Technical Reference #913

913.

Wnt-3a-Dependent Cell Motility Involves RhoA Activation and Is Speciffically Regulated by Dishevelled-2 Yoshimi Endo; Vladimir Wolf; Kanae Muraiso; Keiju Kamijo; Lilian Soon; Aykut Uren; Michal Barshishat-Kupper; Jeffrey Rubin, National Insitutes of Health, The The Journal of Biological Chemistry, 280(913), (2005)
Link To Paper

Abstract:
Wnts stimulate cell migration although the mechanismsresponsible for this effect are not fully understood.To investigate the pathways that mediate Wnt-dependentcell motility we treated Chinese hamster ovary cells withWnt-3a-conditioned medium an

Materials & Methods:
Reagents—A murine Wnt-3a cDNA kindly provided by Dr. Shinji Takada was transfected into L cells and a stable clonal line was selected that released -catenin stabilizing activity into serum-free CM. Wnt-3a CM and CM from nontransfected L cells were harvested from confluent monolayers that had been grown in serum-containing medium but subsequently were incubated for 72 h in serum-free EMEM supplemented with nonessential amino acids L-glutamine (2 mM) sodium pyruvate (1 mM) penicillin (100 units/ml) and streptomycin (100 g/ml). Typically CM was stored at 80 °C; once thawed medium was kept refrigerated and retained activity for several weeks (activity was lost if material was subjected to another round of freeze-thawing). Recombinant secreted Frizzled-related protein-1 (sFRP-1) was purified as previously described (30). Human Dickkopf-1 (Dkk-1) was obtained from R&D Systems (Minneapolis MN). ROCK inhibitor Y-27632 was from Calbiochem (La Jolla CA) human fibronectin was from Roche Applied Science (catalog number 1080938) calf intestine phosphatase was from Promega (Madison WI; catalog number M1821) antibody against the C terminus of -catenin was from Transduction Laboratories (San Diego CA; catalog number C19220) and antibodies to Dvl-2 (sc-13974 for immunoblotting and immunoprecipitation and sc-7399 for immunofluorescent staining) Dvl-3 (sc-8027) RhoA (sc-418) and heat shock protein-70 (HSP-70 sc-7298) were from Santa Cruz Biotechnology (Santa Cruz CA). Cell Culture and Transfection—Early passage CHO-K1 cells were maintained in Ham’s F-12 medium supplemented with 10% fetal bovine serum glutamine (2 mM) penicillin (100 units/ml) and streptomycin (100 g/ml). Transfections were performed with GenePorter 2 (Gene Therapy Systems San Diego CA) according to the manufacturer’s protocol using 1 g of DNA/well in a 6-well plate (2 105 cells/well). A plasmid encoding a dominant-negative form of TCF-4E (DN-TCF) was kindly provided by Drs. Frank McCormick and Osamu Tetsu (31). The TCF/lymphoid enhancer-binding factor-responsive reporter plasmid pTOPFLASH was a gift from Dr. Hans Clevers (32). Immunofluorescent Staining and Assessment of Cell Morphology— The cells were seeded on glass coverslips in complete Ham’s F-12 medium. After 4–6 h the medium was replaced with either Wnt-3a or L CM and cells were incubated for the indicated times. Then they were fixed with freshly prepared 3.7% formaldehyde for 15 min at room temperature and permeabilized with 0.1% Triton X-100 with PBS for 5 min. After blocking with 5% BSA with PBS for 1 h at room temperature the cells were incubated overnight at 4 °C with goat anti-Dvl-2 diluted 1:400 in 2% BSA with PBS followed by fluorescent labeled donkey anti-goat Alexa Fluor 488 (1:1000; Molecular Probes) for 30 min at room temperature. Phalloidin (Alexa Fluor 568; Molecular Probes) and 4 6- diamidino-2-phenylindole (dihydrochloride; Sigma-Aldrich) were included during this 30-min period for F-actin and nuclear staining respectively. In some experiments the cells were only stained with phalloidin. A Leica DM IRB fluorescent microscope with 63 objective lens (Germany) and