Identification and analysis of type II TGF-β receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells
Identification and analysis of type II TGF-β receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 mesenchymal stem cells
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DOI：
10.1093/abbs/gmq075
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发表时间：
2010-10-01
影响因子：
3.7
通讯作者：
Luo, Jinyong
中科院分区：
文献类型：
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作者：
Wu, Ningning;Zhao, Yingze;Luo, Jinyong
我们前期的研究表明，骨形态发生蛋白9（BMP-9）是诱导间充质干细胞（MSCs）向成骨细胞分化的最有效的BMP之一。然而，BMP-9诱导MSCs成骨分化的分子机制仍有待充分阐明。本研究构建了显性负性（DN）II型TGF-β受体，并将其导入C3 H10 T1/2干细胞中，进行了体外和体内试验，以分析和鉴定BMP-9诱导成骨所需的II型TGF-β受体。我们发现，三种DN II型TGF-β受体DN-BMPRII、DN-ActRII和DN-ActRIIB降低BMP-9诱导的碱性磷酸酶（ALP）活性，导致BMP-9诱导的Smad结合元件（SBE）控制的报告活性降低，BMP-9诱导的Smad 6和Smad 7表达降低，BMP-9诱导的体外矿化和体内异位骨形成降低，最终导致骨量减少和骨生成不成熟。这些发现有力地表明，三种野生型II型TGF-β受体，BMPRII，ActRII和ActRIIB，可能在BMP-9诱导的C3 H10 T1/2细胞的成骨分化中发挥功能性作用。然而，C3 H10 T1/2干细胞可以表达BMPRII和ActRII，但不表达ActRIIB。利用RNA干扰（RNAi）技术，我们发现BMP-9诱导的荧光素酶报告基因活性和ALP活性随着BMPRII和ActRII的敲低而沿着受到抑制。综上所述，我们的结果表明，BMPRII和ActRII是BMP-9诱导的C3 H10 T1/2细胞成骨分化的功能性II型TGF-β受体。
Our previous studies have demonstrated that bone morphogenetic protein 9 (BMP-9) is one of the most efficacious BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanism underlying the BMP-9-induced osteogenic differentiation of MSCs remains to be fully elucidated. In this study, dominant negative (DN) type II TGF-beta receptors were constructed and introduced into C3H10T1/2 stem cells, then in vitro and in vivo assays were carried out to analyze and identify the type II TGF-beta receptors required for BMP-9-induced osteogenesis. We found that three DN type II TGF-beta receptors, DN-BMPRII, DN-ActRII, and DN-ActRIIB, diminished BMP-9-induced alkaline phosphatase (ALP) activity, led to a decrease in BMP-9-induced Smad binding element (SBE)-controled reporter activity, reduced BMP-9-induced expressions of Smad6 and Smad7, and decreased BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, finally resulted in decreased bone masses and immature osteogenesis. These findings strongly suggested that three wild-type II TGF-beta receptors, BMPRII, ActRII and ActRIIB, may play a functional role in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells. However, C3H10T1/2 stem cells can express BMPRII and ActRII, but not ActRIIB. Using RNA interference (RNAi), we found that luciferase reporter activity and ALP activity induced by BMP-9 were accordingly inhibited along with the knockdown of BMPRII and ActRII. Taken together, our results demonstrated that BMPRII and ActRII are the functional type II TGF-beta receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells.