Activations of ERK1/2 and JNK by transforming growth factor beta negatively regulate Smad3-induced alkaline phosphatase activity and mineralization in mouse osteoblastic cells.
Transforming growth factor (TGF) beta inhibits alkaline phosphatase (ALP) activity and mineralization in mouse osteoblastic MC3T3-E1 cells, whereas local administration of TGF-beta stimulates bone formation in vivo. We recently demonstrated that Smad3, a TGF-beta signaling molecule, promotes ALP activity and mineralization in MC3T3-E1 cells. Moreover, the target disruption of Smad3 in mouse is reported to cause a decrease in bone mineral density. These findings indicate that Smad3 plays an important role in the regulation of bone formation. However, why the effects of TGF-beta and Smad3 on ALP activity and mineralization are different remains unknown. The purpose of the present study is to clarify the role of mitogen-activated protein kinase (MAPK) in TGF-beta and Smad3 pathways in osteoblast. TGF-beta activated extracellular signal-regulated kinases/p42/p44 (ERK1/2), p38 MAPK, and c-Jun N-terminal kinase (JNK) in mouse osteoblastic MC3T3-E1 cells. The expression of dominant negative type Smad3, Smad3DeltaC, affected neither TGF-beta-activated MAPKs nor TGF-beta-inhibited ALP activity. Specific inhibitors of ERK1/2 activation (PD98059 and U0126), as well as JNK inhibitors (curcumin and dicumarol) antagonized the inhibitory effects of TGF-beta on ALP activity and mineralization, whereas the specific inhibitor of p38 MAPK (SB203580) did not affect them. PD98059 and curcumin enhanced Smad3-induced ALP activity and mineralization, whereas SB203580 inhibited them. In the luciferase reporter assay using 3TP-lux with the specific Smad3-responsive element, PD98059, and curcumin enhanced TGF-beta- and Smad3-induced transcriptional activity in MC3T3-E1 cells. On the other hand, TGF-beta-induced production of type I collagen was antagonized by curcumin but not by PD98059. The present study indicated that TGF-beta-responsive ERK1/2 and JNK cascades negatively regulate Smad3-induced transcriptional activity as well as ALP activity and mineralization in osteoblasts.
Division of Endocrinology/Metabolism, Neurology and Hematology/Oncology, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-Cho, Chuo-ku, Kobe 650-0017, Japan.
Hideaki
H
Sowa
Hiroshi
H
Kaji
Toru
T
Yamaguchi
Toshitsugu
T
Sugimoto
Kazuo
K
Chihara
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003474:Curcumin / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005799:Genes, Dominant; D048031:JNK Mitogen-Activated Protein Kinases; D020935:MAP Kinase Signaling System; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009570:Nitriles / Q000494:pharmacology; D010006:Osteoblasts / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction; D051900:Smad3 Protein; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism
D002070:Butadienes; D016895:Culture Media, Serum-Free; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D009570:Nitriles; D012333:RNA, Messenger; D051900:Smad3 Protein; C497791:Smad3 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; C113580:U 0126; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D000469:Alkaline Phosphatase; D003474:Curcumin; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one