Mechanisms of VE-cadherin Processing and Degradation in Microvascular Endothelial Cells

Abstract

VE-cadherin is an endothelial-specific cadherin that plays important roles in vascular morphogenesis and growth control. To investigate the mechanisms by which endothelial cells regulate cadherin cell surface levels, a VE-cadherin mutant containing the non-adhesive interleukin-2 (IL-2) receptor extracellular domain and the VE-cadherin cytoplasmic tail (IL-2R-VE-cad cyto ) was expressed in microvascular endothelial cells. Expression of the IL-2R-VE-cad cyto mutant resulted in the internalization of endogenous VE-cadherin and in a dramatic decrease in endogenous VE-cadherin levels. The internalized VE-cadherin co-localized with early endosomes, and the lysosomal inhibitor chloroquine dramatically inhibited the down-regulation of VE-cadherin in cells expressing the IL-2R-VE-cad cyto mutant. Chloroquine treatment also resulted in the accumulation of a VEcadherin fragment lacking the ␤-catenin binding domain of the VE-cadherin cytoplasmic tail. The formation of the VE-cadherin fragment could be prevented by treating endothelial cells with proteasome inhibitors. Furthermore, inhibition of the proteasome prevented VE-cadherin internalization and inhibited the disruption of endothelial intercellular junctions by the IL-2R-VE-cad cyto mutant. These results provide new insights into the mechanisms of VE-cadherin processing and degradation in microvascular endothelial cells.Endothelial adherens junctions are adhesive intercellular contacts that are crucial for the maintenance and regulation of normal microvascular function (1-3). Alterations in adherens junction assembly influence endothelial cell motility, vascular morphogenesis, and permeability. Moreover, recent studies indicate that components of adherens junctions also function in intracellular signaling, leading to the current view that these complexes are plasma membrane domains that integrate chemical and mechanical signaling information (4). The major cellcell adhesion molecule at endothelial adherens junctions is VE-cadherin, a cadherin family member that is specifically expressed in endothelial cells (5). The cytoplasmic tail of the classic cadherins, including VE-cadherin, comprises two wellcharacterized domains. The juxtamembrane domain (JMD) 1 binds to the catenin p120, an armadillo family protein that is thought to regulate cadherin adhesive interactions by modulating the activity of Rho family GTPases (6 -8). At the carboxyl-terminal region of the cadherin cytoplasmic tail, a domain termed the catenin binding domain (CBD) interacts with ␤-catenin or plakoglobin (9). ␤-Catenin and plakoglobin both interact with ␣-catenin, which links cadherins to the actin cytoskeleton and to other actin-binding proteins such as ␣-actinin (10 -13). VE-cadherin also associates with the vimentin cytoskeletal network in endothelial cells through interactions with plakoglobin and the intermediate filament-binding protein desmoplakin (14). These unique intercellular junctions, containing both actin and vimentin-binding proteins, have been referred to as complexus adhaerentes (15-...

Titanium dioxide nanomaterials cause endothelial cell leakiness by disrupting the homophilic interaction of VE–cadherin

“…Images were obtained using Nikon A1 confocal microscope (Nikon, Japan). VE-cadherin internalization assay was performed in accordance to previous report 39 . Briefly, confluent HMVECs were serum starved for 6 h. At 3 h into the serum starvation, chloroquine (150 mM) was added to prevent lysosomal degradation of the internalized VE-cadherin.…”

Making and breaking contacts: the cellular biology of cadherin regulation

“…Following internalisation, cadherins are transported to early or sorting endosomes. Two alternative routes are available from this compartment: recycling back to the cell surface (3), a path may commonly involve an intermediate step in the recycling endosomal compartment; or transport through late endosomes for degradation in the lysosome (4). Which of these alternatives is taken can influence surface expression and metabolic stability of the cadherin.…”

“…However, E-cadherin that was internalised by EGF-induced macropinocytosis appeared to be recycled to the cell surface [5]. Moreover, cadherins that are internalised by either clathrin-mediated or clathrin-independent entry mechanisms often appear in early endosomes that stain for EEA-1 [4,11]. This suggests that cadherins may enter a common compartment shortly after endocytosis, implying that the choice of fates is decided at, or after, exist from this early endosomal compartment.…”

“…Constitutive endocytosis of a number of classical cadherins has been observed in cells that display apparently stable cell-cell contacts [3,4]. Basal levels of cadherin internalisation would be expected to support their metabolic turnover and perhaps contribute to local remodelling of contacts.…”

Emerging roles for p120-catenin in cell adhesion and cancer

“…Thus, p120 directly influences adhesive strength by controlling the amount of cadherin available at the cell surface for adhesion. Under normal circumstances, cadherin turnover occurs constitutively as part of an ongoing endocytosis mechanism that participates in the control of cadherin-mediated adhesion (Le et al, 1999;Le et al, 2002;Xiao et al, 2003b) (reviewed in Bryant and Stow, 2004). It now appears that p120 is a determining factor in regulating this dynamic process (Ireton et al, 2002;Davis et al, 2003;Xiao et al, 2003a).…”