DDR1 triggers epithelial cell differentiation by promoting cell adhesion through stabilization of E-cadherin

Discoidin domain receptor 1 (DDR1) promotes E-cadherin–mediated adhesion. The underlying mechanism and its significance, however, have not been elucidated. Here we show that DDR1 overexpression augmented, whereas dominant negative mutant (DN-DDR1) or knockdown of DDR1 inhibited E-cadherin localized in cell-cell junctions in epithelial cells. DDR1 changed the localization and abundance of E-cadherin, as well as epithelial plasticity, as manifested by enhancement of microvilli formation and alteration of cytoskeletal organization. DDR1 also reduced protein abundance of mesenchymal markers, whereas DN-DDR1 and sh-DDR1 showed opposite effects. These results suggest that expression of DDR1 increases epithelial plasticity. Expression of DDR1 augmented E-cadherin protein levels by decreasing its degradation rate. Photobleaching and photoconversion of E-cadherin conjugated with Eos fluorescence protein demonstrated that DDR1 increased the stability of E-cadherin on the cell membrane, whereas sh-DDR1 decreased it. Pull-down assay and expression of constitutively active or dominant-negative Cdc42 showed that DDR1 stabilized E-cadherin through inactivation of Cdc42. Altogether, our results show that DDR1 promotes cell-cell adhesion and differentiation through stabilization of E-cadherin, which is mediated by Cdc42 inactivation.

Download Full-text

Role of Nectin in Formation of E-Cadherin–based Adherens Junctions in Keratinocytes: Analysis with the N-Cadherin Dominant Negative Mutant

Molecular Biology of the Cell ◽

10.1091/mbc.e02-10-0632 ◽

2003 ◽

Vol 14 (4) ◽

pp. 1597-1609 ◽

Cited By ~ 19

Author(s):

Yoshinari Tanaka ◽

Hiroyuki Nakanishi ◽

Shigeki Kakunaga ◽

Noriko Okabe ◽

Tomomi Kawakatsu ◽

...

Keyword(s):

Cell Adhesion ◽

Adhesion Molecule ◽

Cell Adhesion Molecule ◽

Adherens Junctions ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Adhesion Activity ◽

Negative Mutant ◽

E Cadherin ◽

Cell Cell

E-Cadherin is a Ca2+-dependent cell-cell adhesion molecule at adherens junctions (AJs) of epithelial cells. A fragment of N-cadherin lacking its extracellular region serves as a dominant negative mutant (DN) and inhibits cell-cell adhesion activity of E-cadherin, but its mode of action remains to be elucidated. Nectin is a Ca2+-independent immunoglobulin-like cell-cell adhesion molecule at AJs and is associated with E-cadherin through their respective peripheral membrane proteins, afadin and catenins, which connect nectin and cadherin to the actin cytoskeleton, respectively. We showed here that overexpression of nectin capable of binding afadin, but not a mutant incapable of binding afadin, reduced the inhibitory effect of N-cadherin DN on the cell-cell adhesion activity of E-cadherin in keratinocytes. Overexpressed nectin recruited N-cadherin DN to the nectin-based cell-cell adhesion sites in an afadin-dependent manner. Moreover, overexpression of nectin enhanced the E-cadherin–based cell-cell adhesion activity. These results suggest that N-cadherin DN competitively inhibits the association of the endogenous nectin-afadin system with the endogenous E-cadherin-catenin system and thereby reduces the cell-cell adhesion activity of E-cadherin. Thus, nectin plays a role in the formation of E-cadherin–based AJs in keratinocytes.

Download Full-text

Immediate and Delayed Effects of E-Cadherin Inhibition on Gene Regulation and Cell Motility in Human Epidermoid Carcinoma Cells

Molecular and Cellular Biology ◽

10.1128/mcb.25.20.9138-9150.2005 ◽

2005 ◽

Vol 25 (20) ◽

pp. 9138-9150 ◽

Cited By ~ 39

Author(s):

Henriette Andersen ◽

Jakob Mejlvang ◽

Shaukat Mahmood ◽

Irina Gromova ◽

Pavel Gromov ◽

...

