Expression and activity of Runx2 mediated by hyaluronan during chondrocyte differentiation.
During endochondral ossification, the production of hyaluronan (HA) is strictly and selectively regulated by chondrocytes, with a temporal peak at the hypertrophic stage. This study was conducted to clarify the effects of HA on expression and activity of runt-related gene 2 (Runx2), a potent transcription factor for chondrocyte differentiation in hypertrophic chondrocytes. Immature chondrocytes from an ATDC5 cell line were cultured and differentiated in DMEM/Ham's F12 with pre-defined supplements. Using real-time PCR, the gene expressions of type II collagen, MMP-13, HAS2, and Runx2 in cultured chondrocytes were analysed from days 0 to 18 of cell differentiation. The activity and expression of Runx2 in hypertrophic chondrocytes were analysed after the treatment with HA oligosaccharide (HAoligo) using AML-3/Runx2 binding, real-time PCR and Western blot analysis. The effects of pre-incubation of anti-CD44 antibody on Runx2 expression were also examined. Expression of type X collagen and Runx2 mRNAs reached a maximum at the terminal differentiation of chondrocytes. The activity and expression of Runx2 was significantly inhibited in hypertrophic chondrocytes treated with HAoligo compared to the untreated controls. High molecular weight-HA did not affect the expression or activity of Runx2. The expression of Runx2 mRNA was significantly decreased in hypertrophic chondrocytes treated with anti-CD44 antibody. These results suggest that HAoligo may affect the terminal differentiation of chondrocytes during the endochondral ossification by inhibiting the expression and activity of Runx2.
Tanne
Y
Y
Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Minami-Ku, Hiroshima 734-8553, Japan. yukitann@hiroshima-u.ac.jp
Tanimoto
K
K
Tanaka
N
N
Ueki
M
M
Lin
Y Y
YY
Ohkuma
S
S
Kamiya
T
T
Tanaka
E
E
Tanne
K
K
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D045744:Cell Line, Tumor / Q000187:drug effects / Q000502:physiology; D019902:Chondrocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D024121:Collagen Type X / Q000235:genetics / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism; D018960:Hyaluronan Receptors / Q000378:metabolism; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000494:pharmacology*; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D012313:RNA / Q000378:metabolism; D000077278:RNA, Mitochondrial; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D024043:Collagen Type II; D024121:Collagen Type X; D050659:Core Binding Factor Alpha 1 Subunit; D018960:Hyaluronan Receptors; D000077278:RNA, Mitochondrial; C493709:RUNX2 protein, human; D014157:Transcription Factors; D012313:RNA; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; C101754:HAS2 protein, human; D000076002:Hyaluronan Synthases; D053509:Matrix Metalloproteinase 13