IQGAP1 Protein Regulates Nuclear Localization of β-Catenin via Importin-β5 Protein in Wnt Signaling

In the canonical Wnt signaling pathway, the translocation of β-catenin is important for the activation of target genes in the nucleus. However, the molecular mechanisms underlying its nuclear localization remain unclear. In the present study, we found IQGAP1 to be a regulator of β-catenin function via importin-β5. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of β-catenin and expression of Wnt target genes during early embryogenesis. Depletion of endogenous importin-β5 associated with IQGAP1 also reduced expression of Wnt target genes and the nuclear localization of IQGAP1 and β-catenin. Moreover, a small GTPase, Ran1, contributes to the nuclear translocation of β-catenin and the activation of Wnt target genes. These results suggest that IQGAP1 functions as a regulator of translocation of β-catenin in the canonical Wnt signaling pathway.     

Nuclear Dishevelled targets gene regulatory regions and promotes tumor growth

Dishevelled (DVL) critically regulates Wnt signaling and contributes to a wide spectrum of diseases and is important in normal and pathophysiological settings. However, how it mediates diverse cellular functions remains poorly understood. Recent discoveries have revealed that constitutive Wnt pathway activation contributes to breast cancer malignancy, but the mechanisms by which this occurs are unknown and very few studies have examined the nuclear role of DVL. Here, we have performed DVL3 ChIP‐seq analyses and identify novel target genes bound by DVL3. We show that DVL3 depletion alters KMT2D binding to novel targets and changes their epigenetic marks and mRNA levels. We further demonstrate that DVL3 inhibition leads to decreased tumor growth in two different breast cancer models in vivo. Our data uncover new DVL3 functions through its regulation of multiple genes involved in developmental biology, antigen presentation, metabolism, chromatin remodeling, and tumorigenesis. Overall, our study provides unique insight into the function of nuclear DVL, which helps to define its role in mediating aberrant Wnt signaling.     

DVL mutations identified from human neural tube defects and Dandy-Walker malformation obstruct the Wnt signaling pathway

Wnt signaling pathways,including the canonical Wnt/β-catenin pathway,planar cell polarity pathway,and Wnt/Ca~(2+) signaling pathway,play important roles in neural development during embryonic stages.The DVL genes encode the hub proteins for Wnt signaling pathways.The mutations in DVL2 and DVL3 were identified from patients with neural tube defects(NTDs),but their functions in the pathogenesis of human neural diseases remain elusive.Here,we sequenced the coding regions of three DVL genes in 176 stillborn or miscarried fetuses with NTDs or Dandy-Walker malformation(DWM) and 480 adult controls from a Han Chinese population.Four rare mutations were identified:DVL1 p.R558 H,DVL1 p.R606 C,DVL2 p.R633 W,and DVL3 p.R222 Q.To assess the effect of these mutations on NTDs and DWM,various functional analyses such as luciferase reporter assay,stress fiber formation,and in vivo teratogenic assay were performed.The results showed that the DVL2 p.R633 W mutation destabilized DVL2 protein and upregulated activities for all three Wnt signalings(Wnt/β-catenin signaling,Wnt/planar cell polarity signaling,and Wnt/Ca~(2+) signaling) in mammalian cells.In contrast,DVL1 mutants(DVL1 p.R558 H and DVL1 p.R606 C) decreased canonical Wnt/β-catenin signaling but increased the activity of Wnt/Ca~(2+)signaling,and DVL3 p.R222 Q only decreased the activity of Wnt/Ca~(2+) signaling.We also found that only the DVL2 p.R633 W mutant displayed more severe teratogenicity in zebrafish embryos than wild-type DVL2.Our study demonstrates that these four rare DVL mutations,especially DVL2 p.R633 W,may contribute to human neural diseases such as NTDs and DWM by obstructing Wnt signaling pathways.     

