Investigating Bone Morphogenetic Protein (BMP) Signaling in a Newly Established Human Cell Line Expressing BMP Receptor Type II
Investigating Bone Morphogenetic Protein (BMP) Signaling in a Newly Established Human Cell Line Expressing BMP Receptor Type II
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DOI：
10.1620/tjem.222.121
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发表时间：
2010-10-01
影响因子：
2.2
通讯作者：
Hayashi, Haruhide
中科院分区：
文献类型：
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作者：
Kudo, Tada-aki;Kanetaka, Hiroyasu;Hayashi, Haruhide
骨形态发生蛋白 (BMP) 是转化生长因子 β 细胞因子超家族的成员，可在不同组织中引起各种生物效应。 II 型 BMP 受体 (BMPRII) 包含一个独特的羧基末端区域，可与多种信号分子相互作用。然而，内源性 BMPRII 在各种哺乳动物细胞系中表达较低，这阻碍了 BMP 信号传导的分析。因此，我们使用四环素控制的 Flp-In T-REx 基因表达系统建立了表达带有 Flag 表位标记的 BMPRII 的人类细胞系 (BMPRII-Flag)。将 BMPRII-Flag 基因导入 Flp-In T-REx 293 (FT293) 细胞系，该细胞系是人 293 胚胎肾成纤维细胞的衍生物。然后，我们通过定量实时 PCR 分析了亲代 FT293 细胞和已建立的细胞系 FT293-BMPRII 中关键 BMP 靶基因、DNA 结合 (Id) 家族成员 (Id1、Id2 和 Id3) 抑制剂以及抑制性 Smads Smad6 和 Smad7 的表达。四环素处理显着增加了 FT293-BMPRII 细胞中 BMPRII-Flag mRNA 和蛋白的表达，但未引起 Id1、Id2、Id3、Smad6 或 Smad7 mRNA 表达的显着变化。相反，用BMPRII配体BMP2处理诱导亲代FT293细胞和FT293-BMPRII细胞中Id1、Id2、Id3和Smad6的表达。四环素诱导的 BMPRII-Flag 表达显着增强了用 BMP2 处理的 FT293-BMPRII 细胞中 Id1、Id3 和 Smad6 mRNA 表达的诱导。这些发现提供的证据表明，尽管BMPRII对代表性BMP靶基因的表达没有明显影响，但它差异性地调节靶基因对BMP2的反应性。
Bone morphogenetic proteins (BMPs), members of the transforming growth factor beta cytokine superfamily, elicit various biological effects in different tissues. BMP receptor type II (BMPRII) contains a unique carboxyl-terminal region that interacts with multiple signaling molecules. However, expression of endogenous BMPRII is low in various mammalian cell lines, which hampers the analysis of BMP signaling. Therefore, we established a human cell line expressing BMPRII tagged with a Flag epitope (BMPRII-Flag) using the tetracycline-controlled Flp-In T-REx gene expression system. The BMPRII-Flag gene was introduced into the Flp-In T-REx 293 (FT293) cell line, a derivative of human 293 embryonic kidney fibroblasts. Then we analyzed the expression of key BMP target genes, inhibitors of DNA binding (Id) family members (Idl, Id2, and Id3) and the inhibitory Smads Smad6 and Smad7, in parental FT293 cells and an established cell line, FT293-BMPRII, by quantitative real-time PCR. Tetracycline treatment significantly increased the expression of BMPRII-Flag mRNA and protein in FT293-BMPRII cells, but induced no significant changes in expression of Id1, Id2, Id3, Smad6, or Smad7 mRNA. In contrast, treatment with a BMPRII ligand BMP2 induced the expression of Id1, Id2, Id3, and Smad6 in parental FT293 cells and FT293-BMPRII cells. Tetracycline-induced BMPRII-Flag expression significantly enhanced the induction of Id1, Id3, and Smad6 mRNA expression in FT293-BMPRII cells treated with BMP2. These findings provide evidence that although BMPRII has no obvious effect on the expression of representative BMP target genes, it differentially modulates the responsiveness of target genes to BMP2.