title: Nkx3.2-induced suppression of Runx2 is a crucial mediator of hypoxia-dependent maintenance of chondrocyte phenotypes.
abstract: Hypoxia is a key factor in the maintenance of chondrocyte identity. However, crucial chondrogenic transcription factors in the Sox families are not activated in this phenomenon, indicating that other pathways are involved. Nkx3.2 is a well-known chondrogenic transcription factor induced by Sonic hedgehog (Shh); it suppresses a key osteogenic transcriptional factor, Runt-related transcription factor 2 (Runx2), to maintain the chondrogenic phenotype in mesenchymal lineages. The purpose of this study was to examine the function of Nkx3.2 in hypoxia-dependent maintenance of chondrocyte identity. C3H10T1/2 pluripotent mesenchymal cells were cultured with rh-BMP2 (300 ng/ml) to induce chondrogenesis under normoxic (20% O(2)) or hypoxic (5% O(2)) conditions. Immunohistological detection of Nkx3.2 in a micromass cell culture system revealed that hypoxia promoted expression of the Nkx3.2 protein. Real-time RT-PCR analysis revealed that hypoxia promoted Nkx3.2 mRNA expression and suppressed Runx2 mRNA expression; however, Sox9 mRNA expression was not altered by oxygen conditions, as previously described. Over-expression of exogenous Nkx3.2 promoted glycosaminoglycan (GAG) production and inhibited Runx2 mRNA expression and, based on a dual luciferase assay, Runx2 promoter activity. Interestingly, downregulation of Nkx3.2 using RNAi abolished hypoxia-dependent GAG production and restored Runx2 mRNA expression and promoter activity. These results demonstrated that Nkx3.2-dependent suppression of Runx2 was a crucial factor in hypoxia-dependent maintenance of chondrocyte identity.
authors.affiliation: Department of Orthopaedics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Kawato
authors.forename: Makoto
authors.initials: M
authors.lastname: Hirao
authors.forename: Kosuke
authors.initials: K
authors.lastname: Ebina
authors.forename: Noriyuki
authors.initials: N
authors.lastname: Tamai
authors.forename: Kenrin
authors.initials: K
authors.lastname: Shi
authors.forename: Jun
authors.initials: J
authors.lastname: Hashimoto
authors.forename: Hideki
authors.initials: H
authors.lastname: Yoshikawa
authors.forename: Akira
authors.initials: A
authors.lastname: Myoui
mesh_terms: D000693:Anaerobiosis; D000818:Animals; D002460:Cell Line; D019902:Chondrocytes / Q000378:metabolism / Q000502:physiology*; D020219:Chondrogenesis / Q000235:genetics*; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics*; D015536:Down-Regulation; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D010100:Oxygen / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D015854:Up-Regulation
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D018398:Homeodomain Proteins; C107965:Nkx3-2 protein, mouse; D012333:RNA, Messenger; C493716:Runx2 protein, mouse; D014157:Transcription Factors; D010100:Oxygen