|Molecular Vision 2007;
Received 15 May 2007 | Accepted 23 July 2007 | Published 24 July 2007
Mechanism of Src kinase induction of cortical cataract following exposure to stress: destabilization of cell-cell junctions
Bockstaele,2 A. Sue
1Department of Pathology, Anatomy and Cell Biology, 2Department of Neurosurgery and Farber Institute for Neuroscience, Thomas Jefferson University, Philadelphia, PA
Correspondence to: Sue Menko, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 571 Jefferson Alumni Hall, 1020 Locust Street, Philadelphia, PA, 19107; Phone: (215) 503-2166. FAX: (215) 923-3808; email: email@example.com
Purpose: Activation of stress pathways is a primary cause of age-related cataracts. Our laboratory previously developed a lens cataract model in which activation of the p38 kinase/Src family kinase (SFK) stress-signaling pathway is responsible for the induction of cortical opacities. Here, we use this model to investigate further the mechanism of stress-induced cataract.
Methods: Cortical cataracts were induced by mechanical stress and prevented by exposure to the SFK inhibitor PP1. Organization of the actin cytoskeleton, cadherin junctions and membrane structure was determined by fluorescence imaging. Apoptosis was examined by both terminal dUTP nick-end labeling (TUNEL) and DEVDase assays. N-cadherin cleavage was examined by western blot analysis.
Results: In this cortical cataract model, activation of SFKs in the lens epithelium caused increased cleavage of N-cadherin, the loss of cadherin cell-cell junctions, and the disorganization of the actin cytoskeleton. As the function of cadherin junctions and the cytoskeleton are required for assembling a polarized epithelium and protecting it against apoptotic cell death, the SFK-dependent disassembly of these structures was likely responsible for the observed loss of integrity and apoptosis of the lens epithelium. Apoptotic death of lens epithelial cells preceded the appearance of cortical opacities, suggesting that the lens epithelium provided an important line of defense for the lens fiber cells. Opacification was related to defects in the membrane structure of cortical fiber cells. Linearity of fiber cell membranes was lost. Extensive undulations and interdigitations of these membranes occurred as well as large separations between fiber cells as is common to many types of cataract.
Conclusions: Src kinase activation-induced loss of cadherin junctions and apoptosis of the lens epithelium leads to altered fiber cell organization and lens opacification.
The lens is a complex tissue, the function of which is to focus an image on the retina. To do this, the lens must be transparent. There are many factors that contribute to the establishment of lens transparency during development. One is the coordinated loss of nuclei and organelles [1-3]. Another is the construction of a unique cytoarchitecture in which the differentiating lens fiber cells are arranged into a highly ordered hexagonal packed structure [4-6]. Among the many factors important to the maintenance of normal fiber cell structure and function is the protective role provided by the cells of the anterior lens epithelium [7,8]. These epithelial cells are coupled to the lens fiber cells [9,10] and provide the mature lens with its first line of defense against environmental insults [7,11,12].
There are many examples in which dysfunction of the lens epithelium leads to the formation of cataract. For instance, in lenses of aquaporin-1 knockout mice  or Crygs mutant mice , and in lenses exposed to ultraviolet B (UVB) [11,12], damage to the lens epithelium is responsible for cataracts that form in the fiber cell region. Apoptosis of lens epithelial cells is often an early feature of cataractogenesis, preceding the development of opacities in the lens fiber region [15-18]. Increased apoptosis of lens epithelial cells is observed in cataracts induced by ultraviolet (UV), H2O2, and galactose stress [15,16,19].
Cortical cataracts are a common form of age-related cataract, typically induced by stressors such as reactive oxygen, UV, and diabetes. In order to investigate the mechanism of stress-induced cataract, our laboratory developed a cataract culture model in which the development of cortical opacities is dependent on the activation of a classical stress-signaling pathway. Principal components of this signaling pathway are the stress-activated mitogen-activated protein (MAP) kinase p38 and its downstream signaling effector, Src [20,21]. The cortical opacities in this ex vivo cataract culture model are prevented with pharmacological inhibitors to either p38 or Src kinases, validating the connection between the activation of this stress-signaling pathway and the development of the lens opacity [20,21]. The opacification itself is due to the disorganization of fiber cells in the cortical zone .
For epithelial cells such as in the lens, form often dictates function. A major factor in defining epithelial cell cytoarchitecture is the assembly and stabilization of cadherin cell-cell junctions. There is strong evidence that cadherin junctions are important to both the formation and the stability of the lens epithelium. In the presumptive lens, the conditional loss of β-catenin, a component of the cadherin adherens junction, prevents lens epithelial cell morphogenesis . Stabilization of the cadherin junctions in epithelial cells of the mature lens is an important factor in the prevention of their epithelial to mesenchymal transition (EMT) [23,24]. Interestingly, there is direct evidence that β-catenin is lost from cell borders in some human cataracts and moves to a cytoplasmic or nuclear localization . Such altered localization of β-catenin can result from the disassembly of cadherin junctions. Important to this study, the inappropriate activation of Src kinases in lens epithelial cells targets cadherin junctions, causing their disassembly [26,27]. Blocking the loss of cadherin junctions in cells in which stress pathways are activated could be a potential approach for the prevention of some cataracts.
