RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity
Jun Kawasaki, … , Steven J. Fishman, Joanne Chan
Jun Kawasaki, … , Steven J. Fishman, Joanne Chan
Published May 16, 2014
Citation Information: J Clin Invest. 2014;124(6):2774-2784. https://doi.org/10.1172/JCI67084.
View: Text | PDF
Research Article Vascular biology

RASA1 functions in EPHB4 signaling pathway to suppress endothelial mTORC1 activity

Abstract

Vascular malformations are linked to mutations in RAS p21 protein activator 1 (RASA1, also known as p120RasGAP); however, due to the global expression of this gene, it is unclear how these mutations specifically affect the vasculature. Here, we tested the hypothesis that RASA1 performs a critical effector function downstream of the endothelial receptor EPHB4. In zebrafish models, we found that either RASA1 or EPHB4 deficiency induced strikingly similar abnormalities in blood vessel formation and function. Expression of WT EPHB4 receptor or engineered receptors with altered RASA1 binding revealed that the ability of EPHB4 to recruit RASA1 is required to restore blood flow in EPHB4-deficient animals. Analysis of EPHB4-deficient zebrafish tissue lysates revealed that mTORC1 is robustly overactivated, and pharmacological inhibition of mTORC1 in these animals rescued both vessel structure and function. Furthermore, overexpression of mTORC1 in endothelial cells exacerbated vascular phenotypes in animals with reduced EPHB4 or RASA1, suggesting a functional EPHB4/RASA1/mTORC1 signaling axis in endothelial cells. Tissue samples from patients with arteriovenous malformations displayed strong endothelial phospho-S6 staining, indicating increased mTORC1 activity. These results indicate that deregulation of EPHB4/RASA1/mTORC1 signaling in endothelial cells promotes vascular malformation and suggest that mTORC1 inhibitors, many of which are approved for the treatment of certain cancers, should be further explored as a potential strategy to treat patients with vascular malformations.

Authors

Jun Kawasaki, Sandrine Aegerter, R. Dawn Fevurly, Akiko Mammoto, Tadanori Mammoto, Mustafa Sahin, John D. Mably, Steven J. Fishman, Joanne Chan

×

Figure 2

EPHB4 receptor rescue requires RASA1- binding motifs.

Options: View larger image (or click on image) Download as PowerPoint
EPHB4 receptor rescue requires RASA1- binding motifs. The ephb4a MO (500...
The ephb4a MO (500 μM) was injected in double-transgenic line; refs. 32, 33), to visualize endothelial (green; A and C) and blood cells (red; B and D) in the same embryo at 48 hpf. (A–D) Caudal vessel phenotypes. (B and D) Blood flow in control (magnified boxed region in [A], or ephb4a morphant [C]). Yellow arrow indicates the end point of blood flow. Dotted arrows indicate direction of flow. Scale bars: 500 μm (A), 100 μm (B–D). (E–G) Expression of EPHB4 constructs and RASA1 in HEK293T cells. Control and EPHB4 construct–transfected cells were stimulated with clustered ephrin-B2–Fc (EfnB2a) (2 μg/ml). EPHB4 and RASA1 cotransfected (co-TF) cell lysates were immunoprecipitated (IP) using antibodies to EPHB4 or RASA1 (E, expression confirmed in whole cell lysate [WCL]). Receptor activation was determined by tyrosine phosphorylation using 4G10 antibody (F). EPHB4 construct–transfected cells were biotinylated and immunoprecipitated with streptavidin, followed by anti-EPHB4 blotting to determine cell-surface localization (G). (H) The ephb4a MO (500 μM) and ephb4a mRNA (30 ng/μl) coinjection phenotypes at 48 hpf (severe, mild, or normal) were converted to percentages. Caudal functional assay was used to score blood flow (n = 100 per condition).