Rho and Rho-kinase (ROCK) signaling in adherens and gap junction assembly in corneal epithelium.
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PURPOSE: To examine whether Rho and its downstream target, a Rho-associated kinase (ROCK), are involved in the regulation of the assembly of cadherin-mediated cell adhesion and connexin 43 (Cx43) gap junctions in corneal epithelium. METHODS: Rho and ROCK activities in rabbit corneal epithelial cells in culture were inhibited by microinjection of a Clostridium botulinum ADP-ribosyltransferase (C3) and treatment with a ROCK specific inhibitor (Y-27632), respectively. Immunocytochemical and Western blot techniques were used to study the distribution and relative concentrations of E-cadherin and Cx43. Intercellular communication via gap junctions was measured by a dye transfer assay. RESULTS: Inhibition of Rho activity in the primary cultures of rabbit corneal epithelial cells by microinjecting them with C3 resulted in an inhibition of the assembly of E-cadherin-based cell-cell adhesion and Cx43 gap junctions. However, inhibition of the ROCK activity by treatment with Y-27632 inhibited the assembly of E-cadherin-based cell-cell adhesions but not Cx43 gap junctions. In fact, inhibition of ROCK resulted in an increase in the number of Cx43 gap junctions and in cell-cell communication. Culturing corneal epithelial cells in a low calcium medium prevented the formation of E-cadherin adherens junctions but not the Cx43 gap junctions. CONCLUSIONS: E-cadherin adherens junctions are not a prerequisite for the assembly of Cx43 gap junctions in corneal epithelial cells. Different Rho signaling pathways are involved in the regulation of the assembly of E-cadherin mediated cell-cell adhesion and Cx43 gap junctions. Although a Rho/ROCK signaling pathway influences the assembly of E-cadherin adherens junctions, its downregulation influences Cx43 gap junction assembly. (+info)
A presenilin-1/gamma-secretase cleavage releases the E-cadherin intracellular domain and regulates disassembly of adherens junctions.
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E-cadherin controls a wide array of cellular behaviors including cell-cell adhesion, differentiation and tissue development. Here we show that presenilin-1 (PS1), a protein involved in Alzheimer's disease, controls a gamma-secretase-like cleavage of E-cadherin. This cleavage is stimulated by apoptosis or calcium influx and occurs between human E-cadherin residues Leu731 and Arg732 at the membrane-cytoplasm interface. The PS1/gamma-secretase system cleaves both the full-length E-cadherin and a transmembrane C-terminal fragment, derived from a metalloproteinase cleavage after the E-cadherin ectodomain residue Pro700. The PS1/gamma-secretase cleavage dissociates E-cadherins, beta-catenin and alpha-catenin from the cytoskeleton, thus promoting disassembly of the E-cadherin-catenin adhesion complex. Furthermore, this cleavage releases the cytoplasmic E-cadherin to the cytosol and increases the levels of soluble beta- and alpha-catenins. Thus, the PS1/gamma-secretase system stimulates disassembly of the E-cadherin- catenin complex and increases the cytosolic pool of beta-catenin, a key regulator of the Wnt signaling pathway. (+info)
Localization of mLin-7 at nectin-based cell-cell junctions.
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In C. elegans, lin-7 as well as lin-2/lin-10 is involved in the proper localization of the LET-23 receptor tyrosine kinase that regulates vulval induction. The mammalian homologue, mLin-7, forms a ternary complex with the mammalian homologues of LIN-2 and LIN-10 and localizes at cell-cell junctions in epithelial cells, but the mechanism of this localization of mLin-7 is unknown. Nectin is an immunoglobulin-like cell-cell adhesion molecule that is involved in organization of adherens and tight junctions in epithelial cells. Nectin is indirectly associated with the cadherin-catenin system and the actin cytoskeleton through afadin, an actin filament-binding protein. We showed here that mLin-7 localized at the nectin-based cell-cell junctions. This localization of mLin-7 required the interaction of nectin with afadin, but not the cadherin-catenin system or the actin cytoskeleton. mLin-7 did not directly interact with nectin or afadin. The results indicate that mLin-7 localizes at cell-cell junctions through the nectin-afadin system. (+info)
Functional polycystin-1 expression is developmentally regulated during epithelial morphogenesis in vitro: downregulation and loss of membrane localization during cystogenesis.
