Aim: 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.
Conclusion: 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.
© 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.