Desmosomal adhesiveness is developmentally regulated in the mouse embryo and modulated during trophectoderm migration

During embryonic development tissues remain malleable to participate in morphogenetic movements but on completion of morphogenesis they must acquire the toughness essential for independent adult life. Desmosomes are cell-cell junctions that maintain tissue integrity especially where resistance to mechanical stress is required. Desmosomes in adult tissues are termed hyper-adhesive because they adhere strongly and are experimentally resistant to extracellular calcium chelation. Wounding results in weakening of desmosomal adhesion to a calcium-dependent state, presumably to facilitate cell migration and wound closure. Since desmosomes appear early in mouse tissue development we hypothesised that initial weak adhesion would be followed by acquisition of hyper-adhesion, the opposite of what happens on wounding. We show that epidermal desmosomes are calcium-dependent until embryonic day 12 (E12) and become hyper-adhesive by E14. Similarly, trophectodermal desmosomes change from calcium-dependence to hyper-adhesiveness as blastocyst development proceeds from E3 to E4.5. In both, development of hyper-adhesion is accompanied by the appearance of a midline between the plasma membranes supporting previous evidence that hyper-adhesiveness depends on the organised arrangement of desmosomal cadherins. By contrast, adherens junctions remain calcium-dependent throughout but tight junctions become calcium-independent as desmosomes mature. Using protein kinase C (PKC) activation and PKCα-/- mice, we provide evidence suggesting that conventional PKC isoforms are involved in developmental progression to hyper-adhesiveness. We demonstrate that modulation of desmosomal adhesion by PKC can regulate migration of trophectoderm. It appears that tissue stabilisation is one of several roles played by desmosomes in animal development.     

Role of boundary conditions in an experimental model of epithelial wound healing

Coordinated cell movements in epithelial layers are essential for proper tissue morphogenesis and homeostasis, but our understanding of the mechanisms that coordinate the behavior of multiple cells in these processes is far from complete. Recent experiments with Madin-Darby canine kidney epithelial monolayers revealed a wave-like pattern of injury-induced MAPK activation and showed that it is essential for collective cell migration after wounding. To investigate the effects of the different aspects of wounding on cell sheet migration, we engineered a system that allowed us to dissect the classic wound healing assay. We studied Madin-Darby canine kidney sheet migration under three different conditions: 1) the classic wound healing assay, 2) empty space induction, where a confluent monolayer is grown adjacent to a slab of polydimethylsiloxane and the monolayer is not injured but allowed to migrate upon removal of the slab, and 3) injury via polydimethylsiloxane membrane peel-off, where an injured monolayer migrates onto plain tissue culture surface, as in the case of empty space induction allowing for direct comparison. By tracking the motion of individual cells within the sheet under these three conditions, we show how the dynamics of the individual cells' motion is responsible for the coordinated migration of the sheet and is coordinated with the activation of ERK1/2 MAPK. In addition, we demonstrate that the propagation of the waves of MAPK activation depends on the generation of reactive oxygen species at the wound edge.     

Pervanadate stabilizes desmosomes

Desmosomes are intercellular junctions responsible for strong cell-cell adhesion in epithelia and cardiac muscle. Numerous studies have shown that the other major type of epithelial cell adhesion, the adherens junction, is destabilized by src-induced tyrosine phosphorylation of two of its principal components, E-cadherin and β-catenin. Here we show that treatment of epithelial cells with the potent tyrosine phosphatase inhibitor sodium pervanadate causes tyrosine phosphorylation of the major desmosomal components desmoglein 2 and plakoglobin in both the non-ionic detergent soluble and insoluble cell fractions and, surprisingly, stabilizes desmosomal adhesion, inducing the hyper-adhesive form normally found in tissues and confluent cell sheets. Taken together with the few other studies on desmosomes these results suggest that the effects of tyrosine phosphorylation on desmosomal adhesion are complex.     

