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First published online January 10, 2008
doi: 10.1242/10.1242/jcs.021964

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G_o mediates WNT-JNK signaling through Dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells

Department of Pharmacology, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, NY 11794-8651, USA

View larger version (35K): [in this window] [in a new window]   Fig. 1. WNT3a stimulates activation of JNK and the AP-1–luciferase reporter in F9 cells. (A) Confluent wild-type F9 clones (F9) or F9 cells transfected with pCDNA3.1 alone (pcDNA) or F9 cells stably expressing rat Frizzled-1 (rFZ1) were treated with purified WNT3a for 0 to 60 minutes and JNK activation was measured by immunoblotting total lysates with phospho-specific antibodies against Jun. (B) rFZ1-expressing cells were transfected with 30 ng of AP-1–luciferase reporter plasmid in a 12-well plate for 24 hours, followed by 24 hours of serum starvation. The cells then were treated with or without WNT3a for 7 hours and luciferase gene reporter assays were performed, as described in the Materials and Methods. The upper panel displays the mean values±s.e.m. obtained from three independent experiments; the lower panel displays corresponding representative blots. For the AP-1-luciferase assay, the data represent the mean values±s.e.m. obtained from three independent replicate experiments. *P<0.05; **P<0.01 versus the time `zero' control.

View larger version (44K): [in this window] [in a new window]   Fig. 2. Suppression of Go, but not Gq or G11, abolishes, whereas expression of constitutively active Go mimics, the activation of JNK by WNT3a. (A) Confluent F9 cells expressing rFZ1 were treated with pertussis toxin (50 ng/ml) for 1 hour followed by WNT3a treatment for 15 minutes. Cell lysates were collected and JNK activity was assayed by probing Jun phosphorylation. (B-D) F9 cells expressing rFZ1 were treated with siRNAs specific for Go (B), Gq (C) or G11 (D) for 72 hours before treatment with WNT3a for 15 minutes. JNK activity was then determined by probing Jun phosphorylation. (E) F9 cells expressing rFZ1 were transfected with an expression vector harboring the Q205L Go mutant (0.25 µg/well in a 12-well plate). 48 hours after transfection, cells were treated with WNT3a for 15 minutes, cell lysates collected and probed with phospho-Jun-specific antibodies. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays corresponding representative blots. *P<0.05; **P<0.01 versus the –WNT3a control; #, P<0.05; ##, P<0.01 versus the +WNT3a control. The extent of knockdown of the expression of the G-protein alpha-subunit routinely was 70% or more.

View larger version (53K): [in this window] [in a new window]   Fig. 3. Suppression of DVL1 and DVL3, but not of DVL2, abolishes the ability of WNT3a to stimulate JNK activation, whereas expression of exogenous human DVL1 and DVL3 rescues the effect. F9 cells expressing rFZ1 were treated with siRNAs designed to suppress the expression of DVL1 (A), DVL2 (B) and DVL3 (C) for 48 hours, and JNK activity was measured by probing Jun phosphorylation, as described in the Materials and Methods. Specific suppression of individual DVL isoforms was demonstrated by immunoblotting with isoform-specific antibodies. The extent of suppression of DVL isoforms is as follows; DVL1 (71%), DVL2 (81%) and DVL3 (85%). The siRNAs were specific to the particular isoform as no cross-reaction was detected (D). Rescue experiments were performed by transfection of human (h) DVL1 (E) or DVL3 (F) into rFZ1-expressing cells in which DVL1 and DVL3 were knocked-down, respectively, by siRNA treatment as described in the Materials and Methods. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays the corresponding representative blots.*P<0.05; **P<0.01 versus –WNT3a control; #, P<0.05; ##, P<0.01 versus +WNT3a control.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Expression of Dapper1 abolishes the ability of WNT3a, as well as the ability of expression of Q205L Go to stimulate JNK activation. F9 cells expressing rFZ1 were either transfected (1 µg/well in a 12-well plate) with an expression vector harboring Myc-tagged mouse Dapper1 (A) or co-transfected with Myc-Dapper1 and Q205L Go (B) for 24 hours, and JNK activity was determined, as described in the Materials and Methods. The expression of Dapper1 was monitored by immunoblotting with antibodies against Myc. (C) F9 cells were either transfected (1 µg/well in a 6-well plate) with individual HA-DVL-GFP2 or co-transfected with HA-DVL-GFP2 and MycDapper1 (1 µg/well of each plasmid) for 24 hours followed by cell lysis and affinity pull-downs with anti-HA-sepharose beads. The interaction of Dapper1 with individual isoforms of DVL was made visible by probing the blots with the antibody against Myc. The expression of the exogenous DVL-GFP2 was established by stripping the Myc blot and probing with an antibody against HA. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays the corresponding representative blots. The blot of the DVL-Dapper1 interaction is representative of two independent experiments that proved highly reproducible. *P<0.05; **P<0.01 versus –WNT3a control; #, P<0.05; ##, P<0.01 versus +WNT3a control. The expression of DVL3 appears low in the absence of Dapper 1, but this was not a consistent observation.

