In this study, we have identified the canonical Wnt/β-catenin pathway as a previously unrecognized yet critical regulator of osteoclastogenesis. β-Catenin protein is induced during the quiescence-to-proliferation switch of the osteoclast progenitors in response to M-CSF but downregulated during the proliferation-to-differentiation switch in response to RANKL (). Genetically, β-catenin deletion impairs osteoclast precursor proliferation, while β-catenin constitutive activation sustains precursor proliferation, both blocking osteoclast differentiation and causing osteopetrosis. In contrast, β-catenin dosage reduction by heterozygosity enhances osteoclast differentiation and causes osteoporosis. Biochemically, β-catenin activation by GSK3β inhibitors or a Wnt ligand attenuates osteoclastogenesis, whereas β-catenin suppression by a Wnt inhibitor or a PPARγ agonist stimulates osteoclastogenesis. Mechanistically, β-catenin activation promotes precursor proliferation by elevating GATA2 and Evi1 expression and blocks osteoclast differentiation by preventing c-Jun phosphorylation. Therefore, β-catenin exerts a biphasic regulation of osteoclastogenesis: its induction is required for M-CSF-mediated precursor proliferation, yet its degradation is required for RANKL-mediated osteoclast differentiation. Moreover, β-catenin controls osteoclast differentiation in a dosage-dependent manner: a minimum threshold of β-catenin is required for osteoclast progenitors to proliferate, yet above this threshold, β-catenin activation suppresses osteoclast differentiation, while β-catenin attenuation accelerates osteoclast differentiation.
In order to understand the full spectrum of β-catenin functions in osteoclastogenesis, it is essential to target the entire osteoclast lineage from osteoclast progenitors to mature osteoclasts. However, the intrinsic multipotent property of stem/progenitor cells renders it impossible to specifically target the osteoclast lineage. Therefore, by establishing a suite of genetic models using different cre drivers targeting different stages of osteoclastogenesis, we were able to extract the cell-autonomous regulation of osteoclastogenesis by β-catenin. The PPARγ-driven β-catenin models permit osteoclast progenitor targeting without the embryonic lethality seen in the Tie2-driven β-catenin models. Importantly, the bone phenotype in the PPARγ-driven β-catenin models was not due to any MSC or osteoblast targeting, because (i) the reported MSC-bKO mice were embryonic lethal (8
) while, in contrast, the PTbKO mice were viable; (ii) the reported osteoblast-bCA mice died a few days after weaning with a BV/TV ratio of 23% (11
) while, in contrast, the PTbCA mice lived for >12 months with a BV/TV ratio of >80%; (iii) osteoblast numbers and bone formation were largely unaltered in these mice. This strongly supports the notion that the bone phenotype in these mice was mainly the result of osteoclast-autonomous defects. Furthermore, although the Lyz-Cre- and Ctsk-cre-driven β-catenin models cannot target osteoclast progenitors, they permit more specific targeting to macrophage precursors and osteoclasts, respectively. The results, with Lyz-bCA and Ctsk-bCA mice exhibiting osteopetrosis while Lyz-bKO and Ctsk-bKO mice exhibited osteoporosis, confirmed that β-catenin constitutive activation inhibits osteoclastogenesis and β-catenin dosage reduction in the macrophage precursors stimulates osteoclastogenesis. However, Lyz-bKO and Ctsk-bKO mice did not reveal the critical requirement of β-catenin in the quiescence-to-proliferation switch of the osteoclast progenitors, which was uncovered in the PTbKO and Tie2-bKO mice. Therefore, these studies also highlight the inducible PPARγ-tTA-TRE-cre driver as a novel strategy for targeting osteoclast progenitors and the entire osteoclast lineage that is complementary to the existing cre drivers.
Wnt activation is a promising therapeutic strategy for treating bone diseases based on its currently known bone formation-stimulating anabolic effects. For example, a neutralizing monoclonal antibody (Scl-Ab) against the Wnt antagonist sclerostin (sost), which is secreted specifically from osteocytes (27
), has been shown to markedly increase bone formation and reverse estrogen deficiency-induced bone loss (19
). Currently, Scl-Ab is being developed by Amgen as a new anabolic treatment for bone disorders, such as postmenopausal osteoporosis. Furthermore, a neutralizing monoclonal antibody (BHQ880) against the Wnt antagonist Dickkopf-1 (DKK-1), which is expressed predominantly in adult bone and upregulated in multiple myeloma (33
), has been shown to increase bone formation in murine models of human multiple myeloma and rheumatoid arthritis (9
). Currently, BHQ880 is being developed by Novartis as a new approach to promote bone formation and thereby inhibit tumor-induced osteolysis. Our findings provide strong evidence that Wnt activation also inhibits osteoclast differentiation and bone resorption, illuminating a previously unrecognized additional anticatabolic benefit. Consistent with this notion, in a recent first-in-humans study, a sclerostin monoclonal antibody (AMG 785) not only increased bone formation marker but also decreased bone resorption marker in a dose-dependent manner (24
). Therefore, bone-specific activators of Wnt/β-catenin signaling may promise an exciting new class of drugs that can more effectively prevent and treat skeletal fragility.
In summary, the discovery of novel roles of β-catenin in osteoclastogenesis in this study opens an exciting new path to future investigations of the ligands, receptors, signal transducers, and transcription factors that orchestrate the regulation of osteoclast physiology and pharmacology by the Wnt pathway.