Abstract
Wnt11 signals through both canonical (beta-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases beta-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/beta-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through beta-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.
Highlights
Grants R01DE017471 and R01AR028922. □S The on-line version of this article contains supplemental Figs
Wnt11 Increases -Catenin Levels in MC3T3 E1.14 Cells— Given that canonical Wnt signaling plays a prominent role in osteoblast differentiation, and that Wnt11 has been shown to signal in a canonical manner, we evaluated the levels of cytosolic and nuclear -catenin
R-spondin 2 (Rspo2) Enhances Osteoblast Maturation and Mineralization and Stabilizes -Catenin—Because R-spondin expression is both induced by Wnt signaling [33] and an activator of -catenin [17, 33], we examined if some of the osteogenic effects of Wnt11 were mediated by Rspo2
Summary
Grants R01DE017471 and R01AR028922. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. Levels of nuclear and cytosolic -catenin were elevated in Wnt11ϩ cells relative to controls (Fig. 1C), indicative of increased canonical Wnt signaling. Dmp1 (dentin matrix acidic phosphoprotein 1) is among the extracellular matrix genes showing increased expression in Wnt11 cells relative to control cells (supplemental Table S2).
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