title: Recombinant WNTs differentially activate β-catenin-dependent and -independent signalling in mouse microglia-like cells.
abstract: OBJECTIVE
  The objective of this study was to compare the efficacy of different recombinant, commercially available Wingless/Int-1 (WNTs) with regard to WNT/β-catenin signalling, dishevelled (DVL) and G protein activation and the induction of cell proliferation in a microglia-like cell line called N13.
  
  
  METHODS
  For detection of activated signalling molecules, cell lysates are analysed by immunoblotting. Furthermore, we used a [γ(35)S] GTP binding assay to monitor the exchange of GDP for GTP in heterotrimeric G proteins in N13 membrane preparations. Cell proliferation was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay measuring mitochondrial function, which is proportional to the amount of viable cells.
  
  
  RESULTS
  Of the WNTs tested (WNT-3A, -4, -5A, -5B, -7A,-9B), only WNT-3A activated WNT/β-catenin signalling in N13 cells. All WNTs induced the formation of phosphorylated and shifted DVL (PS-DVL) and the activation of heterotrimeric G proteins with variable efficacies. WNT-5A and WNT-9B, which had the highest efficacy in the G protein assay, also induced N13 cell proliferation.
  
  
  CONCLUSIONS
  WNTs show significant differences in their efficacy to activate β-catenin-dependent and -independent signalling. The WNTs tested are present during maturation of the central nervous system and/or in the adult brain and are thus potential regulators of microglia-mediated neuroinflammation.
authors.affiliation: Section Receptor Biology & Signalling, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: M B C
authors.initials: MB
authors.lastname: Kilander
authors.forename: C
authors.initials: C
authors.lastname: Halleskog
authors.forename: G
authors.initials: G
authors.lastname: Schulte
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D000072261:Dishevelled Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000378:metabolism; D051379:Mice; D017628:Microglia / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D013462:Sulfur Radioisotopes; D013997:Time Factors; D051153:Wnt Proteins / Q000378:metabolism*; D060449:Wnt Signaling Pathway; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C495267:CTNNB1 protein, mouse; D000072261:Dishevelled Proteins; D060488:Low Density Lipoprotein Receptor-Related Protein-6; C555476:Lrp6 protein, mouse; D010750:Phosphoproteins; D013462:Sulfur Radioisotopes; D051153:Wnt Proteins; D051176:beta Catenin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D020962:Heterotrimeric GTP-Binding Proteins