Regulation of VEGFR3 signaling in lymphatic endothelial cells

Abstract

The receptor tyrosine kinase vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3) is the principal transmembrane receptor responsible for sensing and coordinating cellular responses to environmental lymphangiogenic stimuli in lymphatic endothelial cells (LECs). VEGFC and D (VEGFC/D) function as the cognate ligands to VEGFR3 by stimulating autophosphorylation of intracellular VEGFR3 tyrosine kinase domains that activate signal cascades involved in lymphatic growth and survival. VEGFR3 primarily promotes downstream signaling through the phosphoinositide 3-kinase (PI3K) and Ras signaling cascades that promote functions including cell proliferation and migration. The importance of VEGFR3 cascades in lymphatic physiology is underscored by identification of dysfunctional VEGFR3 signaling across several lymphatic-related diseases. Recently, our group has shown that intracellular modification of VEGFR3 signaling is a potent means of inducing lymphangiogenesis independent of VEGFC. This is important because long-term treatment with recombinant VEGFC may have deleterious consequences due to off-target effects. A more complete understanding of VEGFR3 signaling pathways may lead to novel drug development strategies. The purpose of this review is to 1) characterize molecular mediators of VEGFC/VEGFR3 downstream signaling activation and their functional roles in LEC physiology and 2) explore molecular regulation of overall VEGFR3 expression and activity within LECs.

Keywords: LECs; VEGFC; VEGFR3; lymphangiogenesis; lymphatics; lymphedema.

FIGURE 1

Initiation and co-modulation of transmembrane VEGFR3 signaling; Transmembrane receptors and intracellular adapter proteins regulate VEGFR3 tyrosine kinase domain activation and signal transduction. Neuropilin-2 and EphB4 receptors promote internalization of VEGF-C/VEGFR3 complexes, which is necessary for downstream signal propagation (A). In contrast, Plexin A1 signaling attenuates VEGFR3 via Neuropilin-2 inhibition, while uPARAP inhibits VEGFR2-VEGFR3 heterodimer formation, thereby fine-tuning lymphangiogenic activity (B). Other transmembrane proteins including the PECAM-1/VE-Cadherin mechanosensory complex and membrane integrins, enhance downstream VEGFR3 signaling (A). Intracellular adapters including Shc and Grb2 activate principal downstream VEGFR3 effectors AKT and ERK, which can be enhanced or attenuated by cytoplasmic proteins ARRB1 and SPREDS (A, B).

FIGURE 2

Cellular regulation of VEGFR3 expression; (A) The FLT4 (VEGFR3) gene promoter is primarily under Prox-1 transcription factor control. Environmental stressors such as hypoxia and inflammation can stimulate lymphangiogenesis by increasing Prox-1 activation. MAFB and SOX18 transcription factors, which play important roles in early lymphatic development, can also directly increase Prox-1 activity, while Yap/Taz transcription factors decrease Prox-1 activity and downstream VEGFR3 transcription (B) Transmembrane and intracellular proteins control VEGFR3 trafficking within lymphatic endothelial cells, which alters lymphangiogenic stimulatory capacity. Robo4 and EphB4 receptors promote VEGFR3 internalization, while VE-Cadherin maintains surface VEGFR3 availability. Cytoplasmic adapter proteins Epsin 1/2 and AIP1 promote VEGFR3 internalization/degradation, while ARRB1 and AGS8 promote surface VEGFR3 availability.

FIGURE 3

Intracellular VEGFR3 signaling; VEGFR3 signaling can be strategically enhanced or inhibited at various levels via targeting of molecular proteins located upstream or downstream from AKT and ERK. Downstream ERK effectors CREB and Ets-2 promote cell proliferation and differentiation, while downstream AKT effectors mTORC1 and eNOS primarily promote cell survival and growth functions. Crosstalk between the AKT and ERK signaling cascades allows context-dependent fine-tuned control of lymphatic endothelial cell function.