View larger version (38K): [in this window] [in a new window]   Fig. 5. WNT3a stimulates activation of small-molecular-weight GTPases, while expression of dominant-negative (DN) versions of small-molecular-weight GTPases or treatment of rFZ1-expressing cells with specific siRNAs targeting the same small GTPases abolish WNT3a stimulation of JNK activity. (A) F9 cells expressing rFZ1 were treated with WNT3a (100 ng/ml) for 10 minutes and activation of each of the small-molecular-weight GTPases was monitored as described in the Materials and Methods. (B,C) F9 cells expressing rFZ1 were transfected (1 µg/well in a 12-well plate) with dominant-negative versions of small-molecular-weight GTPases (B) or treated with siRNAs specific for RhoA, Rac1 and Cdc42 (C) for 24 hours and JNK activity was determined. (D) F9 cells stably expressing rFZ1 were transfected with DVL3-GFP2 alone or together with DN-RhoA for 24 hours, and JNK activity was determined. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays the corresponding representative blots. The DVL3-RhoA epistasis experiment is representative of two independent experiments whose results were in strong agreement. The results displayed for the activation of small-molecular-weight GTPases are from a single experiment performed with triplicate sampling. These results are a representative of two separate experiments whose data were in high agreement. **P<0.01 versus –WNT3a control; ##, P<0.01 versus +WNT3a control.

View larger version (39K): [in this window] [in a new window]   Fig. 6. Expression of dominant-negative (DN) versions of either MEKK 1 or MEKK 4 as well as use of siRNAs specifically targeted to MEKK 1/MEKK 4 blocks the activation of JNK in response to WNT3a stimulation. (A,B) F9 cells expressing rFZ1 were transfected (1 µg/well in a 12-well plate) with an expression vector harboring either DN-MEKK 1 or DN-MEKK 4 (A) or treated with siRNAs specific to either MEKK 1 or MEKK 4 (B) for 24 hours, and JNK activity was determined. (C) Confluent F9 cells expressing rFZ1 were treated with or without inhibitors of JNK (SP600125, 0.4 µM) or p38 (SB203580, 6 µM) for 1 hour before stimulation with WNT3a, followed by determination of JNK activity. (D) F9 cells stably expressing rFZ1 were transfected with DVL3-GFP2 either alone or together with DN-MEKK 1 for 24 hours, followed by determination of JNK activity. (E) F9 cells stably expressing rFZ1 were transfected with constitutively active (CA)-Cdc42 either alone or together with DN-MEKK 1 for 24 hours, followed by determination of JNK activity. The upper panel displays mean values±s.e.m. obtained from three independent experiments; the lower panel displays the corresponding representative blots. The DVL3–DN-MEKK 1 and CA-Cdc42–DN-MEKK 1 epistasis experiments are representative of two independent experiments whose results were in strong agreement. *P<0.05; **P<0.01; versus –WNT3a control; #, P<0.05; ##, P<0.01 versus +WNT3a control.

View larger version (25K): [in this window] [in a new window]   Fig. 7. A JNK inhibitor blocks WNT3a-stimulated LEF/TCF-sensitive transcription. (A,B) F9 cells stably expressing rFZ1 and pTOPFLASH luciferase reporter were transfected with dominant-negative (DN) versions of either RhoA, Rac1 or Cdc42 (A) and either DN-MEKK 1 or DN-MEKK 4 (B) for 24 hours, followed by overnight serum starvation. The cells then were treated with or without WNT3a for 7 hours and luciferase gene reporter assays were performed. (C) Confluent F9 cells stably expressing rFZ1 and pTOPFLASH luciferase reporter were serum-starved overnight and treated with JNK (SP600125, 0.4 µM) or p38 (SB203580, 6 µM) inhibitors for 1 hour, followed by treatment with or without WNT3a for 7 hours. Lysates were collected and a luciferase assay was performed, as described in the Materials and Methods. The data represent mean values±s.e.m. from a single experiment performed in triplicate and are representative of three separate experiments whose results were in strong agreement. **P<0.01; versus –WNT3a control; #, P<0.05; ##, P<0.01 versus +WNT3a control.

View larger version (61K): [in this window] [in a new window]   Fig. 8. Activation of the JNK pathway by WNT3a is not obligate for formation of primitive endoderm. F9 cells stably expressing rFZ1 were treated with a JNK inhibitor (0.4 µM SP600125) or transiently transfected with DN-RhoA or DN-MEKK 1 before stimulation with WNT3a for 4 days. Subsequently, the cells were prepared for immunocytochemistry and stained with a monoclonal antibody against the cytokeratin endo A (TROMA-1), a marker protein for primitive endoderm. Alexa-Fluor-488-conjugated secondary antibodies were employed together with indirect epifluorescence to detect the immune complexes. Typical phase-contrast images (PC) and the indirect immunofluorescence images (IIF) are shown from a single experiment, representative of three independent experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Schematic representation of mammalian WNT3a-sensitive signaling pathways. The scheme represents a work-in-progress of our understanding of WNT3a-stimulated pathways in mammalian cells. JNK activation, crosstalk between the WNT3a–β-catenin canonical and WNT3a-JNK pathways in mouse F9 embryonal stem cells are highlighted. Constitutively active mutants of signaling molecules whose expression provokes activation of JNK in the absence of WNT3a are shown in green. Dominant-negative versions of signaling molecules, siRNAs and inhibitors that can effectively attenuate or block WNT3a-induced JNK activation or WNT3a-stimulated LEF/TCF-sensitive transcription are shown in red. The vectoral flow of information from WNT3a to JNK activation and from WNT3a to LEF/TCF transcription is displayed with arrows.

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