RESUMEN
E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8-mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site-directed mutagenesis, and single-molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped ß-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.
Asunto(s)
Cadherinas , Simulación de Dinámica Molecular , Cadherinas/metabolismo , Unión Proteica , Adhesión CelularRESUMEN
E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8 mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site directed mutagenesis and single molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped ß-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.
RESUMEN
Given the role of E-cadherin (E-cad) in holding epithelial cells together, an inverse relationship between E-cad levels and cell invasion during the epithelial-mesenchymal transition and cancer metastasis has been well recognized. Here we report that E-cad is necessary for the invasiveness of RasV12-transformed intestinal epithelial cells in Drosophila. E-cad/ß-catenin disassembles at adherens junctions and assembles at invasive protrusions--the actin- and cortactin-rich invadopodium-like protrusions associated with the breach of the extracellular matrix (ECM)--during dissemination of RasV12-transformed intestinal epithelial cells. Loss of E-cad impairs the elongation of invasive protrusions and attenuates the ability of RasV12-transformed cells to compromise the ECM. Notably, E-cad and cortactin affect each other's localization to invasive protrusions. Given the essential roles of cortactin in cell invasion, our observations indicate that E-cad plays a role in the invasiveness of RasV12-transformed intestinal epithelial cells by controlling cortactin localization to invasive protrusions. Thus our study demonstrates that E-cad is a component of invasive protrusions and provides molecular insights into the unconventional role of E-cad in cell dissemination in vivo.
Asunto(s)
Cadherinas , Cortactina , Animales , Cortactina/metabolismo , Cadherinas/metabolismo , Células Epiteliales/metabolismo , Actinas/metabolismo , Uniones Adherentes/metabolismo , Drosophila/metabolismoRESUMEN
E-cadherin adhesion is regulated at the cell surface, a process that can be replicated by activating antibodies. We use cryo-electron microscopy (EM) and X-ray crystallography to examine functional states of the cadherin adhesive dimer. This dimer is mediated by N-terminal beta strand-swapping involving Trp2, and forms via a different transient X-dimer intermediate. X-dimers are observed in cryo-EM along with monomers and strand-swap dimers, indicating that X-dimers form stable interactions. A novel EC4-mediated dimer was also observed. Activating Fab binding caused no gross structural changes in E-cadherin monomers, but can facilitate strand swapping. Moreover, activating Fab binding is incompatible with the formation of the X-dimer. Both cryo-EM and X-ray crystallography reveal a distinctive twisted strand-swap dimer conformation caused by an outward shift in the N-terminal beta strand that may represent a strengthened state. Thus, regulation of adhesion involves changes in cadherin dimer configurations.
RESUMEN
E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface. Molecular dynamics simulations and single-molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes: one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped ß-strand and its complementary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.
Asunto(s)
Anticuerpos Monoclonales , Cadherinas , Adhesión Celular , Anticuerpos Monoclonales/química , Cadherinas/química , Cadherinas/inmunología , Cristalografía por Rayos X , Humanos , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Dominios ProteicosRESUMEN
The dynamic regulation of epithelial adherens junctions relies on all components of the E-cadherin-catenin complex. Previously, the complexes have been partially reconstituted and composed only of α-catenin, ß-catenin, and the E-cadherin cytoplasmic domain. However, p120-catenin and the full-length E-cadherin including the extracellular, transmembrane, and intra-cellular domains are vital to the understanding of the relationship between extracellular adhesion and intracellular signaling. Here, we reconstitute the complete and full-length cadherin-catenin complex, including full-length E-cadherin, α-catenin, ß-catenin, and p120-catenin, into nanodiscs. We are able to observe the cadherin in nanodiscs by cryo-EM. We also reconstitute α-catenin, ß-catenin, and p120-catenin with the E-cadherin cytoplasmic tail alone in order to analyze the affinities of their binding interactions. We find that p120-catenin does not associate strongly with α- or ß-catenin and binds much more transiently to the cadherin cytoplasmic tail than does ß-catenin. Overall, this work creates many new possibilities for biochemical studies understanding transmembrane signaling of cadherins and the role of p120-catenin in adhesion activation.
