Fasudil-induced hypoxia-inducible factor-1α degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells

Abstract

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)–dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1α induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1α expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1α protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1α mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1α expression without altering HIF-1α mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1α expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1α expression is through preventing HIF-1α degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1α signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1α signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis. [Mol Cancer Ther 2008;7(6):1551–61]