RhoA Activation Destabilizes ?-catenin During Canonical Wnt Signaling Pathway

Background:Signaling by the Wnt family of secreted glycolipoproteins is one of the fundamental mechanisms that direct cell proliferation,polarity,death and cell fate determination during embryonic development and adult tissue homeostasis.Due to its critical role in the process of dorsoventral patterning,formation of tissues and organs during embryonic development,further study of the molecular regulation mechanism of Wnt signaling pathway has important theoretical and practical significance to fully understand the development process of tissues and organs and the pathogenesis of related diseases.The canonical Wnt signaling pathway mainly through nuclear translocation of?-catenin to achieve activation on the transcriptional activity of target genes,so the canonical Wnt signaling is also called Wnt/?-catenin pathway.In the absence of Wnt ligands,cytoplasmic p-catenin is recruited by a destruction complex assembled by the scaffolding protein Axin,adenomatous polyposis coli(APC)gene product,casein kinase 1(CK1)and glycogen synthase kinase 3(GSK3).CK1? phosphorylates P-catenin at Ser45,which primes the subsequent phosphorylation of ?-catenin at Thr41/Ser37/Ser33 by GSK3?.After these processes,phosphorylated ?-catenin could be recognized and bound by an E3 Ubiquitin ligase subunit,?-transducin repeats containing proteins(?-Trcp),which targeting ?-catenin for ubquitination and subsequent proteasomal degradation,so ?-catenin is unable to translocate into the nucleus.Upon Wnt stimulation,binding of Wnt to Frizzled receptors and the low-density lipoprotein receptor-related protein 5 or 6(LRP5/6)activates the cytoplasmic signaling protein Dishevelled(Dvl)and recruits Axin to the intracellular terminal of LRP5/6,which destroyed the destruction complex and stabilize cytosolic?-catenin.Upon its entering the nucleus,?-catenin in turn activates the transcription of downstream target genes via lymphoid enhancer-binding factor 1(Lefl)and T cell factors(Tcfl,3,4).Interestingly,the direct up-regulation of the secreted target molecule Dickkopf 1(Dkkl),which antagonizes the pathway by interfering with LRP5/6-Wnt interactions,provides a negative feedback mechanism that limits the range of Wnt signaling.So it is obvious that cytoplasmic ?-catenin accumulation is one of the key molecular events in the canonical Wnt signal transduction process.However,there are many factors influencing the stabilization of cytoplasmic ?-catenin and relevant researches are less and inconclusive.The Rho family of small GTPases including RhoA,Racl,and Cdc42 are molecular switches that represent multiple roles in regulation of cell proliferation,apoptosis,differentiation,cytoskeleton remodeling and cell migration by virtue of cycling between active GTP-bound and inactive GDP-bound forms.It has been reported that Racl activation is a critical component of canonical Wnt signaling.Moreover,we present evidence that Racl interacts genetically with ?-catenin and Dkkl in controlling limb outgrowth in mouse embryos.Despite the importance of Racl,whether other Rho small GTPases also regulate canonical Wnt signaling remains unknown.Objective:1.To explore whether RhoA can regulate Wnt/?-catenin signaling pathway by using in vitro experimental methods.2.To determine the exact mechanisms involving RhoA regulating Wnt/b-catenin signaling pathway.3.To demonstrate the role and function of RhoA in the process of mouse limb outgrowth.Methods:1.In vitro,to investigate the potential role of RhoA during Wnt signaling,we used an established binding assay to determine whether the GTP-bound(active)form of RhoA was increased upon Wnt signaling in C3H10T1/2 cells,a cell line of mouse embryonic fibroblasts.After that,to investigate the molecular mechanism underlying Rho A activation by Wnt3a.2.To examine whether RhoA/ROCK participates in canonical Wnt signaling,C3H10T1/2 cells were transfected with constitutively active form of RhoA or ROCK2(caRhoA OR caROCK2)or RhoA(ROCK)siRNA,and assayed for their responses to Wnt3a in up-regulating expression of a Lef1-luciferase reporter.3.To explore the mechanism underlying the role of RhoA/ROCK in canonical Wnt signaling,western analyses were performed for ?-catenin in cytosolic versus nuclear fractions of C3H10T1/2 cells expressing either caRhoA(caROCK)or RhoA siRNA(or treated with ROCK inhibitor,Y27632).4.To further study the precise molecular mechanisms underlying RhoA/ROCK regulating Wnt/?-catenin signaling pathway with the methods of western blot and luciferse assay.5.To determine the physiological relevance of RhoA in canonical Wnt signaling,we genetically inactivated RhoA from the apical ectodermal ridge(AER)of the mouse embryonic limb bud,where Wnt signaling is critical for limb outgrowth through?