Here, we use an ex vivo cataract model in which the mechanical stress of culture preparation induces cortical opacities by activating a classical Src/p38 kinase stress-signaling pathway to investigate the mechanism by which Src activation causes cataracts to form. Our studies show that the inappropriate activation of Src Family Kinases (SFKs) in the lens epithelium caused N-cadherin cleavage, the disassembly of cadherin junctions, and the disorganization of the actin cytoskeleton. These Src-dependent defects resulted in aberrant morphology and apoptosis of the lens epithelium prior to the appearance of cortical opacities. Morphological analysis of fiber cells following the formation of cortical cataracts show dysmorphology of cells in the cortical fiber zone accompanied by destabilization of membrane structures.
Ex vivo cortical cataract cultures
Lenses were removed from chick embryos at embryonic day 10 and grown in Medium 199 (Gibco BRL, Gaithersburg, MD) containing 10% fetal bovine serum (Gibco BRL) as reported previously . These lenses exhibited cortical opacities by day 5 in culture as a result of the mechanical stress applied at the time of culture preparation. Unless indicated otherwise, lenses were observed at culture day 10. For studies involving Src kinase inhibition, the Src kinase specific inhibitor PP1 (Biomol, Plymouth Meeting, PA) was added at the time of plating. Culture media and the inhibitor were renewed every second day throughout the culture period. Our previous studies document that PP1 is an effective, specific inhibitor of Src kinase activity in the chick embryo lens and that blocking activation of Src kinases prevented opacification in these cultured lenses.
Histology and immunofluorescence
Lenses were fixed in 3.7% paraformaldehyde and processed for cryostat sectioning as previously described ; sections of 6-8 μm were prepared. To evaluate tissue cytoarchitecture, sections were stained with hemotoxylin and eosin (H&E). For immunofluorescence staining, lens sections were permeabilized with a 0.25% Triton X-100 buffer, blocked with a buffer containing 1% BSA and 5% FBS, and incubated with a primary antibody to β-catenin (BD Transduction Laboratories, San Diego, CA) or aquaporin-0 (MP28, prepared as described previously ) followed by a fluorescence-conjugated secondary antibody. To visualize actin filament distribution, lens sections were stained with Alexa488-conjugated phalloidin and the nuclei were visualized by DAPI staining (Molecular Probes, Eugene, OR). Fluorescence stained samples were examined with either a Nikon Eclipse 80i or Nikon Optiphot microscope (Optical Apparatus, Ardmore, PA) and images were acquired using Metamorph software (Molecular Devices Corporation, Downingtown, PA).
Samples were processed for electron microscopic analysis by incubation in 2% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M phosphate buffer for one h, further washing in 0.1 M phosphate buffer, and by dehydration in an ascending series of ethanol followed by propylene oxide and flat embedded in Epon 812 (Electron Microscopy Sciences). Thin sections of approximately 50-100 nm in thickness were cut with a diamond knife (Diatome-US, Fort Washington, PA) using a Leica Ultracut (Leica Microsystems, Wetzlar, Germany). Sections were collected on copper mesh grids, examined with an electron microscope (Morgagni, Fei Company, Hillsboro,OR), and the digital images were captured using the AMT advantage HR/HR-B CCD camera system (Advance Microscopy Techniques Corp., Danvers, MA). Figures were assembled and adjusted for brightness and contrast in Adobe Photoshop.
Terminal dUTP nick-end labeling (TUNEL) assay
Fragmented DNA of apoptotic cells was detected in lens sections by labeling with the In Situ Cell Death Detection Kit, TMR Red from Roche Molecular Biochemicals (Indianapolis, IN) and nuclei were labeled by counterstaining with DAPI (Molecular Probes).
DEVDase activity assay
CaspACE Assay System (colorimetric from Promega Labs, Madison, WI) was used to measure DEVDase activity. For these studies, lens epithelia were isolated by microdissection  and were extracted in cell lysis buffer provided by the manufacturer. The DEVDase activity assay was performed according to the manufacturer's instructions on equal amounts of protein from each sample. Protein concentrations were determined using a modified Bradford assay (BioRad, Hercules, CA).
The lens epithelium was removed by microdissection and extracted in OG buffer (44.4 mM n-Octyl β-D glucopyranoside, 1% Triton X-100, 100 mM NaCl, 1 mM MgCl2, 5 mM EDTA, 10 mM imidazole) containing 1 mM sodium vanadate, 0.2 mM H2O2, and a protease inhibitor cocktail. Protein concentrations were determined using the BCA assay (Pierce, Rockford, IL). Equal amounts of protein (15 μg) were separated on Tris-glycine gels obtained from Novex (San Diego, CA), electrophoretically transferred to Immobilon-P membrane (Millipore Corp., Bedford, MA), and immunoblotted as described previously  using antibodies to the N-cadherin cytoplasmic domain (BD Transduction Laboratories), to activated Src (phospho-Src418, Biosource, Camarillo, CA), and to c-Src (Santa Cruz Biotechnology, Santa Cruz, CA). For detection, ECL reagent from Amersham Life Sciences (Arlington Heights, IL) was used. Immunoblots were scanned and densitometry analysis was performed using Kodak 1D software (Eastman Kodak Company, Rochester, NY). All gels were run under reducing conditions.
