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RANKL signaling in bone marrow mesenchymal stem cells negatively regulates osteoblastic bone formation.

ABSTRACT: RANKL signaling is essential for osteoclastogenesis. Its role in osteoblastic differentiation and bone formation is unknown. Here we demonstrate that RANK is expressed at an early stage of bone marrow mesenchymal stem cells (BMSCs) during osteogenic differentiation in both mice and human and decreased rapidly. RANKL signaling inhibits osteogenesis by promoting ?-catenin degradation and inhibiting its synthesis. In contrast, RANKL signaling has no significant effects on adipogenesis of BMSCs. Interestingly, conditional knockout of rank in BMSCs with Prx1-Cre mice leads to a higher bone mass and increased trabecular bone formation independent of osteoclasts. In addition, rank flox/flox: Prx1-Cre mice show resistance to ovariectomy-(OVX) induced bone loss. Thus, our results reveal that RANKL signaling regulates both osteoclasts and osteoblasts by inhibition of osteogenic differentiation of BMSCs and promotion of osteoclastogenesis.

SUBMITTER: Chen X

PROVIDER: S-EPMC6255918 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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RANKL signaling in bone marrow mesenchymal stem cells negatively regulates osteoblastic bone formation.

Chen Xiao X Zhi Xin X Wang Jun J Su Jiacan J

Bone research 20181127

RANKL signaling is essential for osteoclastogenesis. Its role in osteoblastic differentiation and bone formation is unknown. Here we demonstrate that RANK is expressed at an early stage of bone marrow mesenchymal stem cells (BMSCs) during osteogenic differentiation in both mice and human and decreased rapidly. RANKL signaling inhibits osteogenesis by promoting β-catenin degradation and inhibiting its synthesis. In contrast, RANKL signaling has no significant effects on adipogenesis of BMSCs. Int ...[more]

PMID: 30510839

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Project description:BackgroundOsteogenesis Imperfecta (OI) (OMIM %259450) is a heterogeneous group of inherited disorders characterized by increased bone fragility, with clinical severity ranging from mild to lethal. The majority of OI cases are caused by mutations in COL1A1 or COL1A2. Bruck Syndrome (BS) is a further recessively-inherited OI-like phenotype in which bone fragility is associated with the unusual finding of pterygia and contractures of the large joints. Notably, several studies have failed to show any abnormalities in the biosynthesis of collagen 1 in BS patientes. Evidence was obtained for a specific defect of the procollagen telopeptide lysine hydroxylation in BS, whereas mutations in the gene PLOD2 have been identified. Recently, several studies described FKBP10 mutations in OI-like and BS patients, suggesting that FKBP10 is a bonafide BS locus.MethodsWe analyzed the coding region and intron/exon boundaries of COL1A1, COL1A2, PLOD2 and FKBP10 genes by sequence analysis using an ABI PRISM 3130 automated sequencer and Big Dye Terminator Sequencing protocol. Mononuclear cells obtained from the bone marrow of BS, OI patients and healthy donors were cultured and osteogenic differentiation was induced. The gene expression of osteoblast specific markers were also evaluated during the osteoblastic differentiation of mesenchymal stem cell (MSC) by qRT-PCR using an ABI7500 Sequence Detection System.ResultsNo mutations in COL1A1, COL1A2 or PLOD2 were found in BS patient. We found a homozygous 1-base-pair duplication (c.831dupC) that is predicted to produce a translational frameshift mutation and a premature protein truncation 17 aminoacids downstream (p.Gly278ArgfsX95). The gene expression of osteoblast specific markers BGLAP, COL1A1, MSX2, SPARC and VDR was evaluated by Real Time RT-PCR during differentiation into osteoblasts and results showed similar patterns of osteoblast markers expression in BS and healthy controls. On the other hand, when compared with OI patients, the expression pattern of these genes was found to be different.ConclusionsOur work suggests that the gene expression profiles observed during mesenchymal stromal cell differentiation into osteoblast are distinct in BS patients as compared to OI patients. The present study shows for the first time that genes involved in osteogenesis are differentially expressed in BS and OI patients.

Project description:BackgroundSkeletal unloading usually induces severe disuse osteoporosis (DOP), which often occurs in patients subjected to prolonged immobility or in spaceflight astronauts. Increasing evidence suggests that exosomes are important mediators in maintaining the balance between bone formation and resorption. We hypothesized that exosomes play an important role in the maintenance of bone homeostasis through intercellular communication between bone marrow mesenchymal stem cells (BMSCs) and osteoclasts under mechanical loading.MethodsCells were divided into cyclic mechanical stretch (CMS)-treated BMSCs and normal static-cultured BMSCs, and exosomes were extracted by ultracentrifugation. After incubation with CMS-treated BMSC-derived exosomes (CMS_Exos) or static-cultured BMSC-derived exosomes (static_Exos), the apoptosis rates of bone marrow macrophages (BMMs) were determined by flow cytometry, and cell viability was detected with a Cell Counting Kit-8 (CCK-8) assay. Osteoclast differentiation was determined with an in vitro osteoclastogenesis assay. Signaling pathway activation was evaluated by western blotting and immunofluorescence staining. Hindlimb unloading (HU)-induced DOP mouse models were prepared to evaluate the function of exosomes in DOP.ResultsBoth CMS_Exos and static_Exos could be internalized by BMMs, and CMS_Exos did not affect BMM viability or increase apoptosis. The CMS_Exos effectively suppressed receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclastogenesis and F-actin ring formation. Further molecular investigation demonstrated that CMS_Exos impaired osteoclast differentiation via inhibition of the RANKL-induced nuclear factor kappa-B (NF-κB) signaling pathway. Both CMS_Exos and static_Exos partly rescued the osteoporosis caused by mechanical unloading; however, the CMS_Exo group showed more obvious rescue. Treatment with CMS_Exos significantly decreased the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts. Exosomes derived from CMS-treated BMSCs strongly inhibited osteoclast differentiation by attenuating the NF-κB signaling pathway in vitro and rescued osteoporosis caused by mechanical unloading in an HU mouse model in vivo.ConclusionsIn this research, we demonstrated that Exosomes derived from CMS-treated BMSCs inhibited osteoclastogenesis by attenuating NF-κB signaling pathway activity in vitro and ameliorated bone loss caused by mechanical unloading in an HU mouse model, providing new insights into intercellular communication between osteoblasts and osteoclasts under mechanical loading.

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RANKL signaling in bone marrow mesenchymal stem cells negatively regulates osteoblastic bone formation.

Publications

RANKL signaling in bone marrow mesenchymal stem cells negatively regulates osteoblastic bone formation.

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