CHARACTERIZATION OF NICOTINE ACETYLCHOLINE RECEPTOR SUBUNITS IN THE COCKROACH Periplaneta americana MUSHROOM BODIES REVEALS A STRONG EXPRESSION OF β1 SUBUNIT: INVOLVEMENT IN NICOTINE‐INDUCED CURRENTS

Nicotinic acetylcholine receptors are ligand‐gated ion channels expressed in many insect structures, such as mushroom bodies, in which they play a central role. We have recently demonstrated using electrophysiological recordings that different native nicotinic receptors are expressed in cockroach mushroom bodies Kenyon cells. In the present study, we demonstrated that eight genes coding for cockroach nicotinic acetylcholine receptor subunits are expressed in the mushroom bodies. Quantitative real‐time polymerase chain reaction (PCR) experiments demonstrated that β1 subunit was the most expressed in the mushroom bodies. Moreover, antisense oligonucleotides performed against β1 subunit revealed that inhibition of β1 expression strongly decreases nicotine‐induced currents amplitudes. Moreover, co‐application with 0.5 μM α‐bungarotoxin completely inhibited nicotine currents whereas 10 μM d‐tubocurarine had a partial effect demonstrating that β1‐containing neuronal nicotinic acetylcholine receptor subtypes could be sensitive to the nicotinic acetylcholine receptor antagonist α‐bungarotoxin.     

RIFLE: a novel ring zinc finger-leucine-rich repeat containing protein, regulates select cell adhesion molecules in PC12 cells

Cell adhesion molecules play a critical role in cell contacts, whether cell-cell or cell-matrix, and are regulated by multiple signaling pathways. In this report, we identify a novel ring zinc finger-leucine-rich repeat containing protein (RIFLE) and show that RIFLE, expressed in PC12 cells, enhances the Serine (Ser)21/9 phosphorylation of glycogen synthase kinase-3alpha/beta (GSK-3alpha/beta) resulting in the inhibition of GSK-3 kinase activity and increase of beta-catenin levels. RIFLE expression also is associated with elevated E-cadherin protein levels but not N-cadherin. The regulation of these cell adhesion-associated molecules by RIFLE is accompanied by a significant increase in cell-cell and cell-matrix adhesion. Moreover, increase in cell-cell adhesion but not cell-matrix adhesion by RIFLE can be mimicked by selective inhibition of GSK-3. Our results suggest that RIFLE represents a novel signaling protein that mediates components of the Wnt/wingless signaling pathway and cell adhesion in PC12 cells.     

Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization

Epithelialization, a major component of wound healing, depends on keratinocyte adhesion and migration. Initiation of migration relies upon the ability of keratinocytes to free themselves from neighboring cells and basement membrane. The local cytotransmitter acetylcholine (ACh) controls keratinocyte adhesion and locomotion through different classes of ACh receptors (AChR). In this study, we explored signaling pathways downstream of the alpha9 AChR subtype that had been shown to control cell shape and cytoplasm mobility. Inactivation of alpha9 signaling by pharmacologic antagonism and RNA interference in keratinocyte cultures and null mutation in knockout mice delayed wound re-epithelialization in vitro and in vivo, respectively, and diminished the extent of colony scattering and cell outgrowth from the megacolony. Although keratinocytes at the leading edge elongated, produced filopodia and moved out, most of them remained anchored to the substrate by long cytoplasmic processes that stretched during their migration instead of retracting the uropod. Since the velocity of keratinocyte migration was not altered, we investigated the role of alpha9 in assembly/disassembly of the cell-cell and cell-matrix adhesion complexes. Stimulation of alpha9 upregulated in a time-dependent fashion phosphorylation of the adhesion molecules comprising focal adhesions (FAK, paxillin) and intercellular junctions (beta-catenin, desmoglein 3) as well as cytokeratins. Stimulation of alpha9 was associated with activation of phospholipase C, Src, EGF receptor kinase, protein kinase C, Rac and Rho, whereas inhibition of this receptor interfered with phosphorylation of adhesion and cytoskeletal proteins, and also altered cell-cell cohesion. We conclude that signaling through alpha9 AChR is critical for completion of the very early stages of epithelialization. By activating alpha9 AChR, ACh can control the dynamics and strength of cell-cell cohesion, disabling of a trailing uropod and disassembly and reassembly of focal adhesions, thus facilitating crawling locomotion.     

