Veliceasa Dorina, Ivanovic Marina, Hoepfner Frank Thilo-Schulze, Thumbikat Praveen, Volpert Olga V, Smith Norm D
Department of Urology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA.
FEBS J. 2007 Dec;274(24):6365-77. doi: 10.1111/j.1742-4658.2007.06159.x. Epub 2007 Nov 15.
Angiogenic switch in renal cell carcinoma (RCC) is attributed to the inactivation of the von Hippel-Lindau tumor suppressor, stabilization of hypoxia inducible factor-1 transcription factor and increased vascular endothelial growth factor. To evaluate the role of an angiogenesis inhibitor, thrombopsondin-1 (TSP1), we compared TSP1 production in human RCC and normal tissue and secretion by the normal renal epithelium (human normal kidney, HNK) and RCC cells. Normal and RCC tissues stained positive for TSP1, and the levels of TSP1 mRNA and total protein were similar in RCC and HNK cells. However, HNK cells secreted high TSP1, which rendered them nonangiogenic, whereas RCC cells secreted little TSP1 and were angiogenic. Western blot and immunostaining revealed TSP1 in the cytoplasm of RCC cells on serum withdrawal, whereas, in HNK cells, it was rapidly exported. Seeking mechanisms of defective TSP1 secretion, we discovered impaired calcium uptake by RCC in response to vascular endothelial growth factor. In HNK cells, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, a calcium chelator, simulated TSP1 retention, mimicking the RCC phenotype. Further analysis revealed a profound decrease in transient receptor potential canonical ion channel 4 (TRPC4) Ca(2+) channel expression in RCC cells. TRPC4 silencing in HNK cells caused TSP1 retention and impaired secretion. Double labeling of the secretory system components revealed TSP1 colocalization with coatomer protein II (COPII) anterograde vesicles in HNK cells. In contrast, in RCC cells, TSP1 colocalized with COPI vesicles, pointing to the retrograde transport to the endoplasmic reticulum caused by misfolding. Our study indicates that TRPC4 loss in RCC leads to impaired Ca(2+) intake, misfolding, retrograde transport and diminished secretion of antiangiogenic TSP1, thus enabling angiogenic switch during RCC progression.
肾细胞癌（RCC）中的血管生成开关归因于冯·希佩尔-林道肿瘤抑制因子的失活、缺氧诱导因子-1转录因子的稳定以及血管内皮生长因子的增加。为了评估血管生成抑制剂血小板反应蛋白-1（TSP1）的作用，我们比较了TSP1在人RCC和正常组织中的产生情况，以及正常肾上皮细胞（人正常肾脏，HNK）和RCC细胞的分泌情况。正常组织和RCC组织TSP1染色呈阳性，RCC细胞和HNK细胞中TSP1 mRNA和总蛋白水平相似。然而，HNK细胞分泌大量TSP1，使其具有非血管生成性，而RCC细胞分泌很少的TSP1且具有血管生成性。蛋白质免疫印迹和免疫染色显示，血清撤出时RCC细胞的细胞质中有TSP1，而在HNK细胞中，它会迅速输出。在寻找TSP1分泌缺陷的机制时，我们发现RCC对血管内皮生长因子的钙摄取受损。在HNK细胞中，钙螯合剂1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸乙酰甲酯模拟了TSP1的滞留，模拟了RCC表型。进一步分析显示，RCC细胞中瞬时受体电位阳离子通道4（TRPC4）Ca（2+）通道表达显著降低。HNK细胞中TRPC4沉默导致TSP1滞留和分泌受损。对分泌系统成分的双重标记显示，HNK细胞中TSP1与II型包被蛋白（COPII）顺行囊泡共定位。相反，在RCC细胞中，TSP1与I型包被蛋白（COPI）囊泡共定位，表明由于错误折叠导致逆行转运至内质网。我们的研究表明，RCC中TRPC4的缺失导致Ca（2+）摄取受损以及抗血管生成TSP1的错误折叠、逆行转运和分泌减少，从而在RCC进展过程中促成血管生成开关。
