Zhao Shujie, Ammanamanchi Sudhakar, Brattain Michael, Cao Lin, Thangasamy Amalraj, Wang Jing, Freeman James W
Department of Medicine, Division of Medical Oncology, University of Texas Health Science Center, San Antonio, Texas 78229-3900, USA.
J Biol Chem. 2008 Apr 25;283(17):11293-301. doi: 10.1074/jbc.M800154200. Epub 2008 Feb 29.
Transforming growth factorbeta (TGF-beta) signals through Smad-dependent and Smad-independent pathways. However, Smad signaling is altered by allelic deletion or intragenic mutation of the Smad4 gene in more than half of pancreatic ductal adenocarcinomas. We show here that loss of Smad4-dependent signaling leads to aberrant expression of RON, a phosphotyrosine kinase receptor, and that signaling by RON cooperates with Smad4-independent TGF-beta signaling to promote cell motility and invasion. Restoring Smad4 expression in a pancreatic ductal adenocarcinoma cell line that is deficient in Smad4 repressed RON expression. Conversely, small interference RNA knock down of Smad4 or blocking TGF-beta signaling with a TGF-beta type I receptor kinase inhibitor in Smad4-intact cell lines induced RON expression. TGF-beta-induced motility and invasion were inhibited in cells that express Smad4 and that have low levels of RON compared with isogenically matched cells that were deficient in Smad4. Furthermore, knocking down RON expression in Smad4-deficient cells suppressed TGF-beta-mediated motility and invasion. We further determined that Smad4-dependent signaling regulated RON expression at the transcriptional level by real-time reverse transcription PCR and RON promoter luciferase reporter assays. Functional inactivation by site-directed mutations of two Smad binding sites on the RON promoter inhibited TGF-beta-mediated repression of RON promoter activity. These studies indicate that loss of Smad4 contributes to aberrant RON expression and that cross-talk of Smad4-independent TGF-beta signaling and the RON pathway promotes an invasive phenotype.
转化生长因子β（TGF-β）通过Smad依赖和Smad非依赖途径发出信号。然而，在超过一半的胰腺导管腺癌中，Smad4基因的等位基因缺失或基因内突变会改变Smad信号传导。我们在此表明，Smad4依赖信号的缺失导致磷酸酪氨酸激酶受体RON的异常表达，并且RON信号与Smad4非依赖的TGF-β信号协同作用以促进细胞运动和侵袭。在缺乏Smad4的胰腺导管腺癌细胞系中恢复Smad4表达可抑制RON表达。相反，在Smad4完整的细胞系中，用小干扰RNA敲低Smad4或用TGF-β I型受体激酶抑制剂阻断TGF-β信号会诱导RON表达。与缺乏Smad4的同基因匹配细胞相比，在表达Smad4且RON水平较低的细胞中，TGF-β诱导的运动和侵袭受到抑制。此外，在缺乏Smad4的细胞中敲低RON表达可抑制TGF-β介导的运动和侵袭。我们通过实时逆转录PCR和RON启动子荧光素酶报告基因检测进一步确定，Smad4依赖信号在转录水平上调节RON表达。通过对RON启动子上两个Smad结合位点进行定点突变使其功能失活，可抑制TGF-β介导的RON启动子活性的抑制。这些研究表明，Smad4的缺失导致RON异常表达，并且Smad4非依赖的TGF-β信号与RON途径的相互作用促进了侵袭性表型。
