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 6

Degradation of FBN1 triggers persistent activation of TGF-β signaling, leading to BMSC exhaustion.

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Degradation of FBN1 triggers persistent activation of TGF-β signaling, l...
(A) Schematic diagram showing the ADAMTS4/FBN1/TGF-β/SMAD3/P21 signaling pathway. (B) Immunogold-sliver double-labeling of TGF-β and TGF-BR2 in femurs (n = 3). Scale bars: 500 nm and 100 nm (enlarged insets). (C) Immunofluorescence images of SMAD3 in femurs and quantitative data showing the relative fluorescence intensity of SMAD3 in the nucleus (n = 4). Scale bars: 80 μm and 20 μm (enlarged insets). (D) Western blot analysis and quantitative data of the levels of p-SMAD3, SMAD3, and P21 in femurs over a 1-day period (n = 3). (E) Western blot analysis and quantitative data for p-SMAD3/SMAD3 and P21 protein levels in femurs. Each regression curve refers to normalized mean densitometric values of a proteasome protein from the Western blot above. The curves refer to the average trend of diurnal changes in p-SMAD3 or P21 protein levels, which the sine wave model produced (n = 3). (F) Representative flow cytometry plot and quantitative data showing CD11b–CD45–Sca1+CD29+ BMSCs (n = 3). (G) Representative images of micro-CT reconstruction of femurs, with quantitative data shown. Bmal1fl/fl mice were injected with rAAV-ctrl or rAAV-Cdh5-Cre at 3 months of age, and then injected with IN-1130 at 5 months of age for 1 month (n = 6). (H) Representative images of micro-CT reconstruction of femurs from mice injected with IN-1130 or solvent for 1 month, with quantitative data shown. n = 7 (4 mo solvent), n = 6 (15 mo solvent, 15 mo IN-1130). (I) Western blot analysis of p-SMAD3, SMAD3, and P21 protein levels over a 1-day period in aged femurs treated with rAAV (n = 3). Shadow, night; yellow, day; ZT0, 8 am, light turned on. *P < 0.05, **P < 0.01, and ***P < 0.001. Data in C were analyzed by unpaired, 2-tailed t test with Welch’s correction; data in D and I were analyzed using DiscoRhythm (cosinor analysis); data in F, G, and H were analyzed by 1-way ANOVA with Tukey’s multiple-comparison test; and data in E were analyzed by 2-way ANOVA with multiple comparisons.