The primary response gene ST2 which is also known as IL1RL1 or T1 was suggested as an early marker of differentiation in osteogenic cell lines. It was shown that 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 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 electrophoretic mobility shift assay demonstrated binding of Runx2 to both promoters. These results were further confirmed 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 organized cartilaginous growth plate and thus give a new insight into the process of longitudinal bone growth and skeletal growth disorders.
Enhanced ST2 Expression in Late Stages of Chondrocyte Differentiation in Growth Plate is Regulated by Transcriptional Activity of Runx2
MUSUMECI, GIUSEPPE;
2014-01-01
Abstract
The primary response gene ST2 which is also known as IL1RL1 or T1 was suggested as an early marker of differentiation in osteogenic cell lines. It was shown that 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 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 electrophoretic mobility shift assay demonstrated binding of Runx2 to both promoters. These results were further confirmed 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 organized cartilaginous growth plate and thus give a new insight into the process of longitudinal bone growth and skeletal growth disorders.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