Disorganization of the lens epithelium in lenses with cortical cataract
The task of maintaining and protecting fiber cells in the mature lens falls partly to the epithelial cells that line the lens anterior surface. These epithelial cells form a simple monolayer whose basal surfaces are attached to the lens basement membrane capsule and whose apical surfaces are coupled to the underlying lens fiber cells [9,10]. We examined the status of the epithelium in our ex vivo cataract model in which cortical cataracts arise as a result of mechanical stress activation of the p38/SFK stress-signaling pathway . Both H&E (Figure 1A) and DAPI (Figure 1C) staining of these lenses at day 10 in culture revealed significant loss of integrity of the anterior epithelium. DAPI staining of nuclei provided evidence that these cells were consistently disorganized (Figure 1C) and often misshapen and multilayered (Figure 2A). Although in all cases the lens epithelium was disorganized, the severity of the epithelial cell defects varied even within a single cataractous lens. This dysmorphology of epithelial cells observed in the anterior region of cataractous lenses was also characteristic of cells in the equatorial zone (unpublished data). H&E and DAPI staining revealed that in addition to these defects in the cytoarchitecture of the lens epithelium, many of the lens epithelial cells had pycnotic or fragmented nuclei (Figure 1C).
The polarity of normal epithelia can be defined by the organization of the actin cytoskeleton. In the lens epithelial cells, actin filaments are concentrated at the cells' apical and basal surfaces . Here, we examined the organization of the actin cytoskeleton in lenses in which cortical cataracts were induced by staining with fluorescent-tagged phalloidin. This approach revealed both disorganization and lack of polarity of the actin cytoskeleton throughout the epithelium and confirmed our findings that there was significant disorder of the cells in this region of the lens (Figure 1E). Taken together. these results demonstrated that there was tremendous dysmorphology of the lens epithelium in lenses that develop opacities in the cortical fiber zone.
Maintenance of a normal lens epithelium achieved by suppressing activation of Src kinases
Previous studies from our lab have shown that blocking activation of SFKs with inhibitors such as PP1 effectively blocks induction of cortical cataracts . Here, we show the efficacy of the Src inhibitor PP1 over the first day in culture (Figure 1G); Src inhibition is maintained throughout the culture period (unpublished data and ). A normal epithelium was maintained when lenses were prepared under cataract-inducing conditions but grown in the presence of the SFK inhibitor PP1 (Figure 1B,D). None of the defects observed in the epithelia of lenses that had formed cortical cataracts occurred when activation of the Src kinases was blocked. The epithelium of PP1-treated lenses retained a normal cytoarchitecture (Figure 1B), multilayering was prevented (Figure 1B,D) and the actin cytoskeleton remained highly organized at both the apical and basal aspects of the lens epithelial cells (Figure 1F). Pycnotic nuclei were rarely detected in the epithelium of PP1 treated lenses (Figure 1D). These studies suggest that the ability to prevent cortical cataracts from forming under conditions of stress may be connected to the ability to protect the lens epithelium.
Src kinase-dependent disassembly of cadherin junctions in lens epithelial cells
Cadherin junctions define and maintain epithelial cell structure and function. In lens epithelial cells in culture, expression of a constitutively active v-Src kinase causes loss of cadherin junctions [26,27] and the acquisition of a mesenchymal phenotype . Here, we examined whether the defects in the lens epithelium in the cortical cataract model could result from Src kinase-dependent disassembly of cadherin junctions. For this study the lenses were immunostained for β-catenin, an integral component of classical cadherin cell-cell junctions. β-catenin was absent from most lateral cell-cell interfaces (Figure 2A). While some cells retained low level staining for β-catenin, the majority of this staining was aberrantly located to these cells' basal-apical interfaces (Figure 2A). In this example, the epithelium was highly disorganized and multilayered, and the cells in the anterior-most regions of this dysmorphogenic epithelium contained pycnotic nuclei and lacked staining for β-catenin. No β-catenin staining was detected in the nucleus, as would be expected if the Wnt pathway was activated. Importantly, loss of cadherin junctions from lateral cell borders of lens epithelial cells was prevented when the lenses were grown in the presence of the SFK inhibitor PP1 (Figure 2B). When Src kinase activity was suppressed, β-catenin localization was maintained in adherens junctions at the lateral cell-cell interfaces and the cells maintained their polarized morphology. These results showed a direct correlation between Src kinase-induced loss of cadherin cell-cell junctions, destabilization of the lens epithelium, and the induction of cortical cataract.
While cadherin cleavage is a normal mechanism for cadherin turnover, misregulation of this process destabilizes cell-cell junctions. In a model for anterior subcapsular cataract, TGFβ induces a fibrotic pathology as a result of E-cadherin cleavage that is mediated by matrix metalloproteinases (MMPs) . Here, we examined whether the loss of cadherin junctions in our cataract culture model may involve increased cleavage of N-cadherin, the principle cadherin of chicken lens epithelial cells. For this study, protein extracts of lens epithelial cells were analyzed by western blot using an antibody to the N-cadherin cytoplasmic domain (Figure 2C). The results demonstrated a substantial increase in the generation of a 42 kDa N-cadherin cleavage product in the epithelium of lenses grown in the ex vivo cortical cataract cultures (Figure 2C,D). This increase in cadherin cleavage was blocked when activation of Src kinases was suppressed. These results suggest that the loss of cadherin junctions in the cataractous lenses is likely to result from increased cleavage of N-cadherin.
Inappropriate activation of Src kinases induces apoptosis of lens epithelial cells
The loss of cadherin junctions and the presence of pycnotic nuclei in the epithelia of cataractous lenses were consistent with an apoptotic phenotype. Apoptosis of cells in the lens epithelium is a common characteristic of cortical cataracts and there is evidence that a loss of function of the lens epithelium could be responsible for the induction of cataract. Using the TUNEL assay, which stains fragmented DNA at late stages of apoptosis, we investigated the possibility that development of cortical opacities in our cataract model was linked to apoptosis of lens epithelial cells. These studies revealed extensive apoptosis in both the anterior epithelium (Figure 3B) and equatorial epithelium (Figure 3C) as early as day 2 in culture. The results show that apoptosis of cells in the lens epithelium was induced prior to the appearance of opacities in the cortical zone. Importantly, no TUNEL positive nuclei were detected in cortical fiber cells at culture day 2 (Figure 3D), a feature maintained throughout the 10-day culture period. The result that apoptosis of cells in the lens epithelium preceded the appearance of cortical opacities suggested that the loss of function of the lens epithelium could have an inductive role in the development of cortical cataracts.