Asunto(s)
Cadherinas , Cateninas , Cadherinas/genética , Cadherinas/metabolismo , Cateninas/genética , Cateninas/metabolismo , Adhesión Celular/fisiología , Membrana Celular/metabolismo , Fosfoproteínas , Transducción de Señal , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Deficits in gastrointestinal (GI) paracellular permeability has been implicated in etiology of Inflammatory Bowel Disease (IBD), and E-cadherin, a key component of the epithelial junctional complex, has been implicated in both barrier function and IBD. We have previously described antibodies against E-cadherin that activate cell adhesion, and in this study, we show that they increase transepithelial electrical resistance in epithelial cell monolayers in vitro. We therefore tested the hypothesis that adhesion activating E-cadherin mAbs will enhance epithelial barrier function in vivo and limit progression of inflammation in IBD. Activating mAbs to mouse E-cadherin were tested in different mouse models of IBD including the IL10-/- and adoptive T cell transfer models of colitis. Previously established histological and biomarker measures of inflammation were evaluated to monitor disease progression. Mouse E-cadherin activating mAb treatment reduced total colitis score, individual histological measures of inflammation, and other hallmarks of inflammation compared to control treatment. Activating mAbs also reduced the fecal accumulation lipocalin2 and albumin content, consistent with enhanced barrier function. Therefore, E-cadherin activation could be a potential strategy for limiting inflammation in UC.
Asunto(s)
Colitis , Enfermedades Inflamatorias del Intestino , Animales , Cadherinas/metabolismo , Colitis/metabolismo , Colitis/patología , Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Mucosa Intestinal/metabolismo , RatonesRESUMEN
Excessive vascular permeability occurs in inflammatory disease processes. Vascular endothelial cadherin (VE-cadherin) is an adhesion protein that controls vascular permeability. We identified monoclonal antibodies (mAbs) to human VE-cadherin that activate cell adhesion and inhibit the increased permeability of endothelial cell monolayers induced by thrombin receptor activator peptide-6 (TRAP-6). Two mAbs, 8A12c and 3A5a, reduce permeability, whereas an inhibitory mAb, 2E11d, enhances permeability. Activating mAbs also reduce permeability induced by tumor necrosis factor-α (TNF-α) and vascular endothelial cell growth factor (VEGF). The activating mAbs also stabilize the organization of the adherens junctions that are disrupted by TRAP-6, VEGF, or TNF-α. The activating mAbs act directly on the adhesive function of VE-cadherin because they did not block the accumulation of actin filaments stimulated by TRAP-6 and enhance physical cell-cell adhesion of VE-cadherin-expressing tissue culture cells. Therefore, VE-cadherin function can be regulated at the cell surface to control endothelial permeability.NEW & NOTEWORTHY Excessive vascular permeability is a serious complication of many inflammatory disease conditions. We have developed monoclonal antibodies that inhibit increases in endothelial monolayer permeability induced by several signaling factors by activating VE-cadherin mediated adhesion and stabilizing cell junctions. These antibodies and/or the mechanisms they reveal may lead to important therapeutics to treat vascular leakiness and inflammation.
