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Research Articles: Therapeutics, Targets, and Development

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

¹ Department of Obstetrics and Gynecology, Yamagata University, School of Medicine, Yamagata, Japan and ² Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, Osaka, Japan

Requests for reprints: Ken-ichirou Morishige, Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3354; Fax: 81-6-6879-3359. E-mail: mken{at}gyne.med.osaka-u.ac.jp

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-1 induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1 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-1 protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1 mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1 expression without altering HIF-1 mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1 expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1 expression is through preventing HIF-1 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-1 signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1 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]

Grant support: Ministry of Education, Culture, Sports, Science, and Technology of Japan grants-in-aid for Scientific Research 17390445 (H. Kurachi) and 18591822 (K. Takahashi) and Japan Society for the Promotion of Science grant-in-aid for the 21st Century Center of Excellence program.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/25/07; revised 3/26/08; accepted 4/ 4/08.