Cell Adhesion: Sorting out Cell Mixing with Echinoid?

Cadherins control intercellular adhesion in epithelial cells. This property relies on the ability to recruit actin filaments at adherens junctions via beta-catenin and alpha-catenin. A recent study shows that Echinoid, a member of the immunoglobulin domain containing protein family, is a modulator of intercellular adhesion in Drosophila that controls cell sorting.     

β1 Integrin-Mediated Adhesion Signalling Is Essential for Epidermal Progenitor Cell Expansion

Background

There is a major discrepancy between the in vitro and in vivo results regarding the role of β1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of β1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation.

Methodology/Principal Findings

To elucidate this discrepancy we generated hypomorphic mice expressing reduced β1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with β1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of β1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the β1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of β1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing.

Conclusions/Significance

These data demonstrate that expression of β1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis.     

IGF-I Receptor, Cell–Cell Adhesion, Tumour Development and Progression

The insulin-like growth factor I receptor (IGF-IR) has been implicated in the development and progression of many common cancers and other neoplastic diseases. The tumorigenic potential of IGF-IR relies on its antiapoptotic and transforming activities. The molecular mechanisms by which IGF-IR controls the proliferation and survival of tumour cells have been extensively studied and many pathways have been delineated. However, the role of IGF-IR in the regulation of non-mitogenic cell functions is less well understood. Here we focus on IGF-IR-dependent cell-cell adhesion. Limited studies suggested that IGF-IR can regulate cell aggregation and intercellular adhesion mediated by cadherins and cadherin-associated proteins. We review the mechanisms of this process and discuss the impact of IGF-IR-dependent cell-cell adhesion on the phenotype of tumour cells.     

Epithelial Cell Adhesion Molecule (Ep-CAM) Modulates Cell–Cell Interactions Mediated by Classic Cadherins

