title: PHD/HIF-1 upregulates CA12 to protect against degenerative disc disease: a human sample, in vitro and ex vivo study.
abstract: Intervertebral disc degeneration is a major cause of low back pain. The nucleus pulposus (NP) is an important intervertebral disc component. Recent studies have shown that carbonic anhydrase 12 (CA12) is a novel NP marker. However, the mechanism by which CA12 is regulated and its physiological function are unclear. In our study, CA12, hypoxia-inducible factor 1α (HIF-1α) and HIF-2α expression levels were examined in 81 human degenerated NP samples using real-time RT-PCR, immunohistochemistry and western blot. Rat NP cells were cultured in a hypoxic environment, and hypoxia-induced CA12 expression was examined. Rat NP cells were treated with HIF-1α siRNA or the prolyl hydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG) to evaluate the role of PHD/HIF-1 in regulating CA12 expression. Rat NP cells were treated with CA12 siRNA to determine the function of CA12. A rat ex vivo model was established to confirm that PHD, HIF-1, and CA12 have important roles in disc degeneration. We found that CA12 was significantly downregulated in degenerated human NP samples at the mRNA and protein levels. CA12 expression sharply increased by ~30-fold in response to hypoxia. The expression of HIF-1α, but not HIF-2α, also decreased in degenerated human NP samples and was positively correlated with CA12 expression. HIF-1α knockdown under hypoxia reduced the CA12 mRNA and protein expression levels. DMOG treatment increased HIF-1α and CA12 expression. CA12 knockdown significantly inhibited anabolic protein expression, whereas catabolic enzymes remained unchanged. The ex vivo experiments supported our in vitro studies of the role of PHD/HIF-1/CA12. In conclusion, CA12 is downregulated in degenerated NPs, and its expression may be regulated by the PHD/HIF-1 axis. Decreased CA12 expression may lead to decreased extracellular matrix synthesis, which contributes to degenerative disc disease progression.
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Chen
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Xiang-Qian
authors.initials: XQ
authors.lastname: Fang
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Shao-Wei
authors.initials: SW
authors.lastname: Wang
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Zhi-Jun
authors.initials: ZJ
authors.lastname: Hu
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Zhi-Jie
authors.initials: ZJ
authors.lastname: Zhou
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Wen-Bing
authors.initials: WB
authors.lastname: Xu
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Ji-Ying
authors.initials: JY
authors.lastname: Wang
authors.affiliation: Department of Orthopaedics, Shanghai Key Laboratory of Orthopaedic Implant, Shanghai Ninth People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
authors.forename: An
authors.initials: A
authors.lastname: Qin
authors.affiliation: Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
authors.forename: Shun-Wu
authors.initials: SW
authors.lastname: Fan
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D015687:Cell Hypoxia / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D055785:Gene Knockdown Techniques; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D055959:Intervertebral Disc Degeneration / Q000235:genetics / Q000378:metabolism* / Q000517:prevention & control*; D008297:Male; D008875:Middle Aged; D000070614:Nucleus Pulposus / Q000166:cytology / Q000378:metabolism; D064828:Prolyl Hydroxylases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015854:Up-Regulation; D055815:Young Adult; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D002256:Carbonic Anhydrases; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D064828:Prolyl Hydroxylases; D012333:RNA, Messenger; D000099162:Endothelial PAS Domain-Containing Protein 1; C497442:HIF1A protein, human; C497444:Hif1a protein, rat; C477378:carbonic anhydrase XII