Abstract
The Wnt/β-catenin signaling pathway appears to be particularly important for bone homeostasis, whereas nuclear accumulation of β-catenin requires the activation of Rac1, a member of the Rho small GTPase family. The aim of the present study was to investigate the role of RhoA/Rho kinase (Rock)-mediated Wnt/β-catenin signaling in the regulation of aging-associated bone loss. We find that Lrp5/6-dependent and Lrp5/6-independent RhoA/Rock activation by Wnt3a activates Jak1/2 to directly phosphorylate Gsk3β at Tyr216, resulting in Gsk3β activation and subsequent β-catenin destabilization. In line with these molecular events, RhoA loss- or gain-of-function in mouse embryonic limb bud ectoderms interacts genetically with Dkk1 gain-of-function to rescue the severe limb truncation phenotypes or to phenocopy the deletion of β-catenin, respectively. Likewise, RhoA loss-of-function in pre-osteoblasts robustly increases bone formation while gain-of-function decreases it. Importantly, high RhoA/Rock activity closely correlates with Jak and Gsk3β activities but inversely correlates with β-catenin signaling activity in bone marrow mesenchymal stromal cells from elderly male humans and mice, whereas systemic inhibition of Rock therefore activates the β-catenin signaling to antagonize aging-associated bone loss. Taken together, these results identify RhoA/Rock-dependent Gsk3β activation and subsequent β-catenin destabilization as a hitherto uncharacterized mechanism controlling limb outgrowth and bone homeostasis.
Highlights
Wnt ligands activate both β-catenin-dependent and βcatenin-independent signaling pathways and play important roles in embryonic development and tissue homeostasis (van Amerongen and Nusse 2009)
RhoA/Rock activation by Wnt3a constrains β-catenin signaling To determine the role of RhoA/Rock in Wnt/β-catenin signaling, we used an established binding assay to examine the levels of GTP-bound form in primary murine calvarial osteoblasts (PMCOBs)
We explored the exact role of RhoA in Wnt/βcatenin signaling by crossing mice that conditionally expressed the dominant negative form of RhoA or the constitutively active form of RhoA with the mice expressing Cre recombinase under the control of the 2.3-kb mouse collagen1α1 (Col1) promoter
Summary
Wnt ligands activate both β-catenin-dependent and βcatenin-independent signaling pathways and play important roles in embryonic development and tissue homeostasis (van Amerongen and Nusse 2009). During Wnt/β-catenin signaling, the binding of Wnt to Frizzled (FZD) receptors and to the low-density lipoprotein receptor-related protein 5 or 6 (Lrp5/6) activates. The secreted proteins Dkk and sclerostin (Sost) antagonize the pathway by interfering with Lrp5/6-Wnt interactions, thereby providing a negative feedback mechanism that limits the range of Wnt signaling (Bafico et al 2001; Wu et al 2008). Patients with osteoporosis or age-associated bone loss display a significantly low activity of Wnt/β-catenin signaling in bone marrow mesenchymal stromal cells (BMMSCs), primarily due to the high circulating levels of Dkk and Sost (Coulson et al 2017; Hampson et al 2013). Targeting the Wnt/β-catenin signaling represents a promising therapeutic approach for treatment of osteoporosis or aging-associated bone loss (Jing et al 2018; Manolagas and Almeida 2007)
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