Epithelial proliferation induces novel changes in APC expression.
Epithelial proliferation induces novel changes in APC expression.
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上皮增殖诱导 APC 表达的新变化。
DOI：
10.1038/sj.onc.1208820
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Sellin,JosephH
中科院分区：
文献类型：
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作者：
Umar,Shahid;Wang,Yu;Sellin,JosephH
野生型腺瘤性结肠息肉病（APC）蛋白在天然上皮中的作用知之甚少。本研究在已建立的过度增殖、传染性小鼠结肠增生（TMCH）模型中检查了野生型APC和β-连环蛋白表达之间的关系。从正常或TMCH小鼠分离的远端结肠隐窝：（i）分离成细胞溶质和细胞核组分用于Western印迹和免疫沉淀（IP），（ii）提取总RNA分离用于北方印迹，和（iii）通过共聚焦显微镜化学分析。用N-末端APC抗体连续进行的蛋白质印迹显示，到第6天，p312蛋白的丰度增加（4.0±0.75倍，n= 6），到第9天达到峰值，然后到第12天下降。第9天出现一条约130 kDa（p130）条带，第12天增加（1.5±0.11倍，n= 6）。C-末端抗体仅检测到p312。TMCH过程中APC mRNA水平无明显变化，p130的出现不是由于选择性剪接。与N-末端抗APC抗体的共IP揭示了APC在第6天和第12天与β-连环蛋白的缔合。在与抗β-catenin联合IP期间，在第12天，p130而不是p312主要与β-catenin相关。p130也选择性地积聚在细胞核中，在第12天与细胞核β-连环蛋白结合。N-末端抗体的免疫细胞化学显示，在第6天，顶极/顶侧膜内APC的隐窝基底：表面梯度增加。在第12天，观察到强烈的顶端/细胞质和偶尔的核染色沿着纵向隐窝轴。全长APC在上皮过度增殖期间增加，并且可能代表同源平衡反应。在第12天用N-末端抗体在细胞质和散在核APC染色中的显著增加可能代表p130。p130在细胞核内的积聚可能是TMCH时β-catenin功能调节的一种新机制。
The role of wild-type adenomatous polyposis coli (APC) protein in native epithelia is poorly understood. The present study examined the relationships between wild-type APC and β-catenin expression in an established model of hyperproliferation, transmissible murine colonic hyperplasia (TMCH). Distal colonic crypts isolated from normal or TMCH mice were:(i) fractionated into cytosolic and nuclear components for Western blotting and immunoprecipitation (IP),(ii) extracted for total RNA isolation for Northern blotting and,(iii) analysed immunohistochemically by confocal microscopy. Western blots performed sequentially through day 12 TMCH with N-terminal APC antibodies revealed increased abundance of∼ 312 kDa (p312) protein by day 6 (4.0±0.75-fold, n= 6) that peaked by day 9, before declining by day 12. A∼ 130 kDa (p130) band appeared at day 9 and increased by day 12 (1.5±0.11-fold, n= 6). A C-terminal antibody detected only p312. APC mRNA level did not change during TMCH and appearance of p130 was not due to alternative splicing. Co-IP with N-terminal anti-APC antibodies, revealed APC's association with β-catenin both at day 6 and day 12. p130, but not p312, associated predominantly with β-catenin at day 12 during co-IP with anti-β-catenin. p130 also selectively accumulated in the nucleus, bound to nuclear β-catenin at day 12. Immunocytochemistry with N-terminal antibodies revealed an increasing crypt base: surface gradient of APC within the apical pole/apical-lateral membranes at day 6. At day 12, intense apical/cytoplasmic and occasional nuclear staining along the longitudinal crypt axis was observed. Full-length APC increases during epithelial hyperproliferation and may represent a homoeostatic response. The dramatic increase in cytoplasmic and sporadic nuclear APC staining at day 12 with N-terminal antibodies may represent p130. The nuclear accumulation of p130 may be a novel mechanism regulating nuclear β-catenin function during TMCH.