Identification of Acetylcholinesterase Receptors in Rotifera

We have identified acetylcholinesterase (AChE) receptors in six freshwater rotifers. Using β-bungarotoxin labelled with fluoresceinisothiocyanate (FITC), muscarinic and nicotinic receptors were found in Brachionus quadridentatus (females and males), Lecane luna, Lecane quadridentata, Plationus patulus, and Rotaria neptunia. Using α-bungarotoxin-FITC, nicotinic receptors were identified in B. quadridentatus, Lecane bulla, L. luna, L. quadridentata, P. patulus and R. neptunia. Concentrations as low as 1.5 nM of β-bungarotoxin, and 5 nM of α-bungarotoxin identified receptors in the digestive tract. Higher concentrations of both toxins identified additional receptors associated with the lorica. A preliminary analysis of fluorescence intensity in L. quadridentata showed that response to α-bungarotoxin increases with age from newborn to 48-h old, but not in older individuals, thus suggesting an increase in binding sites, and possibly in number of nicotinic receptors, during the first 48-h of life. Our study extends the number of rotifer species in which AChE receptors have been reported.     

Biochemical interactions among intercellular adhesion molecules expressed by airway epithelial cells

Intercellular adhesion between adjacent airway epithelial cells plays a critical role in maintaining the barrier function of the respiratory mucosa. In the current study, we examined the expression and interaction of cell surface adhesion molecules (E-cadherin, ICAM-1, and MUC1) and their intracellular binding partners (alpha-catenin, beta-catenin, gamma-catenin, and ezrin) in 16HBE14o-, HBE1, 1HAEo-, BEAS-2B, A549, and NCI-H292 human airway epithelial cells. Expression of E-cadherin and MUC1, both in whole cell lysates and biotinylated surface proteins, was observed in 16HBE14o-, HBE1, A549, and NCI-H292 cells, while ICAM-1 was detected only in NCI-H292. In contrast, alpha-, beta-, and gamma-catenin and ezrin were expressed in all of the cells. E-cadherin formed coimmunoprecipitation complexes with beta- and gamma-catenin, whereas MUC1 only associated with beta-catenin. ICAM-1, but not MUC1, coimmunoprecipitated with ezrin in NCI-H292 cells. We conclude that airway epithelial cell-cell adhesion involves a complex network of protein-protein interactions mediated by a diverse array of membrane-bound and cytosolic protein partners.     

alpha3beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell-cell adhesion

The beta1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for alpha3beta1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTPmu and by organizing a multimolecular complex containing PKCbetaII, RACK1, PTPmu, beta-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.     

Muscle-specific expression of insulin-like growth factor I counters muscle decline in mdx mice

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell-cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of beta- and gamma-catenin, which is associated with the establishment of keratinocyte cell-cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell-cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of beta- and gamma-catenin and p120ctn. The positive effects of activated Rho on cell-cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell-cell adhesion and induces tyrosine phosphorylation of beta- and gamma-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell-cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.     

Distinct ligand binding sites in integrin alpha3beta1 regulate matrix adhesion and cell-cell contact

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.     