Keyword(s):

Cell Adhesion ◽

Cell Motility ◽

Family Members ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Short Term ◽

Mesenchymal Transition ◽

Negative Mutant ◽

E Cadherin ◽

Cell Cell

ABSTRACT The invasion suppressor protein, E-cadherin, plays a central role in epithelial cell-cell adhesion. Loss of E-cadherin expression or function in various tumors of epithelial origin is associated with a more invasive phenotype. In this study, by expressing a dominant-negative mutant of E-cadherin (Ec1WVM) in A431 cells, we demonstrated that specific inhibition of E-cadherin-dependent cell-cell adhesion led to the genetic reprogramming of tumor cells. In particular, prolonged inhibition of cell-cell adhesion activated expression of vimentin and repressed cytokeratins, suggesting that the effects of Ec1WVM can be classified as epithelial-mesenchymal transition. Both short-term and prolonged expression of Ec1WVM resulted in morphological transformation and increased cell migration though to different extents. Short-term expression of Ec1WVM up-regulated two AP-1 family members, c-jun and fra-1, but was insufficient to induce complete mesenchymal transition. AP-1 activity induced by the short-term expression of Ec1WVM was required for transcriptional up-regulation of AP-1 family members and down-regulation of two other Ec1WVM-responsive genes, S100A4 and igfbp-3. Using a dominant-negative mutant of c-Jun (TAM67) and RNA interference-mediated silencing of c-Jun and Fra-1, we demonstrated that AP-1 was required for cell motility stimulated by the expression of Ec1WVM. In contrast, Ec1WVM-mediated changes in cell morphology were AP-1-independent. Our data suggest that mesenchymal transition induced by prolonged functional inhibition of E-cadherin is a slow and gradual process. At the initial step of this process, Ec1WVM triggers a positive autoregulatory mechanism that increases AP-1 activity. Activated AP-1 in turn contributes to Ec1WVM-mediated effects on gene expression and tumor cell motility. These data provide novel insight into the tumor suppressor function of E-cadherin.

Download Full-text

Expression of a dominant negative cadherin mutant inhibits proliferation and stimulates terminal differentiation of human epidermal keratinocytes

Journal of Cell Science ◽

10.1242/jcs.109.13.3013 ◽

1996 ◽

Vol 109 (13) ◽

pp. 3013-3023 ◽

Cited By ~ 17

Author(s):

A.J. Zhu ◽

F.M. Watt

Keyword(s):

Cell Adhesion ◽

Terminal Differentiation ◽

Intercellular Adhesion ◽

Dominant Negative ◽

Epidermal Keratinocytes ◽

Dominant Negative Mutant ◽

Beta Catenin ◽

Human Epidermal Keratinocytes ◽

Negative Mutant ◽

E Cadherin

Cell adhesion molecules are not only required for maintenance of tissue integrity, but also regulate many aspects of cell behaviour, including growth and differentiation. While the regulatory functions of integrin extracellular matrix receptors in keratinocytes are well established, such functions have not been investigated for the primary receptors that mediate keratinocyte intercellular adhesion, the cadherins. To examine cadherin function in normal human epidermal keratinocytes we used a retroviral vector to introduce a dominant negative E-cadherin mutant, consisting of the extracellular domain of H-2Kd and the transmembrane and cytoplasmic domains of E-cadherin. As a control a vector containing the same construct, but with the catenin binding site destroyed, was prepared. High levels of expression of the constructs were achieved; the dominant negative mutant, but not the control, formed complexes with alpha-, beta- and gamma-catenin. In cells expressing the dominant negative mutant there was a 5-fold decrease in the level of endogenous cadherins and a 3-fold increase in the level of beta-catenin. Cell-cell adhesion and stratification were inhibited by the dominant negative mutant and desmosome formation was reduced. Expression of the mutant resulted in reduced levels of the alpha 2 beta 1 and alpha 3 beta 1 integrins and increased cell motility, providing further evidence for cross-talk between cadherins and the beta 1 integrins. In view of the widely documented loss of E-cadherin in keratinocyte tumours it was surprising that the dominant negative mutant had an inhibitory effect on keratinocyte proliferation and stimulated terminal differentiation even under conditions in which intercellular adhesion was prevented. These results establish a role for cadherins in regulating keratinocyte growth and differentiation and raise interesting questions as to the relative importance of cell adhesion-dependent and -independent mechanisms.

Download Full-text

Cdx1 or Cdx2 expression activates E-cadherin-mediated cell-cell adhesion and compaction in human COLO 205 cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00484.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. G104-G114 ◽

Cited By ~ 37

Author(s):

Matthew S. Keller ◽

Toshihiko Ezaki ◽

Rong-Jun Guo ◽

John P. Lynch

Keyword(s):

