Osteocytic Wnt generates RANKL for osteoclastogenesis via TGFβ signaling

Wnt signaling is generally believed to inhibit osteoclast differentiation (OcD) by upregulating its target Opg expression; however, we unexpectedly found that Wnt mice with activation of Wnt/β-catenin signaling in osteocytes have higher OcD and bone resorption while generating more bone (PNAS 2015,112: E478). Here, we show cellular and molecular mechanisms for this unpredictable function of osteocytic Wnt on OcD. Isolated osteocytes (daCOt) of the Wnt mice display higher expression of RANKL and Opg with increased RANKL/Opg ratio, promoting OcD of bone marrow monocytes/macrophages to enhance bone resorption. RANKL-siRNA knocks down RANKL to reduce this OcD. In daCOt transcriptome analysis, the enrichment of KEGG pathways highlights the largest correlation of OcD with TGFβ signaling, which is activated by highly expressed Tgfb1 and Tgfb2, identified by heat-map analysis, and verified in a dose-dependent manner in Wnt agonist C91-treated wild-type osteocytes. Moreover, TGFβ signaling increases RANKL expression in dose and time-dependent manners, respectively with higher RANKL/Opg ratio and OcD. Conversely, TGFβ signaling inhibition reverses these effects. Importantly, mice with osteocytic deletion of the only TGFβRII decreases osteoclast number by 48.2% to 3.0/mm in Oc.N/B.Pm with markedly reduced expression of RANKL both at protein and mRNA levels in the osteocytes of cancellous bone in distal femurs. Furthermore, luciferase assay with 3kb-RANKL promoter region reveals that TGFβ signaling activates RANKL transcription. In summary, osteocytic Wnt promotes OcD mainly by activating TGFβ signaling, which activates RANKL gene transcription, opening up a novel avenue for the treatment or application of osteoclast or bone resorption-related diseases.