TGF-β inhibits osteogenesis by upregulating the expression of ubiquitin ligase SMURF1 via MAPK-ERK signaling.
High incidence of osteoporotic fractures emphasizes the necessity of developing effective measures to promote osteogenesis. In our study, we investigated a possible role of MAPK-ERK signaling in the TGF-β-mediated osteoblastic differentiation. Our results indicated that TGF-β activated the MAPK-ERK pathway and inhibited osteogenesis in mesenchymal pluripotent cell line, C3H10T1/2, and preosteoblastic cell line, MC3T3 cells. And the downregulation of MAPK-ERK signaling using pharmacological inhibitor U0126 and RNA interference rescued osteoblast differentiation suppressed by TGF-β, which was confirmed by Alkaline phosphatase (ALP) staining and alizarrn red staining, and the enhanced expression of osteogenesic markers. Western blotting analysis indicated that TGF-β induced protein expression of E3 ubiquitin-protein ligase SMURF1, which contributed to the degradation of RUNX2 and SMAD1 as evidenced by SMURF1 inhibition using RNA interference and proteasome inhibitor MG132. Moreover, we observed that the expression of SMURF1 was decreased, while that of SMAD1 and RUNX2 increased by MAPK-ERK inhibitor U0126 in TGF-β-treated differentiating preosteoblasts, suggesting that MAPK-ERK regulated the transcription of osteogenesis-related genes. Furthermore, a synergistic effect between U0126 and bone morphogenic protein (BMP)-2 on osteoblast differentiation and bone formation was observed both in cell cultures and experimental animals. In conclusion, our results revealed that TGF-β inhibited osteoblastic differentiation by inducing the MAPK-ERK pathway which upregulated the expression of ubiquitin ligase SMURF1 and resulted in reduced presence of osteogenic proteins. In addition, the potentiation of BMP-2 on osteogenic activity by ERK1/2 inhibitor U0126 suggests that it may have potential clinical utility for promoting osteogenesis in bone fracture repair.
Department of Orthopaedics, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
Xuewu
http://orcid.org/0000-0003-4669-2658
X
Sun
Department of Orthopaedics, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
Ziang
Z
Xie
Department of Orthopaedics, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
Yan
Y
Ma
Department of Orthopaedics, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
Xin
X
Pan
Department of Orthopaedics, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
Jiying
J
Wang
Department of Orthopaedics, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
Zhijun
Z
Chen
Department of Orthopaedics, Sir Run Run Shaw Hospital, Medical College of Zhejiang University, Hangzhou, China.|Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou, China.
Peihua
P
Shi
D016475:3T3 Cells; D000818:Animals; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects*; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000187:drug effects* / Q000201:enzymology; D010012:Osteogenesis / Q000187:drug effects*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D051898:Smad1 Protein / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D055396:Bone Morphogenetic Protein 2; D050659:Core Binding Factor Alpha 1 Subunit; D061988:Proteasome Inhibitors; D047428:Protein Kinase Inhibitors; C493716:Runx2 protein, mouse; D051898:Smad1 Protein; C497401:Smad1 protein, mouse; D016212:Transforming Growth Factor beta; C492381:Smurf1 protein, mouse; D044767:Ubiquitin-Protein Ligases; D048049:Extracellular Signal-Regulated MAP Kinases; D048369:MAP Kinase Kinase 1; C482127:Map2k1 protein, mouse; D046988:Proteasome Endopeptidase Complex
RUNX2; alkaline phosphatase; bone morphogenic protein; extracellular signal-regulated kinase; osteogenic differentiation