Kim K, Pang K M, Evans M, Hay E D
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mol Biol Cell. 2000 Oct;11(10):3509-23. doi: 10.1091/mbc.11.10.3509.
beta-Catenin promotes epithelial architecture by forming cell surface complexes with E-cadherin and also interacts with TCF/LEF-1 in the nucleus to control gene expression. By DNA transfection, we overexpressed beta-catenin and/or LEF-1 in NIH 3T3 fibroblasts, corneal fibroblasts, corneal epithelia, uveal melanoma cells, and several carcinoma cell lines. In all cases (with or without LEF-1), the abundant exogenous beta-catenin localizes to the nucleus and forms distinct nuclear aggregates that are not associated with DNA. Surprisingly, we found that with time (5-8 d after transfection) cells overexpressing beta-catenin all undergo apoptosis. LEF-1 does not need to be present. Moreover, LEF-1 overexpression in the absence of exogenous beta-catenin does not induce apoptosis, even though some endogenous beta-catenin moves with the exogenous LEF-1 into the nucleus. TOPFLASH/FOPFLASH reporter assays showed that full-length beta-catenin is able to induce LEF-1-dependent transactivation, whereas Arm beta-catenin totally abolishes the transactivating function. However, Arm beta-catenin, containing deletions of known LEF-1-transactivating domains, has the same apoptotic effects as full-length beta-catenin. Overexpressed beta-catenin also induces apoptosis in cells transfected with nuclear localization signal-deleted LEF-1 that localizes only in the cytoplasm. Thus, the apoptotic effects of overexpressed exogenous beta-catenin do not rely on its transactivating function with nuclear LEF-1. Overexpressed delta-catenin, containing 10 Arm repeats, induces only minor apoptosis, suggesting that the major apoptotic effect may be due to domains specific to beta-catenin as well as to Arm repeats. The absence of p53, Rb, cyclin D1, or E2F1 does not affect the apoptotic effect of overexpressed beta-catenin, but Bcl-x(L) reduces it. We hypothesize that in vivo apoptosis of cells overexpressing beta-catenin might be a physiological mechanism to eliminate them from the population.
β-连环蛋白通过与E-钙黏蛋白形成细胞表面复合物来促进上皮结构,并且还在细胞核中与TCF/LEF-1相互作用以控制基因表达。通过DNA转染,我们在NIH 3T3成纤维细胞、角膜成纤维细胞、角膜上皮细胞、葡萄膜黑色素瘤细胞和几种癌细胞系中过表达了β-连环蛋白和/或LEF-1。在所有情况下(无论有无LEF-1),过量表达的外源β-连环蛋白都定位于细胞核并形成与DNA无关的独特核聚集体。令人惊讶的是,我们发现随着时间推移(转染后5 - 8天),过表达β-连环蛋白的细胞都会发生凋亡。LEF-1无需存在。此外,在没有外源β-连环蛋白的情况下过表达LEF-1不会诱导凋亡,尽管一些内源性β-连环蛋白会与外源LEF-1一起进入细胞核。TOPFLASH/FOPFLASH报告基因检测表明,全长β-连环蛋白能够诱导LEF-1依赖的反式激活,而Armβ-连环蛋白则完全消除了反式激活功能。然而,含有已知LEF-1反式激活结构域缺失的Armβ-连环蛋白具有与全长β-连环蛋白相同的凋亡作用。过表达β-连环蛋白也会在转染了仅定位于细胞质的核定位信号缺失的LEF-1的细胞中诱导凋亡。因此,过表达的外源β-连环蛋白的凋亡作用并不依赖于其与核LEF-1的反式激活功能。过表达的含有10个Arm重复序列的δ-连环蛋白仅诱导轻微凋亡,这表明主要的凋亡作用可能归因于β-连环蛋白特有的结构域以及Arm重复序列。p53、Rb、细胞周期蛋白D1或E2F1的缺失并不影响过表达β-连环蛋白的凋亡作用,但Bcl-x(L)会使其减弱。我们推测,过表达β-连环蛋白的细胞在体内发生凋亡可能是一种将它们从群体中清除的生理机制。