Asunto(s)
Uniones Adherentes/efectos de los fármacos , Anticuerpos Monoclonales/farmacología , Cadherinas/agonistas , Permeabilidad Capilar/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Uniones Adherentes/metabolismo , Antígenos CD/metabolismo , Cadherinas/antagonistas & inhibidores , Cadherinas/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Nocodazol/farmacología , Oligopéptidos/farmacología , Receptores de Trombina/agonistas , Receptores de Trombina/metabolismo , Transducción de Señal , Moduladores de Tubulina/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
p120-catenin is considered to be a tumor suppressor because it stabilizes E-cadherin levels at the cell surface. p120-catenin phosphorylation is increased in several types of cancer, but the role of phosphorylation in cancer is unknown. The phosphorylation state of p120-catenin is important in controlling E-cadherin homophilic binding strength which maintains epithelial junctions. Because decreased cell-cell adhesion is associated with increased cancer metastasis we hypothesize that p120-catenin phosphorylation at specific Serine and Threonine residues alters the E-cadherin binding strength between tumor cells and thereby affect the ability of tumor cells to leave the primary tumor and metastasize to distant sites. In this study we show that expression of the p120-catenin phosphorylation dead mutant, by converting six Serine and Threonine sites to Alanine, leads to enhanced E-cadherin adhesive binding strength in tumor cells. We observed a decrease in the ability of tumor cells expressing the p120-catenin phosphorylation mutant to migrate and invade using in-vitro models of cancer progression. Further, tumor cells expressing the phosphorylation mutant form of p120-catenin demonstrated a decrease in ability to metastasize to the lungs using an in-vivo orthotopic mammary fat pad injection model of breast cancer development and metastasis. This suggests that regulation of p120-catenin phosphorylation at the cell surface is important in mediating cell-adhesion, thereby impacting cancer progression and metastasis.
Asunto(s)
Cadherinas/metabolismo , Cateninas/metabolismo , Adhesión Celular , Animales , Cateninas/genética , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Ratones , Mutación , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación , Catenina deltaRESUMEN
E-cadherin is a tumor suppressor protein, and the loss of its expression in association with the epithelial mesenchymal transition (EMT) occurs frequently during tumor metastasis. However, many metastases continue to express E-cadherin, and a full EMT is not always necessary for metastasis; also, positive roles for E-cadherin expression in metastasis have been reported. We hypothesize instead that changes in the functional activity of E-cadherin expressed on tumor cells in response to environmental factors is an important determinant of the ability of the tumor cells to metastasize. We find that E-cadherin expression persists in metastatic lung nodules and circulating tumor cells (CTCs) in two mouse models of mammary cancer: genetically modified MMTV-PyMT mice and orthotopically grafted 4T1 tumor cells. Importantly, monoclonal antibodies that bind to and activate E-cadherin at the cell surface reduce lung metastasis from endogenous genetically driven tumors and from tumor cell grafts. E-cadherin activation inhibits metastasis at multiple stages, including the accumulation of CTCs from the primary tumor and the extravasation of tumor cells from the vasculature. These activating mAbs increase cell adhesion and reduce cell invasion and migration in both cell culture and three-dimensional spheroids grown from primary tumors. Moreover, activating mAbs increased the frequency of apoptotic cells without affecting proliferation. Although the growth of the primary tumors was unaffected by activating mAbs, CTCs and tumor cells in metastatic nodules exhibited increased apoptosis. Thus, the functional state of E-cadherin is an important determinant of metastatic potential beyond whether the gene is expressed.
Asunto(s)
Neoplasias de la Mama/metabolismo , Cadherinas/antagonistas & inhibidores , Cadherinas/metabolismo , Neoplasias Pulmonares/metabolismo , Metástasis de la Neoplasia , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Cadherinas/genética , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica , Metástasis de la Neoplasia/tratamiento farmacológico , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Procesos NeoplásicosRESUMEN
The Hippo pathway is involved in regulating contact inhibition of proliferation and organ size control and responds to various physical and biochemical stimuli. It is a kinase cascade that negatively regulates the activity of cotranscription factors YAP and TAZ, which interact with DNA binding transcription factors including TEAD and activate the expression of target genes. In this study, we show that the palmitoylation of TEAD, which controls the activity and stability of TEAD proteins, is actively regulated by cell density independent of Lats, the key kinase of the Hippo pathway. The expression of fatty acid synthase and acetyl-CoA carboxylase involved in de novo biosynthesis of palmitate is reduced by cell density in an Nf2/Merlin-dependent manner. Depalmitoylation of TEAD is mediated by depalmitoylases including APT2 and ABHD17A. Palmitoylation-deficient TEAD4 mutant is unstable and degraded by proteasome through the activity of the E3 ubiquitin ligase CHIP. These findings show that TEAD activity is tightly controlled through the regulation of palmitoylation and stability via the orchestration of FASN, depalmitoylases, and E3 ubiquitin ligase in response to cell contact.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Acido Graso Sintasa Tipo I/metabolismo , Lipoilación , Neurofibromina 2/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Humanos , Proteínas Musculares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción de Dominio TEARESUMEN
E-cadherin is a key component of the adherens junctions that are integral in cell adhesion and maintaining epithelial phenotype of cells. Homophilic E-cadherin binding between cells is important in mediating contact inhibition of proliferation when cells reach confluence. Loss of E-cadherin expression results in loss of contact inhibition and is associated with increased cell motility and advanced stages of cancer. In this review we discuss the role of E-cadherin and its downstream signaling in regulation of contact inhibition and the development and progression of cancer.
