Abstract
Syntenin, a tandem PDZ-domain-containing scaffold protein, is involved in the regulation of diverse biological functions, including protein trafficking, exosome biogenesis, and cancer metastasis. Here, we present the first study to explore the significance of syntenin in endothelial cells. Syntenin knockdown in human umbilical vein endothelial cells (HUVECs) impaired vascular endothelial growth factor (VEGF)-mediated proliferation, migration, invasion, vascular permeability, and nitric oxide (NO) production. Syntenin knockdown also suppressed expression of the VEGFR2 target genes VEGF, MMP2, and Nurr77 as well as VEGF-induced angiogenesis in vitro and in vivo. And it decreased cell-surface levels of ephrin-B2. Biochemical analyses revealed that syntenin exists in complex with VEGFR2 and ephrin-B2. Syntenin knockdown abolished the association between VEGFR2 and ephrin-B2, suggesting syntenin functions as a scaffold protein facilitating their association in HUVECs. Consistent with these observations, knocking down syntenin or ephrin-B2 abolished VEGF-induced endocytosis and VEGFR2 phosphorylation and activation of its downstream signaling molecules. Treatment with MG132, a proteasome inhibitor, rescued the downregulation of ephrin-B2 and VEGFR2 signaling induced by syntenin knockdown. These findings demonstrate that syntenin promotes VEGF signaling and, through its PDZ-dependent interaction with ephrin-B2, enhances VEGF-mediated VEGFR2 endocytosis and subsequent downstream signaling and angiogenesis in endothelial cells.
Highlights
Angiogenesis is a complex, multi-step process, which involves destabilization of the established vessel, followed by EC proliferation, migration, and tubulogenesis [1, 2]
We first determined that syntenin was abundantly expressed in human umbilical vein endothelial cells (HUVECs) (Figure 1A), comparable to the level in A549 human adenocarcinoma cells, and that stimulation with vascular endothelial growth factor (VEGF) did not alter the level of syntenin (Figure 1B)
We performed a wound-healing migration assay on HUVEC monolayers transfected with control Small interfering RNA (siRNA) or syntenin siRNA
Summary
Angiogenesis is a complex, multi-step process, which involves destabilization of the established vessel, followed by EC proliferation, migration, and tubulogenesis [1, 2]. Ephrin-B2, a transmembrane protein belonging to the B-class ephrin subfamily, and its intracellular interactors, clathrin-associated protein disabled 2 (Dab2) and cell polarity regulator PAR-3, promote clathrin-dependent VEGFR internalization and thereby downstream signaling and angiogenesis [12, 13]. Disruption of this interaction by silencing of Dab or PAR-3 reduces VEGFR2 internalization and impairs VEGF-induced angiogenesis [12]. Ephrin-B2 has a C-terminal PDZ (PSD-95/Dlg/ZO-1)-binding motif for the binding of PDZ domain containing proteins [14] Mutation of this C-terminal PDZ-binding motif impairs endocytosis of VEGFR2, which reduces sprouting angiogenesis under physiological and pathological conditions, and VEGFR2 downstream signaling [13, 15]. The mechanism by which ephrin-B2 promotes the initiation of VEGFR2 endocytosis is unclear, the interaction of ephrin-B2 with its PDZ domain effectors through the C-terminal PDZ binding motif may be required for its regulation of VEGFR2 endocytosis, signaling, and angiogenesis
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