There is an unmet medical need for anabolic treatments to restore lost bone. Human genetic bone disorders provide insight into bone regulatory processes. Sclerosteosis is a disease typi®ed by high bone mass due to the loss of SOST expression. Sclerostin, the SOST gene protein product, competed with the type I and type II bone morphogenetic protein (BMP) receptors for binding to BMPs, decreased BMP signaling and suppressed mineralization of osteoblastic cells. SOST expression was detected in cultured osteoblasts and in mineralizing areas of the skeleton, but not in osteoclasts. Strong expression in osteocytes suggested that sclerostin expressed by these central regulatory cells mediates bone homeostasis. Transgenic mice overexpressing SOST exhibited low bone mass and decreased bone strength as the result of a signi®cant reduction in osteoblast activity and subsequently, bone formation. Modulation of this osteocyte-derived negative signal is therapeutically relevant for disorders associated with bone loss.

High bone mass diseases are caused both by activating mutations in the Wnt pathway and by loss of SOST, a bone morphogenetic protein (BMP) antagonist, leading to the activation of BMP signaling. Given the phenotypic similarity between mutations that activate these signaling pathways, it seems likely that BMPs and Wnts operate in parallel or represent components of the same pathway, modulating osteoblast differentiation. In this study, we show that in C3H10T1/2 cells, Wnt-3A and BMP-6 proteins were inducers of osteoblast differ- Sclerostin, the protein product of the sost gene associated with the high bone mass sclerosteosis phenotype, was predicted to be a secreted glycoprotein with homology to the DAN family of bone morphogenetic protein (BMP) 1 antagonists and more distantly to the BMP antagonist noggin (1, 4). We have shown that sclerostin behaved as a BMP antagonist and blocked BMPinduced osteoblastic activity such as alkaline phosphatase (ALP) activity in human and rodent bone cell models (3). In a broader context, BMPs and BMP antagonists have described skeletal roles specifically in chondrocyte differentiation, joint formation, and osteogenesis (5-7). Recently, Fischer et al. (8) and Bain et al. (9) reported that BMP-2 mediated osteoblast differentiation was modulated by a member of the Wnt protein family (8, 9). A novel role for Wnts was implicated in osteogenesis and BMP signaling.Wnt proteins are cysteine-rich, secreted glycoproteins that have been implicated in embryonic development and cellular differentiation, in particular limb patterning and chondrogenesis (10, 11). Wnt proteins activate downstream signaling pathways in target cells through interactions with Frizzled and low density lipoprotein receptors (LRP) co-receptors. The identification of the high bone mass gene, an activating mutation in LRP5, revealed the role of the Wnt pathway in bone formation (2,12).Given the interaction between the BMP and Wnts in osteoblastic cells described in the literature (8, 9), we were interested in investigating whether BMP antagonists, in particular sclerostin, could modify Wnt activity. Our findings show that the sclerostin antagonism of Wnt-3A-induced activity was not due to a direct interaction between the proteins or between sclerostin and the Wnt signaling pathway. We conclude that sclerostin inhibition of Wnt activity is mediated by BMP proteins and that Wnt induction of ALP in C3H10T1/2 cells is dependent on the expression of BMPs. EXPERIMENTAL PROCEDURES Effects

Noggin and sclerostin are bone morphogenetic protein (BMP) antagonists that modulate mitogenic activity through sequestering BMPs. Little is known of the interactions among this class of proteins. We show that recombinant sclerostin and noggin bound to each other with high affinity (K D ‫؍‬ 2.92 nM). This observation has been extended to naturally expressed noggin and sclerostin from the rat osteosarcoma cell line, ROS 17/ 2.8, supporting a role for the complex in natural systems. The noggin-sclerostin complex was competitive with BMP binding and mutually attenuated the activity of each BMP antagonist. Collectively, the data demonstrate a novel and exquisite paradigm for the regulation of BMP activity through direct neutralization of the BMP and activation by co-localized BMP antagonist expression. The pleiotrophic nature of noggin and sclerostin represents a novel mechanism for the fine-tuning of BMP activity in bone homeostasis.Sclerostin, the protein product of the SOST gene, is absent in sclerosteosis, a skeletal disease characterized by bone overgrowth and strong dense bones (1, 2). We have demonstrated that sclerostin, a bone morphogenetic protein (BMP) 1 antagonist highly expressed by osteocytes, is a key regulator of bone formation in vivo and in vitro (3). A number of BMP antagonists have been described that historically were associated with important regulatory activity in developmental systems. Recent studies have expanded the roles of these proteins beyond the described embryonic roles. Noggin has been shown to be expressed in mineralizing tissue, and in in vivo experiments an essential role in joint formation was established (4 -9). The overexpression of either sclerostin or noggin in bone cells leads to osteopenia in rodents, demonstrating the importance of BMP proteins in bone homeostasis and suggesting that sclerostin and noggin function as negative regulators of bone density (3,4,9).BMPs have been shown to induce the expression of BMP antagonists in mesenchymal (preosteoblastic) and osteoblastic cell lines (10,11). In vivo, the highest expression of sclerostin in adult human bone was seen in hypertrophic chondrocytes and osteocytes throughout the skeleton (3). In comparison, in vivo, noggin expression was reported to be associated with chondrocytes (12) and osteoblasts of cranial sutures (7). Given the close localization of these two BMP antagonist proteins and the similarity of their activities in vitro, we asked how these proteins might interact in regulating bone formation via limiting BMP activity. We report that these proteins coordinate their activity but do so through an unexpected pathway. Sclerostin and noggin bind to each other with high affinity and attenuate the BMP antagonist activity of each other. We propose that these BMP antagonists are pleiotrophic. EXPERIMENTAL PROCEDURESThe Binding of Noggin and Sclerostin-Anti-FLAG M2-agarose beads (Sigma) were washed with IP buffer (20 mM Tris, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1.4 mM ␤ME, 10% glycerol) before incubation in t...