Next, we investigated whether apoptosis of cells in the lens epithelium under conditions of cataract induction resulted from activation of the canonical mitochondrial death pathway. Since this apoptotic pathway involves activation of DEVDases (caspase 3-like proteases), we measured DEVDase activity in the epithelium of lenses cultured in the ex vivo cataract model and compared it to DEVDase activity in normal embryonic day 10 lens epithelia (Figure 4A). A greater than four-fold increase in DEVDase activity occurred in the epithelia of lenses in cataract culture and elevated DEVDase activity was maintained throughout the culture period, although at reduced levels at late times in culture.
Lastly, we examined whether cell death in the lens epithelium was induced by the inappropriate activation of Src kinases. To investigate the correlation between SFK activation and apoptotic death in the lens epithelium, we performed both DEVDase assays and TUNEL staining on lenses grown in cataract culture in the presence of the SFK inhibitor PP1. Inhibition of SFK activity completely blocked the increased activation of caspase 3-like proteases in the ex vivo culture model (Figure 4A). For TUNEL analysis, lenses were grown in cataract culture conditions for 10 days in the presence and absence of the Src kinase inhibitor PP1 (Figure 4B). Cortical cataracts were well developed by culture day 10  at which point extensive apoptosis had occurred in the lens epithelium (Figure 4B, top panel, right). Note that even at this late time in culture, no TUNEL positive cells were detected in the cortical fiber cells (unpublished data). When the lenses were grown under the same ex vivo culture conditions but in the presence of the Src kinase inhibitor PP1, apoptotic cell death was effectively blocked in the lens epithelium (Figure 4B, bottom panel, right). These results suggest that the inappropriate activation of Src kinases under conditions of stress initiates a signaling pathway in the lens epithelium that leads to apoptotic cell death of which cortical opacity is a likely downstream consequence.
Loss of cellular organization and membrane structure in the cortical zone of cataractous lenses
In the cortical region of the lens, the highly elongated fiber cells align with one another to form a tight, hexagonally packed cellular network (Figure 5A and [4-6]). The formation of cortical opacities has been associated with the disorganization of the cortical fiber cells and the appearance of large holes or vacuoles between them. Our initial analysis of the cortical cataract cultures showed significant disorganization of cells specifically in the cortical fiber region . Here, we examine in more detail the phenotype of the fiber cells in the cortical zone of these cataractous lenses, concentrating on the organization, morphology and structure of the cortical fiber cells and their membranes. These cells were imaged following immunostaining for the major intrinsic lens membrane protein, aquaporin-0 (MP28), which in the lens functions as both a water channel and an adhesion molecule . In the normal lens, immunostaining for aquaporin-0 demonstrated the highly ordered structure and hexagonal packing of the cortical fiber cells (Figure 5A, transverse section). Both cell and membrane organization failed to be maintained in the cortical fiber cells of lenses that developed cataracts in culture. The immunolocalization of aquaporin-0 revealed tremendous irregularities in the membrane organization of these cortical fiber cells including loss of linearity, extensive undulations of the membranes, and disorganized regions where neighboring cell membranes overlapped (Figure 5B,C) as well as areas with significant separations between neighboring fiber cells (see Figure 5C with an asterisk). Membrane structure of fiber cells more central to the lens appeared unaffected (unpublished data). This analysis showed that cortical fiber cells had lost their ability to maintain the cell-cell interactions that dictate the linearity of their membrane structure.
Inhibiting inappropriate activation of Src kinases in cataract culture preserves cortical fiber organization
Our previous electron microscopy studies of the normal chick embryo lens show that extensive cell-cell junctions define the highly linear membrane interfaces of cortical fiber cells in the chick embryo lens [28,32]. Ultrastructural analysis of cortical fiber cells in lenses with cortical cataracts revealed a highly irregular membrane structure (Figure 6A,C). Undulating membranes were commonly observed (Figure 6A,C), directly paralleling the membrane defects observed by immunostaining for aquaporin-0 (see Figure 5B,C). In addition to the failure to maintain normal membrane linearity, junctional contacts between cortical fiber cells were greatly diminished, and large separations between the cells often occurred (Figure 6A,C). Importantly, these membrane defects could be prevented if the lenses were cultured in the presence of the SFK inhibitor PP1, creating the conditions which prevent cataract-induction. In these PP1-treated lenses, the cell-cell junctions between cortical fiber cells remained largely intact and the linearity of the membrane interfaces was maintained (Figure 6B,D). These results indicate that the opacities observed in our cataract culture model result from the disorganization of membrane structure and dysmorphology of fiber cells in the cortical fiber zone.
