Regulation of vascular endothelial growth factor transcription by endothelial PAS domain protein 1 (EPAS1) and possible involvement of EPAS1 in the angiogenesis of renal cell carcinoma

BACKGROUND. Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop- helix/PAS domain transcription factor that expressed most abundantly in hig hly vascularized organs. The authors examined the effect of transfection of EPAS1 cDNA on the endogenous expression of vascular endothelial growth fac tor (VEGF) in the 293 Tet-Off cell line and the possible involvement of EPA S1 in the angiogenesis of renal cell carcinoma (RCC). METHODS. Complete cDNA of EPAS1 was cloned and transfected to cells from th e 293 Tet-Off fetal kidney cell line, in which the expression of EPAS1 can be inhibited by doxycycline. The subsequent changes in expression pattern o f VEGF and transferrin receptor (TfR), a target gene of hypoxia-inducible f actor 1 alpha (HIF-1 alpha), were examined by semiquantitative reverse tran scription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorben t assay. In addition, expression of EPAS1, HIF-1 alpha, and VEGF were analy zed by semiquantitative RT-PCR in five RCC cell lines and in 13 RCC tissue samples. In situ hybridization was performed on 7 of the 13 RCC tissue samp les. RESULTS. Endogenous VEGF was increased significantly by the introduction of EPAS1 cDNA at both the mRNA level and the protein level. With the inhibiti on of EPAS1 by doxycycline treatment, the expression of VEGF was significan tly decreased accordingly, whereas the expression of TfR was not affected. EPAS1 was detected in all of the RCC cell lines examined. In RCC tissue sam ples, EPAS1 mRNA and VEGF mRNA were increased significantly in tumor tissue s compared with normal adjacent kidney tissues. In situ hybridization showe d that EPAS1 and VEGF were coexpressed topographically in tumor tissues. CONCLUSIONS, These results suggest that endogenous VEGF can be up-regulated transcriptionally by EPAS1, and EPAS1 may be involved in the angiogenesis of RCC. Cancer 2001;91:1429-36. (C) 2001 American Cancer Society.