Induction of hyper-adhesion attenuates autoimmune-induced keratinocyte cell-cell detachment and processing of adhesion molecules via mechanisms that involve PKC

In confluent keratinocyte monolayers, desmosomal adhesion gradually becomes calcium-independent and this is associated with an increase in the strength of intercellular adhesion (hyper-adhesion). In this study, we investigated the functional and molecular significance of hyper-adhesion in a system challenged by autoimmune sera from patients with Pemphigus Vulgaris (PV), a disease primarily targeting desmosomal adhesion. The results show that keratinocytes with calcium-independent desmosomes are resistant to disruption of intercellular contacts (acantholysis) in experimental PV. Furthermore, both the desmosomal cadherins desmoglein (Dsg) 1 and Dsg3 and the adherens junction protein E-cadherin were decreased in confluent keratinocytes at Day 1, but not in hyper-adhesive cells (Day 6) after incubation with PV serum. Pharmacological induction of the hyper-adhesive state with the PKC inhibitor Go6976 reduced both the acantholysis rate and the processing of cell adhesion molecules induced by PV serum. When the establishment of the hyper-adhesive state was prevented by cell adhesion recognition (CAR) peptides that perturbed desmosomal interactions, Go6976 could still partially attenuate PV acantholysis. Taken together, these data demonstrate that keratinocyte hyper-adhesion decreases the morphological, functional and biochemical dys-cohesive effects of PV serum via mechanisms that involve, at least in part, the function of PKC. This suggests that reinforcing keratinocyte adhesion may be a promising way to inhibit the effects of this most debilitating disorder.     

Pervanadate stabilizes desmosomes

Desmosomes are intercellular junctions responsible for strong cell-cell adhesion in epithelia and cardiac muscle. Numerous studies have shown that the other major type of epithelial cell adhesion, the adherens junction, is destabilized by src-induced tyrosine phosphorylation of two of its principal components, E-cadherin and β-catenin. Here we show that treatment of epithelial cells with the potent tyrosine phosphatase inhibitor sodium pervanadate causes tyrosine phosphorylation of the major desmosomal components desmoglein 2 and plakoglobin in both the non-ionic detergent soluble and insoluble cell fractions and, surprisingly, stabilizes desmosomal adhesion, inducing the hyper-adhesive form normally found in tissues and confluent cell sheets. Taken together with the few other studies on desmosomes these results suggest that the effects of tyrosine phosphorylation on desmosomal adhesion are complex.Key words: desmosome, cell-cell adhesion, intercellular junction, tyrosine phosphorylation, pervanadate, desmoglein, plakoglobin     

Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC,Microtubules and Lysosomal/Proteasomal Degradation

Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds.     

Actomyosin organization in stationary and migrating sheets of cultured human endothelial cells

We used immunofluorescence microscopy to study the organization of actin, myosin and vinculin in confluent endothelial cells and in cells migrating into an experimental wound and interference reflection microscopy to assess the cell-substratum adhesion pattern in these cells. In confluent stationary endothelial cell monolayers actin showed a distinct cell-to-cell organization. Myosin, on the other hand, was diffusely distributed and was clearly absent from cell peripheries. Vinculin was confined as linear arrays to cell-cell contact areas. Interference reflection microscopy revealed areas of close and distant adhesion but no focal adhesion sites in these cultures. Twelve hours after experimental wounding a distinct zone of advancing cells was seen at the wound edge. These cells showed a spreadout morphology and, in contrast to stationary cells, had a stress fibre-type organization of both actin and myosin. Vinculin was in the migrating cells seen as plaques at the ventral cell surface. In interference reflection microscopy numerous focal adhesions were seen. The results indicate that the actomyosin system forms the structural basis for monolayer organization of endothelial cells and responds by reorganization upon cell migration.     

