Strict regulation of CAIXG250/MN by HIF-1α in clear cell renal cell carcinoma

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 G250(MN) protein, an isoenzyme of the carbonic anhydrase family. High expression of G250(MN) 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 CAIX(G250) gene in RCC. Previous studies identified Sp1 and hypoxia-inducible factor (HIF) as main regulatory transcription factors of G250(MN) in various non-RCC backgrounds. However, G250(MN) 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 G250(MN) promoter constructs revealed strong promoter activity in G250(MN) -positive RCC cell lines, but no activity in G250(MN) -negative cell lines. DNase-I footprint and handshift 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 G250(MN) expression was observed. In contrast, no relationship between HIF-2alpha expression and G250(MN) 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 G250(MN) expression in ccRCC, and CAIX(G250) can be regarded as a unique HIF-1alpha: target gene in ccRCC.