B‐cell lymphoma‐3 controls mesenchymal stem cell commitment and senescence during skeletal aging

Dear Editor, Skeletal aging is characterized by progressive bone loss and increased marrow adiposity.1,2 Age-related bone loss impairs the exercise activity of patients and increases the risk of fracture. Bone marrow mesenchymal stem cells (BMSCs) could differentiate into osteoblasts and adipocytes.3,4 The senescence and differentiation shift of BMSCs plays a critical role in skeletal aging and osteoporosis. In our study, we demonstrated that B-cell lymphoma 3 (Bcl-3), amember of the inhibitor of κB (IκB),5,6 attenuated BMSCs senescence and regulated BMSCs differentiation fate through manipulating Wnt signalling.7,8 First, we showed that the level of Bcl-3 from 1 to 18 months in mice was significantly decreased with aging (Figure 1A). Then, we generated Bcl-3–/– mice which displayed slightly smaller size than that of wild-type (WT) mice and results of WB and qRT-PCR confirm that Bcl-3 was deleted in Bcl-3–/– mice (Figure S1A–C). Microcomputed tomography (μCT) analysis (Figure 1B,C and Figure S1D) and H&E staining (Figure S1E) showed a significant bone loss in 10-week-old Bcl-3–/– mice compared with control. Bcl-3 knockout also significantly increased bone loss following ovariectomy (OVX) by μCT (Figure 1D and Figure S1F). Calcein double labelling verified Bcl3–/– mice had less endosteal, periosteal and trabecular bone formation than that of the WT counterparts, mineral apposition and bone formation was lower in Bcl-3–/– group (Figure 1E,F). In addition, the number of trabecular OCN-positive cells in Bcl-3–/– mice was less than that in WT mice, while the number of FABP4-positive adipocytes and trap-positive cells were increased in Bcl-3–/– mice (Figure 1G–I and Figure S1G). Then,we examined the differentiation potential, namely osteogenesis and adipogenesis of BMSCs after Bcl-3 was silenced in vitro. BMSCs were isolated from 5-week-mice and identified by flow cytometry (Figure S2A,B). The osteogenesis of BMSCs was decreased when Bcl-3 was knockdown verified by staining of alkaline phosphatase

