The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity
Michal Dudek, Nicole Gossan, Nan Yang, Hee-Jeong Im, Jayalath P.D. Ruckshanthi, Hikari Yoshitane, Xin Li, Ding Jin, Ping Wang, Maya Boudiffa, Ilaria Bellantuono, Yoshitaka Fukada, Ray P. Boot-Handford, Qing-Jun Meng
Michal Dudek, Nicole Gossan, Nan Yang, Hee-Jeong Im, Jayalath P.D. Ruckshanthi, Hikari Yoshitane, Xin Li, Ding Jin, Ping Wang, Maya Boudiffa, Ilaria Bellantuono, Yoshitaka Fukada, Ray P. Boot-Handford, Qing-Jun Meng
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Research Article Aging Bone biology

The chondrocyte clock gene Bmal1 controls cartilage homeostasis and integrity

Abstract

Osteoarthritis (OA) is the most prevalent and debilitating joint disease, and there are currently no effective disease-modifying treatments available. Multiple risk factors for OA, such as aging, result in progressive damage and loss of articular cartilage. Autonomous circadian clocks have been identified in mouse cartilage, and environmental disruption of circadian rhythms in mice predisposes animals to OA-like damage. However, the contribution of the cartilage clock mechanisms to the maintenance of tissue homeostasis is still unclear. Here, we have shown that expression of the core clock transcription factor BMAL1 is disrupted in human OA cartilage and in aged mouse cartilage. Furthermore, targeted Bmal1 ablation in mouse chondrocytes abolished their circadian rhythm and caused progressive degeneration of articular cartilage. We determined that BMAL1 directs the circadian expression of many genes implicated in cartilage homeostasis, including those involved in catabolic, anabolic, and apoptotic pathways. Loss of BMAL1 reduced the levels of phosphorylated SMAD2/3 (p-SMAD2/3) and NFATC2 and decreased expression of the major matrix-related genes Sox9, Acan, and Col2a1, but increased p-SMAD1/5 levels. Together, these results define a regulatory mechanism that links chondrocyte BMAL1 to the maintenance and repair of cartilage and suggest that circadian rhythm disruption is a risk factor for joint diseases such as OA.

Authors

Michal Dudek, Nicole Gossan, Nan Yang, Hee-Jeong Im, Jayalath P.D. Ruckshanthi, Hikari Yoshitane, Xin Li, Ding Jin, Ping Wang, Maya Boudiffa, Ilaria Bellantuono, Yoshitaka Fukada, Ray P. Boot-Handford, Qing-Jun Meng

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Figure 6

Disrupted circadian control of the NFATC2 pathway in the articular cartilage of Col2a1 Bmal1–/– mice.

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Disrupted circadian control of the NFATC2 pathway in the articular carti...
(A and B) Representative IHC images of NFATC2 and SOX9 in mouse knee joint (n = 6 mice). Scale bars: 50 μm. (C) Quantification of the percentage of IF-positive cells. Data represent the mean ± SEM (n = 6 mice). *P < 0.05 and **P < 0.01, by 2-tailed Student’s t test; 2-way significance was calculated by nonparametric Mann-Whitney U test. (D) qPCR quantification of Nfatc2, Sox9, Acan, and Col2a1 in WT and KO hip cartilage. Data represent the mean ± SEM (n = 4 mice). *P < 0.05 and ***P < 0.001, by 2-tailed Student’s t test. (E) Binding of CLOCK or BMAL1 to the E-box–containing region of the Nfatc2 gene was evaluated in WT and Bmal1-cKO femoral head cartilage using CLOCK- or BMAL1-specific ChIP, followed by qPCR. IgG served as a negative control. Data (percentage of input) were normalized to their respective IgG control and are expressed as the mean ± SEM. **P < 0.01, by 2-tailed Student’s t test. (F) Effects of CLOCK and BMAL1 overexpression on WT or E-box–mutant (MUT) Nfatc2::luc activity. Data represent the mean ± SEM and were normalized to pCDNA3.1 control. Avp::luc reporter (a classical E-box–regulated promoter) served as a positive control. ***P < 0.001, by 2-tailed Student’s t test. (G) qPCR quantification of BMAL1 and NFATC2 mRNA levels in primary human articular chondrocytes (n = 3 individuals) after treatment with siRNA targeting BMAL1. Scrambled siRNA was used as a control. ***P < 0.001, by 2-tailed Student’s t test.