The lens is a major target for mechanical, oxidative, UV, and diabetic stress. These stressors are capable of altering lens structure and function, causing the pathologies associated with cataract including age-related cataract. Many of these stressors are either oxidants themselves or catalyze the production of reactive oxygen species (ROS). ROS, in turn, activate stress signaling pathways such as those involving the p38 stress-activated MAP kinase [33-35] and the Src kinases [36-39]. In addition, the generation of ROS and the subsequent activation of downstream signaling effectors such as p38 can induce apoptosis . While the lens has antioxidants and other factors that protect it from these stress pathways, they are limited in their capacity. Cataractogenesis may ensue when lens defenses are overwhelmed by high levels of ROS production and activation of stress signaling pathways. In order to investigate the mechanism of stress-induced cataract we previously developed an ex vivo model in which cortical cataracts are induced through activation of a mechanical stress-induced p38/Src stress-signaling pathway [20,21]. p38 kinase, not normally active in the lens epithelium, is activated within the first day of cataract culture and maintained at a high level up through the appearance of cortical opacities . p38 induces cataractogenesis by activation of SFKs and inhibitors of this signaling pathway effectively prevent lens opacification [20,21]. The p38/Src cataract-inducing pathway closely mimics stress pathways described in vivo, providing strong evidence that the lens cataract cultures are a good model for investigating the mechanism of stress-induced cataract.
In tissues, there is often a close connection between form and function. This is particularly true of the lens where maintenance of its unique cytoarchitecture is crucial to the preservation of its transparency. Our previous studies show that the establishment of normal lens epithelial cell morphology is dependent on the assembly of cadherin cell-cell junctions . In addition, we show that the expression of a constitutively active Src kinase causes cultured lens cells to lose their epithelial phenotype as a result of the loss of their cell-cell junctions . The susceptibility of N-cadherin junctions in lens epithelial cells to activated Src kinases also has been shown by others . In the cataract model (the subject of this study) activation of Src kinases caused disassembly of cadherin cell-cell junctions and loss of epithelial morphology. Biochemical analysis suggested that the mechanism by which Src kinases disrupt these cadherin junctions involves enhanced cleavage of N-cadherin.
There is substantial evidence in the literature that normal cadherin turnover occurs as a result of its proteolytic cleavage. In fact, N-cadherin is targeted for cleavage by a number of different proteases, a subset of which is activated by the Src kinases. These include MMP-2 [41-44], calpain [45-47], and γ-secretase . Other MMPs not yet implicated in N-cadherin cleavage such as MMP-9  and MT1-MMP, an MMP activator [49,50], are also activated by Src. Supporting our observations of a link between cadherin cleavage, N-cadherin turnover and cataract induction are studies showing that exposure of lenses to stress activates MMP-2 and MMP-9 , that inhibitors of these MMPs prevent formation of TGFβ-induced subcapsular cataract , and that calpain inhibitors suppress formation of selenite-induced cataract [52,53].
While a number of the molecular components of cadherin junctions are targets for Src tyrosine kinases [54-57] and their phosphorylation can affect junctional stability, there is compelling evidence that the tyrosine phosphorylation of N-cadherin itself induces its cleavage and turnover . In addition, proteases that cleave within the cadherin cytoplasmic domain and leave the cadherin receptor in the membrane prevent the binding of catenins necessary to stabilize cadherin cell-cell junctions. This would lead to diminished association of β-catenin with cell-cell junctions as we have observed in the epithelium of lenses with cortical cataracts. We suggest that cadherin cleavage is a likely mechanism for the loss of cadherin junctions under conditions that induce formation of cataracts.
In keratinocytes, E-cadherin cleavage in response to UV irradiation disrupts cadherin-catenin interactions in cells that are to undergo apoptosis . The literature is replete with evidence that the loss of E-cadherin-mediated cell adhesion can be a trigger for apoptosis [60-65]. Our own findings regarding the mechanism of lens cataract induction support these studies. The loss of cadherin junctions in the lens epithelium under cataract-inducing conditions was coincident with their apoptotic cell death and both resulted from the activation of SFKs. The literature shows that many factors protect lens epithelial cells from apoptosis, likely because these cells are the first line of defense for the underlying fiber cells. Prominent among these other factors is the small heat-shock chaperone protein, αA-crystallin [8,66,67], possibly related to its function in PI3K/Akt survival signaling . Since PI3K can be recruited to and activated at cadherin junctions [63,69], future studies will examine whether apoptosis of lens epithelial cells in the cataract cultures involves inhibition of this survival signaling pathway. However, relevant to this pathway, previous studies from this lab show that the actin cytoskeleton has anti-apoptotic function in lens epithelial cells  and that the formation and maintenance of cortical actin filaments requires PI3K signaling . We now provide evidence that the dysmorphology and instability of the lens epithelium under cataract-inducing conditions may involve disruption of the cortical actin cytoskeleton. The actin cytoskeleton may be an important integrator of N-cadherin and the PI3K signaling pathway.
Our studies using the ex vivo cortical cataract model show that programmed cell death resulting from the inappropriate activation of Src kinases was restricted to lens epithelial cells and that this Src-induced functional loss of the lens epithelium preceded the appearance of cortical opacities. While many studies in the literature show a direct connection between apoptosis of cells in the lens epithelium and the development of opacities in the fiber zone, this is the first demonstration that stress-signaling involving the activation of Src kinases is responsible for this phenomenon. Since Src kinases are activated in all known stress-signaling pathways, our findings suggest that inappropriate activation of Src kinases may be a common link between the loss of function of the lens epithelium and the development of lens cataract observed in many different cataract models as well as in age-related human cortical cataract. Note that our studies implicating the stress-activation of Src kinases in the epithelium with the mechanism of induction of lens cortical cataract do not rule out a contributing role for Src kinases in the cortical fiber zone.