Gene Expression ◽

Cell Adhesion ◽

Gene Expression Pattern ◽

Cell Junctions ◽

Specific Gene ◽

Specific Response ◽

Specific Gene Expression ◽

Cdx2 Expression ◽

E Cadherin ◽

Cell Cell

A mature columnar intestinal epithelium develops late in embryogenesis and is maintained throughout the life of the organism. Although the mechanisms driving intestine-specific gene expression have been well studied, those promoting the acquisition of cell-cell junctions, columnar morphogenesis, and polarization have been less studied. The Cdx homeodomain transcription factors (Cdx1 and Cdx2) regulate intestine-specific gene expression and intestinal epithelial differentiation. We report here that Cdx expression induces E-cadherin activity and cell-cell adhesion in human COLO 205 cancer cells. Within days of Cdx1 or Cdx2 expression, a new homotypic cell-cell adhesion phenotype is induced. This is a specific response to Cdx, inasmuch as a Cdx1 mutant failed to elicit the effect. Additionally, Cdx-expressing COLO 205 cells demonstrate a reduced proliferative capacity and an increase in the mRNA expression of differentiation-associated genes. Electron micrographs of these cells demonstrate induction of tight, adherens, and desmosomal junctions, as well as a columnar shape and apical microvilli. Investigations of the adhesion phenotype determined that it was Ca2+dependent and could be blocked by an E-cadherin-blocking antibody. However, E-cadherin protein levels and intracellular distribution were unchanged. Cdx expression restored the ability of the cell membranes to adhere and undergo compaction. We conclude that Cdx1 or Cdx2 expression is sufficient to induce an E-cadherin-dependent adhesion of COLO 205 cells. This adhesion is associated with polarization and cell-cell membrane compaction, as well as induction of a differentiated gene-expression pattern. Ascertaining the mechanism for this novel Cdx effect may yield insight into the development of mature colonic epithelium.

Download Full-text

Actin protrusions push on E-cadherin to maintain epithelial cell-cell adhesion

10.1101/644302 ◽

2019 ◽

Author(s):

John Xiao He Li ◽

Vivian W. Tang ◽

William M. Brieher

Keyword(s):

Cell Adhesion ◽

Actin Polymerization ◽

Mdck Cells ◽

Myosin Ii ◽

Cell Junctions ◽

Apical Junctions ◽

Actin Protrusions ◽

Adhesive Contacts ◽

E Cadherin ◽

Cell Cell

AbstractCadherin mediated cell-cell adhesion is actin dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough together to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized MDCK cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells while reformation of cadherin clusters across the cell-cell boundary triggers microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as three actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNAi results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

Download Full-text

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0429 ◽

2016 ◽

Vol 27 (18) ◽

pp. 2844-2856 ◽

Cited By ~ 31

Author(s):

Megha Vaman Rao ◽

Ronen Zaidel-Bar

Keyword(s):

Cell Adhesion ◽

Wound Repair ◽

Actin Polymerization ◽

Cell Junctions ◽

Epithelial Tissue ◽

Collective Cell Migration ◽

Epithelial Junctions ◽

Major Factors ◽

E Cadherin ◽

Cell Cell

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.

Download Full-text

Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes

Journal of Cell Science ◽

10.1242/jcs.113.10.1803 ◽

2000 ◽

Vol 113 (10) ◽

pp. 1803-1811

Author(s):

Y. Hanakawa ◽

M. Amagai ◽

Y. Shirakata ◽

K. Sayama ◽

K. Hashimoto

Keyword(s):

Cell Adhesion ◽

Adherens Junctions ◽

Dominant Negative ◽

Cell Contact ◽

Beta Catenin ◽

Hacat Cells ◽

Subsequent Formation ◽

Contact Sites ◽

E Cadherin ◽

Cell Cell

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.

Download Full-text

Cellular crowd control: overriding endogenous cell coordination makes cell migration more susceptible to external programming

10.1101/2021.01.23.427700 ◽

2021 ◽

Author(s):

Gawoon Shim ◽

Danelle Devenport ◽

Daniel J. Cohen

Keyword(s):

Cell Migration ◽

Cell Adhesion ◽

Mouse Skin ◽

Cellular Systems ◽

Cell Junctions ◽

External Control ◽

Collective Behaviors ◽

Primary Mouse ◽

E Cadherin ◽

Cell Cell

AbstractAs collective cell migration is essential in biological processes spanning development, healing, and cancer progression, methods to externally program cell migration are of great value. However, problems can arise if the external commands compete with strong, pre-existing collective behaviors in the tissue or system. We investigate this problem by applying a potent external migratory cue—electrical stimulation and electrotaxis—to primary mouse skin monolayers where we can tune cell-cell adhesion strength to modulate endogenous collectivity. Monolayers with high cell-cell adhesion showed strong natural coordination and resisted electrotactic control, with this conflict actively damaging the leading edge of the tissue. However, reducing pre-existing coordination in the tissue by specifically inhibiting E-cadherin-dependent cell-cell adhesion, either by disrupting the formation of cell-cell junctions with E-cadherin specific antibodies or rapidly dismantling E-cadherin junctions with calcium chelators, significantly improved controllability. Finally, we applied this paradigm of weakening existing coordination to improve control to demonstrate accelerated wound closure in vitro. These results are in keeping with those from diverse, non-cellular systems, and confirm that endogenous collectivity should be considered as a key, quantitative design variable when optimizing external control of collective migration.

Download Full-text

The Protein Tyrosine Phosphatase Pez Is a Major Phosphatase of Adherens Junctions and Dephosphorylates β-Catenin

Molecular Biology of the Cell ◽

10.1091/mbc.e02-09-0577 ◽

2003 ◽

Vol 14 (6) ◽

pp. 2520-2529 ◽

Cited By ~ 71

Author(s):

Carol Wadham ◽

Jennifer R Gamble ◽

Mathew A Vadas ◽

Yeesim Khew-Goodall

Keyword(s):

Cell Adhesion ◽

Tyrosine Phosphorylation ◽

Adherens Junctions ◽

Tyrosine Phosphatase ◽

Adherens Junction ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Adherens Junction Proteins ◽

Cell Cell

Cell-cell adhesion regulates processes important in embryonal development, normal physiology, and cancer progression. It is regulated by various mechanisms including tyrosine phosphorylation. We have previously shown that the protein tyrosine phosphatase Pez is concentrated at intercellular junctions in confluent, quiescent monolayers but is nuclear in cells lacking cell-cell contacts. We show here with an epithelial cell model that Pez localizes to the adherens junctions in confluent monolayers. A truncation mutant lacking the catalytic domain acts as a dominant negative mutant to upregulate tyrosine phosphorylation at adherens junctions. We identified β-catenin, a component of adherens junctions, as a substrate of Pez by a “substrate trapping” approach and by in vitro dephosphorylation with recombinant Pez. Consistent with this, ectopic expression of the dominant negative mutant caused an increase in tyrosine phosphorylation of β-catenin, demonstrating that Pez regulates the level of tyrosine phosphorylation of adherens junction proteins, including β-catenin. Increased tyrosine phosphorylation of adherens junction proteins has been shown to decrease cell-cell adhesion, promoting cell migration as a result. Accordingly, the dominant negative Pez mutant enhanced cell motility in an in vitro “wound” assay. This suggests that Pez is also a regulator of cell motility, most likely through its action on cell-cell adhesion.

Download Full-text

Early Events in Actin Cytoskeleton Dynamics and E-Cadherin-Mediated Cell-Cell Adhesion during Epithelial-Mesenchymal Transition

Cells ◽

10.3390/cells9030578 ◽

2020 ◽

Vol 9 (3) ◽

pp. 578 ◽

Cited By ~ 6

Author(s):

Irina Y. Zhitnyak ◽

Svetlana N. Rubtsova ◽

Nikita I. Litovka ◽

Natalya A. Gloushankova

Keyword(s):

Cell Adhesion ◽

Epithelial Mesenchymal Transition ◽

Immunofluorescent Staining ◽

Actin Binding ◽

Mesenchymal Transition ◽

Actin Bundle ◽

Cell Contacts ◽

The Stability ◽

E Cadherin ◽

Cell Cell

Epithelial-mesenchymal transition (EMT) plays an important role in development and also in initiation of metastasis during cancer. Disruption of cell-cell contacts during EMT allowing cells to detach from and migrate away from their neighbors remains poorly understood. Using immunofluorescent staining and live-cell imaging, we analyzed early events during EMT induced by epidermal growth factor (EGF) in IAR-20 normal epithelial cells. Control cells demonstrated stable adherens junctions (AJs) and robust contact paralysis, whereas addition of EGF caused rapid dynamic changes at the cell-cell boundaries: fragmentation of the circumferential actin bundle, assembly of actin network in lamellipodia, and retrograde flow. Simultaneously, an actin-binding protein EPLIN was phosphorylated, which may have decreased the stability of the circumferential actin bundle. Addition of EGF caused gradual replacement of linear E-cadherin–based AJs with dynamic and unstable punctate AJs, which, unlike linear AJs, colocalized with the mechanosensitive protein zyxin, confirming generation of centripetal force at the sites of cell-cell contacts during EMT. Our data show that early EMT promotes heightened dynamics at the cell-cell boundaries—replacement of stable AJs and actin structures with dynamic ones—which results in overall weakening of cell-cell adhesion, thus priming the cells for front-rear polarization and eventual migration.

Download Full-text