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Polycystin-1 is a protein mutated in the majority of cases of autosomal dominant polycystic kidney disease (ADPKD), but its role in the molecular pathway of tubulogenesis and cystogenesis is not understood. To define the role of polycystin-1 during dynamic changes in formation of intercellular contacts and cell polarity accompanying epithelial morphogenesis, we have utilized a 3D MDCK in vitro model of tubulogenesis and cystogenesis. Here we demonstrate that polycystin-1 is a novel component of desmosomal junctions of epithelial cells. A striking downregulation of polycystin-1 mRNA was detected in cysts as compared to tubules, leading to altered protein expression and localization. While polycystin-1 is localized to basolateral membranes of MDCK tubules, it is only detected in cytoplasmic pools in cystic cells. Furthermore, the expression of polycystin-1 is modulated during distinct stages of HGF-induced tubulogenesis from MDCK cysts. Thus, polycystin-1 is not detected in intercellular contacts at early steps of tubulogenesis, but assumes its basolateral localization at the time of cell polarization and lumen formation. An important role of polycystin-1 is further demonstrated using the pancreatic ductal epithelial cell line SU.86.86 which undergoes in vitro differentiation resulting in the formation of domes. Dome formation is thought to parallel tubular differentiation and morphogenesis in vivo. Our data reveal significant upregulation of polycystin-1 mRNA and protein levels in domes. Collectively, our results demonstrate a critical importance of controlled level of polycystin-1 expression for proper tubular differentiation and maturation. We suggest that the loss of polycystin-1 from its basolateral location in tubular epithelium may alter critical pathways controlling normal tubulogenesis leading to cystic transformation. (+info)
Selective targeting of angiogenic tumor vasculature by vascular endothelial-cadherin antibody inhibits tumor growth without affecting vascular permeability.
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Vascular endothelial-cadherin (VE-cadherin) is an endothelial cell-specific adhesion molecule that is localized exclusively at cell-cell contacts referred to as adherens junctions. VE-cadherin-mediated adhesion is crucial for proper assembly of vascular structures during angiogenesis as well as for maintenance of a normal vascular integrity. We have shown previously that a monoclonal antibody (BV13) to VE-cadherin not only inhibits the formation of vascular tubes during tumor angiogenesis but also disrupts adherens junctions of normal vasculature with a concomitant increase in vascular permeability. The goal of the current studies was to block VE-cadherin function during angiogenesis without disrupting existing junctions on normal endothelium. Using in vitro screening assays to test for functional blocking of adherens junction formation and in vivo assays to detect antibody effects on vascular permeability in normal tissues, we have identified a novel blocking antibody (E4G10) that inhibits VE-cadherin function during angiogenesis but does not disrupt existing adherens junctions on normal vasculature. E4G10 inhibited formation of vascular tubes in vivo in the Matrigel plug and corneal micropocket assays. E4G10 also inhibited tumor growth in three models of mouse and human tumors via an antiangiogenic mechanism. Examination of normal mouse and tumor tissues showed that E4G10 bound to endothelial cells in a subset of tumor vasculature but not to normal vasculature. Bromodeoxyuridine labeling experiments showed that E4G10 specifically targeted a subset of tumor endothelium that is undergoing active cell proliferation, which likely reflects the activated, angiogenic endothelium. These findings indicate that VE-cadherin can be selectively targeted during states of pathological angiogenesis, despite its ubiquitous distribution throughout the entire vasculature. Our data also suggest that antibody E4G10 recognizes VE-cadherin epitopes that are only accessible on endothelial cells forming new adherens junctions, such as in angiogenic tumor vasculature. (+info)
Interaction between the transforming growth factor-beta type II receptor/Smad pathway and beta-catenin during transforming growth factor-beta1-mediated adherens junction disassembly.
