title: Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-β signaling in chondrocytes.
abstract: 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.
  
  
  
  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 transforming growth factor beta (TGF-β) pathway.
  
  
  
  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.
  
  
  
  The CR 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.
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA; Department of Orthopaedic Surgery, School of Medicine, Chiba University, 1-8-1, Inohana, Chuou, Chiba, 260-8677, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Akagi
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA; Department of Orthopaedic Surgery, School of Medicine, Chiba University, 1-8-1, Inohana, Chuou, Chiba, 260-8677, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Akatsu
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Fisch
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA.
authors.forename: O
authors.initials: O
authors.lastname: Alvarez-Garcia
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA.
authors.forename: T
authors.initials: T
authors.lastname: Teramura
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Muramatsu
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA; Department of Orthopaedic Surgery, Toho University Sakura Medical Center, 564-1 Shimoshizu, Sakura, Chiba, 285-8741, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Saito
authors.affiliation: Department of Orthopaedic Surgery, School of Medicine, Chiba University, 1-8-1, Inohana, Chuou, Chiba, 260-8677, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Sasho
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA.
authors.forename: A I
authors.initials: AI
authors.lastname: Su
authors.affiliation: Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, USA. Electronic address: mlotz@scripps.edu.
authors.forename: M K
authors.initials: MK
authors.lastname: Lotz
mesh_terms: D056930:ARNTL Transcription Factors / Q000235:genetics* / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D002358:Cartilage, Articular / Q000378:metabolism*; D019902:Chondrocytes / Q000378:metabolism*; D002940:Circadian Rhythm / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D008297:Male; D008875:Middle Aged; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1 / Q000235:genetics* / Q000378:metabolism; D020370:Osteoarthritis, Knee / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D015398:Signal Transduction; D016212:Transforming Growth Factor beta / Q000235:genetics*; D055815:Young Adult
chemical_list: D056930:ARNTL Transcription Factors; C538960:BMAL1 protein, human; C064353:NR1D1 protein, human; D056953:Nuclear Receptor Subfamily 1, Group D, Member 1; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta
keywords: BMAL1; Circadian rhythm; NR1D1; Osteoarthritis; TGF-β