Dapper1 is a nucleocytoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus

Wnt signaling, via the activation of the canonical beta-catenin and lymphoid enhancer factor (LEF)/T-cell factor pathway, plays an important role in embryogenesis and cancer development by regulating the expression of genes involved in cell proliferation, differentiation, and survival. Dapper (Dpr), as a Dishevelled interactor, has been suggested to modulate Wnt signaling by promoting Dishevelled degradation. Here, we provide evidence that Dpr1 shuttles between the cytoplasm and the nucleus. Although overexpressed Dpr1 was mainly found in the cytoplasm, endogenous Dpr1 was localized over the cell, and Wnt1 induced its nuclear export. Treatment with leptomycin B induced nuclear accumulation of both endogenous and overexpressed Dpr1. We further identified the nuclear localization signal and the nuclear export signal within Dpr1. Using reporter assay and in vivo zebrafish embryo assay, we demonstrated that the forced nuclearly localized Dpr1 possessed the ability to antagonize Wnt signaling. Dpr1 interacted with beta-catenin and LEF1 and disrupted their complex formation. Furthermore, Dpr1 could associate with histone deacetylase 1 (HDAC1) and enhance the LEF1-HDAC1 interaction. Together, our findings suggest that Dpr1 negatively modulates the basal activity of Wnt/beta-catenin signaling in the nucleus by keeping LEF1 in the repressive state. Thus, Dpr1 controls Wnt/beta-catenin signaling in both the cytoplasm and the nucleus.     

Rho guanine nucleotide exchange factor xNET1 implicated in gastrulation movements during Xenopus development

During Xenopus development, embryonic cells dramatically change their shape and position. Rho family small GTPases, such as RhoA, Rac, and Cdc42, play important roles in this process. These GTPases are generally activated by guanine nucleotide exchange factors (GEFs); however, the roles of RhoGEFs in Xenopus development have not yet been elucidated. We therefore searched for RhoGEF genes in our Xenopus EST database, and we identified several genes expressed during embryogenesis. Among them, we focused on one gene, designated xNET1. It is similar to mammalian NET1, a RhoA-specific GEF. An in vitro binding assay revealed that xNET1 bound to RhoA, but not to Rac or Cdc42. In addition, transient expression of xNET1 activated endogenous RhoA. These results indicated that xNET1 is a GEF for RhoA. Epitope-tagged xNET1 was localized mainly to the nucleus, and the localization was regulated by nuclear localization signals in the N-terminal region of xNET1. Overexpression of either wild-type or a mutant form of xNET1 severely inhibited gastrulation movements. We demonstrated that xNET1 was co-immunoprecipitated with the Dishevelled protein, which is an essential signaling component in the non-canonical Wnt pathway. This pathway has been shown to activate RhoA and regulate gastrulation movements. We propose that xNET1 or a similar RhoGEF may mediate Dishevelled signaling to RhoA in the Wnt pathway.     

AMPK Activators Suppress Cervical Cancer Cell Growth through Inhibition of DVL3 Mediated Wnt/β-Catenin Signaling Activity

Recent evidence has suggested that AMPK activators may be applied as therapeutic drugs in suppressing cancer cell growth. However, the molecular mechanism of their suppressive function in cancer cells is still unclear. Here we show that AMPK activators impair cervical cancer cell growth through the reduction of DVL3, a positive regulator in Wnt/β-catenin signaling and an oncogenic player in cervical cancer tumorigenesis. By western blot and immunohistochemical analyses, we demonstrated that DVL3 was frequently upregulated and significantly associated with elevated β-catenin (P = 0.009) and CyclinD1 (P = 0.009) expressions in cervical cancer. Enforced expression of DVL3 elevated β-catenin and augmented cervical cancer cell growth, verifying that DVL3-mediated Wnt/β-catenin activation is involved in cervical cancer oncogenesis. On the other aspect, we noted that the cervical cancer cell growth was remarkably suppressed by AMPK activators and such cell growth inhibition was in concomitant with the reduction of DVL3 protein level in dose- and time-dependent manners. Besides, impaired mTOR signaling activity also reduced DVL3 expression. In contrast, co-treatment with Compound C (AMPK inhibitor) could significantly abrogate metformin induced DVL3 reduction. In addition, co-treatment with AM114 or MG132 (proteosomal inhibitors) could partially restore DVL3 expression under the treatment of metformin. Further in vivo ubiquitination assay revealed that metformin could reduce DVL3 by ubiquitin/proteasomal degradation. To our knowledge, this is the first report showing the probable molecular mechanisms of that the AMPK activators suppress cervical cancer cell growth by impairing DVL3 protein synthesis via AMPK/mTOR signaling and/or partially promoting the proteasomal degradation of DVL3.     

