Wnt4 induces nephronic tubules in metanephric mesenchyme by a non-canonical mechanism

Wnt4 and beta-catenin are both required for nephrogenesis, but studies using TCF-reporter mice suggest that canonical Wnt signaling is not activated in metanephric mesenchyme (MM) during its conversion to the epithelia of the nephron. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of beta-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of beta-catenin/FCF-dependent transcription, we developed synthetic cell-permeable analogs of beta-catenin's helix C, which is required for transcriptional activation, in efforts to specifically inhibit canonical Wnt signaling. One inhibitor blocked TCF-dependent transcription and induced degradation of beta-catenin but did not affect tubule formation and stimulated the expression of Lim 1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca2+-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca2+ and caused phosphorylation of CaMKII. Moreover, lonomycin, a Ca2+-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal-epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney. Published by Elsevier Inc.