gms | German Medical Science

Objectives: The molecular mechanisms of highly orchestrated process of longitudinal bone growth and growth related disorders happening in the children's growth plate have not yet been fully understood. A novel signaling pathway, i.e. the ST2/IL-33 pathway has lately been shown to be involved in bone metabolism, especially in osteoclastogenesis. It was reported that ST2 is expressed in developing bones and ST2 receptor (ST2L) plays an important role in regulating osteogenic potential of osteosarcoma cells. Therefore we aimed to investigate expression, regulation and possible role of ST2 signaling in the growth plate chondrocytes.

Methods: Reverse Transcription PCR (RT-PCR), Quantitative real time PCR (qPCR), Immunohistochemistry, Gene knockdown (siRNA), Gene Overexpression (cDNA transfection), Electrophoretic Mobility Shift Assay (EMSA), Chromatin Immunoprecipitation Assay (ChIP). Comparisons were performed using unpaired t-test.

Results: Using RT-PCR, we confirmed expression of both ST2L and soluble ST2 (sST2) in murine chondrogenic cell line ATDC5. Immunohistochemistry detected increased to strong ST2 expression at pre-hypertrophic and hypertrophic chondrocytes of the tibial growth plate of euthanized 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 EMSA demonstrated binding of Runx2 to both promoters. These results were further confirmed by ChIP assay.

Conclusions: 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 organized cartilaginous growth plate and thus give a new insight into the process of longitudinal bone growth and skeletal growth disorders.