Differential regulation of cellular maturation in chondrocytes and osteoblasts by glycine

Abstract

Previous studies have demonstrated the functional expression, by osteoblasts, of N-methyl-D-aspartate (NMDA) receptors responsible for the promotion of cellular differentiation in bone. We have now evaluated the possible role of the endogenous co-agonist of NMDA receptors, glycine (Gly), in chondrogenesis. In ex vivo organotypic cultures of fetal mouse tibias, proximal and distal cartilaginous primordia were significantly increased in the presence of Gly, with the osteogenic center being unchanged. Exposure to Gly drastically increased mRNA expression of the calcified chondrocyte marker osteopontin, without markedly affecting that of a proliferating chondrocyte marker or a hypertrophic chondrocyte marker, as shown in organotypic cultures by in situ hybridization analysis. Gly significantly increased Ca2+ accumulation, osteopontin mRNA expression, and alkaline phosphatase activity in cultured rat costal chondrocytes, without significantly affecting those in cultured rat calvarial osteoblasts. The increase induced by Gly was significantly prevented by an NMDA receptor channel blocker and an antagonist at the Gly site on NMDA receptors, but not by an inhibitory Gly receptor antagonist or a Gly transporter inhibitor, in cultured chondrocytes. Constitutive mRNA expression was seen for NR1, NR2D, and NR3A subunits of NMDA receptors, but not for Gly receptors and transporters, in cultured chondrocytes. Corresponding immunoreactive proteins were detected for NR1 and NR2D subunits in cartilaginous zones of fetal mouse tibias. Thus, Gly might, at least in part, play a role as a trophic factor in the mechanisms associated with chondral calcification through the Gly site of NMDA receptors functionally expressed by chondrocytes in rodent cartilage.

Osteoclastogenesis is negatively regulated by D‐serine produced by osteoblasts

“…Osteoblasts were prepared from calvaria of 1‐ to 2‐day‐old neonatal rats by the sequential enzymatic digestion method as described previously (Takahata et al, 2008). In brief, rat calvaria were gently incubated at 37°C for 10 min with 0.2% collagenase in αMEM, followed by collection of cells in supernatants thus obtained.…”

“…Under these culture conditions, osteoblastic cells were in a proliferative phase between 1 and 6 days with formation of several confluent cell layers, and subsequently by 10–11 days cells were more refractive and round than the background cell layers, followed by formation of some nodules and clusters with matrix. The matrix started to mineralize and calcify within 15 days (Takahata et al, 2008).…”

The emerging chondrocyte channelome

“…It again appears likely that NMDA channels are in someway involved in the mechanotransduction pathway, since mechanically induced hyperpolarizations are reduced by NMDA antagonists (Salter et al, 2004). Furthermore, glycine induces a number of changes on chondrocytes in cartilage explants (including accumulation of calcium) and these effects can be reduced with an NMDA antagonist as, presumably, glycine acts via the glycine binding site of the NMDA receptor (Takahata et al, 2008). …”

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

“…For instance, reverse transcription-polymerase chain reaction (RT-PCR) analysis reveals the constitutive expression of mRNA for particular subunits of N-methyl-D-aspartate (NMDA) receptor (NMDAR) in cultured rat chondrocytes Takahata et al, 2008;Piepoli et al, 2009) and in human articular chondrocytes (Salter et al, 2004;Ramage et al, 2008), in addition to different isoforms of excitatory amino acid transporters Wang et al, 2006).…”