Circadian clock regulates granulosa cell autophagy through NR1D1-mediated inhibition of ATG5

• Published in: American Journal of Physiology: Cell Physiology 2022-02, Vol.322 (2), p.C231-C245
• Main Authors: Zhang, Jing, Zhao, Lijia, Li, Yating, Dong, Hao, Zhang, Haisen, Zhang, Yu, Ma, Tiantian, Yang, Luda, Gao, Dengke, Wang, Xiaoyu, Jiang, Haizhen, Li, Chao, Wang, Aihua, Jin, Yaping, Chen, Huatao
• Format: Article
• Language: English
• Subjects: Animals; Autophagy; Autophagy - physiology; Autophagy-Related Protein 5 - antagonists & inhibitors; Autophagy-Related Protein 5 - biosynthesis; Autophagy-Related Protein 5 - genetics; BMAL1 protein; Cells, Cultured; Circadian Clocks - physiology; Circadian rhythm; Circadian Rhythm - physiology; Circadian Rhythm Signaling Peptides and Proteins - biosynthesis; Circadian Rhythm Signaling Peptides and Proteins - genetics; Circadian rhythms; Electrophoretic mobility; Female; Gene expression; Gene regulation; Granulosa cells; Granulosa Cells - metabolism; Granulosa Cells - pathology; HEK293 Cells; Homeostasis; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nuclear Receptor Subfamily 1, Group D, Member 1 - biosynthesis; Nuclear Receptor Subfamily 1, Group D, Member 1 - genetics; Ovaries; Period 2 protein; Rapamycin; Regulatory sequences; Retinoic acid; Transcription
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00267.2021

Autophagy of granulosa cells (GCs) is involved in follicular atresia, which occurs repeatedly during the ovarian development cycle. Several circadian clock genes are rhythmically expressed in both rodent ovarian tissues and GCs. Nuclear receptor subfamily 1 group D member 1 (NR1D1), an important component of the circadian clock system, is involved in the autophagy process through the regulation of autophagy-related genes. However, there are no reports illustrating the role of the circadian clock system in mouse GC autophagy. In the present study, we found that core circadian clock genes ( , , , and ) and an autophagy-related gene ( ) exhibited rhythmic expression patterns across 24 h in mouse ovaries and primary GCs. Treatment with SR9009, an agonist of NR1D1, significantly reduced the expression of , , and in mouse GCs. ATG5 expression was significantly attenuated by SR9009 treatment in mouse GCs. Conversely, knockdown increased ATG5 expression in mouse GCs. Decreased NR1D1 expression at both the mRNA and protein levels was detected in the ovaries of mice, along with elevated expression of ATG5. Dual-luciferase reporter assay and electrophoretic mobility shift assay showed that NR1D1 inhibited transcription by binding to two putative retinoic acid-related orphan receptor response elements within the promoter. In addition, rapamycin-induced autophagy and ATG5 expression were partially reversed by SR9009 treatment in mouse GCs. Taken together, our current data demonstrated that the circadian clock regulates GC autophagy through NR1D1-mediated inhibition of ATG5 expression, and thus, plays a role in maintaining autophagy homeostasis in GCs.