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First published online October 22, 2008
doi: 10.1242/10.1242/jcs.032854

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p38 mitogen-activated protein kinase regulates canonical Wnt–β-catenin signaling by inactivation of GSK3β

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

View larger version (22K): [in this window] [in a new window]   Fig. 1. Stimulation of Frizzled-1 by Wnt3a activates p38 MAPK in F9 and HEK293 cells. (A) F9 cells stably expressing Rfz1 were treated with Wnt3a (20 ng/ml) for indicated periods of time and the lysates were assayed for p38 activation as described in Materials and Methods. The upper panel represents mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with either anti-ATF2-P (p-ATF2), upper panel or with anti-p38 antibody (p38), lower panel. (B) F9 cells alone and F9 cells expressing either Rfz2 or Rfz1 were treated with Wnt3a for 15 minutes and the lysates were assayed for p38 activation. Representative blots of three independent experiments that proved highly reproducible are shown. (C) Confluent HEK293 cells were treated with Wnt3a for 15 minutes and the lysates were assayed for p38 activation. Representative blots of three independent experiments that proved highly reproducible are displayed. *P<0.05; **P<0.01 versus control (+Wnt3a, 0 minutes).

View larger version (31K): [in this window] [in a new window]   Fig. 2. Suppression of Gq and Gs, but not Go or G11, attenuates Wnt3a-induced p38 MAPK activation. F9 cells expressing Rfz1 were treated with siRNAs specific for Go, Gq, G11 or Gs for 48 hours before treatment with Wnt3a for 15 minutes. p38 MAPK activity was then assayed. Upper panel displays mean values ± s.e.m. obtained from densitometer scanning of images from three independent experiments; the lower panel displays representative blots probed with anti-ATF2-P (p-ATF2), anti-p38 (p38), anti-Go(Go), anti-Gq (Gq), anti-G11(G11) and anti-Gs (Gs). *P<0.05 versus –Wnt3a control; #P<0.05 versus +Wnt3a control. The extent of knockdown of the G-protein -subunit expression was routinely 75% or more.

View larger version (34K): [in this window] [in a new window]   Fig. 3. p38 inhibitors block canonical Wnt–β-catenin–Lef/Tcf pathway. Confluent F9 cells stably transfected with pRfz1 and pTOPFLASH (M50) luciferase reporter were treated with either vehicle (DMSO) or p38 MAPK selective inhibitors, SB203580 (6 µM) (A) or SB239063 (10 µM) (B) for 1 hour before addition of Wnt3a for indicated periods of time. After stimulation, the lysates were collected and cytosolic β-catenin levels were assayed. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin); immunoblots probed with anti-actin antibody (actin) were used as loading controls. Confluent F9 cells stably transfected with pRfz1 and pTOPFLASH (M50) luciferase reporter were treated with either vehicle (DMSO), SB203580 (6 µM) (C,D) or SB239063 (10 µM) (E) for 1 hour before addition of Wnt3a for indicated periods of time (C) or 7 hours (D,E). Activity of the luciferase reporter was monitored. The data represent mean values ± s.e.m. from a single experiment performed in triplicate and is representative of three separate experiments whose results were highly similar. (F) Confluent HEK293 cells were treated with Wnt3a for 4 hours and the lysates were assayed for cytosolic β-catenin levels. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin), immunoblots probed with anti-actin antibody (actin) were used as loading controls. (G) HEK293 cells were transfected with pTOPFLASH (M50, 10 ng/well) and phRL-CMV Renilla luciferase control vector (5 ng/well) for 48 hours followed by stimulation with Wnt3a for 7 hours. Lef/Tcf-sensitive transcription was determined. The data represents mean values ± s.e.m. from a single experiment performed in triplicate and is a representative of three separate experiments whose results were in high agreement. (H) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with vehicle (DMSO, control), p38 inhibitor (SB230580, 6 µM), JNK inhibitor (SP600125, 0.4 µM) or MEK inhibitor (PD98059, 20 µM) for 1 hour prior to stimulation with Wnt3a for 4 days. Subsequently, the cells were prepared for immunocytochemistry and stained with a monoclonal antibody to the cytokeratin endo A (TROMA1) marker protein for primitive endoderm. Alexa Fluor 488-conjugated secondary antibodies were used 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. *P<0.05 and **P<0.01 versus –Wnt3a control); #P<0.05 and ##P<0.01 versus +Wnt3a control.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Suppression of p38 by siRNA treatment or expression of dominant-negative mutant of p38 blocks canonical Wnt–β-catenin–Lef/Tcf pathway. (A) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with 100 nM siRNA specific to mouse p38 or JNK for 48 hours, and the lysates were assayed for cytosolic β-catenin levels. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin); immunoblots probed with anti-actin antibody (actin) were used as loading controls. (B) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with 100 nM siRNA specific to mouse p38 for 48 hours followed by stimulation with Wnt3a for 7 hours. Lef/Tcf-sensitive transcription was determined. The data represent mean values ± s.e.m. from a single experiment performed in triplicate and is representative of three separate experiments whose results were in high agreement. (C) Rescue experiment performed by transfection of hp38 into F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter in which p38 MAPK was knocked down by siRNA treatment. Lef/Tcf-sensitive transcription was determined. The data represents mean values ± s.e.m. from a single experiment performed in triplicate and is representative of three separate experiments whose results were in high agreement. (D<E) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were either transfected with empty vector (–) or with indicated amounts of Flag-tagged dominant-negative mutant (DN) of p38 MAPK [p38 (AGF)] for 24 hours and the lysates were assayed either for β-catenin stabilization after 4 hours of Wnt3a treatment (D) or luciferase reporter activity after 7 hours of Wnt3a treatment (E). Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin), anti-p38 antibody (p38) and the immunoblots with anti-actin antibody (actin) were used as loading controls. (F,G) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with either 100 nM siRNA specific to mouse p38 or dominant-negative mutant (DN) of p38 MAPK for 48 hours. The cells were then treated with SB203580 (6 µM) for 1 hour followed by stimulation with Wnt3a for 7 hours. Lef/Tcf-sensitive transcription (F) and p38 MAPK activation (G) was determined as described in Materials and Methods. *P<0.05 and **P<0.01 versus –Wnt3a control; #P<0.05 and ##P<0.01 versus +Wnt3a control.

