Sclerostin Inhibition of Wnt-3a-induced C3H10T1/2 Cell Differentiation Is Indirect and Mediated by Bone Morphogenetic Proteins

Abstract

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

Targeting subchondral bone for treating osteoarthritis: what is the evidence?

“…A loss-of-function mutation of SOST was identified in sclerosteosis patients [64] who show progressive bone thickening and generalised osteosclerosis. SOST was believed to be a bone morphogenic protein (BMP) antagonist because it inhibits BMP-stimulated bone formation, yet it cannot antagonise all BMP responses [47,65]. Moreover, Wnt proteins induce BMPs, a mechanism inhibited by SOST, indicating that SOST may be a canonical Wnt signalling inhibitor, and that BMP activity is required downstream of a Wnt stimulus [65][66][67][68].…”

“…SOST was believed to be a bone morphogenic protein (BMP) antagonist because it inhibits BMP-stimulated bone formation, yet it cannot antagonise all BMP responses [47,65]. Moreover, Wnt proteins induce BMPs, a mechanism inhibited by SOST, indicating that SOST may be a canonical Wnt signalling inhibitor, and that BMP activity is required downstream of a Wnt stimulus [65][66][67][68]. Of note, our group recently reported [48] that a member of the DKK family, DKK2, a Wnt antagonist, was responsible for the altered phenotype of human primary OA osteoblasts.…”

Genetic regulation of bone mass and susceptibility to osteoporosis

“…The mechanisms by which SOST inhibits bone formation has been clarified by recent studies, which have shown that SOST directly interacts with LRP5 and LRP6 and antagonizes Wnt signaling both in vitro and in vivo (Li et al 2005;Semenov et al 2005). However, other workers have proposed that the inhibitory effect of SOST on Wnt signaling is indirect and mediated through BMP production (Winkler et al 2005). While further studies will be required to clarify the mechanisms by which SOST affects bone formation, it seems possible that the increased bone mass that results from SOST deficiency is mediated, at least in part, through the LRP5-␤-catenin pathway.…”

Wnt signaling and skeletal development

“…Wnt3a has been reported to induce ALP activity in C3H10T1/2 cells through transient transfection [53,87], conditional medium treatment [77,[95][96][97], purified protein treatment [96], adenoviral treatment [98], retroviral infection [99]; (in ST2 cells) transient transfection [53,87], conditional medium treatment [86,97]; (in C2C12 cells) transient transfection [87], or conditional medium treatment [97]. Wnt3a can also enhance ALP activity and BSP in conditional medium, enhance osteocalcin expression in ST2 cells in nondifferentiation medium, and increase mineralization in differentiation medium [86].…”