Effects of Tgds knockdown in ATDC5 cells’ chondrogenic differentiation

Catel–Manzke syndrome is a rare autosomal recessive genetic disorder characterized by a symptomatology known as "Pierre-Robin sequence": patients exhibit a small mandible, cleft palate, and a tendency of the tongue to obstruct the airway and digestive tract. Another characteristic is the presence of an additional phalanx affecting the index finger, resulting in finger deviation (clinodactyly). Other malformations have been observed in the cardiac, articular, and other skeletal regions. The syndrome is also associated with growth delay. Catel-Manzke syndrome is related to mutations in the TGDS (dTDP-D-glucose 4,6-dehydratase) gene, located on chromosome 13. The specific function of TGDS in vertebrates is unknown. We used ATDC5 cells, a well-established model for studying chondrogenesis, to evaluate the effect of TGDS knockdown. ATDC5 TGDS knockdown cells were generated using lentiviral shRNA trasduction (shRNA49). Cells were stimulated with ITS (insulin/transferrin/selenium) for up to 21 days to induce chondrogenesis. Chondrogenic differentiation was assessed by analyzing the expression levels of specific marker genes - SOX9, ACAN, COL2A1, and COL10A1- via quantitative real-time PCR (qPCR). Quantitative real-time PCR analysis confirmed efficient knockdown of TGDS expression in sh49 cell line compared to the scrambled control (scr). During chondrogenic induction, sh49 knockdown led to a consistent reduction in the expression of key markers, including SOX9, ACAN, COL2A1, and COL10A1, throughout the differentiation time course (days 7, 14, and 21). This indicates an impairment of the chondrogenic process in sh49 cells. As further confirmation of these findings, Alcian Blue staining revealed a marked decrease in proteoglycan production in sh49 cells compared to the scrambled control, consistent with reduced chondrogenic differentiation. Taken together, these results suggest that TGDS plays a functional role in promoting chondrogenic differentiation.