Two distinct modes of myosin assembly and dynamics during epithelial wound closure

Actomyosin contraction powers the sealing of epithelial sheets during embryogenesis and wound closure; however, the mechanisms are poorly understood. After laser ablation wounding of Madin-Darby canine kidney cell monolayers, we observed distinct steps in wound closure from time-lapse images of myosin distribution during resealing. Immediately upon wounding, actin and myosin II regulatory light chain accumulated at two locations: (1) in a ring adjacent to the tight junction that circumscribed the wound and (2) in fibers at the base of the cell in membranes extending over the wound site. Rho-kinase activity was required for assembly of the myosin ring, and myosin II activity was required for contraction but not for basal membrane extension. As it contracted, the myosin ring moved toward the basal membrane with ZO-1 and Rho-kinase. Thus, we suggest that tight junctions serve as attachment points for the actomyosin ring during wound closure and that Rho-kinase is required for localization and activation of the contractile ring.     

Calcium signalling in wound-responsive normal human urothelial cell monolayers

Epithelial tissue repair requires coordination of migratory and proliferative activity both adjacent to and remote from the wound edge. Although calcium signalling is implicated, the specific mechanisms are poorly understood. This study characterises the calcium signal invoked in response to scratch wounding of normal human urothelial (NHU) cells and relates it to the localised cellular response. Immediately after wounding of confluent NHU cell monolayers, cells adjacent to the wound edge showed a sustained (>30 min) rise in [Ca(2+)](i), while there was an independent, but simultaneous calcium wave that propagated out from the wound edge. The transient signal involved release of calcium from intracellular stores and was not mediated via gap junctions, but by diffusion of extracellular agonists. We demonstrated that ATP was partially responsible for the initiation and propagation of the calcium wave and showed that the calcium release mechanism was mediated in part via activation of inositol-1,4,5-triphosphate (IP(3)) receptors. By contrast, the sustained calcium signal originated from the extracellular milieu and correlated with an increased rate of migration by these cells. The work presented here provides supportive evidence that the calcium signature, defined by its temporal and amplitude characteristics, is important in co-ordinating the response of cells within an epithelial cell monolayer after wounding.     

Desmosomal cadherins

New evidence from blocking desmosomal adhesion with anti-adhesion peptides reveals a role for desmosomes in cell positioning in morphogenesis. Desmosomal adhesion is necessary for the stability of adherens junctions in epithelial cell sheets. Knockout and mis-expression of desmosomal cadherins in mice suggests that they may function directly or indirectly in regulating epidermal differentiation. Protein kinase C signalling and tyrosine phosphorylation appear to regulate desmosomal adhesion. There are new insights into the role of desmosomal cadherins in autoimmune, infectious and genetic disease.     

Hog barn dust slows airway epithelial cell migration in vitro through a PKCalpha-dependent mechanism

Agricultural work and other occupational exposures are responsible for approximately 15% of chronic obstructive pulmonary disease (COPD). COPD involves airway remodeling in response to chronic lung inflammatory events and altered airway repair mechanisms. However, the effect of agricultural dust exposure on signaling pathways that regulate airway injury and repair has not been well characterized. A key step in this process is migration of airway cells to restore epithelial integrity. We have previously shown that agents that activate the critical regulatory enzyme protein kinase C (PKC) slow cell migration during wound repair. Based on this observation and direct kinase measurements that demonstrate that dust extract from hog confinement barns (HDE) specifically activates the PKC isoforms PKCalpha and PKCepsilon, we hypothesized that HDE would slow wound closure time in airway epithelial cells. We utilized the human bronchial epithelial cell line BEAS-2B and transfected BEAS-2B cell lines that express dominant negative (DN) forms of PKC isoforms to demonstrate that HDE slows wound closure in BEAS-2B and PKCepsilon DN cell lines. However, in PKCalpha DN cells, wound closure following HDE treatment is not significantly different than media-treated cells. These results suggest that the PKCalpha isoform is an important regulator of cell migration in response to agricultural dust exposure.     

