Abstract. p120 ctn binds to the cytoplasmic domain of cadherins but its role is poorly understood. Colo 205 cells grow as dispersed cells despite their normal expression of E-cadherin and catenins. However, in these cells we can induce typical E-cadherin–dependent aggregation by treatment with staurosporine or trypsin. These treatments concomitantly induce an electrophoretic mobility shift of p120 ctn to a faster position. To investigate whether p120 ctn plays a role in this cadherin reactivation process, we transfected Colo 205 cells with a series of p120 ctn deletion constructs. Notably, expression of NH 2-terminally deleted p120 ctn induced aggregation. Similar effects were observed when these constructs were introduced into HT-29 cells. When a mutant N-cadherin lacking the p120 ctn-binding site was

Abstract. Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and �1 integrins influence each other using two different �1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of �1A or the cytoplasmic splice variant �1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of �1 integrins caused a dramatic reorganization of the actin

PURPOSE. The goals of this study were to determine whether MAP kinase signaling pathways play a role in the formation of lens cataracts and to examine the potential signaling relationship between Src and MAP kinases in signaling the induction of lens opacities. METHODS. Embryonic day (E)10 chick lenses were cultured in Medium 199 containing 10% fetal bovine serum. The activation state of Src kinases and the MAP kinases extracellular signalregulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 in the lens epithelium was determined over a time course from 10 minutes to 10 days in culture by immunoblot analysis. Src kinase activation was suppressed by exposure to the Src family kinase-specific inhibitor PP1. To examine the role of specific MAP kinases in the development of lens opacities, lenses were grown for 10 days in the presence or absence of inhibitors of ERK (U0126), JNK (SP600125), and p38 (SB203580). Lenses were observed and photographed daily, and the degree of opacification was quantified by using imageanalysis software. RESULTS. Within a short time after placing embryonic lenses in culture conditions that induce the formation of cataracts, there occurred a great increase in the activation state of the MAP kinase ERK. Activation of ERK was both rapid and transient. No activation of the MAP kinase JNK was observed in the cataract cultures beyond that which occurred in normal lens epithelium, even though JNK activation is often linked to the cellular response to stress. In contrast, although p38 activation was barely detected in the normal embryonic lens, this stressactivated protein kinase exhibited a robust activation in cataract cultures that was sustained throughout the culture period. Studies conducted to map the cataract signaling pathways indicate that the p38 MAP kinase functions upstream of the Src kinase. To analyze the potential role of ERK, JNK, and p38 in cataract induction, lenses were cultured in the presence of specific MAP kinase inhibitors. Although the inhibitors of ERK and JNK did not interfere with the formation of cataract, p38 inhibitors blocked the development of lens opacities with an efficacy similar to that of the Src kinase inhibitor PP1. CONCLUSIONS. Activation of both Src and p38 kinases lead to the induction of cataract. (Invest Ophthalmol Vis Sci. 2004;45: 2314 -2323) DOI:10.1167/iovs.03-1210 C ataract is a multifactorial disease. Many stress factors, including oxidative stress, 1-3 osmotic stress, 4 -6 and UV light 7-10 can induce formation of cataracts, but the signaling pathways involved in the induction of cataract by stress are poorly understood. The Src family tyrosine kinases stand out as signaling intermediates common to pathways activated by many distinct stress factors. Src kinases have been shown to be involved in signaling events stimulated by reactive oxygen species, osmotic stress, UV light, and extracellular mechanical stress, in several different cell types. The MAP kinases-extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38 kinase-are key elements in signal-transduction pathways induced by extracellular stimuli. 18 -20 Like the Src kinases, the activation of MAP kinases is characteristic of the cellular response to many distinct stress inducers. Oxidants, radiation, and high glucose can activate ERK, 21-23 JNK, 21,24 -27 and p38 kinases. 21,28 -30 The MAP kinase signaling response in a cell can be specific to the particular extracellular stimulus. Distinct responses have been observed for different growth factors, cytokines, and cellular stresses. 33 Stress factors work through the different MAP kinases to regulate cell proliferation, differentiation, cell structural reorganization, and cell death. 34 -38 Activation of MAP kinases also has been shown to induce formation of cataract. Cataracts develop in transgenic mice that overexpress MEK1, an upstream activator of ERK, through a mechanism that involves abnormalities in glucose metabolism that damage the lens fiber cells. The activation of MAP kinases by extracellular stresses such as UV light, hydrogen peroxide, high osmolarity, and shear stress involves the activation of Src kinases.

PURPOSE. The goal of this study was to determine the role of Src family kinases (SFKs) in the development of lens cataract. This question was particularly significant, because these tyrosine kinases mediate the stress pathways known to lead to cataract formation. The experiments were focused on whether the inhibition of SFK activity suppresses the formation of lens opacities. METHODS. A whole-lens culture system was developed, in which cortical opacities formed within 5 days, in embryonic day (E)10 lenses grown in medium containing 10% fetal bovine serum. SFK activity was blocked in the cultured lenses by growth in the presence of the SFK-specific inhibitor PP1. Control cultures were grown in medium without inhibitor or in the presence of PP3, the inactive analogue of PP1. Lenses were cultured for 10 days, observed, and photographed daily. Opacification was quantified with image-analysis software. Tissue architecture was determined after hematoxylin and eosin staining and cellular organization by fluorescent localization of filamentous actin with fluorescein-conjugated phalloidin. RESULTS. Almost all lenses in the control cultures developed cortical opacities covering approximately 50% of the lens area by day 10. Similar to control cultures, PP1-treated lenses showed mild posterior opacities during the first 5 days in culture, but then became strikingly transparent. Only 7% of the PP1-treated lenses showed development of cortical cataract, and the average area of opacity was just 0.5% by culture day 10. In all cultured lenses, even in the presence of the PP1 inhibitor, the bow region of the lens extended to the posterior pole, and distribution of nuclei from the posterior pole toward the anterior aspects of the lens suggested that newly added fiber cells were misdirected. However, neither this feature, nor the presence of vacuoles appeared to correlate with the development of opacity in the cultured lenses. Instead, the lens opacities appeared to result from gross abnormalities in the shape and organization of cells in the equatorial and cortical fiber zones, as observed by F-actin staining. Culturing the lenses in the presence of the SFK inhibitor prevented these lens cell aberrations as well as the development of lens opacity. CONCLUSIONS. The formation of cataract can involve activation of SFK-mediated pathway(s) leading to disorganization of developing lens fiber cells, and inhibiting these tyrosine kinases blocks cataract progression. (Invest Ophthalmol Vis Sci. 2002; 43:2293-2300 C ataracts are a leading cause of blindness throughout the world. Although many factors have been shown to trigger formation of cataracts, the signaling pathways that mediate the development of lens opacity are not understood. Of particular interest in the current study were cataracts that form as a result of the induction of stress pathways, because stress factors are often the cause of age-related cataracts. Stress-inducers of cataract formation include oxidative stress, 1-3 osmotic stress, 4 -6 and UV stress. 7-11 When stress factors that induce formation of cataract, such as H 2 O 2 or glucose, are blocked, cataract is prevented. 2,12,13 The tyrosine kinase c-Src mediates downstream signaling from osmotic, oxidative and UV stress-induced pathways 25 This is correlated with the inhibition of cell-cell junction formation. It is likely that the activation of SFKs in the lens in response to stress alters the signaling pathways that regulate normal growth controls in this tissue. Organ-lens culture has provided an important in vitro model system for study of both cataractogenesis and the prevention of cataract. 3,26 -31 However, the time that lenses remain transparent in culture is dependent on their age. Lenses from chick embryos remain transparent for just 3 hours, 26 from weaned rats for 5 days, from adult rats for 7 days, 32 and from adult monkeys for 21 days. MATERIALS AND METHODS Whole Embryonic Chick Lens Culture and Exposure of Lenses to SFK Inhibitors White Leghorn embryonated chicken eggs were procured from Truslow Farms (Chestertown, MD) and incubated in a forced-draft incubator at 37°C. Tissue collection conformed to the ARVO Statement of the Use of Animals in Ophthalmic and Vision Research. Lenses were dissected from day 10 (E10) embryos, and vitreous was removed under a dissecting microscope. The embryonic lenses were cultured in medium 199 (Gibco BRL, Gaithersburg, MD), containing 10% fetal bovine serum (Gibco BRL), 0.1 g/mL L-glutamine, and penicillin-streptomycin, in an atmosphere of 5% CO 2 and 95% air. Lenses were observed

Background: Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion. Methodology/Principal Findings: To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin complex of the adherens junctions. Concomitantly these transfected cells exhibited marked migratory capacity and the formation of filopodial protrusions. These latter events are consistent with Src activation and, indeed, Src-specific inhibition reversed these phenotypes. Moreover Dsg3 knockdown, which also reversed the decreased level of E-cadherin, partially blocked Src phosphorylation. Conclusions/Significance: Our data are consistent with the possibility that Dsg3, as an up-stream regulator of Src activity,

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Background: Desmoglein 3 (Dsg3), a desmosomal adhesion protein, is expressed in basal and immediate suprabasal layers of skin and across the entire stratified squamous epithelium of oral mucosa. However, increasing evidence suggests that the role of Dsg3 may involve more than just cell-cell adhesion. Methodology/Principal Findings: To determine possible additional roles of Dsg3 during epithelial cell adhesion we used overexpression of full-length human Dsg3 cDNA, and RNAi-mediated knockdown of this molecule in various epithelial cell types. Overexpression of Dsg3 resulted in a reduced level of E-cadherin but a colocalisation with the E-cadherin-catenin

The extracellular matrix (ECM), including the basement membrane, serves as an interface between epithelial cells and the surrounding mesenchyme. Therefore, the inductive interactions that occur between groups of mesenchymal and

for its role in intercellular adhesion structures called adherens

Src was first identified as the transforming protein encoded by Rous sarcoma virus; to denote its viral origin it was termed v-Src. Subsequent studies revealed that v-Src is a mutant counterpart of a normal protein, c-Src, which is involved in