Crossveinless 2 (CV-2) is an extracellular BMP modulator protein belonging to the Chordin family. During development it is expressed at sites of high BMP signaling and like Chordin CV-2 can either enhance or inhibit BMP activity. CV-2 binds to BMP-2 via its N-terminal Von Willebrand factor type C (VWC) domain 1. Here we report the structure of the complex between CV-2 VWC1 and BMP-2. The tripartite VWC1 binds BMP-2 only through a short N-terminal segment, called clip, and subdomain (SD) 1. Mutational analysis establishes that the clip segment and SD1 together create high-affinity BMP-2 binding. All four receptor-binding sites of BMP-2 are blocked in the complex, demonstrating that VWC1 acts as competitive inhibitor for all receptor types. In vivo experiments reveal that the BMP-enhancing (pro-BMP) activity of CV-2 is independent of BMP-2 binding by VWC1, showing that pro- and anti-BMP activities are structurally separated in CV-2.

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.Sclerosteosis (OMIM accession number 269500) and van Buchem disease (OMIM accession number 239100) are closely related, rare high bone mass disorders that have been linked to a deficiency in expression of sclerostin, encoded by the SOST gene (1-8). Sclerostin is an osteocyte-derived negative regulator of bone formation belonging to the DAN family of secreted glycoproteins. Members of the DAN family were shown to have the capacity to inhibit BMP and/or Wnt activity (9 -13). Because sclerostin binds, albeit weakly, to mature bone morphogenetic proteins (BMPs), 4 it initially was presumed to be a BMP antagonist; however, currently sclerostin is believed to mediate its inhibitory effect on bone formation by directly blocking the Wnt signaling pathway (9, 14, 15).Canonical Wnt signaling has been described to play a crucial role in several bone mass disorders. Wnt proteins transduce their signals via seven-transmembrane-spanning receptors of the frizzled family and lipoprotein receptor-related protein-5/6 (LRP5/6), thereby controlling the stability of cytoplasmic ␤-catenin. In the absence of Wnt ligands, ␤-catenin forms a complex with APC (adenomatous polyposis coli), axin, GSK3 (glycogen synthase kinase 3), and CK1 (casein kinase I). This complex facilitates phosphorylation and subsequent proteasomal degradation of ␤-catenin. In the presence of Wnt ligands, this complex dissociates, and ␤-catenin accumulates and translocates into the nucleus, where it forms complexes with TCF/Lef1 transcription factors and initiates transcription of target genes (16). The importance of Wnt signaling in bone formation is illustrated by the low bone mass osteoporosis-pseudoglioma syndrome or high bone mass phenotype caused by missense loss or gain of function mutations in LRP5,. Sclerostin was found to ac...

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Sclerosteosis is a rare high bone mass disease that is caused by inactivating mutations in the SOST gene. Its gene product, Sclerostin, is a key negative regulator of bone formation and might therefore serve as a target for the anabolic treatment of osteoporosis. The exact molecular mechanism by which Sclerostin exerts its antagonistic effects on Wnt signaling in bone forming osteoblasts remains unclear. Here we show that Wnt3a-induced transcriptional responses and induction of alkaline phosphatase activity, an early marker of osteoblast differentiation, require the Wnt co-receptors LRP5 and LRP6. Unlike Dickkopf1 (DKK1), Sclerostin does not inhibit Wnt-3a-induced phosphorylation of LRP5 at serine 1503 or LRP6 at serine 1490. Affinity labeling of cell surface proteins with [125I]Sclerostin identified LRP6 as the main specific Sclerostin receptor in multiple mesenchymal cell lines. When cells were challenged with Sclerostin fused to recombinant green fluorescent protein (GFP) this was internalized, likely via a Clathrin-dependent process, and subsequently degraded in a temperature and proteasome-dependent manner. Ectopic expression of LRP6 greatly enhanced binding and cellular uptake of Sclerostin-GFP, which was reduced by the addition of an excess of non-GFP-fused Sclerostin. Finally, an anti-Sclerostin antibody inhibited the internalization of Sclerostin-GFP and binding of Sclerostin to LRP6. Moreover, this antibody attenuated the antagonistic activity of Sclerostin on canonical Wnt-induced responses.