Abstract
L-Sox5 is a member of sex-determining region Y-type high mobility group box (SOX) family of transcription factors. We assessed the effects of retroviral overexpression of L-Sox5 on chondrocyte differentiation using the clonal murine cell line ATDC5. We observed a temporal-restricted expression pattern of L-Sox5 in insulin-induced ATDC5 cells differentiating toward chondrocyte lineage. The protein expression levels of L-Sox5 showed a drastic decrease in contrast to unaltered mRNA levels during differentiation. L-Sox5 delayed the differentiation of ATDC5 cells as evidenced by Alcian blue staining for proteoglycan synthesis. The mRNA levels of chondrocyte and hypertrophic/osteoarthritic markers were markedly decreased or delayed in L-Sox5 overexpressing cells. L-Sox5 abrogated the promoter activity of Runx2. These results suggest that L-Sox5 protein expression may diminish along with the progress of chondrogenic differentiation. L-Sox5 may act as a negative regulator if expressed aberrantly at least in part by regulating the critical fate of chondrogenesis.
References
Mackie EJ, Ahmed YA, Tatarczuch L, Chen KS, Mirams M (2008) Endochondral ossification: how cartilage is converted into bone in the developing skeleton. Int J Biochem Cell Biol 40:46–62
Lefebvre V, Smits P (2005) Transcriptional control of chondrocyte fate and differentiation. Birth Defects Res C Embryo Today 75:200–212
Akiyama H, Lefebvre V (2011) Unraveling the transcriptional regulatory machinery in chondrogenesis. J Bone Miner Metab 29:390–395
Lefebvre V, Li P, de Crombrugghe B (1998) A new long form of Sox5 (L-Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene. EMBO J 17:5718–5733
Saito T, Ikeda T, Nakamura K, Chung UI, Kawaguchi H (2007) S100A1 and S100B, transcriptional targets of SOX trio, inhibit terminal differentiation of chondrocytes. EMBO Rep 8:504–509
Atsumi T, Miwa Y, Kimata K, Ikawa Y (1990) A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells. Cell Differ Dev 30:109–116
Yao Y, Wang Y (2013) ATDC5: an excellent in vitro model cell line for skeletal development. J Cell Biochem 114:1223–1229
Cancedda R, Descalzi Cancedda F, Castagnola P (1995) Chondrocyte differentiation. Int Rev Cytol 159:265–358
Murdoch AD, Grady LM, Ablett MP, Katopodi T, Meadows RS, Hardingham TE (2007) Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: generation of scaffold-free cartilage. Stem Cells 25:2786–2796
Hattori T, Kishino T, Stephen S, Eberspaecher H, Maki S, Takigawa M et al (2013) E6-AP/UBE3A protein acts as a ubiquitin ligase toward SOX9 protein. J Biol Chem 288:35138–35148
An CI, Ganio E, Hagiwara N (2013) Trip12, a HECT domain E3 ubiquitin ligase, targets Sox6 for proteasomal degradation and affects fiber type-specific gene expression in muscle cells. Skelet Muscle 3:11
Hall BK, Miyake T (1995) Divide, accumulate, differentiate: cell condensation in skeletal development revisited. Int J Dev Biol 39:881–893
Shukunami C, Ishizeki K, Atsumi T, Ohta Y, Suzuki F, Hiraki Y (1997) Cellular hypertrophy and calcification of embryonal carcinoma-derived chondrogenic cell line ATDC5 in vitro. J Bone Miner Res 12:1174–1188
Chou CH, Lee CH, Lu LS, Song IW, Chuang HP, Kuo SY et al (2013) Direct assessment of articular cartilage and underlying subchondral bone reveals a progressive gene expression change in human osteoarthritic knees. Osteoarthritis Cartilage 21:450–461
Iwamoto M, Kitagaki J, Tamamura Y, Gentili C, Koyama E, Enomoto H et al (2003) Runx2 expression and action in chondrocytes are regulated by retinoid signaling and parathyroid hormone-related peptide (PTHrP). Osteoarthritis Cartilage 11:6–15
Smits P, Li P, Mandel J, Zhang Z, Deng JM, Behringer RR et al (2001) The transcription factors L-Sox5 and Sox6 are essential for cartilage formation. Dev Cell 1:277–290
Nagy A, Kenesi E, Rentsendorj O, Molnar A, Szenasi T, Sinko I et al (2011) Evolutionarily conserved, growth plate zone-specific regulation of the matrilin-1 promoter: L-Sox5/Sox6 and Nfi factors bound near TATA finely tune activation by Sox9. Mol Cell Biol 31:686–699
Li H, Haudenschild DR, Posey KL, Hecht JT, Di Cesare PE, Yik JH (2011) Comparative analysis with collagen type II distinguishes cartilage oligomeric matrix protein as a primary TGFbeta-responsive gene. Osteoarthritis Cartilage 19:1246–1253
Acknowledgments
This work is dedicated to the memory of late Professor S. Hao. The authors thank Drs. T. Komori, V. Lefebvre, and B. de Crombrugghe for providing materials.
Role of funding sources
This work was jointly supported by grants from the National Natural Science Foundation of China (30270660), the Jilin Province Science and Technology Development Project (20040114), the Fok Ying Tung Education Foundation (91025), and the Training Fund of NENU’S Scientific Innovation Project (NENU-STC08015). Neither of the funding agencies played any part in the design or conduct of this study, nor in the analysis of data or preparation of this manuscript.
Conflict of interest
The authors declare that they have no conflicts of interests.
Author information
Authors and Affiliations
Corresponding authors
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Sun, D., Deepak, V., Mu, P. et al. Constitutive L-Sox5 overexpression delays differentiation of ATDC5 cells into chondrocytes and correlates with reduced expression of differentiation markers. Mol Cell Biochem 401, 21–26 (2015). https://doi.org/10.1007/s11010-014-2288-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-014-2288-8