not shown). YW210.09 antibody treatment did not significantly affect expression of either SP5 or Axin2. Serum samples assayed for inhibition or potentiation of Wnt3a-induced signaling in HEK293 cells confirm that injected antibodies and protein retained at least some activity in vivo throughout the 16-h exposure (data not shown). Since activation or potentiation of Wnt signaling can increase bone mass by enhancing osteoblast differentiation and function, as well as by inhibiting osteoclast differentiation indirectly [28], we tested the activity of LRP6 antibodies on mouse calvarial bones in organotypic culture. Microdissected calvaria explants were cultured with antibody or RANK-Fc protein, and then parietal bone volume and density were analyzed by micro-computed tomography. Using histogram analysis of control samples, X-ray attenuation ranges were defined for calcified (bone) and non- calcified (cartilage) tissues. Treatment with YW210.09 antibody significantly increased the mean bone mineral density (BMD) of calcified parietal bone by 7.4%, similar to the 6.8% increase observed with RANK-Fc treatment to inhibit osteoclast differen- tiation (Figure 7C) [29]. Treatment with YW211.31.62 antibody did not significantly change calcified parietal BMD. The volume of total parietal bone region (calcified and non-calcified) and the proportion of calcified bone in this region were not significantly changed by antibody or RANK-Fc treatments, suggesting that YW210.09 antibody may enhance mineralization without gross changes in cell proliferation (data not shown). Discussion We have identified antibodies against LRP6 that can exert both antagonist and potentiating activities on b-catenin signaling, and demonstrate that these activities depend on different interactions between Wnt isoforms and the coreceptor (Figure 8). Since all antibodies screened that antagonize signaling in Wnt3a-stimulated HEK293 cells also inhibit Wnt3a stimulation in all other cell lines tested, and also inhibit autocrine Wnt signaling in teratocarcinoma cell lines, it was intriguing that these antibodies potentiate autocrine Wnt signaling in the other nine cell lines tested. In addition, the YW210.09 antibody potentiates Wnt3a signaling in all cell lines tested and enhances autocrine Wnt signaling in 7 cell lines, but it inhibits endogenous signaling in 3 other lines. We discovered that introduction of different Wnt isoforms into the same cell line determines the activity of the LRP6 antibodies, and that Wnt3a antagonist and potentiating antibodies also have reciprocal effects on most other Wnt proteins. Based on their functional interaction with two LRP6 antibodies, the 14 Wnt isoforms tested can be grouped into three classes: Wnt3 and Wnt3a are inhibited by YW211.31 and potentiated by YW210.09; Wnts 1, 2, 2b, 6, 8a, 9a, 9b, and 10b are potentiated by YW211.31 and antagonized by YW210.09; and Wnts 4, 7a, 7b, and 10a are potentiated by YW211.31 and not inhibited by YW210.09 (Figure 3C). These classifications do not obviously correspond to the proposed phylogeny of Wnt genes, although the Wnt3/3a subfamily is the most evolutionarily divergent [30]. Chimeric proteins that constitutively activate signaling by fusing different Wnt and FZD isoforms confirm that the activities of the antibodies are determined by the isoform of Wnt, and not FZD. Chimeric protein fusions of Wnt isoforms with LRP6, but not FZD, are insensitive to inhibition by the LRP6 antibodies, suggesting that antagonism may be mediated by blocking ligand- coreceptor interactions. This is confirmed by binding studies in vitro for Wnt3a and YW211.31 antibody, which bind competitively within the E3-E4 region of LRP6, and for Wnt9b and YW210.09 antibody, which compete for binding within the E1-E2 region. Wnts Specify LRP6 mAb Activity The epitopes of the two LRP6 antibodies each define a binding site for a different class of Wnt isoforms, one within the E1-E2 and one within the E3-E4 domain. Our data suggest that there may be at least a third binding site for Wnt isoforms that are not inhibited by either antibody or their combination, and it seems likely each of the four repeat domains binds a different subset of Wnt isoforms. This modular organization might allow for structural divergence of different Wnts and their binding sites to accommodate differential regulation by Wnt-binding and coreceptor-binding antagonists such as SFRP and DKK protein isoforms, respectively. Antibody-mediated Wnt potentiation requires coreceptor di- merization, since one-armed and Fab antibody formats fail to enhance Wnt signaling unless crosslinked. In addition, our cell- based and biochemical data indicate that Wnt binding to crosslinked LRP6 is also necessary for potentiation of signaling, presumably reflecting a requirement for Wnt-mediated recruit- ment of FZD into the complex. A small fraction of overexpressed LRP6 can be identified as a homodimer at the cell surface, and dimerization requires the extracellular domain, however it is not clear whether this contributes to either Wnt-dependent or -independent b-catenin signaling induced by LRP6 overexpression [18]. Deletion of the LRP6 extracellular domain potently activates signaling in a Wnt-independent manner, and forced extracellular dimerization of this recombinant protein by different methods can either enhance or inhibit this activity [18,19]. Wnt induces LRP6 aggregation and phosphorylation at the plasma membrane that both require the homo-oligomerization function of intracellular DVL protein [7]. These large aggregates also contain Axin and GSK3, and likely inhibit b-catenin degradation. It will be interesting to determine whether LRP6 dimerization by antibodies or other extracellular agents facilitates the aggregation function of DVL, and whether coreceptor dimerization normally plays a functional role in Wnt signaling. LRP6 antibodies might also potentiate Wnt signaling by inhibiting binding of antagonists such as DKK isoforms and SOST. We show that YW211.31 antibody can antagonize the activity of exogenous DKK1 protein, and that the one-armed YW211.31 antibody, which does not potentiate Wnt, retains only very weak DKK1 antagonism. However, both the whole IgG and the one-armed antibody show potent and similar activity in blocking DKK1 binding to LRP6 protein in vitro. Since antibody inhibition of DKK1 interaction with LRP6 does not necessarily confer Wnt potentiating activity, and DKK1 antagonism seems to require LRP6 dimerization, the inhibition of DKK1 activity is likely mediated predominantly by potentiation of residual signaling from Wnt bound to coreceptor. Two other groups have reported identification of LRP6 antibodies that bind the E1-E2 domains and potentiate Wnt3a signaling, although they did not test effects on signaling induced by other Wnt isoforms [31,32]. Analogous to YW211.31 antibody, one of these antibodies inhibits exogenous DKK1 antagonism of Wnt3a signaling, and also blocks DKK1 binding to LRP6 in vitro [31]. A Fab fragment of this antibody fails to potentiate Wnt3a signaling and antagonizes exogenous DKK1 activity much less potently. Apparently, inhibition of DKK1 binding to LPR6 within the E1 domain, recognized by this antibody, or the E3-E4 domain, bound by YW211.31 antibody, contributes only weakly to the antagonism of DKK1 activity by these antibodies. In Wnt-driven xenograft and allograft tumor models, the LRP6 antibodies inhibit Wnt signaling and tumor growth in a Wnt isoform-specific manner predicted by their properties character- ized in cell culture. It is not clear why we failed to observe enhancement of signaling or growth in tumors by the antibodies, and it is possible in the MMTV-Wnt1 model that Wnt signaling PLoS ONE | www.plosone.org 12 September 2010 | Volume 5 | Issue 9 | e12682