Apc and beta-Catenin in Cell Regulation and Cancer

APC(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种子遗传事件。ApcMin小鼠携带APC基因的胚系突变，并由于腺癌负担而经历寿命缩短。野生型，但没有突变，APC结合并调节β-连环蛋白，β-连环蛋白是一种哺乳动物中的蝉同系物，需要钙粘附素介导的细胞黏附。B-连环蛋白从与膜E-钙粘附素结合后释放出来，与Tcf/Lef形成异源二聚体，发挥转录因子的作用。为了研究这一途径的调控，我们使用了两种条件下的永生小鼠肠道上皮细胞系(“永生小鼠”/“小鼠”结肠上皮细胞，APC+/-；幼年小鼠结肠上皮细胞，APC+/+)。我们已经证明，β-连环素降解缺陷的IMCE细胞在内毒素和干扰素-g的作用下，EMSA检测到的b-连环蛋白/Lef-1转录因子水平和COX-2的表达水平均高于YAMC细胞。一氧化氮(NO)在这一反应中的关键作用体现在内毒素的消除，一氧化氮合酶II的抑制剂对干扰素-g的作用。此外，NO供体增加了EMSA诱导的b-catenin/Lef-1的形成以及COX-2的表达。这种作用是由β-连环蛋白的可获得性介导的，这一点得到了IMCE和YAMC细胞的差异反应的支持，以及对NO处理反应的直接证明，细胞质中的游离的b-连环蛋白。我们目前的工作集中在NO增加自由、未结合的b-连环蛋白的机制上。初步研究结果表明，NO刺激膜结合的E-钙粘附素的降解，同时伴随着b-连环素从细胞质E-钙粘附素结合部位的释放。使用一种识别E-钙粘蛋白胞外区的抗体，我们发现没有供体的治疗显著增加了E-钙粘素降解产物在介质中的积累。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的抑制剂，发现它们不仅可以阻断NO对E-钙粘蛋白降解的作用，而且可以抑制其对b-catenin/Lef-1转录复合体形成的影响。这些结果表明，NO激活金属蛋白酶后，E-钙粘附素释放出游离的b-连环素，形成β-连环素/Lef-1转录复合体。利用人工合成的金属蛋白酶抑制剂和金属蛋白酶的组织抑制物(TIMPs)，我们正在鉴定NO激活的特异性金属蛋白酶，并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.