Cadherin-mediated cell sorting not determined by binding or adhesion specificity.

Cadherin adhesion molecules play important roles in the establishment of tissue boundaries. Cells expressing different cadherins sort out from each other in cell aggregation assays. To determine the contribution of cadherin binding and adhesion specificity to the sorting process, we examined the adhesion of cells to different purified cadherin proteins. Chinese hamster ovary cell lines expressing one of four different cadherins were allowed to bind to the purified cadherin extracellular domains of either human E-cadherin or Xenopus C-cadherin, and the specificity of adhesion was compared with cell-sorting assays. None of the different cadherin-expressing cells exhibited any adhesive specificity toward either of the two purified cadherin substrates, even though these cadherins differ considerably in their primary sequence. In addition, all cells exhibited similar strengthening of adhesion on both substrates. However, this lack of adhesive specificity did not determine whether different cadherin-expressing cells would sort from each other, and the tendency to sort was not predictable by the extent of sequence diversity in their extracellular domains. These results show that cadherins are far more promiscuous in their adhesive-binding capacity than had been expected and that the ability to sort out must be determined by mechanisms other than simple adhesive-binding specificity.     

Functional engineering of hepatocytes via heterocellular presentation of a homoadhesive molecule, E-cadherin.

Engineering functional activity of liver cell cultures requires the modulation of specific cell-cell interactions. We have investigated the quantitative role of systematically varied presentation of the cell-cell adhesion molecule, E-cadherin, on the differentiated function of cocultured parenchymal liver cells, hepatocytes. Specifically, we incorporated different proportions of E-cadherin transfected L-929 chaperone cells and untransfected chaperone cells, within cultures of primary rat hepatocytes on a collagen substrate. By using a strongly adhesive substrate that restricted cadherin-induced variations in cell spreading and growth-arresting chaperone cells, we could carefully isolate the potential role of cell-cell adhesion on cell differentiation. Using immunofluorescence microscopy, we confirmed that cadherins expressed at hepatocyte-hepatocyte contacts as well as hepatocyte-chaperone contacts were crossreactive. However, hepatocytes cocultured with cadherin-presenting chaperone cells had a 55-65% increase in longterm function over hepatocytes cocultured with control, nonpresenting chaperone cells. Notably, the cadherin-induced increase in function occurred over and above the basal, coculture-induced functional elevation. Further, we quantified the stoichiometric importance of cadherin contacts by comparing established markers of hepatocyte functional activity across a graded range of E-cadherin presentation. At low levels of cadherin-mediated contacts, the induction of differentiated function was weak, while high levels of contacts elicited a marked increase in function. Thus, hepatocyte biochemical functions (albumin and urea secretion) were biphasically governed by the degree of cadherin-based contacts presented during culture. Overall, our results demonstrate the unequivocal role of cell-cell adhesion molecules in hepatocyte functional engineering, through the graded use of cadherin presentation from functionally incompetent, heterotypic chaperone cells.     

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.     

Calcium-Induced Intercellular Adhesion of Keratinocytes Does not Involve Accumulation of β1 Integrins at Cell-Cell Contacts and Does not Involve Changes in the Levels or Phosphorylation of Catenins

On initiation of terminal differentiation human epidermal keratinocytes detach from the underlying basement membrane as a result of inactivation and subsequent loss of integrins from the cell surface. Assembly of keratinocytes into multilayered sheets requires functional E-and P-cadherin and when stratification is inhibited in low calcium medium differentiating keratinocytes continue to express functional integrins. Using immunofluorescence microscopy, we found that on addition of calcium ions to keratinocyte monolayers there was colocalisation of the β₁ integrins and E-cadherin along the lateral membranes except for a zone close to the substratum which exclusively contained integrins. Quantitative immunoelectron microscopy showed that on induction of stable cell-cell contacts the density of β₁ integrins was the same on the apical and lateral membranes, suggesting that the accumulation of integrins on the lateral membranes observed by immunofluorescence microscopy is due to the increased area of contact between adjacent cells and not to an increase in receptor density. There were no changes in the levels of catenins and their degree of phosphorylation after induction of cell-cell contacts. These observations provide new sights into the mechanism of calcium-dependent intercellular adhesion of keratinocytes.     

