Apc and beta-Catenin in Cell Regulation and Cancer

Apc（腺瘤性结肠息肉病）基因的生殖系和体细胞突变被认为是人类和小鼠结直肠癌病因学中的遗传事件。ApcMin小鼠在Apc基因中携带种系突变，并且由于腺癌负荷而经历寿命缩短。野生型，但不是突变的，APC结合并调节β-连环蛋白，钙粘蛋白介导的细胞粘附所需的犰狳的哺乳动物同系物。β-连环蛋白从其与膜E-钙粘蛋白的结合中释放出来，与Tcf/LEF形成异源二聚体，并作为转录因子发挥作用。为了研究该途径的调节，我们使用了两种条件永生小鼠肠上皮细胞系，其在Apc基因型上不同（“Immortomouse”/Min Colonic Epithelia，Apc +/-;年轻成年小鼠结肠上皮，Apc +/+）。我们已经证明，IMCE细胞有缺陷的降解β-连环蛋白，有较高水平的β-连环蛋白/LEF-1转录因子的EMSA和更高的表达考克斯-2比YAMC细胞响应脂多糖（LPS）和干扰素-g（IFN-g）。一氧化氮（NO）在这种反应中的关键作用是通过一氧化氮合酶II抑制剂消除LPS，IFN-g效应来显示的。此外，通过EMSA，NO供体增加了b-连环蛋白/LEF-1的形成以及考克斯-2的表达。IMCE和YAMC细胞中的差异反应以及对NO处理的游离细胞质β-连环蛋白的直接证明支持了这种效应是由β-连环蛋白的可用性介导的。我们目前的工作集中在NO增加游离的、未结合的β-连环蛋白的机制上。初步研究结果表明，NO刺激膜结合的E-钙粘蛋白的降解，同时从细胞质E-钙粘蛋白结合位点释放β-连环蛋白。使用识别E-钙粘蛋白胞外结构域的抗体，我们发现用NO供体处理显著增加了E-钙粘蛋白降解产物在培养基中的积累。由于金属蛋白酶介导E-钙粘蛋白的降解，我们测试了几种金属蛋白酶抑制剂，发现它们不仅阻断NO对E-钙粘蛋白降解的作用，而且消除了其对b-连环蛋白/LEF-1转录复合物形成的作用。这些发现表明，NO激活金属蛋白酶，从E-钙粘蛋白中释放游离的β-连环蛋白，形成β-连环蛋白/LEF-1转录复合物。使用合成的金属蛋白酶抑制剂和金属蛋白酶组织抑制剂（TIMP），我们正在确定特定的金属蛋白酶激活NO和表征这种激活的机制。

Germline and somatic mutations in the Apc (Adenomatous polyposis coli ) gene are thought to be seminal genetic events in the etiology of human and murine colorectal cancer. ApcMin mice carry a germline mutation in the Apc gene and experience reduced lifespan due to adenocarcinoma burden. Wild type, but not mutated, APC binds to and regulates beta-catenin, the mammalian homolog of armadillo required for cadherin-mediated cell adhesion. b-catenin released from its binding to membrane E-cadherin forms a heterodimer with Tcf/LEF and functions as a transcriptional factor. To study the regulation of this pathway, we used two conditional immortal murine intestinal epithelial cell lines contrasting in Apc genotype("Immortomouse"/Min Colonic Epithelia, Apc +/-; Young Adult Mouse Colon epithelia, Apc +/+). We have demonstrated that IMCE cells which have defective degradation of beta-catenin, have higher levels of b-catenin/LEF-1 transcriptional factor by EMSA and higher expression of COX-2 than YAMC cells in response to lipopolysaccharide (LPS) and interferon-g (IFN-g). The critical role of nitric oxide (NO) in this response was shown by the abrogation of the LPS, IFN-g effect by inhibitors of nitric oxide synthase II. Additionally, NO donors increased b-catenin/LEF-1 formation by EMSA as well as the expression of COX-2. That the effect was mediated by the availability of beta-catenin was supported by the differential response in IMCE and YAMC cells and by the direct demonstration of free, cytoplasmic b-catenin in response to NO treatment. Our current work is focused on the mechanism by which NO increases free, unbound b-catenin. Preliminary findings suggest that NO stimulates the degradation of membrane bound E-cadherin with the concomitant release of b-catenin from the cytoplasmic E-cadherin binding site. Using an antibody recognizing the extracellular domain of E-cadherin, we found that treatment with NO donors markedly increased E-cadherin degradation products accumulating in the medium. Since metallo- proteinases mediate the degradation of E-cadherin, we tested several inhibitors of metalloproteinases and found that they not only blocked the effect of NO on E-cadherin degradation but also abrogated its effect on the formation of b-catenin/LEF-1 transcriptional complexes. These findings suggest that the activation of metalloproteinases by NO releases free b-catenin from E-cadherin to form beta-catenin/LEF-1 transcriptional complexes. Using both synthetic metalloproteinase inhibitors and tissue inhibitors of metalloproteinases (TIMPs), we are identifying the specific metalloproteinase activated by NO and characterizing the mechanism of this activation.