Opacification is typically caused by light scatter resulting from morphological defects in the lens fiber zones. These defects are often associated with large "vacuoles" that appear in the fiber cell region of the lens, but there is little understanding of the early morphogenetic changes associated with cataractogenesis or the altered morphogenetic pathway in the fiber zone that leads to the development of cataract. Aquaporin-0, originally referred to as MP28 in the chick, is the major integral membrane protein of lens fiber cells where it functions as both a water channel protein and a cell adhesion molecule in what has been named the lens "thin junction". We exploited the properties of this major lens membrane protein to investigate the early morphogenetic defects in lens fiber cells using our ex vivo cultures as a model for stress-induced cataract. Immunofluorescence analysis revealed that the lens opacities associated with stress-induced activation of Src kinases were linked to the loss of membrane integrity of cells in the cortical fiber zone. These membrane defects included inappropriate interdigitations between neighboring cortical fiber cell membranes, dramatic undulations along the length of the lens fiber cell membrane, and large separations between neighboring cortical fiber cells. All of these morphogenetic defects are likely consequences of the inability of cortical fiber cells to maintain a linear membrane structure following the loss of function of lens epithelial cells. These membrane defects are the earliest morphological defects reported for lens fiber cells during cataractogenesis. We suggest that the appearance of large vacuoles typical of cataractogenesis is a downstream consequence of these early defects in lens membrane structure. These findings were confirmed at the ultrastructural level; electron microscopy analysis also showed dramatic loss of membrane linearity, undulating membranes, and separations between neighboring cortical fiber cells. These defects were similar to those reported for dexamethasone-induced cataract, which is associated with a loss of cadherin junctions . Remarkably, blocking the activation of Src kinases in our ex vivo stress-induced cataract model was sufficient to maintain membrane linearity and block the loss of cell-cell adhesion thus preventing the development of cortical cataracts.
We thank Janice Walker for the critical reading of the manuscript. These studies were supported by NIH Grants EY10577, EY014258, and EY014798 to ASM and an ARVO/Alcon Laboratories, Incorporated Research Fellowship Grant to JZ. ML was supported by National Institutes of Health Training Grant ES007282.
1. Bassnett S, Beebe DC. Coincident loss of mitochondria and nuclei during lens fiber cell differentiation. Dev Dyn 1992; 194:85-93.
2. Bassnett S. The fate of the Golgi apparatus and the endoplasmic reticulum during lens fiber cell differentiation. Invest Ophthalmol Vis Sci 1995; 36:1793-803.
3. Bassnett S, Mataic D. Chromatin degradation in differentiating fiber cells of the eye lens. J Cell Biol 1997; 137:37-49.
4. Kuszak JR. The ultrastructure of epithelial and fiber cells in the crystalline lens. Int Rev Cytol 1995; 163:305-50.
5. Bassnett S, Missey H, Vucemilo I. Molecular architecture of the lens fiber cell basal membrane complex. J Cell Sci 1999; 112:2155-65.
6. Straub BK, Boda J, Kuhn C, Schnoelzer M, Korf U, Kempf T, Spring H, Hatzfeld M, Franke WW. A novel cell-cell junction system: the cortex adhaerens mosaic of lens fiber cells. J Cell Sci 2003; 116:4985-95.
7. Andley UP, Song Z, Wawrousek EF, Bassnett S. The molecular chaperone alphaA-crystallin enhances lens epithelial cell growth and resistance to UVA stress. J Biol Chem 1998; 273:31252-61.
8. Xi JH, Bai F, Andley UP. Reduced survival of lens epithelial cells in the alphaA-crystallin-knockout mouse. J Cell Sci 2003; 116:1073-85.
9. Rae JL, Bartling C, Rae J, Mathias RT. Dye transfer between cells of the lens. J Membr Biol 1996; 150:89-103.
10. Bassnett S, Kuszak JR, Reinisch L, Brown HG, Beebe DC. Intercellular communication between epithelial and fiber cells of the eye lens. J Cell Sci 1994; 107:799-811.
11. Hightower KR, Reddan JR, McCready JP, Dziedzic DC. Lens epithelium: a primary target of UVB irradiation. Exp Eye Res 1994; 59:557-64.
12. Hightower KR. The role of the lens epithelium in development of UV cataract. Curr Eye Res 1995; 14:71-8.
13. Ruiz-Ederra J, Verkman AS. Accelerated cataract formation and reduced lens epithelial water permeability in aquaporin-1-deficient mice. Invest Ophthalmol Vis Sci 2006; 47:3960-7.
14. Bu L, Yan S, Jin M, Jin Y, Yu C, Xiao S, Xie Q, Hu L, Xie Y, Solitang Y, Liu J, Zhao G, Kong X. The gamma S-crystallin gene is mutated in autosomal recessive cataract in mouse. Genomics 2002; 80:38-44.
15. Li WC, Kuszak JR, Dunn K, Wang RR, Ma W, Wang GM, Spector A, Leib M, Cotliar AM, Weiss M, Espy J, Howard G, Farris RL, Auran J, Donn A, Hofeldt A, Mackay C, Merriam J, Mittl R, Smith TR. Lens epithelial cell apoptosis appears to be a common cellular basis for non-congenital cataract development in humans and animals. J Cell Biol 1995; 130:169-81.
16. Li WC, Spector A. Lens epithelial cell apoptosis is an early event in the development of UVB-induced cataract. Free Radic Biol Med 1996; 20:301-11.
17. Tamada Y, Fukiage C, Nakamura Y, Azuma M, Kim YH, Shearer TR. Evidence for apoptosis in the selenite rat model of cataract. Biochem Biophys Res Commun 2000; 275:300-6.
18. Maruno KA, Lovicu FJ, Chamberlain CG, McAvoy JW. Apoptosis is a feature of TGF beta-induced cataract. Clin Exp Optom 2002; 85:76-82.
