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.

Figure 6. Disrupted circadian control of the NFATC2 pathway in the articular cartilage of Col2a1 Bmal1^–/– mice.

(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.

Figure 5. Dysregulated TGF-β signaling in Col2a1 Bmal1^–/– mouse cartilage.

(A and B) Representative IF images of p-SMAD2 and p-SMAD1/5 in mouse knee joint (n = 6 mice). Red shows positive staining; blue shows DAPI staining. The strong IF signals in the BM were due to nonspecific autofluorescence. Scale bars: 50 μm. (C) Quantification of the percentage of IF-positive cells. *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. Data represent the mean ± SEM (n = 6 mice). (D) qPCR quantification of mRNA levels of Ctgf, Serpine1, and Id3 in WT and Col2a1 Bmal1^–/– hip cartilage. Data represent the mean ± SEM and were normalized to WT at 5 am and expressed as relative fold changes (n = 4 animals/time point). *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed Student’s t test. (E) Western blotting of p-SMAD3 and p-SMAD1/5 in primary human articular chondrocytes after treatment with siRNA targeting BMAL1, with and without TGF-β stimulation (100 ng/ml). Scrambled siRNA was used as a control (data not shown). GAPDH was used as a loading control. Representative results are shown for 3 individuals. The knockdown of BMAL1 by siRNA was confirmed by Western blotting. The p-SMAD1/5 blot was derived from parallel samples run on a separate gel. (F) ALK1 and ALK5 levels were measured by qPCR in primary human articular chondrocytes following knockdown of BMAL1 (n = 3 individuals). *P < 0.05, by 2-tailed Student’s t test.

Figure 4. RNA-seq results from cartilage of 8- to 10-week-old WT and Col2a1 Bmal1^–/– littermates.

(A) Heatmap depicting expression levels and patterns of the genes identified as significantly different between WT and Col2a1 Bmal1^–/– cartilages. In total, 5,506 genes were dysregulated in KO cartilages at 1 or more of the 4 time points studied (log₂ fold change >1; adjusted P value < 0.05). White bars represent day; black bars represent night. (B) GO term analysis of the differentially expressed genes. Selective BMAL1-regulated pathways that are potentially relevant to cartilage homeostasis and OA were plotted against their significant levels (–log P value). A positive z-score suggests possible upregulation, while a negative z-score suggests possible downregulation. (C) Several classic BMAL1-regulated clock genes identified in this study were validated by qPCR in mouse cartilage tissue. Data represent the mean ± SEM and were normalized to WT at 5 am and are expressed as relative fold changes (n = 4 animals/time point). All genes tested followed the expression pattern identified by RNA-seq. **P < 0.01 and ***P < 0.001, by 2-tailed Student’s t test.

Figure 3. Progressive cartilage degeneration in a Col2a1 Bmal1^–/– mouse model.

(A) Representative images of Safranin O, fast green, and hematoxylin staining of knee joint articular cartilage from mice at various ages (n = 9/group). Arrows indicate degeneration and lesions. Scale bars: 50 μm. (B) Representative TUNEL staining of cartilage tissues from 2-month-old mice (n = 4). Arrows indicate cartilage damage sites. White lines denote the tide mark. Original magnification, ×20.

Figure 2. Deletion of chondrocyte Bmal1 leads to cartilage-specific loss of circadian rhythm.

(A) Representative BMAL1 IHC (brown) in WT (n = 3 animals/group) and Col2a1 Bmal1^–/– (n = 4) mouse knee joints. Graph shows quantification of BMAL1-positive cells. Data represent the mean ± SEM. Cells were counted across the entire tibial plateau of anatomically equivalent sections and are expressed as a percentage of WT littermates. ***P < 0.001, by 2-tailed Student’s t test. Scale bars: 100 μm. (B) PER2::luc bioluminescence recordings from SCN and peripheral tissues of WT and Col2a1 Bmal1^–/– littermates. n = 4 representative traces. cps, counts per second. (C) Wheel-running activity records of WT and Col2a1 Bmal1^–/– littermates under LD or DD conditions. Actograms were double plotted. Upper bar denotes a 12-hour light (gray)/12-hour dark (black) (LD) schedule. Shaded area denotes constant darkness (DD). Black vertical lines denote significant activity in 10-minute bins. Figure shows a representative sample from 1 littermate pair. The experiment was conducted with 6 pairs.

Figure 1. Reduction of BMAL1 expression in articular cartilage from human knees with OA.

(A) IHC of BMAL1 in full-depth human knee articular cartilage from non-OA (grade 0, G0), grade 2 (G2), or grade 4 (G4) OA samples. Arrows indicate cells with negative or very weak BMAL1 expression. Scale bars: 50 μm. Original magnification, ×3 (insets). (B) Quantification of the percentage of cells that stained positive for BMAL1. *P < 0.05 and **P < 0.01, by nonparametric Mann-Whitney U test. n = 6 individuals per group. (C) Representative Western blot of clinical cartilage samples with various OA scores: grade 0/1, non-OA or mild OA samples (n = 6); grade 2/3, moderate OA samples (n = 6); and grade 4, OA (n = 6). Age range, 45–60 years. For densitometric analysis, the band intensity of BMAL1 was normalized to GAPDH. The average of the G0/1 group was set at 1. *P < 0.05 and **P < 0.01; 2-way significance was calculated by nonparametric Mann-Whitney U test.