SPOT camera (Diagnostic Instruments Inc. Sterling Heights MI) were used to detect fluorescence. The images were processed with Adobe Photoshop Elements 2.0 (Adobe Inc. San Jose CA). For measurement of cell surface area in the plane where cells were in contact with the coverslip the images were obtained with a 20 objective lens from 10 random locations on a single coverslip and analyzed with ImageGaugeV4.21 software (Fuji Photo Film Co. Ltd. Japan). Cell Migration Assays—Scratch or wound healing assays were performed with CHO cells seeded on glass-bottomed dishes (MatTek Corporation Ashland MA) that had been precoated with fibronectin (100 g/ml). After cells were 80% confluent the monolayer was scratched with a pipette tip (Continental Laboratory Product San Diego CA; catalog number 2142S). The medium was replaced with Wnt-3a or L CM and cultures were incubated for 6–24 h in a humidified CO2 incubator at 37 °C. The cells were examined with an Axiovert S100 (Zeiss Germany) inverted microscope (10 objective lens) and Photometrix Sensys camera (Tucson AZ). Transwell assays were performed with 6.5-mm-diameter Falcon cell culture inserts (8-m pore size; Becton Dickinson San Jose CA; catalog number (35) 3097) and 24-well cell culture plates. CHO cells were trypsinized resuspended in serum-free EMEM and transferred to the upper chamber (2 104 cells in 500 l); an equal volume of Wnt-3a or L CM was added to the lower chamber. The cells were maintained for 6–8 h in a humidified CO2 incubator at 37 °C. Following incubation the media were aspirated and the cells remaining on the upper surface of the filter were removed with a cotton swab whereas cells that had migrated to the lower surface were stained with 0.5% crystal violet for 30 min and counted (bright field image with Leica DM IRB fluorescent microscope 40 objective lens). In experiments with sFRP-1 (10 g/ml) CM was preincubated with the protein for 20 min at room temperature prior to addition to the lower chamber. For Dkk-1 (1 g/ml) the cells were preincubated with protein for 20 min at room temperature before transfer to the upper chamber. When experiments were performed with Y-27632 the ROCK inhibitor was preincubated for 20 min at room temperature both with the cells and CM in separate test tubes prior to addition to the upper and lower chambers respectively. For DN-TCF and Dvl-2 RNAi experiments the cells were trypsinized 48 h after transfection and transferred to the upper chamber. Immunoblotting Immunoprecipitation and Phosphatase Treatment— CHO monolayers (70–80% confluent) were incubated in serumfree Ham’s F-12 medium overnight and then treated with Wnt-3a or L CM for varying periods. Where indicated CM was preincubated with sFRP-1 for 20 min at room temperature. When Dkk-1 was used the cells were preincubated with the protein for 20 min at 37 °C. Following CM treatment the cells were rinsed twice with PBS and lysed with buffer containing 50 mM HEPES pH 7.5 50 mM NaCl 1 mM EDTA 1% Triton X-100 10 mM sodium pyrophosphate 50 mM NaF 1 mM sodium vanadate 10 g/ml aprotinin 10 g/ml leupeptin and 1 mM phenylmethylsulfonyl fluoride. The cell lysates were clarified in a desktop centrifuge at 14000 rpm for 10 min at 4 °C. Protein concentration was determined with a Bio-Rad protein assay reagent (Bio-Rad). For all of the immunoblots 30 g of protein were loaded per lane. After SDSPAGE the proteins were transferred to an Immobilon P membrane (Millipore Billerica MA) which was blocked with 5% milk in TTBS (20 mM Tris-HCl pH 8.0 0.05% Tween 20 150 mM NaCl) incubated with primary antibody overnight at 4 °C and subsequently incubated with horseradish peroxidase-labeled secondary antibody. The proteins were visualized with SuperSignal Pico or Femto chemiluminescent