The β-catenin HMP-2 functions downstream of Src in parallel with the Wnt pathway in early embryogenesis of C. elegans

The Wnt and Src pathways are widely used signal transduction pathways in development. β-catenin is utilized in both pathways, as a signal transducer and a component of the cadherin cell adhesion complex, respectively. A C. elegans β-catenin HMP-2 is involved in cell adhesion, but its signaling role has been unknown. Here, we report that in early embryogenesis HMP-2 acts as a signaling molecule in the Src signal. During early embryogenesis in C. elegans, the Wnt and Src pathways are redundantly involved in endoderm induction at the four-cell stage and spindle orientation in an ABar blastomere. RNAi experiments demonstrated that HMP-2 functions in the Src pathway, but in parallel with the Wnt pathway in these processes. HMP-2 localized at the cell boundaries and nuclei, and its localization at cell boundaries was negatively regulated by SRC-1. In addition, HMP-2 was Tyr-phosphorylated in a SRC-1-dependent manner in vivo. Taken together, we propose that HMP-2 functions downstream of the Src signaling pathway and contribute to endoderm induction and ABar spindle orientation, in parallel with the Wnt signaling pathway.     

Wnt signaling in heart valve development and osteogenic gene induction

Wnt signaling mediated by β-catenin has been implicated in early endocardial cushion development, but its roles in later stages of heart valve maturation and homeostasis have not been identified. Multiple Wnt ligands and pathway genes are differentially expressed during heart valve development. At E12.5, Wnt2 is expressed in cushion mesenchyme, whereas Wnt4 and Wnt9b are predominant in overlying endothelial cells. At E17.5, both Wnt3a and Wnt7b are expressed in the remodeling atrioventricular (AV) and semilunar valves. In addition, the TOPGAL Wnt reporter transgene is active throughout the developing AV and semilunar valves at E16.5, with more localized expression in the stratified valve leaflets after birth. In chicken embryo aortic valves, genes characteristic of osteogenic cell lineages including periostin, osteonectin, and Id2 are expressed specifically in the collagen-rich fibrosa layer at E14. Treatment of E14 aortic valve interstitial cells (VICs) in culture with osteogenic media results in increased expression of multiple genes associated with bone formation. Treatment of VIC with Wnt3a leads to nuclear localization of β-catenin and induction of periostin and matrix gla protein but does not induce genes associated with later stages of osteogenesis. Together, these studies provide evidence for Wnt signaling as a regulator of endocardial cushion maturation as well as valve leaflet stratification, homeostasis, and pathogenesis.     

BATF2对Wnt信号通路影响的研究

摘要wnt信号通路在各种生物体内高度进化保守,与癌症的发生发展密切相关。BATF2是一个新近发现的基因,研究表明其具有抑癌基因的作用。目前,BATF2与wnt信号通路的关系尚不清楚,该文用荧光素酶报告基因检测、Real－timePCR和Western blot发现BATF2能影响wnt信号通路。过表达BArF2可明显下调TCF4／p－catenin的转录活性和Wnt信号通路下游基因的表达,可以下调细胞核中的β-caenin。推测BATF2可能通过下调细胞核中的p－catenin来实现对wnt信号通路的下调。上述结果为抑制Wnt信号通路用于肿瘤治疗提供了一定的依据。