EPHB4-RASA1-Mediated Negative Regulation of Ras-MAPK Signaling in the Vasculature: Implications for the Treatment of EPHB4- and RASA1-Related Vascular Anomalies in Humans

Abstract

Ephrin receptors constitute a large family of receptor tyrosine kinases in mammals that through interaction with cell surface-anchored ephrin ligands regulate multiple different cellular responses in numerous cell types and tissues. In the cardiovascular system, studies performed in vitro and in vivo have pointed to a critical role for Ephrin receptor B4 (EPHB4) as a regulator of blood and lymphatic vascular development and function. However, in this role, EPHB4 appears to act not as a classical growth factor receptor but instead functions to dampen the activation of the Ras-mitogen activated protein signaling (MAPK) pathway induced by other growth factor receptors in endothelial cells (EC). To inhibit the Ras-MAPK pathway, EPHB4 interacts functionally with Ras p21 protein activator 1 (RASA1) also known as p120 Ras GTPase-activating protein. Here, we review the evidence for an inhibitory role for an EPHB4-RASA1 interface in EC. We further discuss the mechanisms by which loss of EPHB4-RASA1 signaling in EC leads to blood and lymphatic vascular abnormalities in mice and the implications of these findings for an understanding of the pathogenesis of vascular anomalies in humans caused by mutations in EPHB4 and RASA1 genes. Last, we provide insights into possible means of drug therapy for EPHB4- and RASA1-related vascular anomalies.

Keywords: Ephrin receptor B4; RASA1; Ras-MAPK pathway; angiogenesis; capillary malformation arteriovenous malformation; lymphatic malformation; vascular valves.

Figure 1

Domain structure of EPHB4 and RASA1. LBD, ligand-binding domain; FN, Fibronectin homology; JM, juxtamembrane region; PTK, protein tyrosine kinase domain; SAM, sterile alpha motif domain; SH2 and SH3, Src homology-2 and 3 domains; PH, pleckstrin homology domain; C2, protein kinase C2 homology domain; GAP, RasGTPase-activating domain.

Figure 2

Control of EC collagen IV secretion by EPHB4 and RASA1. During developmental angiogenesis, EPHB4 cooperates with RASA1 to limit GFR-induced Ras activation in EC (red arrows). In the absence of functional EPHB4 and RASA1, dysregulated Ras-MAPK activation results in increased transcription and abundance of collagen P3H and P4H proline and PLOD lysine hydroxylases. In the rough ER (rER), the increased abundance of P3H, P4H and PLODs leads to excessive hydroxylation of collagen IV monomers that inhibits their folding and assembly into triple helical collagen IV protomers that are normally exported from the cell by the COPII secretory pathway. Improperly folded collagen IV is retained within the ER resulting in EC apoptosis. Drugs that inhibit the MAPK pathway (AZD6244), drugs that inhibit collagen IV proline and lysine hydroxylases (2,4PDCA and EDHB) and drugs that promote collagen IV folding in the ER (4PBA) all rescue collagen IV export and EC apoptosis in EPHB4- and RASA1-deficient embryos (blue lines and arrows).

Figure 3

Models of EPHB4 and RASA1 cooperation in EC. Left, EPHB4 signaling generates membrane lipids that are ligands of the RASA1 PH domain leading to RASA1 membrane localization and juxtaposition to Ras-GTP. Right, RASA1 membrane localization is mediated through SH2 domain recognition of phosphorylated tyrosine residues in the EPHB4 JM region. Note that this latter mechanism of EPHB4–RASA1 interplay is not required for developmental angiogenesis or neoangiogenesis in mice.

Figure 4

Models of AVM formation resulting from germline plus 2nd hit mutation in EPHB4 and RASA1 genes. See text for details.