Transcription Factor Dmrt2 Forwards Endochondral Ossification by Inhibiting Early Stage and
Promoting Late Stage of Endochondral Ossification
+1,2
Ono, K; 1Hata, K; 1Nakamura, E; 1 Sugita, A; 1Amano, K; 1 Takigawa, Y; 1 Nakanishi, M; 1 Nishimura, R; 2 Takenoshita, S;
1
Yoneda, T
＋1
Osaka University Graduate School of Dentistry, Suita, Osaka, 2Fukushima Medical University, Fukushima, Fukushima, Japan
koichirono@g-mail.com
ABSTRACT INTRODUCTION: Endochondral bone
formation, which is an essential process for skeletal
development in vertebrates, consists of series of events
including an aggregation of mesenchymal cells, their
differentiation into chondrocytes, followed by chondrocyte
proliferation, hypertrophy and apoptosis, leading to the
replacement by bone. Sox9 is an essential transcription factor
in the early stages of these sequential steps. Recent studies
showed that Sox5 and Sox6 served as chondrocyte-specific
transcription cofactors of Sox9 and that the cooperation of
Sox5/6/9 plays important roles in chondrogenesis [1].
However, transcriptional targets of Sox5/6/9 and, more
importantly, biological function of the target molecules in
endochondral bone formation are unknown. Here, we focused
to study Dmrt2 (double-sex and mab-3 related transcription
factor 2), since we found that Dmrt2 expression was markedly
up-regulated in the mesenchymal stem cells that differentiated
to chondrocytes following Sox5/6/9 introduction by
microarray analysis. Moreover, recent genetic studies have
reported that a loss of Dmrt1/2 genes causes growth
retardation and malformation of fingers in human [2] and that
Dmrt2-deficient mice show malformation of rib and sternum
[3]. These results collectively suggest a critical role of Dmrt2
in chondrocyte differentiation and consequent vertebrate
skeletal development.
METHODS: All experiments were conducted according to
the ethical guidelines of the Institutional Review Boards and
approved by the Institutional Animal Use Committee of the
Osaka University Graduate School of Dentistry. C3H10T1/2
cells were introduced with Sox5/6/9 using the adenovirus
system, cultured for 7 days, mRNA was collected and
microarray analysis was carried out using Affymetrix
Genechip® mouse genome 430 2.0. Localization of Dmrt2
proteins was examined by immunohistochemistry in E14.5
mouse embryo hind limbs. To investigate the role of Dmrt2 in
chondrogenesis, we either overexpressed Dmrt2 or suppressed
Dmrt2 expression by siRNA in primary chondrocytes isolated
from 4-week-old ICR mouse costal cartilage. Chondrocytespecific gene expression was examined by quantitative
RT-PCR (qRT-PCR). To study in vivo significance of Dmrt2
during endochondral bone formation, we generated transgenic
(TG) mice carrying the full-length Dmrt2 gene driven by the
Prx1 promoter which allowed us to selectively overexpress
genes of interest in the developing mouse limb bud. E14.5
tibiae of TG and wild type mice were histologically analyzed
by double-staining with alcian blue and alizarin red, and H&E
staining and in situ hybridization (ISH). Statistical analysis
was performed using student t-test. P values less than 0.05
were considered significant.
RESULTS: Microarray analysis using C3H10T1/2
mesenchymal stem cells overexpressing Sox5/6/9 or GFP
showed that Dmrt2 was a transcriptional target of Sox5/6/9.
Dmrt2 was highly expressed in E14.5 mouse embryo hind
limbs compared with other organs and tissues determined by
real-time RT-PCR. Immunohistochemical analysis revealed
that Dmrt2 was strongly expressed in the late proliferating to
pre-hypertrophic chondrocytes. These data led us to
hypothesize that Dmrt2 regulated both proliferating and
hypertrophic stages of chondrocyte differentiation. To prove
this hypothesis, we investigated the effects of Dmrt2 on the
expression of Col10α1, a well-accepted marker of
hypertrophic chondrocytes. Overexpression of Dmrt2
up-regulated Col10α1 mRNA expression in primary
chondrocytes, whereas suppression of Dmrt2 by siRNA
decreased it. Molecular study showed that Dmrt2 increased the
Col10α1 gene promoter activity by directly binding to the
putative Dmrt2 binding elements.
We next examined the role of Dmrt2 in the early stages of
endochondral bone formation. When Dmrt2 was
overexpressed in primary chondrocytes, Col2α1 and aggrecan
mRNA expression were markedly decreased. We also found
that knockdown of Dmrt2 increased these early chondrocyte
markers. Interestingly, overexpression of Dmrt2 reduced the
expression of Sox9 and Sox9-induced Col2α1 expression,
suggesting that Dmrt2 serves as a negative regulator for Sox9
(Fig.1). Finally, we studied in vivo significance of Dmrt2
during endochondral ossification by examining TG mice
carrying Dmrt2 gene that was driven by the Prx1 promoter. Of
note, TG mice exhibited severe defects in limb development
(Fig.2). Hind limbs isolated from E14.5 TG mice showed
reduced Col2α1 and aggrecan mRNA expression compared
with WT mice. ISH analysis further revealed that early stages
of endochondral bone formation were disturbed in TG mice.
DISCUSSION: Dmrt2 regulates cartilage differentiation
through inhibiting early-stages and promoting late-stages of
chondrocyte differentiation. Since our data show Dmrt2 is
expressed in the late stages of proliferating zone and
negatively regulates Sox9 function, it seems likely that Dmrt2
works as a switching regulator to forward endochondral bone
formation. Further elucidation of Dmrt2 function in
chondrogenesis would contribute to better understanding of
the mechanism underlying skeletal development.
REFERENCES:
[1] Lefebvre V., et al, 1998. EMBO J. 17: 5718–5733.
[2] Ounap, K., et al, 2004. Am J Med Genet., A 130, 415–423.
[3] Seo, K.W., et al, 2006.Dev. Biol. 290, 200–210.
Fig.1 Inhibitory effects of Dmrt2 on Sox9 function and
expression (* p<0.05).
Fig.2 Disturbed limb development in E14.5 TG mice.
Poster No. 957 • 55th Annual Meeting of the Orthopaedic Research Society