BACKGROUND: RNF43 and its homolog ZNRF3 are transmembrane E3 ubiquitin ligases frequently mutated in many human cancer types. Their main role relays on the inhibition of canonical Wnt signaling by the negative regulation of frizzled receptors and LRP5/6 co-receptors levels at the plasma membrane. Intracellular RING domains of RNF43/ZNRF3 mediate the key enzymatic activity of these proteins, but the function of the extracellular Protease Associated (PA) fold in the inhibition of Wnt/β-catenin pathway is controversial up-to date, apart from the interaction with secreted antagonists R-spondin family proteins shown by the crystallographic studies. METHODS: In our research we utilised cell-based approaches to study the role of RNF43 lacking PA domain in the canonical Wnt signalling pathway transduction. We developed controlled overexpression (TetON) and CRISPR/Cas9 mediated knock-out models in human cells. RESULTS: RNF43ΔPA mutant activity impedes canonical Wnt pathway, as manifested by the reduced phosphorylation of LRP6, DVL2 and DVL3 and by the decreased β-catenin-dependent gene expression. Finally, rescue experiments in the CRISPR/Cas9 derived RNF43/ZNRF3 double knock-out cell lines showed that RNFΔPA overexpression is enough to inhibit activation of LRP6 and β-catenin activity as shown by the Western blot and Top flash dual luciferase assays. Moreover, RNF43 variant without PA domain was not sensitive to the R-spondin1 treatment. CONCLUSION: Taken together, our results help to understand better the mode of RNF43 tumor suppressor action and solve some discrepancies present in the field. Video Abstract.
- MeSH
- HEK293 buňky MeSH
- LDL receptor related protein 6 metabolismus MeSH
- lidé MeSH
- mutace MeSH
- proteinové domény MeSH
- signální dráha Wnt * MeSH
- thrombospondiny metabolismus MeSH
- ubikvitinligasy genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- audiovizuální média MeSH
- časopisecké články MeSH
- práce podpořená grantem MeSH
Actin-associated proteins regulate multiple cellular processes, including proliferation and differentiation, but the molecular mechanisms underlying these processes are unclear. Here, we report that the actin-binding protein filamin A (FlnA) physically interacts with the actin-nucleating protein formin 2 (Fmn2). Loss of FlnA and Fmn2 impairs proliferation, thereby generating multiple embryonic phenotypes, including microcephaly. FlnA interacts with the Wnt co-receptor Lrp6. Loss of FlnA and Fmn2 impairs Lrp6 endocytosis, downstream Gsk3β activity, and β-catenin accumulation in the nucleus. The proliferative defect in Flna and Fmn2 null neural progenitors is rescued by inhibiting Gsk3β activity. Our findings thus reveal a novel mechanism whereby actin-associated proteins regulate proliferation by mediating the endocytosis and transportation of components in the canonical Wnt pathway. Moreover, the Fmn2-dependent signaling in this pathway parallels that seen in the non-canonical Wnt-dependent regulation of planar cell polarity through the Formin homology protein Daam. These studies provide evidence for integration of actin-associated processes in directing neuroepithelial proliferation.
- MeSH
- beta-katenin metabolismus MeSH
- buněčná diferenciace MeSH
- buněčná membrána fyziologie MeSH
- buněčné linie MeSH
- endocytóza fyziologie MeSH
- filaminy genetika metabolismus MeSH
- GSK3B antagonisté a inhibitory metabolismus MeSH
- HEK293 buňky MeSH
- jaderné proteiny genetika metabolismus MeSH
- LDL receptor related protein 6 metabolismus MeSH
- lidé MeSH
- mikrocefalie genetika MeSH
- mikrofilamentové proteiny genetika metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- proliferace buněk genetika fyziologie MeSH
- proteiny Wnt metabolismus MeSH
- signální dráha Wnt fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Aberrant fibroblast growth factor (FGF) signaling disturbs chondrocyte differentiation in skeletal dysplasia, but the mechanisms underlying this process remain unclear. Recently, FGF was found to activate canonical WNT/β-catenin pathway in chondrocytes via Erk MAP kinase-mediated phosphorylation of WNT co-receptor Lrp6. Here, we explore the cellular consequences of such a signaling interaction. WNT enhanced the FGF-mediated suppression of chondrocyte differentiation in mouse limb bud micromass and limb organ cultures, leading to inhibition of cartilage nodule formation in micromass cultures, and suppression of growth in cultured limbs. Simultaneous activation of the FGF and WNT/β-catenin pathways resulted in loss of chondrocyte extracellular matrix, expression of genes typical for mineralized tissues and alteration of cellular shape. WNT enhanced the FGF-mediated downregulation of chondrocyte proteoglycan and collagen extracellular matrix via inhibition of matrix synthesis and induction of proteinases involved in matrix degradation. Expression of genes regulating RhoA GTPase pathway was induced by FGF in cooperation with WNT, and inhibition of the RhoA signaling rescued the FGF/WNT-mediated changes in chondrocyte cellular shape. Our results suggest that aberrant FGF signaling cooperates with WNT/β-catenin in suppression of chondrocyte differentiation.
- MeSH
- beta-katenin genetika metabolismus MeSH
- biologické modely MeSH
- buněčná diferenciace účinky léků genetika MeSH
- chondrocyty účinky léků metabolismus MeSH
- chrupavka cytologie účinky léků metabolismus MeSH
- fibroblastové růstové faktory farmakologie MeSH
- fibroblastový růstový faktor 2 farmakologie MeSH
- HEK293 buňky MeSH
- končetinové pupeny účinky léků embryologie metabolismus MeSH
- konfokální mikroskopie MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- LDL receptor related protein 6 genetika metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protein Wnt3A farmakologie MeSH
- proteiny Wnt genetika metabolismus farmakologie MeSH
- receptory fibroblastových růstových faktorů genetika metabolismus MeSH
- signální transdukce účinky léků genetika MeSH
- synergismus léků MeSH
- transkriptom účinky léků genetika MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.
- MeSH
- adaptorové proteiny signální transdukční genetika metabolismus MeSH
- arrestiny genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- embryo savčí cytologie MeSH
- fibroblasty cytologie metabolismus MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- fosforylace MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem genetika metabolismus MeSH
- HEK293 buňky MeSH
- konfokální mikroskopie MeSH
- kultivované buňky MeSH
- LDL receptor related protein 6 genetika metabolismus MeSH
- lidé MeSH
- myši knockoutované MeSH
- myši MeSH
- nádorové supresorové proteiny genetika metabolismus MeSH
- protein Wnt3A genetika metabolismus MeSH
- RNA interference MeSH
- vazba proteinů MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/β-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with β-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or β-catenin-induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of β-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling.
- MeSH
- antibiotika antitumorózní farmakologie terapeutické užití MeSH
- beta-katenin metabolismus MeSH
- dánio pruhované MeSH
- experimentální nádory MeSH
- HEK293 buňky MeSH
- kolorektální nádory farmakoterapie patologie MeSH
- LDL receptor related protein 6 metabolismus MeSH
- lidé MeSH
- monensin farmakologie terapeutické užití MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- signální dráha Wnt účinky léků MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- Xenopus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH