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Desmosomes are adhesive intercellular junctions prominent in the skin and heart. Loss of desmosome function is associated with severe congenital and acquired disorders characterized by tissue fragility. Pemphigus vulgaris (PV) is an autoimmune disorder in which antibodies are directed against the desmosomal adhesion molecule Dsg3, resulting in severe mucosal erosions and epidermal blistering. To define the mechanisms by which Dsg3 autoantibodies disrupt keratinocyte adhesion, the fate of PV IgG and various desmosomal components was monitored in primary human keratinocytes exposed to PV patient IgG. PV IgG initially bound to keratinocyte cell surfaces and colocalized with desmosomal markers. Within 6 h after PV IgG binding to Dsg3, electron microscopy revealed that desmosomes were dramatically disrupted and keratinocyte adhesion was severely compromised. Immunofluorescence analysis indicated that PV IgG and Dsg3 were rapidly internalized from the cell surface in a complex with plakoglobin but not desmoplakin. Dsg3 internalization was associated with retraction of keratin filaments from cell-cell borders. Furthermore, the internalized PV IgG-Dsg3 complex colocalized with markers for both endosomes and lysosomes, suggesting that Dsg3 was targeted for degradation. Consistent with this possibility, biotinylation experiments demonstrated that soluble Dsg3 cell surface pools were rapidly depleted followed by loss of detergent-insoluble Dsg3. These findings demonstrate that Dsg3 endocytosis, keratin filament retraction, and the loss of keratinocyte cell-cell adhesion are coordinated responses to PV IgG.Desmosomes are adhesive intercellular junctions that mediate tight adhesion between epithelial cells (1, 2). Desmosomes are particularly prominent in tissues that experience mechanical stress, such as the skin and heart, and function as plasma membrane attachment sites for intermediate filaments. The importance of desmosomes in tissue function and integrity has been revealed by numerous genetic and autoimmune disorders that impact desmosomal components (3, 4). The desmosomal cadherins, the desmogleins and desmocollins, are the transmembrane components of desmosomes responsible for mediating cell-cell adhesion (5). The tails of the desmosomal cadherins interact with the cytoplasmic protein plakoglobin along with other related proteins to couple the cadherins to desmoplakin and the intermediate filament cytoskeleton (2, 6). A number of desmoglein and desmocollin isoforms have been identified, and the genes encoding these proteins are expressed in a tissue-and differentiation-specific manner (5, 7). Mutations in genes encoding desmosomal components lead to heart and skin disorders (3,8,9). Similarly, autoantibodies directed against the desmosomal cadherins lead to a class of severe epidermal blistering disorders termed pemphigus (10). These disorders underscore the importance of understanding how desmosomes assemble, disassemble, and contribute to tissue architecture and function.The mechanisms by which intercellular junctio...

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-...

VE-cadherin is an endothelial-specific cadherin that plays a central role in vascular barrier function and angiogenesis. The cytoplasmic domain of VE-cadherin is linked to the cytoskeleton through interactions with the armadillo family proteins β-catenin and plakoglobin. Growing evidence indicates that β-catenin and plakoglobin play important roles in epithelial growth and morphogenesis. To test the role of these proteins in vascular cells, a replication-deficient retroviral system was used to express intercellular junction proteins and mutants in the human dermal microvascular endothelial cell line (HMEC-1). A mutant VE-cadherin lacking an adhesive extracellular domain disrupted endothelial barrier function and inhibited endothelial growth. In contrast, expression of exogenous plakoglobin or metabolically stable mutants of β-catenin stimulated HMEC-1 cell growth, which suggests that the β-catenin signaling pathway was active in HMEC-1 cells. This possibility was supported by the finding that a dominant-negative mutant of the transcription factor TCF-4, designed to inhibit β-catenin signaling, also inhibited HMEC-1 cell growth. These observations suggest that intercellular junction proteins function as components of an adhesion and signaling system that regulates vascular barrier function and growth.

Rationale p120-catenin (p120) is an armadillo family protein that binds to the cytoplasmic domain of classical cadherins and prevents cadherin endocytosis. The role of p120 in vascular development is unknown. Objective The purpose of this study is to examine the role of p120 in mammalian vascular development by generating a conditionally mutant mouse lacking endothelial p120 and determining the effects of the knockout on vasculogenesis, angiogenic remodeling, and the regulation of endothelial cadherin levels. Methods and Results A conditional Cre/loxP gene deletion strategy was used to ablate p120 expression, using the Tie2 promoter to drive endothelial Cre recombinase expression. Mice lacking endothelial p120 died embryonically beginning at E11.5. Major blood vessels appeared normal at E9.5. However, both embryonic and extraembryonic vasculature of mutant animals were disorganized and displayed decreased microvascular density by E11.5. Importantly, both vascular endothelial (VE)-cadherin and N-cadherin levels were significantly reduced in vessels lacking p120. This decrease in cadherin expression was accompanied by reduced pericyte recruitment and hemorrhaging. Furthermore, p120-null cultured endothelial cells exhibited proliferation defects that could be rescued by exogenous expression of VE-cadherin. Conclusions These findings reveal a fundamental role for p120 in regulating endothelial cadherin levels during vascular development, as well as microvascular patterning, vessel integrity, and endothelial cell proliferation. Loss of endothelial p120 results in lethality due to decreased microvascular density and hemorrhages.