Interference with cellular differentiation by D‐serine through antagonism at N‐methyl‐D‐aspartate receptors composed of NR1 and NR3A subunits in chondrocytes

Abstract

Serine racemase (SR) is responsible for the biosynthesis of D-serine (D-Ser), an endogenous co-agonist for the glycine (Gly)-binding site on N-methyl-D-aspartate (NMDA) receptors, from L-Ser in the brain. We have previously demonstrated high expression of SR by chondrocytes in cartilage. In this study, we attempted to elucidate the possible functional role of D-Ser in chondrogenesis. Expression of mRNA and corresponding protein was seen for SR in cultured rat costal chondrocytes, while the addition of L-Ser significantly increased intracellular and extracellular levels of D-Ser. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, SR mRNA was highly localized in hypertrophic and calcified chondrocytes. Exposure to D-Ser not only suppressed several chondrocytic maturation markers, including alkaline phosphatase (ALP) activity, Ca2+ accumulation, nodule formation, and osteopontin expression, in rat chondrocytes, but also delayed chondral mineralization in mouse metatarsals. Either NMDA or Gly alone significantly increased Ca2+ accumulation in cultured chondrocytes, whereas D-Ser significantly prevented Ca2+ accumulation by Gly, but not by NMDA. Gly alone also significantly increased gene transactivation by the introduction of runt-related transcription factor-2 (Runx2) in COS7 cells transfected with NR1 and NR3A subunits, while D-Ser significantly prevented the increase by Gly without affecting the promoter activity of Runx2. In both cultured chondrocytes and metatarsals from NR1-null mice, significant decreases were seen in ALP activity and chondral mineralization, respectively. These results suggest that D-Ser may negatively regulate cellular differentiation through inhibiting NMDA receptors composed of NR1 and NR3A subunits in a manner related to Runx2 transcriptional activity in chondrocytes.

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

“…NS, not significant. scriptional network and local autocrine/paracrine system, but also by circulating endocrine factors including growth hormone, insulin-like growth factor-1 (IGF1), glucocorticoid, thyroid hormone, and adrenaline (26,30). Among these endocrine factors, growth hormone is a determinant of longitudinal bone elongation by way of a local direct action at the growth plate as well as by transformation into IGF1 in the liver (30) after the rhythmic secretion from the anterior pituitary in a manner highly related to the sleep cycle (31).…”

Osteoclastogenesis is negatively regulated by D‐serine produced by osteoblasts

“…The assay buffer composed of 0.05 M 2‐amino‐2‐methylpropanol, 2 mM MgCl 2 and 10 mM p ‐nitrophenylphosphoric acid was added at a volume of 200 µl into 10 µl of cell suspensions, followed by reaction for 30 min at 37°C and subsequent immediate determination of the absorbance of p ‐nitrophenol at 405 nm. Protein concentration was determined with a Bio‐Rad Protein Assay Kit, and ALP activity was standardized on the basis of both cellular protein content and incubation time for the representation as nmol/min/mg protein (Takarada et al, 2009).…”

“…The NMDA receptor (NMDAR) antagonist dizocilpine markedly diminishes temporal increases in both activities of alkaline phosphatase (ALP) and Ca 2+ accumulation, as well as the expression of the maturation marker osteocalcin, during cellular development through downregulation of the expression of runt‐related transcription factor‐2 (Runx2) essential for the cellular commitment and differentiation during osteoblastogenesis as a master regulator in cultured rat calvarial osteoblasts (Hinoi et al, 2003). Moreover, constitutive expression is seen with mRNA and corresponding proteins for particular subunits of NMDAR in cultured rat chondrocytes in vitro (Wang et al, 2005; Takarada et al, 2009) and in rat cartilage in vivo (Takarada et al, 2009). Similar expression profiles are found with different isoforms of excitatory amino acid transporters in chondrocytes and cartilage (Hinoi et al, 2005; Wang et al, 2006).…”

“…D ‐Ser is shown to be transformed from L ‐Ser by the catalytic action of serine racemase (SR) in many brain areas (Mothet et al, 2000; Oliet and Mothet, 2006), while SR mRNA is ubiquitously found in all chondrocytic layers including proliferating, pre‐hypertrophic and hypertrophic chondrocytic layers in tibia of neonatal rats at 1 day old (Takarada et al, 2008). Moreover, D ‐Ser negatively regulates cellular differentiation in a manner related to Runx2 in chondrocytes (Takarada et al, 2009).…”

The neurobiology of D-amino acid oxidase and its involvement in schizophrenia

“…97 D-serine may also modulate glycinergic transmission by antagonizing NR1/NR3A or NR1/NR3B receptors, which are insensitive to glutamate and activated by glycine. 98,99 D-serine also binds to human platelet 5-HT 3 receptors. 100 D-proline does not act at NMDARs but can activate glycine receptors, 101 whereas D-leucine is a potent regulator of the blood-brain barrier enkephalin 36,45,53 Some values are approximate as they were estimated from graphical data.…”