细胞P120的作用

P120属于连环蛋白家族成员,通常情况下,P120定位于细胞膜,通过钙黏附素介导细胞之间的黏附.当P120异位分布于细胞质和细胞核时,与细胞增殖、炎性反应及肿瘤的发生发展关系密切.文章综述了P120结构、亚型及P120在细胞的不同定位和作用.从分子生物学领域进一步阐明细胞P120的作用,为临床治疗疾病提供新的靶点.     

Differential function of N-cadherin and cadherin-7 in the control of embryonic cell motility.

Similar amounts of N-cadherin and cadherin-7, the prototypes of type I and type II cadherin, induced cell-cell adhesion in murine sarcoma 180 transfectants, Ncad-1 and cad7-29, respectively. However, in the initial phase of aggregation, Ncad-1 cells aggregated more rapidly than cad7-29 cells. Isolated Ncad-1 and cad7-29 cells adhered and spread in a similar manner on fibronectin (FN), whereas aggregated cad7-29 cells were more motile and dispersed than aggregated Ncad-1 cells. cad7-29 cells established transient contacts with their neighbors which were stabilized if FN-cell interactions were perturbed. In contrast, Ncad-1 cells remained in close contact when they migrated on FN. Both beta-catenin and cadherin were more rapidly downregulated in cad7-29 than in Ncad-1 cells treated with cycloheximide, suggesting a higher turnover rate for cadherin-7-mediated cell-cell contacts than for those mediated by N-cadherin. The extent of FN-dependent focal adhesion kinase phosphorylation was much lower if the cells had initiated N-cadherin-mediated rather than cadherin-7-mediated cell adhesion before plating. On grafting into the embryo, Ncad-1 cells did not migrate and remained at or close to the graft site, even after 48 h, whereas grafted cad7-29 cells dispersed efficiently into embryonic structures. Thus, the adhesive phenotype of cadherin-7-expressing cells is regulated by the nature of the extracellular matrix environment which also controls the migratory behavior of the cells. In addition, adhesions mediated by different cadherins differentially regulate FN-dependent signaling. The transient contacts specifically observed in cadherin- 7-expressing cells may also be important in the control of cell motility.     

Desmoglein isoform distribution affects stratum corneum structure and function

Desmogleins are desmosomal cadherins that mediate cell-cell adhesion. In stratified squamous epithelia there are two major isoforms of desmoglein, 1 and 3, with different distributions in epidermis and mucous membrane. Since either desmoglein isoform alone can mediate adhesion, the reason for their differential distribution is not known. To address this issue, we engineered transgenic mice with desmoglein 3 under the control of the involucrin promoter. These mice expressed desmoglein 3 with the same distribution in epidermis as found in normal oral mucous membranes, while expression of other major differentiation molecules was unchanged. Although the nucleated epidermis appeared normal, the epidermal stratum corneum was abnormal with gross scaling, and a lamellar histology resembling that of normal mucous membrane. The mice died shortly after birth with severe dehydration, suggesting excessive transepidermal water loss, which was confirmed by in vitro and in vivo measurement. Ultrastructure of the stratum corneum showed premature loss of cohesion of corneocytes. This dysadhesion of corneocytes and its contribution to increased transepidermal water loss was confirmed by tape stripping. These data demonstrate that differential expression of desmoglein isoforms affects the major function of epidermis, the permeability barrier, by altering the structure of the stratum corneum.     

神经细胞粘附分子与记忆

记忆的形成阶段包含着神经元突触的可塑性变化过程.近年来的研究表明,神经细胞粘附分子可同时增进突触的可塑性和维持突触结构的稳定性.许多研究证实神经细胞粘附分子对与学习和记忆相关的过程起着一定的调节作用.     

Differential structural properties and expression patterns suggest functional significance for multiple mouse desmoglein 1 isoforms

