ECTS Abstracts (2015) 1 P228

Runx2 regulates ST2 expression in the late stages of growth plate chondrocyte differentiation

Ehsan Bonyadi Rad1, Karin Pichler1, Giuseppe Musumeci2, Egon Marth3 & Annelie Weinberg1

1Department of Orthopedics and Orthopedic Surgery, Medical University Graz, Graz, Austria; 2Department of Bio-Medical Science, School of Medicine, University of Catania, Catania, Italy; 3Institute of Hygiene, Microbiology and Environmental Medicine, Medical University Graz, Graz, Austria.

The primary response gene ST2 (IL1RL1 or T1) was suggested as an early marker of differentiation in osteogenic cell lines. ST2 receptor (ST2L) plays an important role in regulating osteogenic potential of osteosarcoma cells as well as regulation of osteoclastogenesis. In this study we aimed to investigate expression, regulation and possible biological role of this gene in the growth plate chondrocytes which is responsible for long bone elongation. We confirmed expression of both ST2L and soluble ST2 (sST2) in murine chondrogenic cell line ATDC5 using reverse transcription PCR. Increased to strong ST2 expression was observed by immunohistochemistry at pre-hypertrophic and hypertrophic chondrocytes of the tibial growth plate of euthanised three week old mice. Surprisingly, consistent with these results we noted several fold upregulation of both ST2L and sST2 mRNA in the late stages of ATDC5 differentiation. ColX and MMP-13, markers of chondrocyte hypertrophy, were also significantly increased in these hypertrophic ATDC5 cells. Master transcription factor Runx2 which controls chondrocyte hypertrophy was shown to be upregulated at differentiated ATDC5 cells. Our results showed that Runx2 upregulation by cDNA transfection or downregulation by siRNA knockdown significantly increase or decrease ST2 expression, respectively. Our results clearly indicated that both ST2 splice variants are transcribed from proximal promoter and low sST2 mRNA is also produced from distal promoter in Runx2 overexpressing cells. In silico promoter analysis identified consensus Runx2 binding sites on distal and proximal promoters and electrophoretic mobility shift assay demonstrated binding of Runx2 to both promoters. These results were confirmed further by chromatin immunoprecipitation assay. Overall, our data suggest that Runx2 is involved in regulating enhanced ST2 expression in pre- and hypertrophic chondrocytes. Therefore, further investigations might lead to elucidate ST2 function in the highly organised cartilaginous growth plate and thus give a new insight into the process of longitudinal bone growth.

Disclosure: The authors declared no competing interests. This work was supported by the MUG Research Units: Division of General Pediatric and Adolescence Surgery; Funded by: Hygienefonds der Med Uni Graz, Austria.

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