Dear Editor, Skeletal aging is characterized by progressive bone loss and increased marrow adiposity. 1,2 Age-related bone loss impairs the exercise activity of patients and increases the risk of fracture. Bone marrow mesenchymal stem cells (BMSCs) could differentiate into osteoblasts and adipocytes. 3,4 The senescence and differentiation shift of BMSCs plays a critical role in skeletal aging and osteoporosis. In our study, we demonstrated that B-cell lymphoma 3 (Bcl-3), a member of the inhibitor of κB (IκB), 5,6 attenuated BMSCs senescence and regulated BMSCs differentiation fate through manipulating Wnt signalling. 7,8 First, we showed that the level of Bcl-3 from 1 to 18 months in mice was significantly decreased with aging ( Figure 1A). Then, we generated Bcl-3 -/mice which displayed slightly smaller size than that of wild-type (WT) mice and results of WB and qRT-PCR confirm that Bcl-3 was deleted in Bcl-3 -/mice (Figure S1A-C). Microcomputed tomography (μCT) analysis ( Figure 1B,C and Figure S1D) and H&E staining ( Figure S1E) showed a significant bone loss in 10-week-old Bcl-3 -/mice compared with control. Bcl-3 knockout also significantly increased bone loss following ovariectomy (OVX) by μCT ( Figure 1D and Figure S1F). Calcein double labelling verified Bcl-3 -/mice had less endosteal, periosteal and trabecular bone formation than that of the WT counterparts, mineral apposition and bone formation was lower in Bcl-3 -/group ( Figure 1E,F). In addition, the number of trabecular OCN-positive cells in Bcl-3 -/mice was less than that in WT mice, while the number of FABP4-positive adipocytes and trap-positive cells were increased in Bcl-3 -/mice ( Figure 1G-I and Figure S1G).
Then, we examined the differentiation potential, namely osteogenesis and adipogenesis of BMSCs after Bcl-3 was silenced in vitro. BMSCs were isolated from 5-week-mice and identified by flow cytometry (Figure S2A (ALP) and calcium nodules stained by Alizarin Red S (ARS) and the decreased mRNA levels of Ocn, Osterix and Runx2 (Figure 2A,B). On the contrary, adipogenesis was increased in Bcl-3-knockdown BMSCs validated by oil red staining and the mRNA levels of Fabp4, Adipoq and Pparγ ( Figure 2C,D). When Bcl-3 was knocked down in BMSCs, the expression of FABP4 was increased, accompanied by decreased OSTERIX expression ( Figure 2E). On the other hand, we found that Bcl-3 overexpression could promote osteoblastic differentiation and inhibit adipogenic differentiation in senescent BMSCs treated by H 2 O 2 ( Figure  S3A-F).
In aging BMSCs, the expression level of Bcl-3 was decreased ( Figure 2F and Figure S4A). For self-renewable capacities, BMSCs of Bcl-3 -/mice showed fewer colonyforming unit-fibroblasts (CFU-Fs) ( Figure 2G,H). The number of SA-β-gal-positive blue cells in Bcl-3 knockdown BMSCs was significantly increased than control BMSCs ( Figure 2I). The expression of aging makers, P21 and P16, was significantly upregulated when Bcl-3 was knockdown in BMSCs ( Figure 2J). Moreover, Bcl-3 overexpression reduced senescent cells numbers and the expression of aging makers after treated with H 2 O 2 ( Figure S4B,C). To sum up, Bcl-3 was decreased during BMSCs senescence, and the supply of Bcl-3 could protect BMSCs from aging.
To explore the molecular mechanisms, we performed RNA sequencing in shBcl-3 and shCtrl BMSCs. The results of gene ontology (GO) revealed that the downregulated genes following Bcl-3 knockdown were mainly enriched in ossification, bone mineralization, bone growth, etc. Kyoto encyclopaedia of genes and genomes (KEGG) revealed that Bcl-3-knockdown BMSCs influenced Wnt/βcatenin signalling pathways ( Figure 3A,B). Heat map showed that relative expression levels of genes in the Wnt pathway were decreased in shBcl-3 BMSCs compared to shCtrl BMSCs ( Figure 3C). In addition, we found that Wnt/β-catenin signalling was downregulated in Bcl-3 knockdown BMSCs indicated by gene set enrichment  The data are presented as the mean ± SD. *P <.05; **P <.01 vs. control group. Statistical analysis was performed using two-way ANOVA analysis (GSEA, Figure 3D). The expression levels of genes in Wnt signalling were decreased when Bcl-3 was knockdown in BMSCs ( Figure S5A,B) and increased in Bcl-3-overexpressed BMSCs ( Figure S5C,D).
The degradation of Ac-K49 β-catenin was significant in Bcl-3-silenced BMSCs after treated with cycloheximide (CHX, Figure 3H and Figure S5H). In addition, TOP/FOP flash assays showed that Bcl-3 depletion reduced the transcriptional activity of β-catenin and decreased the combination of β-catenin with promotors of Runx2 and Osterix ( Figure 3I-K).
Osteoporosis is common among aged individuals, which could be attributed to the senescence and bone-fat imbalance of BMSCs. During osteogenetic differentiation in BMSCs, Wnt/β-catenin signalling functions positively. In the previous study, Bcl-3 was proved to interact with βcatenin and regulate Wnt signalling in colorectal tumour cells. 9 However, the potential correlations between Bcl-3 and Wnt /β-catenin in osteoporosis are unclear. Bcl-3 has been proven to activate the matrix metalloproteinase 1 expression in chondrocytes and synovial fibroblast, being the limited research on the skeletal system. 10 Our study revealed that loss of Bcl-3 led to bone loss, while the overexpression could be therapeutic, unveiling the substantial role of Bcl-3 in skeletal aging. Nevertheless, the investigations into BMSCs transcriptional alterations could reflect the regulative effects of Bcl-3 partially, calling for further explorations in the future.

A C K N O W L E D G E M E N T
We thank SHANGHAI OE BIOTECH to perform the RNA sequencing.