Altered distribution and co-localization of polycystin-2 with polycystin-1 in MDCK cells after wounding stress

Polycystin-1 and -2 are integral membrane glycoproteins defective in autosomal dominant polycystic kidney disease (ADPKD). Recent studies showed a coupled polycystin-1 and -2 action in cell signaling and channel activation suggesting an important biological role for the two proteins at the plasma membrane. To gain a better understanding about the (co)-distribution and dynamics of the polycystin-1 and -2 complex under stress conditions, we used a wound-healing model of Madine Darby canine kidney (MDCK) renal epithelial cells. In this model, cells near the wound edge undergo a process of reorganization to active migration, while cells further from the edge are unaffected and remain confluent. For the first time, endogenous polycystin-1 and -2 were found to partly co-localize in the plasma membrane of confluent monolayers, and both proteins co-localized in the primary cilium. Upon wound healing, the association of polycystin-2 to the membrane was greatly reduced at the wound edge and the submarginal cells. Polycystin-1 remained incorporated to the membrane at the edge of the cell sheet at all time points, although strongly reduced in lamellipodia-forming cells. Adherens junctions and desmosomes, and respective connected actin and keratin cytoskeleton were also disturbed in lamellipodia-forming cells. We propose that altered subcellular localization of polycystin-1 and -2 as a result of stress will affect signaling and other cellular processes mediated by these proteins.     

Pemphigus vulgaris antigen, a desmoglein type of cadherin, is localized within keratinocyte desmosomes

Pemphigus vulgaris antigen (PVA) is a member of the desmoglein subfamily of cadherin cell adhesion molecules. Because autoantibodies in this disease cause blisters due to loss of epidermal cell adhesion, and because desmoglein is found in the desmosome cell adhesion junction, we wanted to determine if PVA is also found in desmosomes. By immunofluorescence, PV IgG bound, in a dotted pattern, to the cell surface of cultured human keratinocytes induced to differentiate with calcium, suggesting junctional staining. However, by preembedding, immunogold electron microscopic studies only slight labeling could be detected in desmosomes, presumably because of difficulty in gold penetration of intact desmosomes. We therefore treated the keratinocytes with 0.01% trypsin in 1 mM calcium, conditions known to preserve cadherin antigenicity but that caused slight separation of desmosomes, before immunogold staining. In this case there was extensive labeling of the extracellular part of desmosomes but not of the interdesmosomal cell membrane which was stained with anti-beta 2- microglobulin antibodies. To confirm the specificity of this binding we showed that antibodies raised in rabbits against the extracellular portions of PVA also bound desmosomes in these cultures. In intact mouse epidermis we could also show slight, but specific, immunogold desmosomal labeling with PV IgG. Furthermore, neonatal mice injected with PV IgG affinity purified on PVA showed desmosomal separation with the IgG localized to desmosomal cores. These results indicate that PVA is organized and concentrated within the desmosome where it presumably functions to maintain the integrity of stratifying epithelia.     

Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility

Protein kinase C (PKC) alpha has been implicated in beta1 integrin-mediated cell migration. Stable expression of PKCalpha is shown here to enhance wound closure. This PKC-driven migratory response directly correlates with increased C-terminal threonine phosphorylation of ezrin/moesin/radixin (ERM) at the wound edge. Both the wound migratory response and ERM phosphorylation are dependent upon the catalytic function of PKC and are susceptible to inhibition by phosphatidylinositol 3-kinase blockade. Upon phorbol 12,13-dibutyrate stimulation, green fluorescent protein-PKCalpha and beta1 integrins co-sediment with ERM proteins in low-density sucrose gradient fractions that are enriched in transferrin receptors. Using fluorescence lifetime imaging microscopy, PKCalpha is shown to form a molecular complex with ezrin, and the PKC-co-precipitated endogenous ERM is hyperphosphorylated at the C-terminal threonine residue, i.e. activated. Electron microscopy showed an enrichment of both proteins in plasma membrane protrusions. Finally, overexpression of the C-terminal threonine phosphorylation site mutant of ezrin has a dominant inhibitory effect on PKCalpha-induced cell migration. We provide the first evidence that PKCalpha or a PKCalpha-associated serine/threonine kinase can phosphorylate the ERM C-terminal threonine residue within a kinase-ezrin molecular complex in vivo.     

