Antiproliferative factor regulates connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells.
Antiproliferative factor regulates connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells.
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DOI：
10.1091/mbc.e11-08-0714
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发表时间：
2012-05
影响因子：
3.3
通讯作者：
Planey SL
中科院分区：
文献类型：
--
作者：
Matika CA;Wasilewski M;Arnott JA;Planey SL
结缔组织生长因子(CTGF/CNN2)是一个新的APF靶基因。报道了APF细胞受体细胞骨架相关蛋白4(CKAP4)介导APF诱导的CTGF转录的新机制。抗增殖因子(APF)是间质性膀胱炎(IC)患者尿液中升高的一种唾液糖肽，IC是一种病因不明的慢性痛性膀胱病。APF通过与细胞骨架相关蛋白4(CKAP4)结合，改变与细胞增殖、细胞黏附和肿瘤发生有关的基因转录，在体外抑制正常膀胱上皮细胞和T24膀胱癌细胞的增殖，但控制APF抗增殖作用的具体分子机制和效应基因尚不清楚。在本研究中，我们发现APF作用于T24膀胱癌细胞后，结缔组织生长因子(CTGF/CCN2)的表达上调了7.5倍。Western印迹显示，APF处理后CCN2蛋白水平呈剂量依赖性增加，并分泌到培养上清液中。CCN2过表达增强了APF的抗增殖活性，而CCN2基因敲除则降低了APF诱导的P53表达。使用荧光素酶报告结构，我们发现APF处理导致CCN2近端启动子的五倍激活，重要的是，小干扰RNA介导的CKAP4的敲除抑制了CCN2的上调。此外，我们还证明了CKAP4以APF依赖的方式移位到细胞核并与CCN2近端启动子结合，这为APF调控CCN2涉及CKAP4核移位和与CCN2启动子结合提供了证据。
Connective tissue growth factor (CTGF/CNN2) is a novel APF target gene. A novel mechanism is described by which the APF cellular receptor, cytoskeleton-associated protein 4 (CKAP4), mediates APF-induced CTGF transcription. Antiproliferative factor (APF) is a sialoglycopeptide elevated in the urine of patients with interstitial cystitis (IC)—a chronic, painful bladder disease of unknown etiology. APF inhibits the proliferation of normal bladder epithelial and T24 bladder carcinoma cells in vitro by binding to cytoskeleton-associated protein 4 (CKAP4) and altering the transcription of genes involved in proliferation, cellular adhesion, and tumorigenesis; however, specific molecular mechanisms and effector genes that control APF's antiproliferative effects are unknown. In this study, we found that there was a 7.5-fold up-regulation of connective tissue growth factor (CTGF/CCN2) expression in T24 bladder carcinoma cells treated with APF. Western blot revealed a dose-dependent increase in CCN2 protein levels, with secretion into the culture medium after APF treatment. CCN2 overexpression enhanced APF's antiproliferative activity, whereas CCN2 knockdown diminished APF-induced p53 expression. Using a luciferase reporter construct, we found that APF treatment resulted in fivefold activation of the CCN2 proximal promoter and, of importance, that small interfering RNA–mediated knockdown of CKAP4 inhibited CCN2 upregulation. In addition, we demonstrate that CKAP4 translocates to the nucleus and binds to the CCN2 proximal promoter in an APF-dependent manner, providing evidence that CCN2 regulation by APF involves CKAP4 nuclear translocation and binding to the CCN2 promoter.