Feng X H, Filvaroff E H, Derynck R
Department of Growth and Development, University of California, San Francisco 94143, USA.
J Biol Chem. 1995 Oct 13;270(41):24237-45. doi: 10.1074/jbc.270.41.24237.
Transforming growth factor-beta (TGF-beta) inhibits the proliferation of epithelial cells by altering the expression or function of various components of the cell cycle machinery. Expression of one of these components, cyclin A, is inhibited by TGF-beta treatment. We have identified a 760-base pair fragment of the human cyclin A gene promoter that is sufficient to confer TGF-beta responsiveness. Using this promoter fragment, we have developed a cyclin A-based luciferase reporter assay that quantitates the growth inhibitory effect of TGF-beta in transient transfection assays. This assay was used to determine which domains of the type I (RI) and type II (RII) receptors were required for the antiproliferative effect of TGF-beta. In parallel, the functionality of chimeric receptors, between RI and RII (RI-RII or RII-RI), was tested for TGF-beta effect on gene expression using a reporter assay based on the plasminogen activator inhibitor type 1 (PAI-1) promoter. We found that TGF-beta-induced inhibition of cyclin A expression was absent in RI or RII-deficient Mv1Lu cells and that this response was restored by expression of wild-type type I or type II receptors in these cells. Furthermore, expression of a single chimeric receptor, either RI-RII or RII-RI, did not confer cyclin A regulation by TGF-beta. However, expression of two reciprocal chimeras (RI-RII and RII-RI) resulted in growth inhibition, similarly to wild-type receptors. In addition, chimeric receptors as well as mutant receptors with a deleted cytoplasmic domain and kinase-negative receptors inhibited TGF-beta responsiveness in the cyclin A reporter assay in a dominant negative fashion. Finally, in both receptor types, the juxtamembrane domain preceding the kinase domain was essential for receptor function but the cytoplasmic tail was dispensable. Our results suggest that a functional TGF-beta receptor complex is required for TGF-beta-dependent down-regulation of cyclin A gene expression and illustrate the identical receptor requirements for TGF-beta-induced growth inhibition and gene expression.
转化生长因子-β（TGF-β）通过改变细胞周期机制中各种成分的表达或功能来抑制上皮细胞的增殖。细胞周期蛋白A是这些成分之一，其表达受到TGF-β处理的抑制。我们鉴定出了人细胞周期蛋白A基因启动子的一个760碱基对的片段，该片段足以赋予对TGF-β的反应性。利用这个启动子片段，我们开发了一种基于细胞周期蛋白A的荧光素酶报告基因检测方法，该方法可在瞬时转染实验中定量TGF-β的生长抑制作用。此检测方法用于确定TGF-β的抗增殖作用所需的I型（RI）和II型（RII）受体的哪些结构域。同时，使用基于纤溶酶原激活物抑制剂1型（PAI-1）启动子的报告基因检测，测试RI和RII之间的嵌合受体（RI-RII或RII-RI）对TGF-β基因表达的功能。我们发现，在缺乏RI或RII的Mv1Lu细胞中，TGF-β诱导的细胞周期蛋白A表达抑制不存在，而在这些细胞中表达野生型I型或II型受体可恢复这种反应。此外，单独表达一种嵌合受体，无论是RI-RII还是RII-RI，都不能赋予TGF-β对细胞周期蛋白A的调节作用。然而，表达两种相互的嵌合体（RI-RII和RII-RI）会导致生长抑制，类似于野生型受体。此外，嵌合受体以及具有缺失细胞质结构域的突变受体和激酶阴性受体在细胞周期蛋白A报告基因检测中以显性负性方式抑制TGF-β反应性。最后，在两种受体类型中，激酶结构域之前的近膜结构域对于受体功能至关重要，但细胞质尾巴是可有可无的。我们的结果表明，功能性TGF-β受体复合物是TGF-β依赖性下调细胞周期蛋白A基因表达所必需的，并说明了TGF-β诱导的生长抑制和基因表达对受体的相同要求。