150th Anniversary Series: Desmosomes and Autoimmune Disease,Perspective of Dynamic Desmosome Remodeling and Its Impairments in Pemphigus

Abstract

Desmosomes are the most important intercellular adhering junctions that adhere two adjacent keratinocytes directly with desmosomal cadherins, that is, desmogleins (Dsgs) and desmocollins, forming an epidermal sheet. Recently, two cell–cell adhesion states of desmosomes, that is, “stable hyper-adhesion” and “dynamic weak-adhesion” conditions have been recognized. They are mutually reversible through cell signaling events involving protein kinase C (PKC), Src and epidermal growth factor receptor (EGFR) during Ca²⁺-switching and wound healing. This remodeling is impaired in pemphigus vulgaris (PV, an autoimmune blistering disease), caused by anti-Dsg3 antibodies. The antibody binding to Dsg3 activates PKC, Src and EGFR, linked to generation of dynamic weak-adhesion desmosomes, followed by p38MAPK-mediated endocytosis of Dsg3, resulting in the specific depletion of Dsg3 from desmosomes and acantholysis. A variety of pemphigus outside-in signaling may explain different clinical (non-inflammatory, inflammatory, and necrolytic) types of pemphigus. Pemphigus could be referred to a “desmosome-remodeling disease involving pemphigus IgG-activated outside-in signaling events”.     

Alignment of desmosomes in stratifying human epidermis

Summary Cultured human epithelial cells stained with antibody to desmosomal proteins by indirect immunofluorescence showed linear arrays of desmosomes en face between stratified cells. To confirm that an extensive linear pattern existed on the cell surface, subconfluent cultures were viewed using scanning electron microscopy. Aligned arrays of blunt protrusions lying parallel to each other and extending in the direction of the long axis of the cell were observed on the surface of groups of superficial cells in intact cultures. That this pattern was indeed related to desmosomal distribution was verified by transmission microscopy of thin sections cut in a plane between the upper and lower surfaces of flattened stratified cells to view desmosomes directly. A similar arrangement of desmosomes was seen in intact tissue, using epidermal sheets separated from newborn foreskin. The same pattern found in flattened cells was sometimes apparent in more rounded basal cells where the cytoplasm was beginning to extend. Since desmosomal plaques are associated with keratin filaments, the alignment of desmosomes must occur in association with cytoskeletal changes as cells become flattened toward the distal epithelial surface. The primary initiation of desmosomal alignment remains to be investigated. However, the present findings demonstrate an increasingly regular membrane-cytoskeletal spatial interaction as stratified epithelial cells of skin mature.     

Identification of PSF as a protein kinase Calpha-binding protein in the cell nucleus

Protein kinase C (PKC) isoforms are present in the cell nucleus in diverse cell lines and tissues. Since little is known about proteins interacting with PKC inside the cell nucleus, we used Neuro-2a neuroblastoma cells, in which PKCalpha is present in the nucleus, to screen for nuclear binding partners for PKC. Applying overlay assays, we detected several nuclear proteins which bind to PKCalpha. Specificity of binding was shown by its dependence on PKC activation by phorbol ester, calcium, and phosphatidylserine. The PKC-binding proteins were partially purified and analyzed by microsequencing and mass spectrometry. Four proteins could be identified: PTB-associated splicing factor (PSF), p68 RNA helicase, and the heterogeneous nuclear ribonucleoprotein (hnRNP) proteins A3 and L. In the case of PSF, binding to PKC could also be demonstrated in a GST-pull-down assay using GST-PKCalpha, expressed in insect cells. Phosphorylation experiments revealed that PSF is a weak in vitro substrate for PKCalpha.     

Early stages of endothelial wound repair: conversion of quiescent to migrating endothelial cells involves tyrosine phosphorylation and actin microfilament reorganization

