Background/Purpose

NR1D1 is a negative regulator of BMAL1, a core clock gene that regulate circadian rhythmicity. Involvement of NR1D1 in inflammatory process has been reported, but data regarding its function in articular cartilage is limited. Since IL-1β induced inflammation is one of the key factors that disturb cartilage homeostasis and lead to cartilage degradation, we examined the function of NR1D1 in articular cartilage in relation to IL-1β stimulation.

Methods

RNA was extracted from human cartilage tissues harvested from normal and osteoarthritis (OA) knees (n=15 each), and expression levels of NR1D1 and BMAL1 mRNA were assessed by quantitative PCR. NR1D1 protein expression was confirmed by immunohistochemistry in normal and OA human cartilage, as well as in normal knees and knees with surgically induced OA of mice. To examine the circadian rhythmicity of gene expression in cultured chondrocytes isolated from normal human cartilage, chondrocytes were synchronized by dexamethasone and harvested at 4-hour intervals up to 48 hours for RNA and protein extraction. Chondrocytes were then treated with small interfering RNA (siRNA) for NR1D1 or BMAL1, followed by IL-1β stimulation to test the effect of knock down on response to IL-1β.

Results

Both NR1D1 and BMAL1 mRNA levels were significantly reduced in OA cartilage, compared to normal cartilage. NR1D1 protein was predominantly expressed in the superficial and upper-mid zone of normal cartilage, both in human and in mouse knees. The protein expression was reduced in OA cartilage, although high expression was observed in cluster cells. In surgically induced mice OA, NR1D1 protein expression was significantly reduced before cartilage loss occurred. In cultured human chondrocytes, a clear circadian rhythmicity was observed for NR1D1 and BMAL1 mRNA levels. NR1D1 expression was at its lowest level at T12 and T36, whereas highest expression was observed at T24 and T48. The expression pattern of BMAL1 displayed the reversed pattern. Treatment with siRNA significantly suppressed levels of both genes at all time points, but the rhythmic expression pattern was preserved. Increase in BMAL1 expression was observed at T24 and T48 after knocking down NR1D1, and decreased NR1D1 levels were observed at all time points after knocking down BMAL1. IL-1β treatment significantly induced IL6, COX2, iNOS, MMP13 and ADAMTS4. NR1D1 knock down further increased the expression levels of iNOS, MMP13 and ADAMTS4, while by contrast the IL-1 induction of IL6 and COX2 was blunted. Genome-wide sequencing of RNA from chondrocytes treated with NR1D1 siRNA identified 330 genes that were significantly different and this affected predominantly TGFβ signaling pathway and protein processing in endoplasmic reticulum (ER), as well as ER stress response.

Conclusion

NR1D1 and BMAL1 expression are reduced in OA cartilage. NR1D1 and BMAL1 present circadian rhythmicity in cultured chondrocytes with an opposite phase, indicating a bidirectional regulation between the two genes. Reduced expression of NR1D1 in chondrocyte leads to altered response to IL-1β stimulation, affects TGFβ signaling and ER function, thus suggesting an important role of NR1D1 in cartilage homeostasis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/reduced-expression-of-circadian-rhythm-genes-in-human-osteoarthritis-cartilage-nr1d1-suppression-alters-chondrocyte-response-to-il-1%ce%b2-stimulation/