The four isoforms of desmosomal cadherin desmogleins (Dsg1-4) are expressed in epithelial tissues in a differentiation-specific manner. Extensive sequencing of the human genome has revealed only one copy of the Dsg1 gene. However, we recently cloned two novel additional mouse Dsg1 genes, Dsg1-beta and -gamma, which flank the original Dsg1-alpha on chromosome 18. Sequence conservation between the Dsg1 isoforms diverged significantly at exon 11, particularly in the region that encodes for the extracellular anchoring (EA) domains. Computational analysis revealed very low hydrophilic potential of the Dsg1-gamma EA compared with the corresponding sequences of Dsg1-alpha and -beta, suggesting that the Dsg1-gamma EA domain may have a stronger affinity to the cell membrane. We generated antibodies using synthetic peptides or recombinant proteins localized within the EA domains. These antibodies were tested for their specificity and were then used to demonstrate expression of Dsg1 isoforms in various tissues. In the epidermis, all Dsg1 isoforms were differentially expressed in the differentiating cell layers. In the hair follicle, all Dsg1 isoforms were present throughout the entire process of its development and cycling but the expression of Dsg1 isoforms is subject to significant hair cycle-dependent changes. Dsg1-beta and -gamma, but not Dsg1-alpha, were detected in the sebaceous gland epithelium and the stratified epithelium of the stomach. Finally, Dsg1-alpha and Dsg1-beta, but not Dsg1-gamma, are proteolytically cleaved by exfoliative toxin A. These results suggest that the developmental complexity of mouse tissues, including skin and hair, may play a significant role in the evolutionary driving force to maintain multiple Dsg1 genes in mouse.     

Desmosomal Cadherins and Signaling: Lessons from Autoimmune Disease

Abstract

Autoantibodies from patients suffering from the autoimmune blistering skin disease pemphigus can be applied as tools to study desmosomal adhesion. These autoantibodies targeting the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 cause disruption of desmosomes and loss of intercellular cohesion. Although pemphigus autoantibodies were initially proposed to sterically hinder desmosomes, many groups have shown that they activate signaling pathways which cause disruption of desmosomes and loss of intercellular cohesion by uncoupling the desmosomal plaque from the intermediate filament cytoskeleton and/or by interfering with desmosome turnover. These studies demonstrate that desmogleins serve as receptor molecules to transmit outside-in signaling and demonstrate that desmosomal cadherins have functions in addition to their adhesive properties. Two central molecules regulating cytoskeletal anchorage and desmosome turnover are p38MAPK and PKC. As cytoskeletal uncoupling in turn enhances Dsg3 depletion from desmosomes, both mechanisms reinforce one another in a vicious cycle that compromise the integrity and number of desmosomes.     

Structure and Functions of Classical Cadherins

Cadherins are a family of membrane receptors that mediate calcium-dependent homophilic cell–cell adhesion. Cadherins play a key role in the regulation of organ and tissue development during embryogenesis. In adult organisms, these proteins are responsible for formation of stable cell–cell junctions and maintenance of normal tissue structure. Disruption in expression or function of cadherins may cause uncontrolled cell migration and proliferation during tumor development. This review focuses on the structure and physiological functions of classical cadherins.     

Cadmium (Cd2+) disrupts E-cadherin-dependent cell-cell junctions in MDCK cells

Summary Previous studies from our laboratory have shown that Cd²⁺ can selectively disrupt E-cadherin-dependent cell-cell junctions in the porcine renal epithelial cell line, LLC-PK₁. The objective of the present studies was to determine whether or not Cd²⁺ could produce similar effects in Madin-Darby canine kidney (MDCK) cells, an immortal epithelial cell line derived from dog kidney. This is an important issue because MDCK cells have been used extensively as a model system to study the basic mechanisms of E-cadherin-dependent cell-cell adhesion. MDCK cells on permeable membrane supports were exposed to Cd²⁺ by adding CdCl₂ to either the apical or the basolateral compartment. The integrity of cell-cell junctions was assessed by morphologic observation of the cells and by monitoring the transepithelial electrical resistance. The results showed that exposure to 10–40 μM Cd²⁺ for 15 min-4 h caused the cells to separate from each other without detaching from the growing surface. The separation of the cells was accompanied by a marked drop in the transepithelial electrical resistance, a loss of E-cadherin from the cell-cell contacts, and a reorganization of the actin cytoskeleton. These effects were much more pronounced when Cd²⁺ was added basolaterally than when it was added apically. Moreover, the effects of Cd²⁺ were qualitatively similar to those observed when the cells were incubated in Ca²⁺-free medium. These results show that Cd²⁺ can disrupt E-cadherin-dependent cell-cell junctions in MDCK cells, and they indicate that this cell line would be an appropriate model for further mechanistic studies in this area.     