Endothelial repair to reestablish structural integrity following wounding is a complex process. Since the actin cytoskeleton undergoes specific changes in distribution as quiescent endothelial cells switch to activated migrating cells over a 6-h period following wounding (Lee et al. 1996), we studied tyrosine phosphorylation in association with actin microfilaments and adhesion proteins using double immunofluorescent confocal microscopy. We showed that in a confluent monolayer phosphotyrosine localized at the periphery of the cell at vinculin cell-cell adhesion sites within the actin-dense peripheral band (DPB) and centrally at talin/vinculin cell-substratum adhesion sites at the ends of central microfilaments. Over a period of 6 h following in vitro wounding there was a reduction of peripheral phosphotyrosine associated with the loss of both cell-cell adhesion sites and the DPB (stage I). Concomitantly, an increase in central phosphotyrosine was associated with an increase in cell-substratum adhesion sites and central microfilaments parallel to the wound edge (stage II), which subsequently redistributed perpendicular to the wound edge (stage III). We also localized FAK and paxillin at the ends of parallel and perpendicular central microfilaments. Immunoprecipitation of paxillin showed increased phosphotyrosine and protein levels when prominent central microfilaments were present and underwent remodeling. Inhibition of tyrosine kinases by genistein and tyrosine phosphatases by sodium orthovanadate resulted in reduced endothelial repair associated with disruption of adhesion site formation and central microfilament formation/redistribution in each stage of repair. We suggest that tyrosine phosphorylation of adhesion proteins, such as paxillin, may be important in regulating the early stages of endothelial wound repair. Received: 22 March 1999 / Accepted: 24 March 1999     

Overexpression of protein kinase Calpha in MCF-10A human breast cells engenders dramatic alterations in morphology, proliferation, and motility.

A nonmetastatic human mammary epithelial cell line (MCF-10A) was engineered to overproduce protein kinase Calpha (PKCalpha) so as to investigate a role for this isoform in the metastatic phenotype. PKCalpha transfectants (clone 26alpha) expressed an 8-fold higher level of PKCalpha protein without compensatory alterations in other isoforms. Clone 26alpha proliferated slowly (accumulating in G1 of the cell cycle) but exhibited pronounced increases in motility and adhesion. Elevated expression of cell cycle inhibitor p27 and focal adhesion proteins was observed, whereas E-cadherin expression decreased to undetectable levels. These observations were consistent with the morphology of PKCalpha transfectants (large, disaggregated, and flat, with lamellipodia and extensive actin fibers) and control cells (small, aggregated, and refractile). Treatment with PKC inhibitors or transfection of a dominant negative (dn) mutant of Rac1, but neither dn RhoA nor dn cdc42, reduced the motility of clone 26alpha, implicating PKCalpha catalytic activity and endogenous Rac1, respectively, in the PKCalpha-induced phenotype. Overall, PKCalpha overexpression suppresses proliferation while endowing MCF-10A cells with properties consistent with the metastatic phenotype.     

Different regulation of Purkinje cell dendritic development in cerebellar slice cultures by protein kinase Calpha and -beta

Activity of protein kinase C (PKC), and in particular the PKCgamma-isoform, has been shown to strongly affect and regulate Purkinje cell dendritic development, suggesting an important role for PKC in activity-dependent Purkinje cell maturation. In this study we have analyzed the role of two additional Ca(2+)-dependent PKC isoforms, PKCalpha and -beta, in Purkinje cell survival and dendritic morphology in slice cultures using mice deficient in the respective enzymes. Pharmacological PKC activation strongly reduced basal Purkinje cell dendritic growth in wild-type mice whereas PKC inhibition promoted branching. Purkinje cells from mice deficient in PKCbeta, which is expressed in two splice forms by granule but not Purkinje cells, did not yield measurable morphological differences compared to respective wild-type cells under either experimental condition. In contrast, Purkinje cell dendrites in cultures from PKCalpha-deficient mice were clearly protected from the negative effects on dendritic growth of pharmacological PKC activation and showed an increased branching response to PKC inhibition as compared to wild-type cells. Together with our previous work on the role of PKCgamma, these data support a model predicting that normal Purkinje cell dendritic growth is mainly regulated by the PKCgamma-isoform, which is highly activated by developmental processes. The PKCalpha isoform in this model forms a reserve pool, which only becomes activated upon strong stimulation and then contributes to the limitation of dendritic growth. The PKCbeta isoform appears to not be involved in the signaling cascades regulating Purkinje cell dendritic maturation in cerebellar slice cultures.