Requirement of protein kinase Calpha, extracellular matrix remodeling, and cell-matrix interaction for transforming growth factorbeta-regulated expression of E-cadherin and catenins

A hallmark of transforming growth factorbeta (TGFbeta) action is the induction of the synthesis and secretion of extracellular-matrix adhesion molecules and induction of the cell-surface expression of integrin receptors for these molecules (termed extracellular-matrix remodeling). The signal pathways leading to extracellular-matrix remodeling and the significance of extracellular-matrix remodeling in TGFbeta function is not well-understood. In the epithelium-derived human colon cancer cell line Moser, TGFbeta induces extracellular-matrix remodeling in a protein kinase Calpha-dependent manner. In this study we showed that TGFbeta was a potent inducer of the homotypic cell-cell adhesion molecule E-cadherin and its undercoat-associated proteins, the catenins and dramatically increased the amount of E-cadherin/gamma-catenin complex formation. We found that the induction of E-cadherin and alpha- and beta-catenin by TGFbeta was also dependent on protein kinase Calpha, whereas the induction of gamma-catenin was independent of protein kinase Calpha but dependent on other protein kinase C isoforms. We also found that protein kinase Calpha-dependent induction of extracellular-matrix remodeling and subsequent cell-matrix interaction requiring both fibronectin and laminin were a prerequisite for the induction of E-cadherin (and alpha- and beta-catenin but not gamma-catenin) by TGFbeta. We therefore concluded that two signal pathways exist in TGFbeta-regulated expression of E-cadherin and the catenins. We also concluded that a functional significance of TGFbeta-induced extracellular matrix remodeling is the activation of signal transduction mechanisms through increased interaction between extracellular matrix fibronectin and laminin and their cell-surface integrin receptors, which lead to the induction of E-cadherin (and alpha- and beta-catenin).     

Cholinergic receptor alterations in the cerebral cortex of spinal cord injured rat

Many areas of the cerebral cortex process sensory information or coordinate motor output necessary for control of movement. Disturbances in cortical cholinergic system can affect locomotor coordination. Spinal cord injury causes severe motor impairment and disturbances in cholinergic signalling can aggravate the situation. Considering the impact of cortical cholinergic firing in locomotion, we focussed the study in understanding the cholinergic alterations in cerebral cortex during spinal cord injury. The gene expression of key enzymes in cholinergic pathway - acetylcholine esterase and choline acetyl transferase showed significant upregulation in the cerebral cortex of spinal cord injured group compared to control with the fold increase in expression of acetylcholine esterase prominently higher than cholineacetyl transferase. The decreased muscarinic receptor density and reduced immunostaining of muscarinic receptor subtypes along with down regulated gene expression of muscarinic M1 and M3 receptor subtypes accounts for dysfunction of metabotropic acetylcholine receptors in spinal cord injury group. Ionotropic acetylcholine receptor alterations were evident from the decreased gene expression of alpha 7 nicotinic receptors and reduced immunostaining of alpha 7 nicotinic receptors in confocal imaging. Our data pin points the disturbances in cortical cholinergic function due to spinal cord injury; which can augment the locomotor deficits. This can be taken into account while devising a proper therapeutic approach to manage spinal cord injury.     

Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with beta-catenin

Carbonic anhydrase IX (CA IX) is a cancer-associated transmembrane isoform of zinc metalloenzymes that catalyse interconversion between carbon dioxide and bicarbonate. CA IX is strongly induced by tumor hypoxia and has been proposed to participate in acidification of tumor microenvironment and in cell adhesion. To elucidate the cell adhesion-related role of CA IX, we investigated its subcellular localization and relationship to E-cadherin, a key adhesion molecule whose loss or destabilization is linked to tumor invasion. For this purpose, we generated MDCK cells with constitutive expression of human CA IX protein. During the monolayer formation, CA IX was localized to cell-cell contacts and its distribution in lateral membranes overlapped with E-cadherin. Calcium switch-triggered disruption and reconstitution of cell contacts resulted in relocalization of both CA IX and E-cadherin to cytoplasm and back to plasma membrane. A similar phenomenon was observed in hypoxia-treated and reoxygenated cells. Moreover, CA IX-expressing MDCK cells exhibited reduced cell adhesion capacity and lower levels of Triton-insoluble E-cadherin. Finally, CA IX was found to coprecipitate with beta-catenin. We conclude that CA IX has a capacity to modulate E-cadherin-mediated cell adhesion via interaction with beta-catenin, which could be of potential significance in hypoxia-induced tumor progression.     