19. Murata M, Ohta N, Sakurai S, Alam S, Tsai J, Kador PF, Sato S. The role of aldose reductase in sugar cataract formation: aldose reductase plays a key role in lens epithelial cell death (apoptosis). Chem Biol Interact 2001; 130-132:617-25.
20. Zhou J, Menko AS. The role of Src family kinases in cortical cataract formation. Invest Ophthalmol Vis Sci 2002; 43:2293-300.
21. Zhou J, Menko AS. Coordinate signaling by Src and p38 kinases in the induction of cortical cataracts. Invest Ophthalmol Vis Sci 2004; 45:2314-23.
22. Smith AN, Miller LA, Song N, Taketo MM, Lang RA. The duality of beta-catenin function: a requirement in lens morphogenesis and signaling suppression of lens fate in periocular ectoderm. Dev Biol 2005; 285:477-89.
23. Stump RJ, Lovicu FJ, Ang SL, Pandey SK, McAvoy JW. Lithium stabilizes the polarized lens epithelial phenotype and inhibits proliferation, migration, and epithelial mesenchymal transition. J Pathol 2006; 210:249-57.
24. Dwivedi DJ, Pino G, Banh A, Nathu Z, Howchin D, Margetts P, Sivak JG, West-Mays JA. Matrix metalloproteinase inhibitors suppress transforming growth factor-beta-induced subcapsular cataract formation. Am J Pathol 2006; 168:69-79.
25. Rungger-Brandle E, Conti A, Leuenberger PM, Rungger D. Expression of alphasmooth muscle actin in lens epithelia from human donors and cataract patients. Exp Eye Res 2005; 81:539-50.
26. Menko AS, Boettiger D. Inhibition of chicken embryo lens differentiation and lens junction formation in culture by pp60v-src. Mol Cell Biol 1988; 8:1414-20.
27. Volberg T, Geiger B, Dror R, Zick Y. Modulation of intercellular adherens-type junctions and tyrosine phosphorylation of their components in RSV-transformed cultured chick lens cells. Cell Regul 1991; 2:105-20.
28. Menko AS, Klukas KA, Liu TF, Quade B, Sas DF, Preus DM, Johnson RG. Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression. Dev Biol 1987; 123:307-20.
29. Walker JL, Menko AS. alpha6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching. Dev Biol 1999; 210:497-511.
30. Weber GF, Menko AS. Actin filament organization regulates the induction of lens cell differentiation and survival. Dev Biol 2006; 295:714-29.
31. Lindsey Rose KM, Gourdie RG, Prescott AR, Quinlan RA, Crouch RK, Schey KL. The C terminus of lens aquaporin 0 interacts with the cytoskeletal proteins filensin and CP49. Invest Ophthalmol Vis Sci 2006; 47:1562-70.
32. Menko AS, Klukas KA, Johnson RG. Chicken embryo lens cultures mimic differentiation in the lens. Dev Biol 1984; 103:129-41.
33. Igarashi M, Wakasaki H, Takahara N, Ishii H, Jiang ZY, Yamauchi T, Kuboki K, Meier M, Rhodes CJ, King GL. Glucose or diabetes activates p38 mitogen-activated protein kinase via different pathways. J Clin Invest 1999; 103:185-95.
34. Alikhani M, Maclellan CM, Raptis M, Vora S, Trackman PC, Graves DT. Advanced glycation end products induce apoptosis in fibroblasts through activation of ROS, MAP kinases, and the FOXO1 transcription factor. Am J Physiol Cell Physiol 2007; 292:C850-6.
35. Van Laethem A, Nys K, Van Kelst S, Claerhout S, Ichijo H, Vandenheede JR, Garmyn M, Agostinis P. Apoptosis signal regulating kinase-1 connects reactive oxygen species to p38 MAPK-induced mitochondrial apoptosis in UVB-irradiated human keratinocytes. Free Radic Biol Med 2006; 41:1361-71.
36. Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol 2000; 20:2175-83.
37. Dasari A, Bartholomew JN, Volonte D, Galbiati F. Oxidative stress induces premature senescence by stimulating caveolin-1 gene transcription through p38 mitogen-activated protein kinase/Sp1-mediated activation of two GC-rich promoter elements. Cancer Res 2006; 66:10805-14.
38. Lahair MM, Howe CJ, Rodriguez-Mora O, McCubrey JA, Franklin RA. Molecular pathways leading to oxidative stress-induced phosphorylation of Akt. Antioxid Redox Signal 2006; 8:1749-56.
39. Meurer S, Pioch S, Gross S, Muller-Esterl W. Reactive oxygen species induce tyrosine phosphorylation of and Src kinase recruitment to NO-sensitive guanylyl cyclase. J Biol Chem 2005; 280:33149-56.
40. Ferreira-Cornwell MC, Veneziale RW, Grunwald GB, Menko AS. N-cadherin function is required for differentiation-dependent cytoskeletal reorganization in lens cells in vitro. Exp Cell Res 2000; 256:237-47.
41. Chen JM, Aimes RT, Ward GR, Youngleib GL, Quigley JP. Isolation and characterization of a 70-kDa metalloprotease (gelatinase) that is elevated in Rous sarcoma virus-transformed chicken embryo fibroblasts. J Biol Chem 1991; 266:5113-21.
