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 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 circadian rhythm 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.

Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice

“…More importantly, the phosphorylated Smad2/3 and Serpine1 expressions are also significantly reduced in Bmal1 cKO mice. While, previous reports showed that TGF-β signaling could also upregulate the Bmal1 mRNA expression in human articular cartilage 47 , whether circadian rhythm is involved in the lesions of Alk5 cKO mice needs to be studied.…”

Osteoarthritis year in review 2017: biology

“…SIRT1 was also linked to circadian rhythm by Yang et al 43 , showing that siRNAmediated knockdown or pharmacologic inhibition of SIRT1 in chondrocytes results in lower levels of transcripts for the clock gene Bmal1. It was already known that loss of Bmal1 increases cartilage damage and disrupted circadian rhythm in chondrocytes 44 , as highlighted in last year's Year in Review 45 . Interestingly, similar methods used to knock down Bmal1 expression reciprocally reduced SIRT1 expression.…”

Timing metabolism in cartilage and bone: links between circadian clocks and tissue homeostasis

“…Analysis of gene expression patterns in human cartilage revealed that circadian rhythm pathways were among the most disrupted in OA patients (Akagi et al 2017, Soul et al 2018. Several studies have also demonstrated that expression of BMAL1/BMAL1 is significantly reduced in human osteoarthritic cartilage (Dudek et al 2016, Snelling et al 2016, Yang et al 2016a, Akagi et al 2017. Knockdown of BMAL1 in human chondrocytes altered TGF-β signalling and was associated with increased expression of catabolic factors (e.g.…”

“…Knockdown of BMAL1 in human chondrocytes altered TGF-β signalling and was associated with increased expression of catabolic factors (e.g. MMP1, MMP3, MMP13 and ADAMTS5) (Snelling et al 2016, Yang et al 2016a, Akagi et al 2017, Khurana et al 2019.…”