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Osteocytic Wnt generates RANKL for osteoclastogenesis via TGFβ signaling

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE301026 | GEO | 2025/12/02

REPOSITORIES: GEO

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Project description:Spinal cord injury (SCI) causes severe bone loss and disrupts connections between higher centers in the central nervous system (CNS) and bone. Muscle contraction elicited by functional electrical stimulation (FES) partially protects against loss of bone but cellular and molecular events by which this occurs are unknown. Here, using a rat model, we characterized effects of 7 days of contraction-induced loading of tibia and fibula due to FES when begun 16 weeks after SCI. SCI reduced tibial and femoral BMD by 12-17% and promoted bone resorption, as indicated by increased serum CTX; SCI-related changes in CTX were reversed by FES. In cultures of bone marrow cell-derived cells, SCI increased the number of osteoclasts and mRNA levels of the several osteoclast differentiation markers; these changes were significantly reversed by FES. The number of osteoblasts was also reduced by SCI as was the ratio of OPG/RANKL mRNAs therein; the unfavorable change in OPG/RANKL ratio was partially reversed by FES. cDNA microarray analysis revealed that alterations in genes involved in signaling through Wnt, FSH/LH, PTH and calcineurin/NFAT pathways may be linked to the favorable action of FES on SCI-induced bone resorption. In particular, SCI increased levels of the Wnt inhibitors DKK1, sFRP2 and SOST in osteoblasts, These effects were completely or partially reversed by FES. Our results demonstrate an anti-bone resorptive activity of acute FES in bone loss after SCI and suggest potential underlying mechanisms, among them involving increased Wnt signaling to cause more favorable ratios of OPG and RANKL for the inhibition of osteoclastogenesis. The present study indicates that the effects of bone reloading on SCI- related bone remodeling occurred independently of the effects of higher CNS centers on bone. Implantation of the FES microstimulators was performed 14 weeks after SCI. The FES was begun during the 16th week following spinal cord transection. Stimulation was provided for 60 minutes on each training day and consisted of brief periods of contraction (2 seconds) at 40 Hz at 1.5 V with longer periods of rest (18 seconds). Animals received FES on 7 consecutive days; collection of blood and tissues occurred at day at after initiating FES, as described below. The SCI-Sham FES animals received the implant surgery and gastrocnemius ablation during the 14th week after the spinal cord transection, but did not have a stimulator unit inserted; samples were collected from these animals at week 17. To provide age-matched non-SCI controls, additional animals underwent a sham-SCI surgery identical to that for the SCI animals, except that the spinal cord was not transected. Tissues were collected from these animals at 12-14 weeks after surgery. Of note, at this age, animals were sexually mature and their growth minimal. To collect blood and tissues, animals were anesthetized by inhalation of isofluorane followed by removal of the soleus and plantaris muscles after careful dissection and collection of blood by ventricular puncture and aspiration. Animals were euthanized by aortic transaction and tibia and femur were removed as a single piece, leaving the knee joint intact, and placed in a-MEM for isolation of bone marrow cells. Muscles were weighed; weights are expressed after being normalized to body weight before SCI or sham-SCI surgeries to control for individual variations in size.

Project description:Background This work focuses on the computational modelling of osteomyelitis, a bone pathology caused by bacteria infection (mostly Staphylococcus aureus). The infection alters the RANK/RANKL/OPG signalling dynamics that regulates osteoblasts and osteoclasts behaviour in bone remodelling, i.e. the resorption and mineralization activity. The infection rapidly leads to severe bone loss, necrosis of the affected portion, and it may even spread to other parts of the body. On the other hand, osteoporosis is not a bacterial infection but similarly is a defective bone pathology arising due to imbalances in the RANK/RANKL/OPG molecular pathway, and due to the progressive weakening of bone structure. Results Since both osteoporosis and osteomyelitis cause loss of bone mass, we focused on comparing the dynamics of these diseases by means of computational models. Firstly, we performed meta-analysis on a gene expression data of normal, osteoporotic and osteomyelitis bone conditions. We mainly focused on RANKL/OPG signalling, the TNF and TNF receptor superfamilies and the NF-kB pathway. Using information from the gene expression data we estimated parameters for a novel model of osteoporosis and of osteomyelitis. Our models could be seen as a hybrid ODE and probabilistic verification modelling framework which aims at investigating the dynamics of the effects of the infection in bone remodelling. Finally we discuss different diagnostic estimators defined by formal verification techniques, in order to assess different bone pathologies (osteopenia, osteoporosis and osteomyelitis) in an effective way. Conclusions We present a modeling framework able to reproduce aspects of the different bone remodeling defective dynamics of osteomyelitis and osteoporosis. We report that the verification-based estimators are meaningful in the light of a feed forward between computational medicine and clinical bioinformatics Model is encoded by Ruby and submitted and curated to BioModels by Ahmad Zyoud

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Osteocytic Wnt generates RANKL for osteoclastogenesis via TGFβ signaling

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