Apc and beta-Catenin in Cell Regulation and Cancer

Apc 和 β-连环蛋白在细胞调节和癌症中的作用

• 批准号: 6559083
• 负责人: JAMES M PHANG
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6559083
• 关键词: adenomatous polyps; cadherins; carcinogenesis; cell adhesion; cytoskeletal proteins; enzyme inhibitors; gastrointestinal epithelium; genetic regulation; intermolecular interaction; metalloendopeptidases; neoplasm /cancer genetics; nitric oxide; oncoproteins; protein degradation; protein structure function; tissue /cell culture

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.

Apc（大肠腺瘤性息肉病）基因的种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重要遗传事件。ApcMin小鼠携带Apc基因的种系突变，并且由于腺癌负担而缩短寿命。野生型，但未突变，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源物。b-连环蛋白与膜e -钙粘蛋白结合后释放，与Tcf/LEF形成异源二聚体，并作为转录因子发挥作用。为了研究这一途径的调控，我们使用了两种Apc基因型比较的条件不朽小鼠肠上皮细胞系（“不朽小鼠”/Min结肠上皮，Apc +/-；年轻成年小鼠结肠上皮，Apc +/+）。我们已经证明，在脂多糖（LPS）和干扰素g （IFN-g）的作用下，具有β -连环蛋白降解缺陷的IMCE细胞比YAMC细胞具有更高水平的b-连环蛋白/LEF-1转录因子和更高水平的COX-2表达。一氧化氮（NO）在这种反应中的关键作用通过一氧化氮合酶II抑制剂消除LPS， IFN-g效应而得到证明。此外，NO供体通过EMSA增加b-catenin/ lev -1的形成以及COX-2的表达。IMCE和YAMC细胞的差异反应，以及对NO处理的细胞质中游离的b-catenin的直接证明，支持了β -catenin的可用性介导的作用。我们目前的工作重点是NO增加游离、未结合的b-连环蛋白的机制。初步结果表明，NO刺激膜结合E-cadherin的降解，同时从细胞质E-cadherin结合位点释放b-catenin。使用一种识别E-cadherin细胞外结构域的抗体，我们发现NO供体治疗显著增加了E-cadherin降解产物在培养基中的积累。由于金属蛋白酶介导E-cadherin的降解，我们测试了几种金属蛋白酶抑制剂，发现它们不仅阻断了NO对E-cadherin降解的影响，而且消除了NO对b-catenin/LEF-1转录复合物形成的影响。这些发现表明，NO激活金属蛋白酶释放E-cadherin中的游离b-catenin，形成β -catenin/ lev -1转录复合物。利用合成金属蛋白酶抑制剂和组织金属蛋白酶抑制剂（TIMPs），我们确定了NO激活的特定金属蛋白酶，并表征了这种激活的机制。