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

Abstract

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 NR1 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.

Population genomics in a disease targeted primary cell model

“…SOX9 regulates COL2A1 expression and other genes involved in endochondral bone formation, and thus is considered as a key player in chondrogenesis. Here, we show an association of the SRR rs1885987 G allele with low expression of the gene itself while the same allele was associated with high BMD in both our studies supporting a negative effect of SRR in bone cells as shown by Takarada et al (2008). The large size-effect of the cis-eQTL detected for SRR (r 2 = 0.5) allowed efficient fine-mapping to the proximal promoter along with validation by independent expression methods.…”

“…through the inhibition of the SOX9 transcriptional activity (Takarada et al 2008). SOX9 regulates COL2A1 expression and other genes involved in endochondral bone formation, and thus is considered as a key player in chondrogenesis.…”

Clock Genes Influence Gene Expression in Growth Plate and Endochondral Ossification in Mice

“…Staining intensity was finally quantified using the computer program Scion Image. Determination of ALP activity was done as described previously (24). In brief, cells were solubilized with 0.1% Triton X-100 followed by determination of the ALP activity in lysates using p-nitrophenol phosphate as a substrate.…”

Predominant Promotion by Tacrolimus of Chondrogenic Differentiation to Proliferating Chondrocytes

“…Cells were washed with 3% acetic acid for 30 s 3 times, and then photographed with an IMT-2-21 dissecting microscope (Olympus, Tokyo). Staining intensity was finally quantified using the computer program Scion image as described previously (18).…”

“…Metatarsals were then dissected for frozen sections with a thickness of 10 μm in a cryostat. In situ hybridization was carried out as described previously (18). In brief, sections were fixed with 4% paraformaldehyde freshly prepared in 0.1 M phosphate buffer (pH 7.4) for 10 min at room temperature, followed by washing and treatment with Proteinase K. Sections were subjected to acetylation in 0.1 M triethanolamine / 0.25% acetic anhydride, followed by washing and stepwise dehydration with ethanol for subsequent hybridization with a DIG-labeled cRNA probe at 65°C for 16 h. The area of positive signals was finally quantified using the computer program Scion image.…”