Liu X, Yue J, Frey R S, Zhu Q, Mulder K M
Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey 17033, USA.
Cancer Res. 1998 Oct 15;58(20):4752-7.
Previous results have suggested that Smad1 transduces signals in response to bone morphogenetic proteins (BMPs), but not in response to transforming growth factor beta (TGF-beta). Here we investigated the ability of TGF-beta to regulate Smad1 phosphorylation, hetero-oligomerization with Smad4, translocation to the nucleus, and transcriptional activation of 3TP-luciferase reporter activity in TGF-beta- and BMP-responsive Hs578T human breast cancer cells. We demonstrate that Smad1 was rapidly phosphorylated in vivo in response to both TGF-beta3 and BMP2 as determined using an antibody against the epitope-tagged Smad1 being expressed. In addition, both TGF-beta3 and BMP2 increased Smad1-Smad4 hetero-oligomerization in Hs578T cells. Visualization of Smad1 nuclear translocation with the aid of green fluorescent protein (GFP) in live cells demonstrated nuclear accumulation of GFP-Smad1 fluorescence in response to either TGF-beta or BMP2 stimulation. After ligand stimulation, approximately 60-70% of transfected cells displayed prominent nuclear fluorescence. Expression of Smad1 in Hs578T cells increased the activity of the TGF-beta-responsive reporter 3TP-Lux. Moreover, TGF-beta treatment further potentiated the effect of Smad1 on 3TP-luciferase activity. Collectively, our results demonstrate that TGF-beta as well as BMP can signal through Smad1.
先前的研究结果表明，Smad1可转导骨形态发生蛋白（BMP）信号，但不转导转化生长因子β（TGF-β）信号。在此，我们研究了TGF-β在TGF-β和BMP应答性Hs578T人乳腺癌细胞中调节Smad1磷酸化、与Smad4异源寡聚化、转运至细胞核以及激活3TP-荧光素酶报告基因活性的能力。我们证明，使用针对所表达的表位标记Smad1的抗体测定，Smad1在体内可对TGF-β3和BMP2作出快速磷酸化反应。此外，TGF-β3和BMP2均可增加Hs578T细胞中Smad1-Smad4异源寡聚化。借助绿色荧光蛋白（GFP）在活细胞中观察Smad1核转位，结果显示，在TGF-β或BMP2刺激下，GFP-Smad1荧光在细胞核中积累。配体刺激后，约60-70%的转染细胞呈现明显的核荧光。Hs578T细胞中Smad1的表达增加了TGF-β应答性报告基因3TP-Lux的活性。此外，TGF-β处理进一步增强了Smad1对3TP-荧光素酶活性的影响。总体而言，我们的结果表明，TGF-β以及BMP均可通过Smad1发出信号。