Antisense inhibition of neuronal nicotinic receptors in the tobacco-feeding insect,Manduca sexta

Acetylcholine is the predominant excitatory transmitter in the insect central nervous system with many of its effects mediated by nicotinic acetylcholine receptors. These receptors are present at very high density and are structurally heterogeneous, although little is known about functional distinctions between them. An interesting system for examining these receptors is the larval stage of Manduca sexta, a nicotine-resistant tobacco-feeding insect. The nicotinic responses of cultured neurons were found to be blocked by mecamylamine and curare but highly resistant to alpha-bungarotoxin. The responses were also unaffected by the reducing agent dithiothreitol and the alkylating agent bromoacetylcholine suggesting that the alpha-subunit dicysteine agonist binding site is protected. To begin determining the functional roles of different subunits in these receptors, cultured neurons were treated with oligonucleotides based on the gene sequence of the alpha subunit, MARA1. Antisense DNA caused a significant downward shift in the amplitude distribution of nicotinic responses compared to sense or reverse antisense treatments. These treatments did not affect currents mediated by the application of GABA. The reduction in the nicotinic depolarization and inward currents did not affect the rate of current onset or recovery, suggesting that antisense MARA1 causes a partial block of all nicotinic responses in these neurons. These results demonstrate that receptor gene expression in insect neurons can be manipulated in a sequence-specific manner by antisense treatment and they provide evidence that MARA1 is important for normal nicotinic responses in Manduca.     

The recruitment of phosphatidylinositol 3-kinase to the E-cadherin-catenin complex at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and human keratinocyte differentiation

Calcium induces epidermal keratinocyte differentiation, but the mechanism is not completely understood. We have previously demonstrated that calcium-induced human keratinocyte differentiation requires an intracellular calcium rise caused by phosphatidylinositol 3-kinase (PI3K)-dependent activation of phospholipase C-gamma1. In this study we sought to identify the upstream signaling pathway necessary for calcium activation of PI3K and its subsequent activation of phospholipase C-gamma1. We found that calcium induces the recruitment of PI3K to the E-cadherin-catenin complex at the plasma membrane of human keratinocytes. Knocking-down E-cadherin, beta-catenin, or p120-catenin expression blocked calcium activation of PI3K and phospholipase C-gamma1 and calcium-induced keratinocyte differentiation. However, knocking-down gamma-catenin expression had no effect. Calcium-induced PI3K recruitment to E-cadherin stabilized by p120-catenin at the plasma membrane requires beta-catenin but not gamma-catenin. These data indicate that the recruitment of PI3K to the E-cadherin/beta-catenin/p120-catenin complex via beta-catenin at the plasma membrane is required for calcium-induced phospholipase C-gamma1 activation and, ultimately, keratinocyte differentiation.     

Regulation of E-cadherin-mediated adhesion by muscarinic acetylcholine receptors in small cell lung carcinoma

We present the first evidence that adhesion mediated by a member of the cadherin gene family can be regulated by a G protein-coupled receptor. We show that activating the M3 muscarinic acetylcholine receptor (mAChR) rapidly induces E-cadherin-mediated adhesion in a small cell lung carcinoma (SCLC) cell line. This response is inhibited by E- cadherin antibodies, and does not occur in another SCLC cell line which expresses functional mAChR but reduced levels of E-cadherin. Protein kinase C may be involved, since phorbol 12-myristate 13-acetate also induces E-cadherin-mediated aggregation. Immunofluorescence analyses indicate that mAChR activation does not grossly alter E-cadherin surface expression or localization at areas of cell-cell contact, suggesting mAChR activation may increase E-cadherin binding activity. Our findings suggest that G protein-coupled receptors may regulate processes involving cadherin-mediated adhesion, such as embryonic development, neurogenesis, and cancer metastasis.     

