The circadian clock in mammals temporally coordinates physiological and behavioural processes to anticipate daily rhythmic changes in their environment. Chronic disruption to circadian rhythms (e.g., through ageing or shift work) is thought to contribute to a multitude of diseases, including degeneration of the musculoskeletal system. The intervertebral disc (IVD) in the spine contains circadian clocks which control ∼6% of the transcriptome in a rhythmic manner, including key genes involved in extracellular matrix (ECM) homeostasis. However, it remains largely unknown to what extent the local IVD molecular clock is required to drive rhythmic gene transcription and IVD physiology. In this work, we identified profound age-related changes of ECM microarchitecture and an endochondral ossification-like phenotype in the annulus fibrosus (AF) region of the IVD in the Col2a1-Bmal1 knockout mice. Circadian time series RNA-Seq of the whole IVD in Bmal1 knockout revealed loss of circadian patterns in gene expression, with an unexpected emergence of 12 h ultradian rhythms, including FOXO transcription factors. Further RNA sequencing of the AF tissue identified region-specific changes in gene expression, evidencing a loss of AF phenotype markers and a dysregulation of ECM and FOXO pathways in Bmal1 knockout mice. Consistent with an up-regulation of FOXO1 mRNA and protein levels in Bmal1 knockout IVDs, inhibition of FOXO1 in AF cells suppressed their osteogenic differentiation. Collectively, these data highlight the importance of the local molecular clock mechanism in the maintenance of the cell fate and ECM homeostasis of the IVD. Further studies may identify potential new molecular targets for alleviating IVD degeneration.

哺乳动物的生物钟在时间上协调生理和行为过程，以预测其环境中的每日节律变化。昼夜节律的慢性破坏（例如，由于衰老或轮班工作）被认为会导致多种疾病，包括肌肉骨骼系统的退化。脊柱中的椎间盘 (IVD) 包含生物钟，以有节奏的方式控制约 6% 的转录组，其中包括参与细胞外基质 (ECM) 稳态的关键基因。然而，目前仍不清楚局部 IVD 分子时钟在多大程度上需要驱动节律性基因转录和 IVD 生理学。在这项工作中，我们在Col2a1 - Bmal1敲除小鼠中发现了 ECM 微结构与年龄相关的深刻变化，以及 IVD 纤维环 (AF) 区域的软骨内骨化样表型。Bmal1敲除中整个 IVD 的昼夜节律时间序列 RNA-Seq显示基因表达的昼夜节律模式丢失，意外出现 12 小时超昼夜节律，包括 FOXO 转录因子。对 AF 组织的进一步 RNA 测序发现了基因表达的区域特异性变化，证明Bmal1敲除小鼠中 AF 表型标记物的丢失以及 ECM 和 FOXO 通路的失调。与Bmal1敲除 IVD中 FOXO1 mRNA 和蛋白质水平上调一致，抑制 AF 细胞中的 FOXO1 抑制了其成骨分化。总的来说，这些数据强调了局部分子时钟机制在维持 IVD 细胞命运和 ECM 稳态中的重要性。进一步的研究可能会确定减轻 IVD 退化的潜在新分子靶点。