reagents (Pierce) using BioMax films (Eastman Kodak Co. Rochester NY). For immunoprecipitation the cell lysate (500 g) was incubated with Dvl antibody (2 g) overnight at 4 °C in a rotary shaker followed by the addition of protein A/G-agarose (Santa Cruz Biotechnology) and incubation for 1–2 h at 4 °C. The beads were pelleted and washed twice with cell lysis buffer prior to SDS-PAGE. Phosphatase treatment was performed as previously described (33). Rho A Activation Assay—RhoA activation was demonstrated with a GST-RBD pull-down assay essentially as previously described (22). Because the recombinant fusion protein was labile aliquots of bacteria expressing the protein were snap-frozen on liquid nitrogen and stored at 80 °C until the day of the assay when the protein was bound to glutathione-agarose beads (Santa Cruz Biotechnology). CHO cells were grown to 80% confluence serum-starved overnight and then treated with Wnt-3a or L CM for the indicated times. The cells were washed and lysed and 1.0–1.5-mg lysates were incubated with GST-RBD agarose (20 l) for 1 h at 4 °C in a rotary shaker. The beads were pelleted and washed and proteins recovered for immunoblotting with RhoA antibody. Whole cell lysates were similarly immunoblotted for comparison. Similar results were obtained with the Rho Activation Kit from Upstate Cell Signaling (Lake Placid NY; catalog number 17-294). Small Interference RNA—pSilencer 1.0-U6 (Ambion Austin TX) was used as a vector to introduce hairpin loop double-stranded RNA into cells to disrupt endogenous Dvl expression. The targeted sequence of Dvl-2 was: 5 -CUUUGAGAACAUGAGCAAC-3 . The following oligonucleotides were made to order by Invitrogen and annealed according to instructions from Ambion: sense 5 -CTTTGAGAACATGAGCAACTTCAAGAGAGTTGCTCATGTTCTCAAAGTTTTTT- 3 ; antisense 5 -AATTAAAAAACTTTGAGAACATGAGCAACTCTCTTGAAGTTGCTCATGTTCTCAAAGGGCC- 3 . The oligonucleotide duplex was ligated into ApaI/EcoRI-digested pSilencer vector according to the protocol from Ambion. One g of the Dvl-2 RNAi construct or vector control was transfected into HEK293 (4 105) or CHO (2 105) cells with Gene- Porter2 following the manufacturer’s instructions. The cells were harvested 48 h after transfection for expression analysis or functional studies. Reverse Transcription-PCR Analysis—RNA was prepared from CHO cells with TRIzol (Invitrogen) according to the manufacturer’s instructions and stored at 80 °C. Complementary DNA was generated with the SuperScriptTM first strand synthesis system from Invitrogen. Sub- sequent PCR was performed with the following pairs of primers: Dvl-1 forward: 5 -GCGGAGACCAAAATCATCTACCAC-3 Dvl-1 reverse: 5 - GACCATAGACTCTGTGCCTGTCTC-3 ; Dvl-2 forward: 5 -CGCGTCGGTTTGCGGGTGTG- 3 Dvl-2 reverse: 5 -GAAGGATGGAGGCCTTGAGTC- 3 ; Dvl-3 forward: 5 -GAGCCATGGGCGAGACCAAG-3 Dvl-3 reverse: 5 -GTTCTGTGGAGCTGCTGAACCTGC-3 ; and -actin forward: 5 -CCACTGGCATCGTGATGGAC-3 -actin reverse: 5 -GCGGATGTCCACGTCACACT- 3 . The predicted sizes of PCR products were 420 bp (Dvl-1) 496 bp (Dvl-2) 624 bp (Dvl-3) and 428 bp ( -actin). All of the PCR experiments were performed with the same set of conditions: denaturation at 94 °C for 3 min; amplification for 30 cycles each consisting of incubation at 94 °C for 10 s 58 °C for 30 s and 72 °C for 45 s and extension at 72 °C for 8 min. Statistical Analysis—The Wilcoxon rank-sum test was used to evaluate the significance of differences in cell spreading (area of cell/coverslip contact) among the treatment groups. The significance of differences in data obtained from transwell and luciferase reporter assays was determined with Student’s two-tailed t test. The differences were considered to be significant when p 0.05.

Microscopic Technique
Fluoroescence Miroscopy

Cell Type(s)
CHO-K1