Canonical WNT Signaling Promotes Osteogenesis by Directly Stimulating Runx2 Gene Expression

Abstract

Both activating and null mutations of proteins required for canonical WNT signaling have revealed the importance of this pathway for normal skeletal development. However, tissue-specific transcriptional mechanisms through which WNT signaling promotes the differentiation of bone-forming cells have yet to be identified. Here, we address the hypothesis that canonical WNT signaling and the bone-related transcription factor RUNX2/CBFA1/ AML3 are functionally linked components of a pathway required for the onset of osteoblast differentiation. Our findings show that, in bone of the SFRP1 (secreted frizzled-related protein-1)-null mouse, which exhibits activated WNT signaling and a high bone mass phenotype, there is a significant increase in expression of T-cell factor (TCF)-1, Runx2, and the RUNX2 target gene osteocalcin. We demonstrate by mutational analysis that a functional TCF regulatory element responsive to canonical WNT signaling resides in the promoter of the Runx2 gene (؊97 to ؊93). By chromatin immunoprecipitation, recruitment of ␤-catenin and TCF1 to the endogenous Runx2 gene is shown. Coexpression of TCF1 with canonical WNT proteins resulted in a 2-5-fold activation of Runx2 promoter activity and a 7-8-fold induction of endogenous mRNA in mouse pluripotent mesenchymal and osteoprogenitor cells. This enhancement was abrogated by SFRP1. Taken together, our results provide evidence for direct regulation of Runx2 by canonical WNT signaling and suggest that Runx2 is a target of ␤-catenin/TCF1 for the stimulation of bone formation. We propose that WNT/TCF1 signaling, like bone morphogenetic protein/transforming growth factor-␤ signaling, activates Runx2 gene expression in mesenchymal cells for the control of osteoblast differentiation and skeletal development.During development of the skeleton and formation of bone tissue, several morphogenic growth factor and hormone signaling pathways impinge upon transcriptional regulators to induce the osteogenic phenotype (1, 2). The challenge is to identify how developmental cues and regulatory factors are integrated to accommodate the requirements for biological control of cell differentiation and tissue formation. Here, we have addressed the interaction of two key signals for osteogenesis: the WNT pathway, which contributes to the development of skeletal structures (3, 4), and the transcription factor RUNX2 (CBFA1/AML3), which is required for embryonic bone formation (5, 6).WNT signaling comprises a family of 19 secreted glycoproteins that have functions related to cell specification, formation of the body plan, cell growth, differentiation and apoptosis (7,8). WNT proteins function through Frizzled receptors, which transduce the signal through either the canonical ␤-catenin pathway or non-canonical pathway (7, 8). Activation of the Frizzled receptor complex results in inhibition of a phosphorylation cascade that stabilizes intracellular ␤-catenin levels. ␤-Catenin is subsequently translocated to the nucleus to form a transcriptionally active heterodimeric ␤-catenin TCF 2...

LRP8 mediates Wnt/β-catenin signaling and controls osteoblast differentiation

“…To further consolidate the effect of LRP8 in Wnt3a-induced osteoblast differentiation, we investigated LRP8's effect in regulating Wnt3/b-catenin-induced expression of RunX2 and collagen type I alpha 1 (Col1a1), which are early-stage makers for osteoblast differentiation. (24,47) As expected, LRP8 depletion inhibited Wnt3a-induced activation of RunX2 and Col1a1 whereas LRP8 overexpression enhanced both basal and Wnt3a-induced activation of RunX2 and Col1a1 (Fig. 4C and D).…”

“…(19,23) Wnt induced the activation of Runt-related transcription factor 2 (RunX2), an osteogenic transcriptional factor, which is essential in osteoblast differentiation. (24)(25)(26) LRP8 (also known as apolipoprotein E receptor 2) also belongs to the LDLR family. (27) LRP8 consists of seven conserved LDL repeats and three EGF receptor-like domains, which are similar to LRP5/6.…”

Novel Wnt Regulator NEL-Like Molecule-1 Antagonizes Adipogenesis and Augments Osteogenesis Induced by Bone Morphogenetic Protein 2

“…29 In osteoblast precursor cells, canonical Wnt signaling directly stimulates the Runx2 P1 promoter to drive osteoblast differentiation. 60 Because we showed that NELL-1 significantly increased the nuclear accumulation of bcatenin, it is possible that Runx2 expression may be likewise stimulated to induce osteoblastogenesis. Thus, NELL-1 is not only regulated by Runx2 and acts as its downstream mediator, but NELL-1 may also regulate Runx2 via canonical Wnt signaling.…”

“…60,61 Endogenous Wnt signaling is also increased in areas of bone healing, and Wnt-filled liposomes can accelerate bone healing. 29 In osteoblast precursor cells, canonical Wnt signaling directly stimulates the Runx2 P1 promoter to drive osteoblast differentiation.…”

Bone Anabolic Agents for the Treatment of Multiple Myeloma

“…WNT3a promotes OB differentiation and function, conversely dickkopf (DKK)1, soluble frizzled receptor-like proteins (sFRP)3 and sclerostin inhibit osteo-genesis [27][28][29]. BM serum of MM patients is often characterized by high levels of WNT inhibitors, which are considered promising therapeutic targets.…”