Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-β signaling in chondrocytes R. Akagi, Y. Akatsu,

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Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-β signaling in chondrocytes R. Akagi, Y. Akatsu, K.M. Fisch, O. Alvarez-Garcia, T. Teramura, Y. Muramatsu, M. Saito, T. Sasho, A.I. Su, M.K. Lotz Osteoarthritis and Cartilage Volume 25, Issue 6, Pages 943-951 (June 2017) DOI: 10.1016/j.joca.2016.11.007 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 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. Six 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. Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Rhythmicity of NR1D1 and BMAL1 expression in cultured chondrocytes. (A), (B) Chondrocytes isolated from five individual donors were analyzed with and without dexamethasone (DEX) synchronization. qPCR and western blotting were performed on samples collected at 4 h intervals. T0 indicates 24 h 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 h delayed phase from mRNA expression. Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Effect of NR1D1 and BMAL1 knock down on expression of NR1D1 and BMAL1. (A) Western blotting was performed on cultured chondrocytes from seven 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. BMAL1 expression was reduced and dysregulated by knocking down BMAL1, whereas knocking down NR1D1 resulted in increased expression levels of BMAL1 after T24. Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 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 three individual donors after siRNA transfection for NR1D1 and BMAL1. (B) qPCR for NR1D1 and BMAL1 were performed on cultured chondrocytes isolated from four 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). Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Supp. 1 Effect of NR1D1 and BMAL1 knock down on expression of CR genes. NR1D1 knock down increased BHLHE40 from T16 to T48, while having subtle effect on PER2, RORA, RORC, and RBX1. In contrast, BMAL1 knock down increased BHLHE40 at T8 and decreased its expression at T24, while the expression level of PER2 was reduced at T0, T24 and T36. RORA, RORC, and RBX1 were not significantly affected by BMAL1 knock down. Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Table S2 Differentially expressed genes after NR1D1 or BMAL1 knock down. RNA sequencing was performed on samples after siRNA transfection for NR1D1 and BMAL1. A large number of differentially expressed genes (n = 50) were altered by both siRNAs treatments. Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Table S3 Differentially expressed genes in TGF-β pathway after NR1D1 or BMAL1 knock down. RNA sequencing performed on samples after siRNA transfection for NR1D1 and BMAL1 revealed altered expression levels of TGF-β pathway genes. Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Supp. 4 Effect of NR1D1 and BMAL1 knock down on expression of TGF-β1 pathway genes. qPCR was performed for TGF-β pathway genes after siRNA transfection for NR1D1 and BMAL1 with and without TGF-β1 stimulation (*P < 0.05, **P < 0.01). NR1D1 knock down increased TGFBR1, TGFB1, TGFB2 and TGFB3, while reducing the expression level of TGFBR2. BMAL1 knock down increased TGFBR1 and TGFB3, while reducing TGFBR2 and TGFBR3. Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Supp. 5 Nuclear receptors expression in normal and OA cartilage. RNA sequencing revealed that NR1D1 expression was highest among the 48 nuclear receptors in both normal and OA cartilage. However, the expression level in OA cartilage was significantly lower than the expression level in normal cartilage. Osteoarthritis and Cartilage 2017 25, 943-951DOI: (10.1016/j.joca.2016.11.007) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions