The R-Spondin (RSpo) family of secreted proteins is implicated in the activation of the Wnt signaling pathway. Despite the high structural homology between the four members, expression patterns and phenotypes in knockout mice have demonstrated striking differences. Here we dissected and compared the molecular and cellular function of all RSpo family members. Although all four RSpo proteins activate the canonical Wnt pathway, RSpo2 and 3 are more potent than RSpo1, whereas RSpo4 is relatively inactive. All RSpo members require Wnt ligands and LRP6 for activity and amplify signaling of Wnt3A, Wnt1, and Wnt7A, suggesting that RSpo proteins are general regulators of canonical Wnt signaling. Like RSpo1, RSpo2-4 antagonize DKK1 activity by interfering with DKK1 mediated LRP6 and Kremen association. Analysis of RSpo deletion mutants indicates that the cysteine-rich furin domains are sufficient and essential for the amplification of Wnt signaling and inhibition of DKK1, suggesting that Wnt amplification by RSpo proteins may be a direct consequence of DKK1 inhibition. Together, these findings indicate that RSpo proteins modulate the Wnt pathway by a common mechanism and suggest that coexpression with specific Wnt ligands and DKK1 may determine their biological specificity in vivo.

The R-Spondin (RSpo) family of secreted proteins act as potent activators of the Wnt/␤-catenin signaling pathway. We have previously shown that RSpo proteins can induce proliferative effects on the gastrointestinal epithelium in mice. Here we provide a mechanism whereby RSpo1 regulates cellular responsiveness to Wnt ligands by modulating the cell-surface levels of the coreceptor LRP6. We show that RSpo1 activity critically depends on the presence of canonical Wnt ligands and LRP6. Although RSpo1 does not directly activate LRP6, it interferes with DKK1/Kremen-mediated internalization of LRP6 through an interaction with Kremen, resulting in increased LRP6 levels on the cell surface. Our results support a model in which RSpo1 relieves the inhibition DKK1 imposes on the Wnt pathway.beta-catenin ͉ DKK1 ͉ Kremen

Objective. During the course of different musculoskeletal diseases, joints are progressively damaged by inflammatory, infectious, or mechanical stressors, leading to joint destruction and disability. While effective strategies to inhibit joint inflammation, such as targeted cytokine-blocking therapy, have been developed during the last decade, the molecular mechanisms of joint damage are still poorly understood. This study was undertaken to investigate the role of the Wnt pathway modulator R-Spondin 1 (RSpo1) in protecting bone and cartilage in a mouse model of arthritis.Methods. Tumor necrosis factor ␣ (TNF␣)-transgenic mice were treated with vehicle or Rspo1. Mice were evaluated for signs of arthritis, and histologic analysis of the hind paws was performed. Moreover, we determined the effect of Rspo1 on Wnt signaling activity and osteoprotegerin (OPG) expression in murine primary osteoblasts.Results. The secreted Wnt pathway modulator RSpo1 was highly effective in preserving the structural integrity of joints in a TNF␣-transgenic mouse model of arthritis by protecting bone and cartilage from inflammation-related damage. RSpo1 antagonized the Wnt inhibitor Dkk-1 and modulated Wnt signaling in mouse mesenchymal cells. In osteoblasts, RSpo1 induced differentiation and expression of OPG, thereby inhibiting osteoclastogenesis in vitro. In vivo, RSpo1 promoted osteoblast differentiation and bone formation while blocking osteoclast development, thereby contributing to the integrity of joints during inflammatory arthritis.Conclusion. Our results demonstrate the therapeutic potential of RSpo1 as an anabolic agent for the preservation of joint architecture.The canonical Wnt/␤-catenin signaling pathway plays a pivotal role in diverse biologic processes such as embryonic development, regulation of mesenchymal cell differentiation, and bone physiology (1-4). The Wnt family of secreted proteins regulates ␤-catenin turnover and promotes accumulation of intracellular ␤-catenin via activation of a coreceptor complex consisting of the Frizzled family of proteins and low-density lipoprotein receptor-related protein 5 (LRP-5) or LRP-6 (5-7). ␤-catenin in turn drives transcription activation by binding to the T cell factor (TCF)/Lef family of transcription factors (8,9). Different extracellular proteins modulate Wnt signaling activity at the receptor level in a negative and positive manner. Among these, the Dkk family of proteins (Dkk-1-Dkk-4) block Wnt signaling by promoting internalization of the LRP-5/6 receptor complex (10,11). More recently, another group of proteins, the R-Spondin family of Wnt modulators (RSpo1-RSpo4), has gained increased attention due to their capacity to amplify ␤-catenin/Wnt signaling activity (12-14) by