Proper expression of helix-loop-helix protein Id2 is important to chondrogenic differentiation of ATDC5 cells.
The process of chondrogenesis can be mimicked in vitro by insulin treatment of mouse ATDC5 chondroprogenitor cells. To identify novel factors that are involved in the control of chondrogenesis, we carried out a large-scale screening through retroviral insertion mutagenesis and isolated a fast-growing ATDC5 clone incapable of chondrogenic differentiation. Inverse-PCR analysis of this clone revealed that the retroviral DNA was inserted into the promoter region of mouse Id2 (inhibitor of DNA-binding protein 2) gene. This retroviral insertion increased Id2 protein levels to twice those found in normal ATDC5 cells. To investigate whether an elevated level of Id2 protein was responsible for inhibition of chondrogenic differentiation, ATDC5 cells were infected with a retrovirus to stably express Id2. ATDC5 cells expressing ectopic Id2 exhibited signs of de-differentiation, such as rapid growth, and insulin failed to induce expression of Sox9 (Sry-type high-mobility-group box 9) or matrix genes such as type II collagen (COL2) in these cells. When endogenous Id2 was knocked down by siRNA (small interfering RNA) in ATDC5 cells, expression of Sox9 and COL2 was increased and chondrogenic differentiation was accelerated. To examine how Id2 is expressed in chondrocytes in vivo, we carried out immunostaining of E16.5 mouse embryos and found that Id2 is expressed in articular chondrocytes and proliferating chondrocytes, but barely detectable in hypertrophic chondrocytes. Our results suggest that proper expression of Id2 is important to achieving a fine balance between growth and differentiation during chondrogenesis.
Yang
Liu
L
Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, U.S.A. lyang@u.washington.edu
Ma
Xiaoyun
X
Lyone
Anne
A
Zou
Junhui
J
Blackburn
Michael L
ML
Pan
Jing
J
Yang
Dingqiao
D
Matsushita
Hiroshi
H
Mei
Bin
B
Zielinska-Kwiatkowska
Anna
A
Chansky
Howard A
HA
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D020219:Chondrogenesis / Q000187:drug effects; D002999:Clone Cells; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D006132:Growth Plate / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2 / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D009154:Mutation / Q000235:genetics; D034741:RNA, Small Interfering / Q000378:metabolism; D012190:Retroviridae; D055758:SOX9 Transcription Factor / Q000378:metabolism
D051792:Basic Helix-Loop-Helix Proteins; D024043:Collagen Type II; D051797:Inhibitor of Differentiation Protein 2; D007328:Insulin; D034741:RNA, Small Interfering; D055758:SOX9 Transcription Factor; C497452:Idb2 protein, mouse; C527933:Sox9 protein, mouse