Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.
Pemphigus vulgaris (PV) is an epidermal blistering disorder caused by antibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). The mechanism by which PV IgG disrupt adhesion is not fully understood. To address this issue, primary human keratinocytes and patient IgG were utilized to define the morphological, biochemical and functional changes triggered by PV IgG. Three phases of desmosome disassembly were distinguished. Analysis of fixed and living keratinocytes demonstrated that PV IgG cause rapid Dsg3 internalization which likely originates from a non-junctional pool of Dsg3. Subsequently, Dsg3 and other desmosomal components rearrange into linear arrays that run perpendicular to cell contacts. Dsg3 complexes localized at the cell surface are transported in a retrograde fashion along these arrays before being released into cytoplasmic vesicular compartments. These changes in Dsg3 distribution are followed by depletion of detergent insoluble Dsg3 pools and by the loss of cell adhesion strength. Importantly, this process of disassembly can be prevented by expressing exogenous Dsg3, thereby driving Dsg3 biosynthesis and desmosome assembly. These data support a model in which PV IgG cause the loss of cell adhesion by altering the dynamics of Dsg3 assembly into desmosomes and the turnover of cell surface pools of Dsg3 through endocytic pathways.
Cadherins are a large family of cell-cell adhesion molecules that tether cytoskeletal networks of actin and intermediate filaments to the plasma membrane. This function of cadherins promotes tissue organization and integrity, as demonstrated by numerous disease states that are characterized by the loss of cadherin-based adhesion. However, plasticity in cell adhesion is often required in cellular processes such as tissue patterning during development and epithelial migration during wound healing. Recent work has revealed a pivotal role for various membrane trafficking pathways in regulating cellular transitions between quiescent adhesive states and more dynamic phenotypes. The regulation of cadherins by membrane trafficking is emerging as a key player in this balancing act and studies are beginning to reveal how this process goes awry in the context of disease. This review summarizes the current understanding of how cadherins are routed and how the interface between cadherins and membrane trafficking pathways regulates cell surface adhesive potential. Particular emphasis is placed on the regulation of cadherin trafficking by catenins and the interplay between growth factor signaling pathways and cadherin endocytosis.
Endocytosis; adherens junctions; desmosomes; cell-cell adhesion; pemphigus vulgaris; Epithelial Mesenchymal Transition; catenin; growth factors
p120-catenin is a cytoplasmic binding partner of cadherins and functions as a set point for cadherin expression by preventing cadherin endocytosis, and degradation. p120 is known to regulate cell motility and invasiveness by inhibiting RhoA activity. However, the relationship between these functions of p120 is not understood. Here, we provide evidence that p120 functions as part of a plasma membrane retention mechanism for VE-cadherin by preventing the recruitment of VE-cadherin into membrane domains enriched in components of the endocytic machinery, including clathrin and the adaptor complex AP-2. The mechanism by which p120 regulates VE-cadherin entry into endocytic compartments is dependent on p120's interaction with the cadherin juxtamembrane domain, but occurs independently of p120's prevention of Rho GTPase activity. These findings clarify the mechanism for p120's function in stabilizing VE-cadherin at the plasma membrane and demonstrate a novel role for p120 in modulating the availability of cadherins for entry into a clathrin-dependent endocytic pathway.
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.
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.
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.
Cadherin; endothelial; adhesion
Endothelial cells (EC) express VE-cadherin and N-cadherin, and recent data suggest that VE-cadherin levels are dependent on N-cadherin expression. While investigating changes in N-cadherin levels during endothelial monolayer maturation, we found that VE-cadherin levels are maintained in EC despite a decrease in N-cadherin, suggesting that VE-cadherin levels may not depend on N-cadherin. Knockdown of N-cadherin did not affect VE-cadherin levels in EC with low endogenous N-cadherin expression. Surprisingly, however, knockdown of N-cadherin in EC with high endogenous N-cadherin expression increased VE-cadherin levels suggesting an inverse relationship between the two. This was further supported by a decrease in VE-cadherin following overexpression of N-cadherin. Experiments in which p120, a catenin that binds N- and VE-cadherin, was knocked down or overexpressed indicate that these two cadherins compete for p120. These data demonstrate that VE-cadherin levels are not directly related to N-cadherin levels but may be inversely related due to competition for p120.
adherens junction; catenin; VE-cadherin; confluence
Desmosomes are intercellular junctions that tether intermediate filaments to the plasma membrane. Desmogleins and desmocollins, members of the cadherin superfamily, mediate adhesion at desmosomes. Cytoplasmic components of the desmosome associate with the desmosomal cadherin tails through a series of protein interactions, which serve to recruit intermediate filaments to sites of desmosome assembly. These desmosomal plaque components include plakoglobin and the plakophilins, members of the armadillo gene family. Linkage to the cytoskeleton is mediated by the intermediate filament binding protein, desmoplakin, which associates with both plakoglobin and plakophilins. Although desmosomes are critical for maintaining stable cell–cell adhesion, emerging evidence indicates that they are also dynamic structures that contribute to cellular processes beyond that of cell adhesion. This article outlines the structure and function of the major desmosomal proteins, and explores the contributions of this protein complex to tissue architecture and morphogenesis.
