Clock Gene Bmal1 Modulates Human Cartilage Gene Expression by Crosstalk With Sirt1

Abstract

The critical regulation of the peripheral circadian gene implicated in osteoarthritis (OA) has been recently recognized; however, the causative role and clinical potential of the peripheral circadian rhythm attributable to such effects remain elusive. The purpose of this study was to elucidate the role of a circadian gene Bmal1 in human cartilage and pathophysiology of osteoarthritis. In our present study, the mRNA and protein levels of circadian rhythm genes, including nicotinamide adenine dinucleotide oxidase (NAD(+)) and sirtuin 1 (Sirt1), in human knee articular cartilage were determined. In OA cartilage, the levels of both Bmal1 and NAD(+) decreased significantly, which resulted in the inhibition of nicotinamide phosphoribosyltransferase activity and Sirt1 expression. Furthermore, the knockdown of Bmal1 was sufficient to decrease the level of NAD(+) and aggravate OA-like gene expression changes under the stimulation of IL-1β. The overexpression of Bmal1 relieved the alteration induced by IL-1β, which was consistent with the effect of the inhibition of Rev-Erbα (known as NR1D1, nuclear receptor subfamily 1, group D). On the other hand, the transfection of Sirt1 small interfering RNA not only resulted in a reduction of the protein expression of Bmal1 and a moderate increase of period 2 (per2) and Rev-Erbα but also further exacerbated the survival of cells and the expression of cartilage matrix-degrading enzymes induced by IL-1β. Overexpression of Sirt1 restored the metabolic imbalance of chondrocytes caused by IL-1β. These observations suggest that Bmal1 is a key clock gene to involve in cartilage homeostasis mediated through sirt1 and that manipulating circadian rhythm gene expression implicates an innovative strategy to develop novel therapeutic agents against cartilage diseases.

Figure 1.

Expression of the key genes of circadian rhythm in human articular cartilage tissue in OA patients. OA cartilage tissues were obtained from the knee joint of patients with or without OA. A, The hematoxylin and eosin (H&E) and Safranin O stainings were performed on articular cartilage. B, The remarkable changes of cartilage genes related to synthetic metabolism and catabolism were examined by real-time PCR, including the col2a1, aggrecan, MMP13, and ADAMTS5 in the knee joint of the patients. C, The protein levels of col2a1, aggrecan, MMP13, and ADAMTS5 were examined by Western blot. D, The expression levels of the key circadian rhythm genes, for example, Bmal1, per1/2, cry1/2, and Rev-erbα were determined by real-time PCR. Bars represent a mean and SE of n = 3–6/group.

Figure 2.

Bmal1 expression level was related with the Sirt1 activity, which is influenced by the level of NAD⁺. A, The representative IHC images of Sirt1 and Bmal1 in patients' knee joints. B, The expression levels of Bmal1 were determined by real-time PCR and Western blot. C, The level of NAD⁺ in knee joint tissue was detected by a colorimetric NAD/NADH quantification kit. D, The expression of NAMPT and Sirt1 was examined by Western blot and real-time PCR. Bars represent a mean and SE of n = 3–6/group.

Figure 3.

Knockdown of Bmal1 inhibited the matrix proteoglycan synthesis in chondrocytes, which was further aggravated by IL-1β. A, The proliferation of chondrocytes was determined using the CCK-8 assay after transfection with Bmal1 siRNA. B and C, Representative images of alcian blue staining and the levels of alcian blue-stained proteoglycans were detected by OD values at 620 nm. D, The chondrocytes were treated with IL-1β for 24 hour after the transfection of Bmal1 siRNA. Quantitative results for col2a1 and aggrecan in chondrocytes were measured by real-time PCR after transfection with Bmal1 siRNA under stimulation with IL-1β (10 ng/mL). E, The expression of MMP1, MMP3, MMP13, and ADAMT5 in chondrocytes was measured by real-time PCR after transfection with Bmal1 siRNA under stimulation of IL-1β (10 ng/mL). F, The representative Western blots of the col2a1 and aggrecan, MMP 13, and ADAMTS5 after transfection with Bmal1 siRNA under stimulation of IL-1β. Bars represent a mean and SE of n = 5/group. control, control siRNA; siBmal1, Bmal1siRNA.

Figure 4.

Overexpression of Bmal1 neutralized the metabolic imbalance of chondrocytes induced by IL-1β. The chondrocytes were treated with IL-1β for 24 hours after transfection with the Bmal1 plasmid. A and B, The representative images of the chondrocyte transfection with the Bmal1 plasmid, and the transfection efficiency was evaluated by real-time PCR and Western blot. C, The quantitative results for col2a1 and aggrecan in chondrocytes transfected with Bmal1 under stimulation with IL-1β was determined by real-time PCR. D, Quantitative results for the expression levels of MMP13 and ADAMTS5 were measured by real-time PCR. Bars represent a mean and SE of n = 5/group. control, control plasmid; plaBmal1, Bmal1 plasmid.

Figure 5.

Effects of Sirt1 siRNA on chondrocytes under stimulation with IL-1β. The chondrocytes were treated with IL-1β for 24 hours after the transfection with Sirt1 siRNA. A, Western blot for Sirt1 and representative data were from repeated experiments. B, Western blots for Bmal1, Per2, and Rev-erbα after Sirt1 siRNA treatment. C, The proliferation of chondrocytes was determined using the CCK-8 assay after the Sirt1 silence or IL-1β treatment. D, Real-time PCR for MMP1, MMP3, MMP13, and ADAMTS5 mRNA in chondrocytes. The values were at a relative expression level to the control expression. E, Western blots for MMP13 and ADAMTS 5 after Sirt1 siRNA treatment. Bars represent a mean and SE of n = 5/group. control, control siRNA; siBmal1, Sirt1 siRNA.

Figure 6.

EX527, a Sirt1 inhibitor, influenced the rhythmic expression levels of Bmal1, per2, and Rev-erbα. A, After 2 hours of EX527 (100 μM) treatment and then at the first 8 hours, the Bmal1 was stained by immunofluorescence. B, The protein expression of Bmal1, Per2, and Rev-erbα was detected after 2 hours of EX527 treatment. Samples were collected every 4 hours for 24 hours. C–E, The circadian rhythms of Bmal1, Per2, and Rev-erbα were detected after 2 hours of EX527 treatment. Samples were collected every 4 hours for 48 hours. Bars represent a mean and SE of n = 5/group.

Figure 7.

Overexpression of Sirt1 restored the metabolic imbalance of chondrocytes caused by IL-1β. The chondrocytes were treated with IL-1β for 24 hours after the transfection with the Sirt1 plasmid. A, Western blot for Sirt1 and representative data from repeated experiments. B, The proliferation of chondrocytes was determined by the CCK-8 assay. C, The expression of MMP1, MMP3, MMP13, and ADAMTS5 mRNA in chondrocytes was measured by real-time PCR after transfection with the Sirt1 plasmid under stimulation with IL-1β (10 ng/mL). The values were at a relative expression level to the control expression. D, Western blots for MMP13 and ADAMTS5. Bars represent a mean and SE of n = 5/group. control, control plasmid; plaSirt1, Sirt1 plasmid.