Serine racemase suppresses chondrogenic differentiation in cartilage in a Sox9-dependent manner

Serine racemase (SR) is responsible for the biosynthesis of D-serine (D-Ser), an endogenous co-agonist for N-methyl-D-aspartate (NMDA) receptors, from L-serine (L-Ser) in the central nervous system. In the present study, we investigated the role of SR in the regulation of chondrogenic differentiation in cartilage. On in situ hybridization analysis of tibia from neonatal rats, SR mRNA was ubiquitously expressed in all cell layers of proliferating to hypertrophic chondrocytes. In the pre-chondrogenic cell line ATDC5 cells, mRNA expression was seen with SR irrespective of the cellular maturity, with no mRNA expression of the NRI subunit essential for the heteromeric assembly of functional NMDA receptor channels. In ATDC5 cells stably overexpressing SR, significant inhibition was found with the maturation-dependent temporal increases in Alcian blue staining, alkaline phosphatase (ALP) activity and mRNA expression of type II and type X collagens. Stable overexpression of SR significantly impaired the sry-type HMG box 9 (Sox9) transcriptinal activity in ATDC5 cells, while Sox9 transcriptional activity was significantly inhibited in COS7 cells with co-introduction of SR and Sox9. However, no significant inhibition was seen with Sox9 transcriptional activity in COS7 cells co-introduced of either SR_K56G defective of D-Ser formation ability or 3-phosphoglycerate dehydrogenase essential for D-Ser biosynthesis. The co-introduction of SR with Sox9 significantly decreased the Sox9 protein level with that of Sox9 mRNA being unchanged. These results suggest that SR may negatively regulate cellular differentiation through the inhibition of Sox9 transcriptional activity in chondrocytes.