Desmosomal proteins link neighboring cells and are anchored to intermediate filaments. They are essential for stable adhesion and play important roles in morphogenesis.
Cell–extracellular matrix (ECM) and cell–cell interactions regulate keratinocyte cell fate and differentiation. In the present analysis, we examined the differentiation of primary human keratinocytes cultured on micropatterned substrates that varied the extent of cell–cell contact while maintaining constant cell–ECM areas. Bowtie-shaped micropatterned areas (75–1600 µm2) were engineered to either permit or prevent cell–cell contact for pairs of adherent keratinocytes. Cell pairs with direct cell–cell contact exhibited enhanced expression of the differentiation markers involucrin and keratin 10 compared to cells with no cell–cell contact. In contrast, available cell-spreading area, as regulated by pattern size, did not alter keratinocyte involucrin expression. Disruption of E-cadherin binding by either antibody blocking or expression of a dominant-negative receptor diminished the ability of micropattern-regulated cell–cell contact to modulate involucrin expression. These results demonstrate that cadherin-mediated cell–cell contact regulates early keratinocyte differentiation independently from changes in cell shape.
VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin–p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.
Cytoskeletal networks control organelle subcellular distribution and function. Herein, we describe a previously unsuspected association between intermediate filament proteins and the adaptor complex AP-3. AP-3 and intermediate filament proteins cosedimented and coimmunoprecipitated as a complex free of microtubule and actin binding proteins. Genetic perturbation of the intermediate filament cytoskeleton triggered changes in the subcellular distribution of the adaptor AP-3 and late endocytic/lysosome compartments. Concomitant with these architectural changes, and similarly to AP-3-null mocha cells, fibroblasts lacking vimentin were compromised in their vesicular zinc uptake, their organellar pH, and their total and surface content of AP-3 cargoes. However, the total content and surface levels, as well as the distribution of the transferrin receptor, a membrane protein whose sorting is AP-3 independent, remained unaltered in both AP-3- and vimentin-null cells. Based on the phenotypic convergence between AP-3 and vimentin deficiencies, we predicted and documented a reduced autophagosome content in mocha cells, a phenotype previously reported in cells with disrupted intermediate filament cytoskeletons. Our results reveal a novel role of the intermediate filament cytoskeleton in organelle/adaptor positioning and in regulation of the adaptor complex AP-3.
The mechanisms by which catenins regulate cadherin function are not fully understood, and the precise function of p120 catenin (p120ctn) has remained particularly elusive. In microvascular endothelial cells, p120ctn colocalized extensively with cell surface VE-cadherin, but failed to colocalize with VE-cadherin that had entered intracellular degradative compartments. To test the possibility that p120ctn binding to VE-cadherin regulates VE-cadherin internalization, a series of approaches were undertaken to manipulate p120ctn availability to endogenous VE-cadherin. Expression of VE-cadherin mutants that competed for p120ctn binding triggered the degradation of endogenous VE-cadherin. Similarly, reducing levels of p120ctn using siRNA caused a dramatic and dose-related reduction in cellular levels of VE-cadherin. In contrast, overexpression of p120ctn increased VE-cadherin cell surface levels and inhibited entry of cell surface VE-cadherin into degradative compartments. These results demonstrate that cellular levels of p120ctn function as a set point mechanism that regulates cadherin expression levels, and that a major function of p120ctn is to control cadherin internalization and degradation.
adhesion; cytoskeleton; endocytosis; cadherin; catenin
Desmocollin 1 (Dsc1) is part of a desmosomal cell adhesion receptor formed in terminally differentiating keratinocytes of stratified epithelia. The dsc1 gene encodes two proteins (Dsc1a and Dsc1b) that differ only with respect to their COOH-terminal cytoplasmic amino acid sequences. On the basis of in vitro experiments, it is thought that the Dsc1a variant is essential for assembly of the desmosomal plaque, a structure that connects desmosomes to the intermediate filament cytoskeleton of epithelial cells. We have generated mice that synthesize a truncated Dsc1 receptor that lacks both the Dsc1a- and Dsc1b-specific COOH-terminal domains. This mutant transmembrane receptor, which does not bind the common desmosomal plaque proteins plakoglobin and plakophilin 1, is integrated into functional desmosomes. Interestingly, our mutant mice did not show the epidermal fragility previously observed in dsc1-null mice. This suggests that neither the Dsc1a- nor the Dsc1b-specific COOH-terminal cytoplasmic domain is required for establishing and maintaining desmosomal adhesion. However, a comparison of our mutants with dsc1-null mice suggests that the Dsc1 extracellular domain is necessary to maintain structural integrity of the skin.
The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.