Endothelial BMAL1 decline during aging leads to bone loss by destabilizing extracellular fibrillin-1
Ying Yin, … , Luoying Zhang, Lili Chen
Ying Yin, … , Luoying Zhang, Lili Chen
Published December 16, 2024
Citation Information: J Clin Invest. 2024;134(24):e176660. https://doi.org/10.1172/JCI176660.
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Research Article Aging Bone biology Article has an altmetric score of 2

Endothelial BMAL1 decline during aging leads to bone loss by destabilizing extracellular fibrillin-1

Abstract

The occurrence of aging is intricately associated with alterations in circadian rhythms that coincide with stem cell exhaustion. Nonetheless, the extent to which the circadian system governs skeletal aging remains inadequately understood. Here, we noticed that skeletal aging in male mice was accompanied by a decline in a core circadian protein, BMAL1, especially in bone marrow endothelial cells (ECs). Using male mice with endothelial KO of aryl hydrocarbon receptor nuclear translocator–like protein 1 (Bmal1), we ascertained that endothelial BMAL1 in bone played a crucial role in ensuring the stability of an extracellular structural component, fibrillin-1 (FBN1), through regulation of the equilibrium between the extracellular matrix (ECM) proteases thrombospondin type 1 domain–containing protein 4 (THSD4) and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), which promote FBN1 assembly and breakdown, respectively. The decline of endothelial BMAL1 during aging prompted excessive breakdown of FBN1, leading to persistent activation of TGF-β/SMAD3 signaling and exhaustion of bone marrow mesenchymal stem cells. Meanwhile, the free TGF-β could promote osteoclast formation. Further analysis revealed that activation of ADAMTS4 in ECs lacking BMAL1 was stimulated by TGF-β/SMAD3 signaling through an ECM-positive feedback mechanism, whereas THSD4 was under direct transcriptional control by endothelial BMAL1. Our investigation has elucidated the etiology of bone aging in male mice by defining the role of ECs in upholding the equilibrium within the ECM, consequently coordinating osteogenic and osteoclastic activities and retarding skeletal aging.

Authors

Ying Yin, Qingming Tang, Jingxi Yang, Shiqi Gui, Yifan Zhang, Yufeng Shen, Xin Zhou, Shaoling Yu, Guangjin Chen, Jiwei Sun, Zhenshuo Han, Luoying Zhang, Lili Chen

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Figure 4

Regulation of BMSCs senescence by endothelial BMAL1 is mediated by the matrix proteases THSD4 and ADAMTS4.

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Regulation of BMSCs senescence by endothelial BMAL1 is mediated by the m...
(A) Volcano plot showing the DEGs in ECs sorted from Bmal1-KD and control mice (n = 3). (B) Gene Ontology (GO) cellular component (CC) analysis of DEGs between Bmal1-KD and control ECs. (C) Logic diagram of screening candidate genes of secretory proteins that have relatively higher expression in ECs than other cells in bone tissue, according to the Human Protein Atlas. Genes are listed in Supplemental Table 5. (D) Confirmation of the candidate genes by qPCR (n = 3). (E) Representative SA–β-gal staining (blue), EdU immunofluorescence, and ARS staining of BMSCs that were indirectly cocultured with EC (n = 3). Scale bars: 20 μm, 50 μm, and 80 μm. (F) Representative images of FISH THSD4 and immunofluorescence staining for CD31 or ADAMTS4 (n = 3). Scale bars: 100 μm and 10 μm (insets). (G) Western blot analysis of the relative levels of THSD4 and ADAMTS4 in femurs over a 1-day period with quantitative data shown (n = 3). (H) Western blot analysis of BMAL1, THSD4, and ADAMTS4 protein levels at the indicated time points in femurs, with the corresponding heatmap shown (n = 3). (I) Schematic diagram of animal experiments with Bmal1fl/fl mice injected with rAAV-ctrl, rAAV-Cdh5-Cre, and rAAV-Thsd4, or the ADAMTS4 inhibitor. (J) Representative images of micro-CT reconstruction of femurs corresponding to the samples in I, with quantitative data shown (n = 6). BV/TV, bone volume over total volume; Tb.N, trabecular number. (K) Representative flow cytometry plot showing CD11b–CD45–Sca1+CD29+ BMSCs in bone marrow corresponding to the samples in I, with quantitative data shown. Shadow, night; yellow, day; ZT0, 8 am, light turned on. n = 4. *P < 0.05, **P < 0.01, and ***P < 0.001. Data in D were analyzed by unpaired, 2-tailed t test with Welch’s correction; data in G were analyzed using DiscoRhythm (cosinor analysis); and data in J and K were analyzed by 1-way ANOVA with Tukey’s multiple-comparison test.