Grabmaier Karin, A de Weijert Mirjam C, Verhaegh Gerald W, Schalken Jack A, Oosterwijk Egbert
Laboratory for Experimental Urology, 190-RT NCMLS, University Medical Center Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Oncogene. 2004 Jul 22;23(33):5624-31. doi: 10.1038/sj.onc.1207764.
Renal cell carcinoma of the clear cell type (ccRCC) is associated with loss of functional von Hippel-Lindau (VHL) protein and high, homogeneous expression of the G250MN protein, an isoenzyme of the carbonic anhydrase family. High expression of G250MN is found in all ccRCCs, but not in most normal tissues, including normal human kidney. We specifically studied the mechanism of transcriptional regulation of the CAIXG250 gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250MN in various non-RCC backgrounds. However, G250MN regulation in RCC has not been studied and may be differently regulated in view of the HIF accumulation under normoxic conditions due to VHL mutations. Transient transfection of different G250MN promoter constructs revealed strong promoter activity in G250MN -positive RCC cell lines, but no activity in G250MN -negative cell lines. DNase-I footprint and band-shift analysis demonstrated that Sp1 and HIF-1alpha proteins in nuclear extracts of RCC cells bind to the CAIX promoter and mutations in the most proximal Sp1 binding element or HIF binding element completely abolished CAIX promoter activity, indicating their critical importance for the activation of G250 expression in RCC. A close correlation between HIF-1alpha expression and G250MN expression was observed. In contrast, no relationship between HIF-2alpha expression and G250MN was seen. The participation of cofactor CBP/p300 in the regulation of G250 transcription was shown. In conclusion, HIF-1alpha and Sp1, in combination with CBP/p300, are crucial elements for G250MN expression in ccRCC, and CAIXG250 can be regarded as a unique HIF-1alpha target gene in ccRCC.
透明细胞型肾细胞癌（ccRCC）与功能性冯·希佩尔-林道（VHL）蛋白缺失以及碳酸酐酶家族同工酶G250MN蛋白的高表达且表达均匀相关。G250MN在所有ccRCC中均有高表达，但在包括正常人类肾脏在内的大多数正常组织中未发现高表达。我们专门研究了肾细胞癌中CAIXG250基因的转录调控机制。先前的研究确定Sp1和缺氧诱导因子（HIF）是各种非肾细胞癌背景下G250MN的主要调节转录因子。然而，尚未对肾细胞癌中G250MN的调控进行研究，鉴于VHL突变导致常氧条件下HIF积累，其调控方式可能有所不同。不同G250MN启动子构建体的瞬时转染显示，在G250MN阳性肾细胞癌细胞系中具有强启动子活性，而在G250MN阴性细胞系中无活性。DNA酶I足迹法和凝胶迁移分析表明，肾癌细胞核提取物中的Sp1和HIF-1α蛋白与CAIX启动子结合，最近端Sp1结合元件或HIF结合元件中的突变完全消除了CAIX启动子活性，表明它们对肾细胞癌中G250表达的激活至关重要。观察到HIF-1α表达与G250MN表达之间存在密切相关性。相比之下，未发现HIF-2α表达与G250MN之间存在关联。研究表明辅因子CBP/p300参与了G250转录的调控。总之，HIF-1α和Sp1与CBP/p300共同作用，是ccRCC中G250MN表达的关键因素，并且CAIXG250可被视为ccRCC中独特的HIF-1α靶基因。
