We and others have shown that N-cadherin (Ncad) physically interacts with low density lipoprotein receptor-related protein-5 or 6 (Lrp5/6) and axin, resulting in negative regulation of canonical Wnt/β-catenin signalling in osteoblasts. We tested whether removal of the N-cadherin gene (Cdh2) alters bone mass accrual in response to Lrp5/6 signalling. We administered a Dickkopf-1 (Dkk1) neutralizing antibody (αDkk1) to activate Lrp5/6 in mice with conditional Cdh2 ablation driven by the 2.3 Col1A1 promoter (Col1-cKO). At the dose of 5 mg/kg body weight i.p., three times/week for 4 weeks, αDkk1 was ineffective in WT mice, but produced a twofold increase of BV/TV (0.496±0.085 P<0.01 vs baseline) in Col-Cdh2 cKO mice. A higher dose (20 mg/kg) was equally effective in both genotypes. At the molecular level, a single dose of αDkk1 produced accelerated accumulation of β-catenin in bone of Col1-cKO relative to WT mice, indicating direct Wnt/β-catenin signalling activation in bone by αDkk1 and enhanced responsiveness in the absence of Ncad. To corroborate this finding, we introduced one Dkk1-resistant Lrp5A214V allele associated with high bone mass (HBM), in conditionally Cdh2 ablated mice driven by Osx1-Cre (Osx1-cKO). Although bone mass (by μCT) was lower in Osx1-cKO than in WT, the compound Lrp5A214V;Cdh2 cKO mutants were osteosclerotic and indistinguishable from Lrp5A214V HBM mice. Despite lower total β-catenin abundance in Osx1-cKO bone marrow stromal cells, steady state cytosolic β-catenin was not decreased. Upon Wnt3a stimulation, N-terminally un-phosphorylated β-catenin was more abundant in Osx1-cKO than in WT cells, suggesting higher levels of active β-catenin in the absence of N-cadherin in response to Wnt3a. In summary, mice lacking Cdh2 in osteolineage cells are hyper-responsive to Wnt signaling activation and to its osteo-anabolic effect. These results provide in vivo proof of the concept that Ncad restrains anabolic Lrp5/6 signalling in bone forming cells.
Disclosure: The authors declared no competing interests. This work has been supported by NIH grant AR055913 (RC).