Abstract
Objectives:
Previously, we showed that Papss2 expression is regulated by Sox9 in primary chondrocytes and a chondrogenic cell line, ATDC5. Here we explore molecular mechanisms whereby Sox9 regulates mouse Papss2 mRNA expression.
Methods:
Luciferase reporter assays were performed in ATDC5 cells to identify Sox9-responsive elements in the mouse Papss2 gene. Electromobility shift assays, mobility shift competition assays, and super shift assays were used to characterize protein binding to a 32bp Sox9-responsive element. Western blot and co-immunoprecipitation assays were used to determine the effects of Sox9 on C/EBPβ protein levels and binding of Sox9 to C/EBPβ.
Results:
An evolutionarily conserved 509bp Sox9-responsive DNA element was identified in the Papss2 gene, which was subsequently narrowed down to 32bp. Putative SoxE and C/EBPβ binding sites were identified within this 32bp. Increasing amounts of C/EBPβ resulted in attenuation of Sox9-mediated activation of the responsive element indicating C/EBPβ acts as a transcriptional repressor. Three protein-DNA complexes containing C/EBPβ were identified on the Sox9-responsive element under conditions when Papss2 expression was low. With high Sox9 expression, when Papss2 expression was stimulated, the formation of C/EBPβ containing protein-DNA complexes was inhibited, Sox9 and C/EBPβ protein-protein binding was observed, and overall cellular C/EBPβ protein levels were reduced.
Conclusion:
We propose that Sox9 acts to derepress C/EBPβ-inhibited transcription of Papss2 by first interacting with C/EBPβ to prevent it from binding DNA, then reducing C/EBPβ expression.
Keywords:
ATDC5; C/EBPβ; Papss2; Sox9; cartilage; chondrocyte; transcription.