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Cadherinas/metabolismo , Inhibición de Contacto/fisiología , Neoplasias/metabolismo , Neoplasias/patología , Animales , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Humanos , Transducción de Señal/fisiologíaRESUMEN
The loss of E-cadherin expression in association with the epithelial-mesenchymal transition (EMT) occurs frequently during tumor metastasis. However, metastases often retain E-cadherin expression, an EMT is not required for metastasis, and metastases can arise from clusters of tumor cells. We demonstrate that the regulation of the adhesive activity of E-cadherin present at the cell surface by an inside-out signaling mechanism is important in cancer. First, we find that the metastasis of an E-cadherin-expressing mammary cell line from the mammary gland to the lung depends on reduced E-cadherin adhesive function. An activating monoclonal antibody to E-cadherin that induces a high adhesive state significantly reduced the number of cells metastasized to the lung without affecting the growth in size of the primary tumor in the mammary gland. Second, we find that many cancer-associated germline missense mutations in the E-cadherin gene in patients with hereditary diffuse gastric cancer selectively affect the mechanism of inside-out cell surface regulation without inhibiting basic E-cadherin adhesion function. This suggests that genetic deficits in E-cadherin cell surface regulation contribute to cancer progression. Analysis of these mutations also provides insights into the molecular mechanisms underlying cadherin regulation at the cell surface.
Asunto(s)
Cadherinas/genética , Cadherinas/metabolismo , Animales , Cadherinas/fisiología , Adhesión Celular/fisiología , Línea Celular Tumoral , Membrana Celular/metabolismo , Movimiento Celular , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Glándulas Mamarias Humanas/metabolismo , Ratones , Ratones Endogámicos BALB C , Mutación Missense , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Receptores de Superficie Celular , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
The Hippo-YAP pathway mediates organ size control, contact inhibition, and tumorigenesis. It is a kinase cascade that inhibits the nuclear localization and transcriptional activities of YAP and TAZ. E-cadherin, cell junctions, polarity proteins, and the merlin/NF2 tumor suppressor activate the pathway to inhibit YAP/TAZ activity, while growth factor signaling inhibits the pathway to activate YAP/TAZ in the nucleus. We examined its role in the development of mouse mammary glands and tumor formation using gland reconstitution by transplantation of genetically modified mammary stem cells (MaSCs). Knockdown of YAP and TAZ with shRNA in MaSCs did not inhibit gland reconstitution. In contrast, knockdown of ß-catenin blocked gland reconstitution, consistent with the known role of Wnt signaling in mammary gland development. However, we find that Hippo signaling is involved in mammary tumor formation. Expression of a constitutively active form of YAP caused rapid formation of large tumors. Moreover, knockdown of YAP/TAZ slowed the development of tumors in polyoma middle T transgenic mice, a well-studied mammary tumor model involving activation of several signaling pathways. YAP accumulated in nuclei of mammary glands in ErbB2/EGFR-transgenic mice, suggesting that EGFR signaling affects YAP in vivo similar to cell culture. ErbB2/EGFR-transgenic mice develop mammary tumors in 7-8 months, but surprisingly, MaSCs from these mice did not form tumors when transplanted into host mice. Nonetheless, expression of dominant-negative Lats, which inhibits Hippo signaling, leads to tumor formation in ErbB2-transgenic mice, suggesting that Hippo signaling is involved in EGFR-induced mammary tumorigenesis.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias de la Mama/genética , Carcinogénesis/genética , Receptores ErbB/genética , Fosfoproteínas/genética , Receptor ErbB-2/genética , Aciltransferasas , Animales , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular , Femenino , Vía de Señalización Hippo , Humanos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Factores de Transcripción/genética , Vía de Señalización Wnt/genética , Proteínas Señalizadoras YAP , beta Catenina/genéticaRESUMEN
