Abstract
Introduction
Cartilage homeoprotein 1 (CART-1) is a homeoprotein which has been suggested to play a role in chondrocyte differentiation and in skeletal development. It is expressed mainly in prechondrocytic mesenchymal condensations. Patients with mutations in the CART-1 gene display several craniofacial abnormalities, suggesting that CART-1 has a functional role in craniofacial skeletal development. However, its target genes and position in the established chondrogenic pathways is poorly documented. Given the fact that CART-1 is expressed predominantly in the chondrocyte lineage and its role in skeletal development, we hypothesized that CART-1 regulates expression of several pivotal genes involved in chondrogenic differentiation.
Methods
The coding sequence of human CART-1 was custom synthesized with optimized codon usage and cloned into a p3XFLAG-CMV-7.1 expression vector. FLAG-CART-1 was transiently overexpressed in SW1353 cells by polyethyleneimine-mediated transfection (1,000 ng of plasmid/well in 12-well plates). FLAG-Empty vector was used as a negative control. FLAG-CART-1 overexpression was confirmed by means of anti-FLAG immunoblotting. To investigate a potential connection between CART-1 and established key chondrogenic pathways, TGFβ3 (10 ng/mL) was added to SW1353 cells in CART-1 overexpression cultures or their appropriate controls. Cells were harvested 48 hours after transfection and mRNA expression of several genes involved in chondrogenic differentiation was determined by qRT-PCR. Data represent three separate experiments performed in technical triplicate.
Results
Overexpression of CART-1 was confirmed on protein level. CART-1 significantly upregulated the expression of hypertrophic markers MMP13 and COLX, while the expression of RUNX2, ALP and COL1 was significantly downregulated. The expression of COL2A1 and SOX9 was not altered in the presence of CART1. TGFβ3 significantly decreased MMP13 expression in SW1353 cultures, but induced the expression of COLX, RUNX2 and COL1. This TGFβ3 dependent behaviour was reversed when CART-1 was overexpressed in these cultures.
Conclusion
Our results implicate a functional role for the homeodomain protein CART-1 in controlling the expression of several markers involved in chondrocyte differentiation and show important interactions with other signaling pathways involved in chondrogenic differentiation. Current efforts focus on further elucidating the connection between CART-1 and other chondrogenic pathways.