-catenin.Specifically,we utilized a mouse strain(CAT-dnRhoA+/-)that carries the CMV gene promoter fused to the CAT gene cassette flanked by two loxP sites and the dnRhoA gene cassette.We generated embryos of Msx2-Cre;CAT-dnRhoA+/+;R26-Dkk1+/+ by crossing the CAT-dnRhoA+/+;Dkkl+/-with Msx2-Cre;R26-Dkkl+/-,taking advantage of the fact that Msx2-Cre is expressed in the limb bud ectoderm that gave rise to the AER.Then observed the limb structures phenotypes of embryos at E16.5.To confirm the specificity of dnRhoA,we generated RhoA floxed mice(RhoAf/f)with CRISPR/Cas9-medited'genome-editing technique.We then crossed the RhoAf/f;Dkk1+/+ with Msx2-Cre;RhoAf/+;R26-Dkk1+/-to generate embryos of Msx2-Cre;RhoAf/f;R26-Dkk1+/+,resulting in conditional knockout of RhoA and overexpression of Dkkl in the limb bud ectoderm.6.To gain further insights about the effect of RhoA inactivation in the limb bud ectoderm,we performed histological assays and in situ hybridization in E10.5 mouse embryos.Results:1.1)rWnt3a time-and dose-dependently activated RhoA in C3H10T1/2 cell line,and this activation effect could be negated by rDkkl;2)the activation of RhoA by Wnt3a in other cell lines including HEK293T cells,L929 cells,and NIH3T3 cells were confirmed;3)Wnt3a activates RhoA through a signaling cascade involving LRP5/6,G?q/11??,Dvl,and Daaml.2.In C3H10T1/2 cell line,knockdown of RhoA or ROCK or stimulated with ROCK inhibitor,Y27632,could synergisticly up-regulate the activity of the Lef1-luciferase reporter gene induced by Wnt3a(p<0.01);overexpression of caRhoA or caROCK2 down-regulated the activity of the Lef1-luciferase reporter gene induced by Wnt3a(p<0.01).3.RhoA siRNA or treatment with Y27632 increased P-catenin in both the cytosolic and nuclear fractions with or without Wnt3a treatment;overexpression of caRhoA or caROCK2 decreased ?-catenin in both the cytosolic and nuclear fractions with or without Wnt3 a treatment.4.Western blot results showed that:1)Wnt3a activated JAK1/2 through RhoA/ROCK;2)JAKs directly phosphorylated GSK3? at Tyr216 and subsequently activated GSK3?;3)activated GSK3? phosphorylated ?-catenin and induecd p-catenin degredation.5.1)Msx2-Cre;R26-Dkkl+/+ embryos lacked all hindlimb structures and exhibited truncations at various levels in the forelimb;2)Msx2-Cre;CAT-dnRhoA+/-;R26-Dkkl+/+ embryos exhibited normal forelimb structures and varying lengths of hindlimbs with femora;3)Msx2-Cre;R26-Dkk1+/+;RhoAf/f embryos exhibited similar phenotypes of Msx2-Cre;CAT-dnRhoA+/-;R26-Dkkl+/+;4)Msx2-Cre;R26-Dkk1+/-;caRhoA+/-)developed no hindlimb,and nearly all forelimbs(10/12 from 6 embryos)lacked structures distal to the scapula;these phenotypes are remarkably similar to those of the Msx2-Cre;R26-Dkkl+/+ or Msx2-Cre;?-cateninn/f embryos;5)Msx2-Cre;CAT-dnRhoA+/-;?-cateninn/f embryos at E16.5 still lacked all hindlimb structures and exhibited truncations at various levels in the forelimbs,and these defects were identical to those in embryos of Msx2-Cre;?-cateninn/f.6.1)Both fore-and hindlimb buds were obviously smaller in the Msx2-Cre;R26-Dkkl+/+ embryos than those of the control littermates;Correlated to the size of limb buds,Msx2-Cre;R26-Dkkl+/+ embryos exhibited the increased cell apoptosis and decreased cell proliferation in AER;2)expression of dnRhoA in the Msx2-Cre;R26-Dkkl+/+ embryos largely restored the cell apoptosis and proliferation,whereas expression of caRhoA in the AER of Msx2-Cre;R26-Dkkl+/-embryos resulted in similar alterations of cell apoptosis and proliferation in AER of Msx2-Cre;R26-Dkkl+/+ embryos;3)the striking morphological rescue by dnRhoA in Msx2-Cre;R26-Dkkl+/+ embryos prompted us to examine the potential regulation of RhoA at the molecular level;4)Consistent with the important roles of fibroblast growth factor 8(FGF8)in the AER and bone morphogenetic protein 4(BMP4)in the distal ventral ectoderm,the Msx2-Cre;R26-Dkkl+/+ embryos expressed only residual BMP4 and no FGF8 expression in the forelimb of E10.5(34-35 somites)embryos;5)in the AER part of Msx2-Cre;caRhoA+/-embryos,the expression level of phosphor-JAK2,phosphor-GSK3?(Y216),phosphor-?-catenin(S33/37/T41)increased,while the protein level of total-?-catenin decreased;6)in the AER part of Msx2-Cre;CAT-dnRhoA+/-embryos,the expression level of phosphor-JAK2,phosphor-GSK3p(Y216),phosphor-?-catenin(S33/37/T41)decreased,while the protein level of total-?-catenin increased and nucleus translocation increased significantly.Conclusions:1.Wnt activates RhoA through FZD/G?q/Dvl/Daaml signaling cascades;2.RhoA phosphorylated and destabilizes ?-catenin through ROCK/JAK/GSK3?(Tyr216)signaling cascades,which downregulated Wnt/?-catenin signaling pathway;3.RhoA interacts genetically with Dkk1 in regulating canonical Wnt signaling and limb outgrowth...