20 µM U0126 was applied 1 hour before treatment with Wnt3a. Cells were labeled with 20 µM BrdU for 5 hours prior to cytochemical analysis using anti-BrdU antibody. Cell nuclei were stained with DAPI. Cells containing BrdU incorporated into the nucleus were scored as BrdU positive and the relative percentage of BrdU-positive cells was determined. Analyses were performed at least three times and 100 cells were counted in each case. Error bars indicate the standard deviations of three independent analyses.

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