Tight regulation of cadherin-mediated intercellular adhesions is critical to both tissue morphogenesis during development and tissue homeostasis in adults. Cell surface expression of the cadherin-catenin complex is often directly correlated with the level of adhesion, however, examples exist where cadherin appears to be inactive and cells are completely non-adhesive. The state of p120-catenin phosphorylation has been implicated in regulating the adhesive activity of E-cadherin but the mechanism is currently unclear. We have found that destabilization of the microtubule cytoskeleton, independent of microtubule plus-end dynamics, dephosphorylates p120-catenin and activates E-cadherin adhesion in Colo 205 cells. Through chemical screening, we have also identified several kinases as potential regulators of E-cadherin adhesive activity. Analysis of several p120-catenin phosphomutants suggests that gross dephosphorylation of p120-catenin rather than that of specific amino acids may trigger E-cadherin adhesion. Uncoupling p120-catenin binding to E-cadherin at the membrane causes constitutive adhesion in Colo 205 cells, further supporting an inhibitory role of phosphorylated p120-catenin on E-cadherin activity.
Asunto(s)
Cadherinas/metabolismo , Cateninas/metabolismo , Neoplasias del Colon/metabolismo , Microtúbulos/metabolismo , Cateninas/genética , Adhesión Celular , Línea Celular Tumoral , Neoplasias del Colon/genética , Humanos , Mutación , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Catenina deltaRESUMEN
The Hippo pathway is involved in the regulation of contact inhibition of proliferation and responses to various physical and chemical stimuli. Recently, several upstream negative regulators of Hippo signaling, including epidermal growth factor receptor ligands and lysophosphatidic acid, have been identified. We show that fibronectin adhesion stimulation of focal adhesion kinase (FAK)-Src signaling is another upstream negative regulator of the Hippo pathway. Inhibition of FAK or Src in MCF-10A cells plated at low cell density prevented the activation of Yes-associated protein (YAP) in a large tumor suppressor homologue (Lats)-dependent manner. Attachment of serum-starved MCF-10A cells to fibronectin, but not poly-d-lysine or laminin, induced YAP nuclear accumulation via the FAK-Src-phosphatidylinositol 4,5-bisphosphate 3-kinase (PI3K) signaling pathway. Attenuation of FAK, Src, PI3K, or PDK1 activity blocked YAP nuclear accumulation stimulated by adhesion to fibronectin. This negative regulation of the Hippo pathway by fibronectin adhesion signaling can, at least in part, explain the effects of cell spreading on YAP nuclear localization and represents a Lats-dependent component of the response to cell adhesion.
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Fibronectinas/metabolismo , Quinasa 1 de Adhesión Focal/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Línea Celular , Proliferación Celular/fisiología , Inhibición de Contacto/fisiología , Quinasa 1 de Adhesión Focal/genética , Vía de Señalización Hippo , Humanos , Laminina/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosfoproteínas/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Polilisina/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Factores de Transcripción , Proteínas Señalizadoras YAP , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genéticaRESUMEN
Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation.
