Wnts Specify LRP6 mAb Activity YW211.31.57 or YW210.09 antibody, respectively, to increase potentiation effects. (B) qPCR expression analysis of AXIN2 mRNA in four cell lines treated with 25 mg/ml YW211.31.57 antibody or Fzd8CRD-Fc protein, with and without (NA) 0.2 mg/ml Wnt3a. (C–D) Expression of Wnt-induced genes in NCI-H23 (C) and M14 (D) cells is potentiated by YW211.31.57 and antagonized by YW210.09 antibody (30 mg/ml). CD4-Fc protein (30 mg/ml) serves as a negative controls (C). For M14 cells (D), AXIN2 and SP5 expression is potentiated more potently by Wnt3a protein or YW211.31.57 antibody than is APCDD1 and ZNRF3 expression. (E–F) In Hs578T cells stably integrated with Wnt luciferase reporter, YW211.31.57 antibody shows concentration- dependent inhibition of Wnt3a-stimulated signaling (E) and potentiation of autocrine Wnt signaling (F). Fzd8CRD-Fc protein inhibits and YW210.09 antibody potentiates signaling with or without 0.1 mg/ml Wnt3a stimulation. RNAi experiments indicate that at least 41% of Wnt3a-induced signaling in Hs578T cells is dependent on LPR5 expression (data not shown), and this signaling is predicted to be inhibited by Fzd8CRD-Fc protein but not YW211.31.57 antibody. In this experiment, SV40-driven luciferase was not transfected for normalization and, instead, antibody and protein treatments were independently confirmed to have no significant effect on viability of this cell line. (G–H) EKVX cells transfected with Wnt luciferase reporter also display potentiation of autocrine Wnt signaling (NA) and antagonism of Wnt3a-induced signaling by YW211.31.57 antibody. Antibody-mediated potentiation of autocrine Wnt signaling is inhibited by 5 mg/ml Fzd8CRD-Fc protein (H). doi:10.1371/journal.pone.0012682.g002 correlates with the Wnt isoform, and not the FZD isoform. Fzd8CRD-Fc protein has no effect on signaling induced by any of the four Wnt-FZD chimeras, consistent with the chimeras functioning independently of the FZD-binding site of Wnts. Expression of chimeras that fuse Wnt1 or Wnt3a to LRP6 induce Wnt signaling much more potently than LRP6 overexpression (data not shown). YW211.31.57 and YW210.09 antibodies are not able to inhibit this induction, consistent with the hypothesis that the Figure 3. Wnt isoforms specify inhibitory or potentiating activities of LRP6 antibodies. (A) YW211.31.57 antibody antagonizes Wnt3a- induced signaling and potentiates Wnt1-induced signaling, whereas YW210.09 antibody exhibits reciprocal activities. HEK293 cells stably integrated with Wnt and SV40 luciferase reporters were transfected with Wnt3a, Wnt1, or empty vector expression constructs and treated with monoclonal antibodies (mAb). (B) Treatment of cultured cells from mouse MMTV-Wnt1 allograft tumors with 10 mg/ml YW210.09 antibody inhibits expression of Wnt target genes Axin2 and Mmp7, as does 10 mg/ml Fzd8CRD-Fc protein. (C–E) Summary tables of the effects of 10 mg/ml LRP6 antibodies, antibody combinations (E), and Fzd8CRD-Fc protein on signaling induced by transfection of expression constructs for Wnt isoforms (C, E) or chimeric proteins fusing Wnt isoforms to FZD isoforms or LRP6 (D) in HEK293 (C–E) or Hs578T (C) cell lines stably integrated with Wnt luciferase reporters. Expression of Wnt luciferase reporter is normalized to cell number and additionally normalized to levels in cells transfected with the same expression construct but not treated with proteins. The fold change in Wnt signaling is highlighted in red or green to indicate inhibition or potentiation, respectively, with p- value ,0.01 by ANOVA. Gray fields indicate conditions that were not tested because Wnt induction was not significant. doi:10.1371/journal.pone.0012682.g003 PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e12682