Fasudil-induced hypoxia-inducible factor-1alpha degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells.
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-1alpha induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1alpha 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-1alpha protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1alpha mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1alpha expression without altering HIF-1alpha mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1alpha expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1alpha expression is through preventing HIF-1alpha 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-1alpha signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1alpha 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.
Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan.
Keiko
K
Takata
Ken-Ichirou
K
Morishige
Toshifumi
T
Takahashi
Kae
K
Hashimoto
Seiji
S
Tsutsumi
Limei
L
Yin
Tsuyoshi
T
Ohta
Jun
J
Kawagoe
Kazuhiro
K
Takahashi
Hirohisa
H
Kurachi
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D019898:Autocrine Communication / Q000187:drug effects*; D015687:Cell Hypoxia / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D018994:Myosin Light Chains / Q000378:metabolism; D009389:Neovascularization, Pathologic / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D021381:Protein Transport / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D054875:Ubiquitination / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D054460:rho-Associated Kinases / Q000378:metabolism
D000577:Amides; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007976:Leupeptins; D018994:Myosin Light Chains; D011725:Pyridines; D042461:Vascular Endothelial Growth Factor A; C108830:Y 27632; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D040301:Vascular Endothelial Growth Factor Receptor-2; D054460:rho-Associated Kinases; C049347:fasudil; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde