Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-β signaling in chondrocytes

Abstract

Objectives: Circadian rhythm (CR) was identified by RNA sequencing as the most dysregulated pathway in human osteoarthritis (OA) in articular cartilage. This study examined circadian rhythmicity in cultured chondrocytes and the role of the CR genes NR1D1 and BMAL1 in regulating chondrocyte functions.

Methods: RNA was extracted from normal and OA-affected human knee cartilage (n = 14 each). Expression levels of NR1D1 and BMAL1 mRNA and protein were assessed by quantitative PCR and immunohistochemistry. Human chondrocytes were synchronized and harvested at regular intervals to examine circadian rhythmicity in RNA and protein expression. Chondrocytes were treated with small interfering RNA (siRNA) for NR1D1 or BMAL1, followed by RNA sequencing and analysis of the effects on the transforming growth factor beta (TGF-β) pathway.

Results: NR1D1 and BMAL1 mRNA and protein levels were significantly reduced in OA compared to normal cartilage. In cultured human chondrocytes, a clear circadian rhythmicity was observed for NR1D1 and BMAL1. Increased BMAL1 expression was observed after knocking down NR1D1, and decreased NR1D1 levels were observed after knocking down BMAL1. Sequencing of RNA from chondrocytes treated with NR1D1 or BMAL1 siRNA identified 330 and 68 significantly different genes, respectively, and this predominantly affected the TGF-β signaling pathway.

Conclusions: The CR pathway is dysregulated in OA cartilage. Interference with circadian rhythmicity in cultured chondrocytes affects TGF-β signaling, which is a central pathway in cartilage homeostasis.

Keywords: BMAL1; Circadian rhythm; NR1D1; Osteoarthritis; TGF-β.

Figure 1. Expression of NR1D1 and BMAL1 mRNA and protein in cartilage

(A) qPCR was performed on 14 normal and 14 OA human articular cartilage samples (* p<0.05, ** p<0.01). (B) NR1D1 and BMAL1 Protein expression was analyzed by immunohistochemistry. Representative images are shown for young normal, aged normal and OA normal appearing and OA lesion area. Images are representative of 5–8 samples for each tissue type. NR1D1 and BMAL1 positive cells were observed predominantly in the superficial and upper-mid zone of normal cartilage. Protein expression was reduced in OA cartilage even in the normal areas, although strong expression was observed in the cluster cells. (C) Immunohistochemistry of mouse cartilage. 6 mouse joints for each sample type were analyzed. Bar graphs indicate percentages of positive cells. NR1D1 and BMAL1 positive cells were distributed mainly in the superficial to upper-mid zone. Expression levels were significantly reduced with aging and with induction of surgical OA.

Figure 2. Rhythmicity of NR1D1 and BMAL1 expression in cultured chondrocytes

(A) (B) Chondrocytes isolated from 5 individual donors were analyzed with and without dexamethasone (DEX) synchronization. qPCR and western blotting were performed on samples collected at 4 hour intervals. T0 indicates 24 hours after DEX was added to the culture medium. (A) NR1D1 mRNA expression peaked at T24 and T48, with lowest expression observed at T8 and T32. BMAL1 expression followed a reversed phase. (B) NR1D1 protein fluctuated in an identical pattern with the mRNA expression. BMAL1 protein expression had less fluctuation, with approximately 4 hours delayed phase from mRNA expression.

Figure 3. Effect of NR1D1 and BMAL1 knock down on expression of NR1D1 and BMAL1

(A) Western blotting was performed on cultured chondrocytes from 7 individual donors after siRNA transfection, collected at T12 and T24 after DEX synchronization. Effective knock down of both NR1D1 and BMAL1 was confirmed at both time points. Knocking down BMAL1 decreased the expression level of NR1D1, while knocking down NR1D1 had less effect on BMAL1 protein level. (B) qPCR was performed on samples after siRNA transfection, and RNA collected at time points as indicated. NR1D1 expression was reduced at all time points by knocking down NR1D1 or BMAL1, while still showing rhythmicity. BM AL1 expression was reduced and dysregulated by knocking down BMAL1, whereas knocking down NR1D1 resulted in increased expression levels of BMAL1 after T24.

Figure 4. Correlation of NR1D1 and BMAL1 knock down and TGF-β signaling

(A) qPCR for TGF-β signaling pathway genes was performed on cultured chondrocytes isolated from 3 individual donors after siRNA transfection for NR1D1 and BMAL1. (B) qPCR for NR1D1 and BMAL1 were performed on cultured chondrocytes isolated from 4 individual donors after TGF-β1 stimulation. TGF-β1 treatment significantly reduced NR1D1 expression and significantly increased BMAL1 expression. (C) qPCR was performed for ELN and TNC after siRNA transfection with and without TGF-β1 stimulation. (* p<0.05, ** p<0.01).