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The aim of the current study was to examine the influence of transforming growth factor (TGF)-beta 1 on proximal tubular epithelial cell-cell interaction, with particular emphasis on the regulation of adherens junction complex formation. Stimulation of the proximal tubular cell line HK-2 cells by TGF-beta 1 led to loss of cell-cell contact and disassembly of both adherens and tight junctional complexes. Adherens junction disassembly was associated with reduction of both Triton-soluble and Triton-insoluble E-cadherin, and an increase in detergent-soluble beta-catenin. Under these conditions, immunoprecipitation and Western analysis demonstrated decreased association of beta-catenin, both with E-cadherin, alpha-catenin, and the cell cytoskeleton. Confocal microscopy after immunostaining, showed decreased intensity of peripheral E-cadherin staining, and redistribution of beta-catenin expression to a perinuclear location. Tight junction disassembly was manifest by a reduction in the expression of Triton-soluble occludin and ZO-1 by Western analysis and their disassociation manifested by immunostaining and confocal microscopy. Loss of cell-cell contact and disassembly of adherens junctions were seen after addition of TGF-beta 1 to the basolateral aspect of the cells. Immunoprecipitation experiments demonstrated co-localization of E-cadherin, beta-catenin, and TGF-beta 1 RII in unstimulated cells. After TGF-beta 1 stimulation, the TGF-beta 1 RII no longer associated with either E-cadherin or beta-catenin. Dissociation of the adherens junction protein from the TGF-beta 1 receptor was associated with increased beta-catenin tyrosine phosphorylation and decreased threonine phosphorylation. Furthermore after receptor ligand binding, beta-catenin became associated with the TGF-beta 1-signaling molecules Smad3 and Smad4. (+info)
Histamine stimulates phosphorylation of adherens junction proteins and alters their link to vimentin.
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Histamine increases microvascular permeability by creating small transitory (100-400 nm) gaps between adjacent endothelial cells at sites of vascular endothelial (VE)-cadherin-based adhesion. We examined the effects of histamine on the proteins within the VE-cadherin-based adherens junction in primary human umbilical vein endothelial cells. VE-cadherin is linked not only by beta- and alpha-catenin to cortical actin but also by gamma-catenin to the intermediate filament vimentin. In mature human umbilical vein cultures, the VE-cadherin immunoprecipitate contained equivalent amounts of alpha- and beta-catenin, 130% as much beta- as gamma-catenin, and 50% as much actin as vimentin. Within 60 s, histamine decreased the fraction of VE-cadherin in the insoluble portion of the cell lysate by 35 +/- 1.5%. At the same time, histamine decreased the amount of vimentin that immunoprecipitated with VE-cadherin by 50 +/- 6%. Histamine did not affect the amount of actin or the amount of alpha-, beta-, or gamma-catenin that immunoprecipitated with VE-cadherin. Within 60 s, histamine simulated a doubling in the phosphorylation of VE-cadherin and beta- and gamma-catenin. The VE-cadherin immunoprecipitate contained kinase activity that phosphorylated VE-cadherin and gamma-catenin in vitro. (+info)
Crk adapter proteins promote an epithelial-mesenchymal-like transition and are required for HGF-mediated cell spreading and breakdown of epithelial adherens junctions.
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Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor (HGF), promotes an epithelial-mesenchymal transition and cell dispersal. However, little is known about the HGF-dependent signals that regulate these events. HGF stimulation of epithelial cell colonies leads to the enhanced recruitment of the CrkII and CrkL adapter proteins to Met-dependent signaling complexes. We provide evidence that signals involving CrkII and CrkL are required for the breakdown of adherens junctions, the spreading of epithelial colonies, and the formation of lamellipodia in response to HGF. The overexpression of a CrkI SH3 domain mutant blocks these HGF-dependent events. In addition, the overexpression of CrkII or CrkL promotes lamellipodia formation, loss of adherens junctions, cell spreading, and dispersal of colonies of breast cancer epithelial cells in the absence of HGF. Stable lines of epithelial cells overexpressing CrkII show enhanced activation of Rac1 and Rap1. The Crk-dependent breakdown of adherens junctions and cell spreading is inhibited by the expression of a dominant negative mutant of Rac1 but not Rap1. These findings provide evidence that Crk adapter proteins play a critical role in the breakdown of adherens junctions and the spreading of sheets of epithelial cells. (+info)