Extracellular HIV-1 Tat induces human beta-defensin-2 production via NF-kappaB/AP-1 dependent pathways in human B cells

Defensins, a family of antimicrobial peptides, are one of the first lines of host defense. Human beta-defensins (hBD) such as hBD-2 and -3 have anti-HIV activity. Previous studies have shown that HIV-1 virion can induce the expression of hBD, although the exact components of HIV-1 virion that are responsible for hBD expression have not yet been elucidated. In this study, we examined the effect of HIV-1 Tat on the expression of hBD in B cells. Stimulation of B cells with HIV-1 Tat protein significantly increased the mRNA and protein levels of hBD-2. HIV-1 Tat also induced the activation of a reporter gene for hBD-2 in a dose-dependent manner in B cells. Pretreatment of B cells with a JNK inhibitor suppressed HIV-1 Tat-induced hBD-2 expression. Pretreatment of B cells with AP-1 inhibitors or NF-κB inhibitors led to a decrease in HIV-1 Tat-induced protein and mRNA expression of hBD-2. Taken together, our results indicate that HIV-1 Tat can up-regulate the expression of hBD-2 via JNK-NF-κB/AP-1-dependent pathways in human B cells.     

Heat Shock Factor 2 Protects against Proteotoxicity by Maintaining Cell-Cell Adhesion

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Inhibition of tyrosine phosphorylation potentiates substrate-induced neurite growth

Protein tyrosine kinases (PTKs) have major roles in signal transduction and growth control. There are several lines of evidence implicating PTKs in the regulation of axon growth, and this has led to the suggestion that they are centrally involved in the transduction of neuronal growth signals. To test this idea, we assayed the effect of the compounds genistein and lavendustin, specific inhibitors of PTKs, on neurite growth. We find that genistein greatly reduces phosphotyrosine in neurons, as expected from its action on other cells. Surprisingly, administration of genistein or lavendustin potentiated substrate-induced neurite growth in at least several different neuronal types. Stimulation of neurite growth by genistein was abolished by vanadate, providing additional evidence that inhibition of PTKs is responsible for this effect. The potentiation of growth is rather general, in that it occurs on several different extracellular matrix substrates and on two different cell adhesion molecules. Both the initiation of neurite growth and the rate of neurite elongation appear to be potentiated. Our results do not provide evidence for models of substrate-induced signal transduction that involve PTKs asa positive and necessary step, but suggest that such kinases play aregulatory role in neurite elongation. © 1992 John Wiley & Sons, Inc.     

Comparison of molecular mechanisms mediating cell contact phenomena in model developmental systems: an exploration of universality

Are there universal molecular mechanisms associated with cell contact phenomena during metazoan ontogenesis? Comparison of adhesion systems in disparate model systems indicates the existence of unifying principles. Requirements for multicellularity are (a) the construction of three‐dimensional structures involving a crucial balance between adhesiveness and motility; and (b) the establishment of integration at molecular, cellular, tissue, and organismal levels of organization. Mechanisms for (i) cell–cell and cell–substrate adhesion, (if) cell movement, (Hi) cell‐cell communication, (iv) cellular responses, (v) regulation of these processes, and (vi) their integration with patterning, growth, and other developmental processes are all crucial to metazoan development, and must have been present for the emergence and radiation of Metazoa. The principal unifying themes of this review are the dynamics and regulation of cell contact phenomena. Our knowledge of the dynamic molecular mechanisms underlying cell contact phenomena remains fragmentary. Here we examine the molecular bases of cell contact phenomena using extant model developmental systems (representing a wide range of phyla) including the simplest i.e. sponges, and the eukaryotic protist Dictyostelium discoideum, the more complex Drosophila melanogaster, and vertebrate systems. We discuss cell contact phenomena in a broad developmental context. The molecular language of cell contact phenomena is complex; it involves a plethora of structurally and functionally diverse molecules, and diverse modes of intermolecular interactions mediated by protein and/or carbohydrate moieties. Reasons for this are presumably the necessity for a high degree of specificity of inter‐molecular interactions, the requirement for a multitude of different signals, and the apparent requirement for an increasingly large repertoire of cell contact molecules in more complex developmental systems, such as the developing vertebrate nervous system. However, comparison of molecular models for dynamic adhesion in sponges and in vertebrates indicates that, in spite of significant differences in the details of the way specific cell–cell adhesion is mediated, similar principles are involved in the mechanisms employed by members of disparate phyla. Universal requirements are likely to include (a) rapidly reversible intermolecular interactions; (b) low‐affinity intermolecular interactions with fast on–off rates; (c) the compounding of multiple intermolecular interactions; (d) associated regulatory signalling systems. The apparent widespread employment of molecular mechanisms involving cadherin‐like cell adhesion molecules suggests the fundamental importance of cadherin function during development, particularly in epithelial morphogenesis, cell sorting, and segregation of cells.