TGF-β inhibits osteogenesis by upregulating the expression of ubiquitin ligase SMURF1 via MAPK-ERK signaling
TGF-β inhibits osteogenesis by upregulating the expression of ubiquitin ligase SMURF1 via MAPK-ERK signaling
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DOI：
10.1002/jcp.25920
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发表时间：
2018-01-01
影响因子：
5.6
通讯作者：
Shi, Peihua
中科院分区：
文献类型：
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作者：
Sun, Xuewu;Xie, Ziang;Shi, Peihua
骨质疏松性骨折的高发强调了制定有效措施促进成骨的必要性。在我们的研究中，我们研究了MAPK-ERK信号在tgf - β介导的成骨细胞分化中的可能作用。我们的研究结果表明，tgf - β激活MAPK-ERK通路，抑制间充质多能细胞系C3H10T1/2和成骨前细胞系MC3T3细胞的成骨。利用药理抑制剂U0126和RNA干扰下调MAPK-ERK信号通路，挽救了tgf - β抑制的成骨细胞分化，碱性磷酸酶（ALP）染色和黑素红染色证实了这一点，并增强了成骨标志物的表达。Western blotting分析表明，tgf - β诱导E3泛素蛋白连接酶SMURF1的蛋白表达，通过RNA干扰和蛋白酶体抑制剂MG132抑制SMURF1，从而促进RUNX2和SMAD1的降解。此外，我们观察到在tgf - β处理的分化成骨前细胞中，MAPK-ERK抑制剂U0126降低了SMURF1的表达，而SMAD1和RUNX2的表达升高，这表明MAPK-ERK调节了成骨相关基因的转录。此外，在细胞培养和实验动物中，U0126和骨形态发生蛋白(BMP)-2对成骨细胞分化和骨形成均有协同作用。总之，我们的研究结果表明，tgf - β通过诱导MAPK-ERK通路抑制成骨细胞分化，该通路上调泛素连接酶SMURF1的表达，导致成骨蛋白的存在减少。此外，ERK1/2抑制剂U0126增强BMP-2对成骨活性的影响提示其在骨折修复中促进成骨可能具有潜在的临床应用价值。
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-beta-mediated osteoblastic differentiation. Our results indicated that TGF-beta 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-beta, 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-beta 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-beta-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-beta 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.