Interference with cellular differentiation by D-serine through antagonism at N-methyl-D-aspartate receptors composed of NR1 and NR3A subunits in chondrocytes.
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.
Takarada
Takeshi
T
Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa 920-1192, Japan.
Takahata
Yoshifumi
Y
Iemata
Mika
M
Hinoi
Eiichi
E
Uno
Kyosuke
K
Hirai
Takao
T
Yamamoto
Tomomi
T
Yoneda
Yukio
Y
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D019556:COS Cells; D002113:Calcification, Physiologic; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000235:genetics; D002522:Chlorocebus aethiops; D019902:Chondrocytes / Q000201:enzymology / Q000378:metabolism*; D020219:Chondrogenesis / Q000235:genetics; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D005865:Gestational Age; D005998:Glycine / Q000378:metabolism; D006801:Humans; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D008682:Metatarsal Bones / Q000196:embryology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016202:N-Methylaspartate / Q000378:metabolism; D053495:Osteopontin / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D019998:Racemases and Epimerases / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013997:Time Factors; D046509:Tissue Culture Techniques; D014162:Transfection; D000099300:Serine Racemase
D000469:Alkaline Phosphatase; D002118:Calcium; D050659:Core Binding Factor Alpha 1 Subunit; D005998:Glycine; D008562:Membrane Glycoproteins; D016202:N-Methylaspartate; D053495:Osteopontin; D012333:RNA, Messenger; D019998:Racemases and Epimerases; D016194:Receptors, N-Methyl-D-Aspartate; D012694:Serine; D000099300:Serine Racemase; C096392:Grin3a protein, rat; C493716:Runx2 protein, mouse; C493717:Runx2 protein, rat; C506897:Spp1 protein, rat