Phenytoin stimulates chondrogenic differentiation in mouse clonal chondrogenic EC cells, ATDC5.
Phenytoin (DPH) is known to affect bone formation. However, the mechanism of this effect has not been well understood. In this study, we evaluated the effects of DPH on cartilage formation in a model system using ATDC5 cells, a clonal murine chondrogenic cell line. Alcian blue staining for cartilage nodules and real-time reverse-transcription polymerase chain reaction for the expression of genes encoding type II collagen, aggrecan, transforming growth factor (TGF)-beta1, bone morphogenetic protein (BMP)-4, parathyroid hormone-related peptide (PTHrP), indian hedgehog (Ihh), and patched (Ptc) were performed in ATDC5 cells cultured with DPH. The ATDC5 cells demonstrated enhanced cartilage formation in cultures with DPH. During promoted chondrogenic differentiation, it was observed that DPH increased the mRNA expression of TGF-beta1, BMP-4, Ihh, and Ptc, in a dose-dependent manner on Days 5 to 15. In contrast, other antiepileptic drugs, phenobarbital and valproic acid had no effects on chondrogenesis in ATDC5 cells and osteogenesis in MC3T3-E1 cells. Our results provide fundamental evidence that DPH has a direct stimulatory effect on cartilage formation by regulating TGF-beta and hedgehog signaling molecules, and that DPH effect on bone formation, including chondrogenesis and osteogenesis, is distinct from other antiepileptic drugs as suggested in clinical settings.
Okada
Akinobu
A
Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., Osaka.
Shiomi
Teruaki
T
Aoki
Yoshinobu
Y
Fujiwara
Michio
M
D016475:3T3 Cells; D053674:Aggrecans; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000235:genetics; D002113:Calcification, Physiologic / Q000187:drug effects; D002356:Cartilage / Q000187:drug effects / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects* / Q000235:genetics; D045744:Cell Line, Tumor; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism; D020219:Chondrogenesis / Q000187:drug effects* / Q000235:genetics; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics; D002999:Clone Cells; D024043:Collagen Type II / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D016326:Extracellular Matrix Proteins / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D053823:Hedgehog Proteins; D037181:Lectins, C-Type / Q000235:genetics; D051379:Mice; D044162:Parathyroid Hormone-Related Protein / Q000235:genetics; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D010634:Phenobarbital / Q000494:pharmacology; D010672:Phenytoin / Q000494:pharmacology*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D015534:Trans-Activators / Q000235:genetics; D014635:Valproic Acid / Q000494:pharmacology
C508320:Acan protein, mouse; D053674:Aggrecans; D000927:Anticonvulsants; C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D011508:Chondroitin Sulfate Proteoglycans; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D053823:Hedgehog Proteins; D037181:Lectins, C-Type; D044162:Parathyroid Hormone-Related Protein; D000072079:Patched Receptors; D000072081:Patched-1 Receptor; D011506:Proteins; C000606240:Ptch1 protein, mouse; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D015534:Trans-Activators; D014635:Valproic Acid; D010672:Phenytoin; D010634:Phenobarbital