Platelet-endothelial cell adhesion molecule-1 (CD31), a scaffolding molecule for selected catenin family members whose binding is mediated by different tyrosine and serine/threonine phosphorylation

Platelet-endothelial cell adhesion molecule (PECAM)-1 is a 130-kDa glycoprotein commonly used as an endothelium-specific marker. Evidence to date suggests that PECAM-1 is more than just an endothelial cell marker but is intimately involved in signal transduction pathways. This is mediated in part by phosphorylation of specific tyrosine residues within the ITAM domain of PECAM-1 and by recruitment of adapter and signaling molecules. Recently we demonstrated that PECAM-1/beta-catenin association functions to regulate beta-catenin localization and, moreover, to modulate beta-catenin tyrosine phosphorylation levels. Here we show that: 1) not only beta-catenin, but also gamma-catenin is associated with PECAM-1 in vitro and in vivo; 2) PKC enzyme directly phosphorylates purified PECAM-1; 3) PKC-derived PECAM-1 serine/threonine phosphorylation inversely correlates with gamma-catenin association; 4) PECAM-1 recruits gamma-catenin to cell-cell junctions in transfected SW480 cells; and 5) gamma-catenin may recruit PECAM-1 into an insoluble cytoskeletal fraction. These data further support the concept that PECAM-1 functions as a binder and modulator of catenins and provides a molecular mechanism for previously reported PECAM-1/cytoskeleton interactions.     

The cadherin-catenin complex is expressed alternately with the adenomatous polyposis coli protein during rat incisor amelogenesis.

E-cadherin, a calcium-dependent cell-cell adhesion molecule, is expressed in highly specific spatiotemporal patterns throughout metazoan development, notably at sites of embryonic induction. E-cadherin also plays a critical role in regulating cell motility/adhesion, cell proliferation, and apoptosis. We have used the continuously erupting rat incisor as a system for examining the expression of E-cadherin and the associated catenins [alpha-, beta-, gamma-catenin (plakoglobin) and p120(ctn)] during amelogenesis. Using immunhistochemical techniques, we observed expression of alpha-catenin and gamma-catenin in ameloblasts throughout amelogenesis. In contrast, expression of E-cadherin, beta-catenin, and p120(ctn) was strong in presecretory, transitional, and reduced stage ameloblasts (Stages I, III, and V) but was dramatically lower in secretory and maturation stage ameloblasts (Stages II and IV). This expression alternates with the expression pattern we previously reported for the adenomatous polyposis coli protein (APC), a tumor suppressor that competes with E-cadherin for binding to beta-catenin. We suggest that alternate expression of APC and the cadherin-catenin complex is critical for the alterations in cell-cell adhesion and other differentiated cellular characteristics, such as cytoskeletal alterations, that are required for the formation of enamel by ameloblasts.     

N-glycosylation affects the adhesive function of E-Cadherin through modifying the composition of adherens junctions (AJs) in human breast carcinoma cell line MDA-MB-435

E-cadherin mediates calcium-dependent cell-cell adhesion between epithelial cells. The ectodomain of human E-cadherin contains four potential N-glycosylation sites at Asn residues 554, 566, 618, and 633. In this study, the role of N-glycosylation in E-cadherin-mediated cell-cell adhesion was investigated by site-directed mutagenesis. In MDA-MB-435 cells, all four potential N-glycosylation sites of human E-cadherin were N-glycosylated. Removal of N-glycan at Asn-633 dramatically affected E-cadherin stability. In contrast, mutant E-cadherin lacking the other three N-glycans showed similar protein stability in comparison with wild-type E-cadherin. Moreover, N-glycans at Asn-554 and Asn-566 were found to affect E-cadherin-mediated calcium-dependent cell-cell adhesion, and removal of either of the two N-glycans caused a significant decrease in calcium-dependent cell-cell adhesion accompanied with elevated cell migration. Analysis of the composition of adherens junctions (AJs) revealed that removal of N-glycans on E-cadherin resulted in elevated tyrosine phosphorylation level of beta-catenin and reduced beta- and alpha-catenins at AJs. These findings demonstrate that N-glycosylation may affect the adhesive function of E-cadherin through modifying the composition of AJs.