View larger version (36K): [in this window] [in a new window]   Fig. 5. p38 MAPK operates downstream of Dishevelleds. (A) F9 cells expressing Rfz1 were treated with siRNAs designed to suppress the expression of Dvl3 for 48 hours and p38 MAPK activity was assayed after 15 minutes of stimulation with Wnt3a. The extent of suppression of Dvl3 is more than 85%. Upper panel represents mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-ATF2-P (p-ATF2), anti-p38 (p38) and anti-Dvl3 (Dvl3) antibodies. (B) F9 cells stably expressing Rfz1 were transfected with Dvl3-GFP2 for 24 hours and p38 MAPK activation was determined. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-ATF2-P (p-ATF2), anti-p38 (p38) and anti-HA (HA) antibodies. (C) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with 100 nM siRNA specific to mouse Dvl3 for 48 hours and the lysates were assayed for cytosolic β-catenin levels after 4 hours of Wnt3a treatment. Upper panel displays mean values ± s.e.m. obtained from three independent experiments; the lower panel displays representative blots probed with anti-β-catenin antibody (β-catenin), anti-Dvl3 (Dvl3); immunoblots probed with anti-actin antibody (actin) were used as loading controls. (D) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were transfected with either empty vector (–) or with HA-Dvl3-GFP2. After 4 hours of transfection, the transfection medium was replaced with serum medium (15%) containing either DMSO or SB203580 (6 µM) and cultures grown for an additional 20 hours. After 20 hours, the lysates were collected and luciferase assay was performed. Upper panel represents mean values ± s.e.m. from three independent experiments performed in triplicate; lower panel shows representative blots probed with anti-HA (HA) and anti-actin (actin) antibodies. *P<0.05 and **P<0.01 versus –Wnt3a control; #P<0.05 and ##P<0.01 versus +Wnt3a control.

View larger version (34K): [in this window] [in a new window]   Fig. 6. p38 suppresses GSK3β activity. (A) Confluent F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with either vehicle (DMSO) or SB203580 (6 µM) for 1 hour prior to the addition of Wnt3a for indicated periods of time. After stimulation, the lysates were collected and subjected to immunoblot analysis with anti-GSK3β Ser9-P antibody. Upper panel represents mean values ± s.e.m. from three independent experiments and the lower panel represents representative blots probed with either anti-GSK3β Ser9-P (p-GSK3β Ser 9) or with anti-GSK3β (GSK3β) antibodies. (B) F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were either transfected with empty vector (–) or with Flag-tagged dominant-negative (DN) mutant of p38 MAPK [p38 (AGF)] for 24 hours. After Wnt3a stimulation for 1 hour, the lysates were collected and subjected to immunoblot analysis with anti-GSK3β Ser9-P antibody as described. Upper panel represents mean values ± s.e.m. from three independent experiments and the lower panel represents representative blots probed with either anti-GSK3β Ser9-P (p-GSK3β Ser 9), anti-p38 (p38) or with anti-GSK3β (GSK3β) antibodies. (C) Confluent F9 cells stably transfected with pRfz1 and pTOPFLASH luciferase reporter were treated with either vehicle (DMSO) or SB203580 (6 µM) for 1 hour prior to the addition of Wnt3a for 10 minutes. GSK3β was immunoprecipitated from whole cell lysates and its activity was measured by an in vitro kinase assay. The data represent mean values ± s.e.m. from two independent experiments that are highly reproducible. *P<0.05 and **P<0.01 versus –Wnt3a control; #P<0.05 and ##P<0.01 versus +Wnt3a control.

View larger version (12K): [in this window] [in a new window]   Fig. 7. Schematic representation of role of p38 MAPK in canonical Wnt–β-catenin signaling pathway. Activation of Fz1 by Wnt3a leads to accumulation of β-catenin, Lef/Tcf-sensitive transcriptional response and primitive endoderm formation in mouse F9 cells through G-proteins and Dishevelleds. p38 MAPK plays a crucial role in canonical signaling by inactivating GSK3β and by operating downstream of Dishevelleds.

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