The contribution of noncadherin-type, Ca²⁺-independent cell–cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca²⁺-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM–positive transfectants behave like cells with a decreased strength of cell–cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM–cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of α- and β-catenins decreased in cells overexpressing Ep-CAM. While the total β-catenin content remains unchanged, a reduction in total cellular α-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell–cell adhesions diminish, Ep-CAM–mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell–cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell–cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.Tissue and organ morphogenesis can be viewed as the result of interactions of various cell populations. One important type of intercellular interaction involved in the processes of tissue morphogenesis, morphogenetic movements of cells, and segregation of cell types, are adhesions mediated by cell adhesion molecules (Steinberg and Pool, 1982; Edelman, 1986; Cunningham, 1995; Takeichi, 1995; Gumbiner, 1996). Except for their direct mechanical role as interconnectors of cells and connectors of cells to substrates, cell adhesion molecules are also believed to be responsible for a variety of dynamic processes including cell locomotion, proliferation, and differentiation. There is also evidence that the adhesion systems within a cell may act as regulators of other cell adhesions, thereby offering a means of signaling that is relevant for rearrangements in cell or tissue organization (Edelman, 1993; Rosales et al., 1995; Gumbiner, 1996).In many tissues, a critical role in the maintenance of multicellular structures is assigned to cadherins, a family of Ca²⁺-dependent, homophilic cell–cell adhesion molecules (Takeichi, 1991, 1995; Gumbiner, 1996). In epithelia this critical role belongs to E-cadherin, which is crucial for the establishment and maintenance of epithelial cell polarity (McNeil et al., 1990; Näthke et al., 1993), morphogenesis of epithelial tissues (Wheelock and Jensen, 1992; Larue et al., 1996), and regulation of cell proliferation and programmed cell death (Hermiston and Gordon, 1995; Hermiston et al., 1996; Takahashi and Suzuki, 1996; Wilding et al., 1996; Zhu and Watt, 1996). Expression of different types of classic cadherin molecules (Nose et al., 1988; Friedlander et al., 1989; Daniel et al., 1995), and even quantitative differences in the levels of the same type of cadherin (Steinberg and Takeichi, 1994), may be responsible for segregation of cell types in epithelial tissues. The phenotype of epithelial cells may be modulated by expression of combinations of different types of cadherins (Marrs et al., 1995; Islam et al., 1996). However, cadherins represent only one of the intercellular adhesion systems that are present in epithelia, along with adhesion molecules of the immunoglobulin superfamily, such as carcinoembryonic antigen (Benchimol et al., 1989), and others. The actual contribution of Ca²⁺-independent nonjunctional adhesion molecules to the formation and maintenance of the epithelial tissue architecture and epithelial cell morphology is not clear.We have recently demonstrated that a 40-kD epithelial glycoprotein, which we have designated epithelial cell adhesion molecule (Ep-CAM)¹ (Litvinov et al., 1994a ), may perform as a homophilic, Ca²⁺-independent intercellular adhesion molecule, capable of mediating cell aggregation, preventing cell scattering, and directing cell segregation. This type I transmembrane glycoprotein consists of two EGF-like domains followed by a cysteine-poor region, a transmembrane domain, and a short (26-amino acid) cytoplasmic tail, and is not structurally related to the four major types of CAMs, such as cadherins, integrins, selectins, and the immunoglobulin superfamily (for review see Litvinov, 1995). Ep-CAM demonstrates adhesion properties when introduced into cell systems that are deficient in intercellular adhesive interactions (Litvinov et al., 1994a ). However, the participation of the Ep-CAM molecule in supporting cell–cell interactions of epithelial cells was not evident (Litvinov et al., 1994b ).Most epithelial cell types coexpress E-cadherin (and sometimes other classic cadherins) and Ep-CAM (for review see Litvinov, 1995) during some stage of embryogenesis. In adult squamous epithelia, which are Ep-CAM negative, de novo expression of this molecule is associated with metaplastic or neoplastic changes. Thus, in ectocervical epithelia, expression of Ep-CAM occurs in early preneoplastic lesions (Litvinov et al., 1996); most squamous carcinomas of the head and neck region are Ep-CAM positive (Quak et al., 1990), and basal cell carcinomas are Ep-CAM positive in contrast to the normal epidermis (Tsubura et al., 1992).In many tumors that express Ep-CAM heterogeneously, an Ep-CAM–positive cell population may be found within an Ep-CAM–negative cell population, with both cell types expressing approximately equal levels of cadherins, as illustrated in Fig. ​Fig.11 A by a case of basal cell carcinoma. In glandular tissues such as gastric epithelium, which are low/ negative for Ep-CAM, expression of Ep-CAM is related to the development of early stages of intestinal metaplasia (our unpublished observation). Even in tissues with relatively high Ep-CAM expression, such as colon, the development of polyps is accompanied by an increase in Ep-CAM expression (Salem et al., 1993). In intestinal metaplasia one may observe Ep-CAM–positive cells bordering morphologically identical normal cells that are Ep-CAM–negative (as illustrated in Fig. ​Fig.11 B) Ep-CAM–positive cells bordering Ep-CAM–negative epithelial cells may also be found in some normal tissues such as hair follicles (Tsubura et al., 1992). Open in a separate windowFigure 1Examples of Ep-CAM expression by some cells within the E-cadherin–positive cell population. (A) Heterogeneous expression of Ep-CAM in a basal cell carcinoma, as detected by immunofluorescent staining with mAb 323/A3 to Ep-CAM (green fluorescence); the red fluorescence indicates the expression of E-cadherin (mAb HECD-1). (B) The de novo expression of Ep-CAM in gastric mucosa in relation to the development of intestinal metaplasia; immunohistochemical staining with mAb 323/A3. Note the bordering Ep-CAM–positive and –negative cells. Bars, 30 μM.From the examples presented, an increased or de novo expression of Ep-CAM is often observed in epithelial tissues in vivo. Expression of an additional molecule that may participate in cell adhesion in the context of other adhesion systems may have certain effects on the cell–cell interactions. Therefore, we have investigated whether the increased/de novo expression of Ep-CAM in epithelial cells (a) has any impact on interactions of positive cells with the parental Ep-CAM–negative cells, and (b) modulates in any way intercellular adhesive interactions of cells interconnected by E-cadherin, which is the major morphoregulatory molecule in epithelia.Here we demonstrate that expression of Ep-CAM by some cells in a mixed cell population expressing classical cadherins induces segregation of the Ep-CAM–positive cells from the parental cell population due to a negative effect on cadherin junctions caused by expression of Ep-CAM. The cadherin-modulating properties observed for Ep-CAM suggest a role for this molecule in the development of a proliferative and metaplastic cell phenotype, and probably in the development and progression of malignancies.     

Cell Adhesion: Separation of p120's Powers?

The catenin p120 is involved in many processes, including cell-cell adhesion and cancer. Recent work explores whether p120 independently regulates two key binding partners, RhoGTPase and cadherin.     

Matrix-dependent Tiam1/Rac Signaling in Epithelial Cells Promotes Either Cell–Cell Adhesion or Cell Migration and Is Regulated by Phosphatidylinositol 3-Kinase

We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin–mediated cell–cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin–mediated cell–cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin–mediated cell– cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase– PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin–mediated cell– cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.     

G Protein �¦� Subunits Regulate Cell Adhesion through Rap1a and Its Effector Radil

The activation of several G protein-coupled receptors is known to regulate the adhesive properties of cells in different contexts. Here, we reveal that Gβγ subunits of heterotrimeric G proteins regulate cell-matrix adhesiveness by activating Rap1a-dependent inside-out signals and integrin activation. We show that Gβγ subunits enter in a protein complex with activated Rap1a and its effector Radil and establish that this complex is required downstream of receptor stimulation for the activation of integrins and the positive modulation of cell-matrix adhesiveness. Moreover, we demonstrate that Gβγ and activated Rap1a promote the translocation of Radil to the plasma membrane at sites of cell-matrix contacts. These results add to the molecular understanding of how G protein-coupled receptors impinge on cell adhesion and suggest that the Gβγ·Rap1·Radil complex plays important roles in this process.     

Pancreatic Cancer Cell Glycosylation Regulates Cell Adhesion and Invasion through the Modulation of α2β1 Integrin and E-Cadherin Function

In our previous studies we have described that ST3Gal III transfected pancreatic adenocarcinoma Capan-1 and MDAPanc-28 cells show increased membrane expression levels of sialyl-Lewis x (SLe^x) along with a concomitant decrease in α2,6-sialic acid compared to control cells. Here we have addressed the role of this glycosylation pattern in the functional properties of two glycoproteins involved in the processes of cancer cell invasion and migration, α2β1 integrin, the main receptor for type 1 collagen, and E-cadherin, responsible for cell-cell contacts and whose deregulation determines cell invasive capabilities. Our results demonstrate that ST3Gal III transfectants showed reduced cell-cell aggregation and increased invasive capacities. ST3Gal III transfected Capan-1 cells exhibited higher SLe^x and lower α2,6-sialic acid content on the glycans of their α2β1 integrin molecules. As a consequence, higher phosphorylation of focal adhesion kinase tyrosine 397, which is recognized as one of the first steps of integrin-derived signaling pathways, was observed in these cells upon adhesion to type 1 collagen. This molecular mechanism underlies the increased migration through collagen of these cells. In addition, the pancreatic adenocarcinoma cell lines as well as human pancreatic tumor tissues showed colocalization of SLe^x and E-cadherin, which was higher in the ST3Gal III transfectants. In conclusion, changes in the sialylation pattern of α2β1 integrin and E-cadherin appear to influence the functional role of these two glycoproteins supporting the role of these glycans as an underlying mechanism regulating pancreatic cancer cell adhesion and invasion.     

Leaders in Cell Adhesion: An Interview with Richard Hynes,Pioneer of Cell–Matrix Interactions

Abstract

On a recent visit Richard O Hynes, FRS, HHMI, Daniel K. Ludwig Professor for Cancer Research at the Koch Institute for Integrative Cancer Research, MIT, graciously agreed to be interviewed in person for the first in Cell Communication and Adhesion's series on “Leaders in Cell Adhesion”. In this interview we discussed three things: 1) the early role of family, mentors, and luck on his career path; 2) his major discoveries of fibronectin, integrins and the evolution of extracellular matrix proteins; and 3) his role in, and thoughts on, current science policy. This interview reveals his characteristic calmness and infectious optimism, his spontaneous and down to earth sense of humor, and his great ability to place scientific questions in perspective. The interview, carried out on April 30^th 2013 is reported here verbatim with only minor editing for clarity.     

Keeping the Vimentin Network under Control: Cell–Matrix Adhesion–associated Plectin 1f Affects Cell Shape and Polarity of Fibroblasts

Focal adhesions (FAs) located at the ends of actin/myosin-containing contractile stress fibers form tight connections between fibroblasts and their underlying extracellular matrix. We show here that mature FAs and their derivative fibronectin fibril-aligned fibrillar adhesions (FbAs) serve as docking sites for vimentin intermediate filaments (IFs) in a plectin isoform 1f (P1f)-dependent manner. Time-lapse video microscopy revealed that FA-associated P1f captures mobile vimentin filament precursors, which then serve as seeds for de novo IF network formation via end-to-end fusion with other mobile precursors. As a consequence of IF association, the turnover of FAs is reduced. P1f-mediated IF network formation at FbAs creates a resilient cage-like core structure that encases and positions the nucleus while being stably connected to the exterior of the cell. We show that the formation of this structure affects cell shape with consequences for cell polarization.     

Role of UDP-N-Acetylglucosamine2-Epimerase/N-Acetylmannosamine Kinase (GNE) in β1-Integrin-Mediated Cell Adhesion

Hereditary inclusion body myopathy (GNE myopathy) is a neuromuscular disorder due to mutation in key sialic acid biosynthetic enzyme, GNE. The pathomechanism of the disease is poorly understood as GNE is involved in other cellular functions beside sialic acid synthesis. In the present study, a HEK293 cell-based model system has been established where GNE is either knocked down or over-expressed along with pathologically relevant GNE mutants (D176V and V572L). The subcellular distribution of recombinant GNE and its mutant showed differential localization in the cell. The effect of mutation on GNE function was investigated by studying hyposialylation of cell membrane receptor, β1-integrin. Hyposialylated β1-integrin localized to internal vesicles that was restored upon supplementation with sialic acid. Fibronectin stimulation caused migration of hyposialylated β1-integrin to the cell membrane and co-localization with focal adhesion kinase (FAK) leading to increased focal adhesion formation. This further activated FAK and Src, downstream signaling molecules and led to increased cell adhesion. This is the first report to show that mutation in GNE affects β1-integrin-mediated cell adhesion process in GNE mutant cells.     

β1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival

Extracellular matrix (ECM) integrity in the central nervous system (CNS) is essential for neuronal homeostasis. Signals from the ECM are transmitted to neurons through integrins, a family of cell surface receptors that mediate cell attachment to ECM. We have previously established a causal link between the activation of the matrix metalloproteinase-9 (MMP-9), degradation of laminin in the ECM of retinal ganglion cells (RGCs), and RGC death in a mouse model of retinal ischemia-reperfusion injury (RIRI). Here we investigated the role of laminin-integrin signaling in RGC survival in vitro, and after ischemia in vivo. In purified primary rat RGCs, stimulation of the β1 integrin receptor with laminin, or agonist antibodies enhanced RGC survival in correlation with activation of β1 integrin’s major downstream regulator, focal adhesion kinase (FAK). Furthermore, β1 integrin binding and FAK activation were required for RGCs’ survival response to laminin. Finally, in vivo after RIRI, we observed an up-regulation of MMP-9, proteolytic degradation of laminin, decreased RGC expression of β1 integrin, FAK and Akt dephosphorylation, and reduced expression of the pro-survival molecule bcl-xL in the period preceding RGC apoptosis. RGC death was prevented, in the context of laminin degradation, by maintaining β1 integrin activation with agonist antibodies. Thus, disruption of homeostatic RGC-laminin interaction and signaling leads to cell death after retinal ischemia, and maintaining integrin activation may be a therapeutic approach to neuroprotection.     

Bacterial Genotoxins Promote Inside-Out Integrin β1 Activation,Formation of Focal Adhesion Complexes and Cell Spreading

Integrins are membrane bound receptors that regulate several cellular processes, such as cell adhesion, migration, survival and proliferation, and may contribute to tumor initiation/progression in cells exposed to genotoxic stress. The extent of integrin activation and its role in cell survival upon intoxication with bacterial genotoxins are still poorly characterized. These toxins induce DNA strand breaks in the target cells and activate the DNA damage response (DDR), coordinated by the Ataxia Telangectasia Mutated (ATM) kinase. In the present study, we demonstrate that induction of DNA damage by two bacterial genotoxins promotes activation of integrin β1, leading to enhanced assembly of focal adhesions and cell spreading on fibronectin, but not on vitronectin. This phenotype is mediated by an ATM-dependent inside-out integrin signaling, and requires the actin cytoskeleton remodeler NET1. The toxin-mediated cell spreading and anchorage-independent survival further relies on ALIX and TSG101, two components of the endosomal sorting complex required for transport (ESCRT), known to regulate integrin intracellular trafficking. These data reveal a novel aspect of the cellular response to bacterial genotoxins, and provide new tools to understand the carcinogenic potential of these effectors in the context of chronic intoxication and infection.     

Cell–Cell Adhesion Molecule CEACAM1 is Expressed in Normal Breast and Milk and Associates with β1 Integrin in a 3D Model of Morphogenesis

CEA cell adhesion molecule-1 (CEACAM1) is a cell-cell adhesion molecule that, paradoxically, is expressed in an apical location in normal breast epithelium. Strong lumenal membrane staining is observed in 100% of normal glands (11/11), low in atypical hyperplasia (2/6), high in cribiform ductal carcinoma in situ (DCIS) (8/8), but low in other types of DCIS (2/15). Although most invasive ductal carcinomas express CEACAM1 (21/26), the staining pattern tends to be weak and cytoplasmic in tumours with minimal lumena formation (grades 2-3), while there is membrane staining in well-differentiated tumours (grade 1). The 'normal' breast epithelial line MCF10F forms acini with lumena in Matrigel with apical membrane expression of CEACAM1. MCF7 cells that do not express CEACAM1 and fail to form lumena in Matrigel, revert to a lumen forming phenotype when transfected with the CEACAM1-4S but not the -4L isoform. CEACAM1 directly associates with and down-regulates the expression of beta1-integrin. Immuno-electron microscopy reveals numerous vesicles coated with CEACAM1 within the lumena, and as predicted by this finding, CEACAM1 is found in the lipid fraction of breast milk. Thus, CEACAM1 is a critical molecule in mammary morphogenesis and may play a role in the absorption of the lipid vesicles of milk in the infant intestinal tract.