Abstract
Angiogenesis is a hallmark of cancer cell malignancy. The role of the RHO family GTPase RHOG in angiogenesis in vascular endothelial cells has recently been elucidated. However, the regulation of RHOG during this process, as well as its cross-talk with other RHO GTPases, have yet to be fully examined. In this study, we found that siRNA-mediated depletion of RHOG strongly inhibits tube formation in vascular endothelial cells (ECV cells), an effect reversed by transfecting dominant active constructs of CDC42 or RAC1 in the RHOG-depleted cells. We also found CDC42 to be upstream from RAC1 in these cells. Inhibiting either Phosphatidyl inositol (3) kinase (PI3K) with Wortmannin or the mitogen-activated protein kinase extracellular-regulated kinase (MAPK ERK) with U0126 leads to the inhibition of tube formation. While knocking down either RHO, GTPase did not affect p-AKT levels, and p-ERK decreased in response to the knocking down of RHOG, CDC42 or RAC1. Recovering active RHO GTPases in U0126-treated cells also did not reverse the inhibition of tube formation, placing ERK downstream from PI3K-RHOG-CDC42-RAC1 in vascular endothelial cells. Finally, RHOA and the Rho activated protein kinases ROCK1 and ROCK2 positively regulated tube formation independently of ERK, while RHOC seemed to inhibit the process. Collectively, our data confirmed the essential role of RHOG in angiogenesis, shedding light on a potential new therapeutic target for cancer malignancy and metastasis.
Highlights
Angiogenesis is the formation of new blood vessels from preexisting blood vessels [1]
The following primary antibodies were used in this study: Mouse monoclonal anti-RHOG, mouse monoclonal anti-RHOA, rabbit polyclonal anti-RHOC, mouse monoclonal anti-RAC1, rabbit monoclonal anti-ROCK1, rabbit monoclonal anti-ROCK2, rabbit polyclonal anti-p-AKT1, rabbit polyclonal anti-pan-AKT, and rabbit polyclonal anti-Actin antibodies, as well as the MEK1/2 inhibitor U0126 and the phosphatidylinositol (3) kinase (PI3K) inhibitor Wortmannin, which were obtained from Abcam (Abcam Inc., Cambridge, UK)
RHOG expression by 60–80% in the transfected cells compared to Luciferase siRNA-transfected control control cells (β−actin was used as loading control) (Figure 1A,B)
Summary
Angiogenesis is the formation of new blood vessels from preexisting blood vessels [1]. It is required during embryonic development, growth, regeneration and wound healing [1,2]. It is implicated in pathological processes including arthritis, muscular dystrophy, and tumorigenesis [3]. The angiogenic process starts when endothelial cells (EC) branch from pre-existing small vessels, forming sprouts of capillaries that can supply the tissue of the tumor [2]. The new blood vessel formation is triggered by angiogenic stimuli, such as the vascular endothelial growth factor (VEGF) [1,4]. New blood vessel formation is regulated by the RHO
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