CD137 Regulates Bone Loss via the p53 Wnt/β-Catenin Signaling Pathways in Aged Mice

Abstract

Senile osteoporosis is a chronic skeletal disease, leading to increased bone brittleness and risk of fragile fractures. With the acceleration of population aging, osteoporosis has gradually become one of the most serious and prevalent problems worldwide. Bone formation is highly dependent on the proper osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in the bone marrow microenvironment, which is generated by the functional relationship among different cell types, including osteoblasts, adipogenic cells, and bone marrow stromal cells in the bone marrow. It is still not clear how osteoporosis is caused by its molecular mechanism. With aging, bone marrow is able to restrain osteogenesis. Discovering the underlying signals that oppose BMSC osteogenic differentiation from the bone marrow microenvironment and identifying the unusual changes in BMSCs with aging is important to elucidate possible mechanisms of senile osteoporosis. We used 3 gene expression profiles (GSE35956, GSE35957, and GSE35959) associated with osteoporosis. And a protein-protein interaction (PPI) network was also built to identify the promising gene CD137. After that, we performed in vivo experiments to verify its function and mechanism. In this experiment, we found that significant bone loss was observed in aged (18-month-old) mice compared with young (6-month-old) mice. The adipose tissue in bone marrow cavity from aged mice reached above 10 times more than young mice. Combining bioinformatics analysis and vivo experiments, we inferred that CD137 might be involved in the p53 and canonical Wnt/β-catenin signaling pathways and thereby influenced bone mass through regulation of marrow adipogenesis. Importantly, osteoporosis can be rescued by blocking CD137 signaling in vivo. Our research will contribute to our understanding not only of the pathogenesis of age-related bone loss but also to the identification of new targets for treating senile osteoporosis.

Keywords: Bioinformatics; CD 137; gene expression; hub genes; osteoporosis.

Figure 1

Identification of DEGs in osteoporosis patients and non-osteoporotic donors. (A–F) Three volcano plots and Heat maps show all the expressed genes from GSE35956, GSE35957 and GSE35959. Blue and red in (A–F) respectively represent the down and up regulated genes. Each column represents a probe, and each row represents a gene. DEGs, differentially expressed genes.

Figure 2

(A) An extended range of DEGs were selected from GSE35956, GSE35957 and GSE35959. |log FC| > 1, P value < 0.05. (B). GO and KEGG enrichment analysis of DEGs. (C) PPI network construction and analysis of hub genes. (D) Hub genes were selected according to the top 5 nodes ranked by degree using cyto-Hubba. DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein–protein interaction.

Figure 3

Bone loss and bone marrow adipogenic differentiation in aged mice. (A) Oil red-O stain of the medullary cavity of femur for intra-bone marrow in mice. (B) A morphometric analysis in the volume of bone marrow adipose tissue (BMAT). (C) Bone volume fraction (BV/TV), trabecular bone number (Tb.N) and trabecular bone space (Tb.Sp) of the proximal tibia from 6- and 18-month-old mice were determined by micro-CT. (D) Representative 3D reconstruction images of the proximal tibia Tb. All the data were obtained from three independent experiments. Data are shown as the means ± SEMs. **p < 0.01, ***p < 0.001, 18 mo vs. 6 mo.

Figure 4

Alleviatation of trabecular bone loss and inhibition of bone marrow adipogenic differentiation in aged mice via blocking of CD137 signaling in vivo. (A) The 16-month-old C57BL/6 mice were intraperitoneally injected with neutralization antibody against CD137 once a week for 8 weeks. (B) Oil red-O stain of the medullary cavity of femur for intra-bone marrow in mice. (C) A morphometric analysis in the volume of bone marrow adipose tissue. (D) BV/TV, Tb.N, and Tb.Sp of the proximal tibia from 18-month-old mice with and without neutralization antibody against 4-1BB were determined by micro-CT. (E) Representative 3D reconstruction images of the proximal tibia Tb. All the data were obtained from three independent experiments. Data are shown as the means ± SEMs. *p < 0.05.

Figure 5

Diagram of the possible mechanism of bone loss via the CD137-p53 Wnt/β-catenin signaling pathways in aged mice. When CD137L binds to CD137 on BMSCs from aged mice, CD137 signaling is activated so that p53 Wnt/β-catenin signaling pathway is activated, then promotes the BMSC adipogenic differentiation potential of aged mice.