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Cadherinas/metabolismo , Regulación Alostérica/efectos de los fármacos , Animales , Anticuerpos Neutralizantes/farmacología , Cateninas/metabolismo , Adhesión Celular/efectos de los fármacos , Perros , Humanos , Imagenología Tridimensional , Cinética , Cloruro de Litio/farmacología , Células MCF-7 , Ratones , Modelos Biológicos , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Multimerización de Proteína , Ratas , Estaurosporina/farmacología , Catenina deltaRESUMEN
Cadherin-6B induces bone morphogenetic protein (BMP) signaling to promote the epithelial mesenchymal transition (EMT) in the neural crest. We have previously found that knockdown of Cadherin-6B inhibits both BMP signaling and the emigration of the early pre-migratory neural crest cells from the dorsal neural tube. In this study, we found that inhibition of BMP signaling in the neural tube, mediated by the ectopic expression of Smad-6 or Noggin, decreased the size of the Islet-1-positive dorsal cell population. Knockdown or loss of function of Cadherin-6B suppressed the generation of Islet-1-expressing cells in the dorsal neural tube, but not the Lim-1/2 positive dorsal cell population. Our results thus indicate that Cadherin-6B is necessary for the generation of Islet-1-positive dorsal interneurons, as well as the initiation of pre-migratory neural crest cell emigration.
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Proteínas Aviares/metabolismo , Cadherinas/metabolismo , Interneuronas/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas Aviares/antagonistas & inhibidores , Proteínas Aviares/genética , Proteínas Morfogenéticas Óseas/metabolismo , Cadherinas/antagonistas & inhibidores , Cadherinas/genética , Proteínas Portadoras/metabolismo , Movimiento Celular , Embrión de Pollo , Transición Epitelial-Mesenquimal , Técnicas de Silenciamiento del Gen , Cresta Neural/citología , Cresta Neural/embriología , Cresta Neural/metabolismo , Tubo Neural/citología , Tubo Neural/embriología , Tubo Neural/metabolismo , Transducción de Señal , Proteína smad6/metabolismoRESUMEN
Epithelial-mesenchymal transition (EMT) of mammary epithelial cells is important in both normal morphogenesis of mammary glands and metastasis of breast cancer. Cadherin switching from E-cadherin to N-cadherin plays important roles in EMT. We found that cadherin switching is important in bone morphogenetic protein 4 (BMP4)-induced EMT in MCF-10A cells. BMP4 increased the phosphorylation of SMAD proteins in MCF-10A cells. Canonical BMP4 signaling decreased the expression of E-cadherin and disrupted the polarity of the tight junction protein ZO-1 in MCF-10A cells. However, the expression of N-cadherin and SNAI2 was up-regulated in BMP4-treated MCF-10A cells. MCF-10A cells that expressed N-cadherin migrated into type I collagen gels in response to BMP4 when evaluated using three-dimensional culture assays. Thus, active canonical BMP4 signaling is important for the migration and EMT of mammary epithelial cells. Moreover, the decrease in E-cadherin and/or increase in N-cadherin may be required for BMP4-induced migration and EMT.
Asunto(s)
Proteína Morfogenética Ósea 4/genética , Neoplasias de la Mama/genética , Cadherinas/biosíntesis , Transición Epitelial-Mesenquimal/genética , Neoplasias de la Mama/patología , Cadherinas/genética , Línea Celular Tumoral , Movimiento Celular/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Humanos , Glándulas Mamarias Humanas/patología , Morfogénesis/genética , Metástasis de la Neoplasia , Proteína de la Zonula Occludens-1/genéticaRESUMEN
The Hippo-YAP pathway mediates the control of cell proliferation by contact inhibition as well as other attributes of the physical state of cells in tissues. Several mechanisms sense the spatial and physical organization of cells, and function through distinct upstream modules to stimulate Hippo-YAP signaling: adherens junction or cadherin-catenin complexes, epithelial polarity and tight junction complexes, the FAT-Dachsous morphogen pathway, as well as cell shape, actomyosin or mechanotransduction. Soluble extracellular factors also regulate Hippo pathway signaling, often inhibiting its activity. Indeed, the Hippo pathway mediates a reciprocal relationship between contact inhibition and mitogenic signaling. As a result, cells at the edges of a colony, a wound in a tissue or a tumor are more sensitive to ambient levels of growth factors and more likely to proliferate, migrate or differentiate through a YAP and/or TAZ-dependent process. Thus, the Hippo-YAP pathway senses and responds to the physical organization of cells in tissues and coordinates these physical cues with classic growth-factor-mediated signaling pathways. This Commentary is focused on the biological significance of Hippo-YAP signaling and how upstream regulatory modules of the pathway interact to produce biological outcomes.