42. Aguirre-Ghiso JA, Frankel P, Farias EF, Lu Z, Jiang H, Olsen A, Feig LA, de Kier Joffe EB, Foster DA. RalA requirement for v-Src- and v-Ras-induced tumorigenicity and overproduction of urokinase-type plasminogen activator: involvement of metalloproteases. Oncogene 1999; 18:4718-25.
43. Frame MC, Fincham VJ, Carragher NO, Wyke JA. v-Src's hold over actin and cell adhesions. Nat Rev Mol Cell Biol 2002; 3:233-45.
44. Hamaguchi M, Yamagata S, Thant AA, Xiao H, Iwata H, Mazaki T, Hanafusa H. Augmentation of metalloproteinase (gelatinase) activity secreted from Rous sarcoma virus-infected cells correlates with transforming activity of src. Oncogene 1995; 10:1037-43.
45. Sato N, Fujio Y, Yamada-Honda F, Funai H, Wada A, Kawashima S, Awata N, Shibata N. Elevated calcium level induces calcium-dependent proteolysis of A-CAM (N-cadherin) in heart--analysis by detergent-treated model. Biochem Biophys Res Commun 1995; 217:649-53.
46. Covault J, Liu QY, el-Deeb S. Calcium-activated proteolysis of intracellular domains in the cell adhesion molecules NCAM and N-cadherin. Brain Res Mol Brain Res 1991; 11:11-6.
47. Robert A, Miron MJ, Champagne C, Gingras MC, Branton PE, Lavoie JN. Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein. J Cell Biol 2002; 158:519-28.
48. Gianni D, Zambrano N, Bimonte M, Minopoli G, Mercken L, Talamo F, Scaloni A, Russo T. Platelet-derived growth factor induces the beta-gamma-secretase-mediated cleavage of Alzheimer's amyloid precursor protein through a Src-Rac-dependent pathway. J Biol Chem 2003; 278:9290-7.
49. Cha HJ, Okada A, Kim KW, Sato H, Seiki M. Identification of cis-acting promoter elements that support expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) in v-src transformed Madin-Darby canine kidney cells. Clin Exp Metastasis 2000; 18:675-81.
50. Kadono Y, Okada Y, Namiki M, Seiki M, Sato H. Transformation of epithelial Madin-Darby canine kidney cells with p60(v-src) induces expression of membrane-type 1 matrix metalloproteinase and invasiveness. Cancer Res 1998; 58:2240-4.
51. Tamiya S, Wormstone IM, Marcantonio JM, Gavrilovic J, Duncan G. Induction of matrix metalloproteinases 2 and 9 following stress to the lens. Exp Eye Res 2000; 71:591-7.
52. Biswas S, Harris F, Dennison S, Singh J, Phoenix DA. Calpains: targets of cataract prevention? Trends Mol Med 2004; 10:78-84.
53. Tamada Y, Fukiage C, Mizutani K, Yamaguchi M, Nakamura Y, Azuma M, Shearer TR. Calpain inhibitor, SJA6017, reduces the rate of formation of selenite cataract in rats. Curr Eye Res 2001; 22:280-5.
54. Qi J, Wang J, Romanyuk O, Siu CH. Involvement of Src family kinases in N-cadherin phosphorylation and beta-catenin dissociation during transendothelial migration of melanoma cells. Mol Biol Cell 2006; 17:1261-72.
55. Coluccia AM, Benati D, Dekhil H, De Filippo A, Lan C, Gambacorti-Passerini C. SKI-606 decreases growth and motility of colorectal cancer cells by preventing pp60(c-Src)-dependent tyrosine phosphorylation of beta-catenin and its nuclear signaling. Cancer Res 2006; 66:2279-86.
56. Lilien J, Balsamo J. The regulation of cadherin-mediated adhesion by tyrosine phosphorylation/dephosphorylation of beta-catenin. Curr Opin Cell Biol 2005; 17:459-65.
57. Castano J, Solanas G, Casagolda D, Raurell I, Villagrasa P, Bustelo XR, Garcia de Herreros A, Dunach M. Specific phosphorylation of p120-catenin regulatory domain differently modulates its binding to RhoA. Mol Cell Biol 2007; 27:1745-57.
58. Lee MM, Fink BD, Grunwald GB. Evidence that tyrosine phosphorylation regulates N-cadherin turnover during retinal development. Dev Genet 1997; 20:224-34.
59. Hung CF, Chiang HS, Lo HM, Jian JS, Wu WB. E-cadherin and its downstream catenins are proteolytically cleaved in human HaCaT keratinocytes exposed to UVB. Exp Dermatol 2006; 15:315-21.
60. Galaz S, Espada J, Stockert JC, Pacheco M, Sanz-Rodriguez F, Arranz R, Rello S, Canete M, Villanueva A, Esteller M, Juarranz A. Loss of E-cadherin mediated cell-cell adhesion as an early trigger of apoptosis induced by photodynamic treatment. J Cell Physiol 2005; 205:86-96.
61. Rios-Doria J, Day ML. Truncated E-cadherin potentiates cell death in prostate epithelial cells. Prostate 2005; 63:259-68.
62. Fouquet S, Lugo-Martinez VH, Faussat AM, Renaud F, Cardot P, Chambaz J, Pincon-Raymond M, Thenet S. Early loss of E-cadherin from cell-cell contacts is involved in the onset of Anoikis in enterocytes. J Biol Chem 2004; 279:43061-9.
63. Pece S, Chiariello M, Murga C, Gutkind JS. Activation of the protein kinase Akt/PKB by the formation of E-cadherin-mediated cell-cell junctions. Evidence for the association of phosphatidylinositol 3-kinase with the E-cadherin adhesion complex. J Biol Chem 1999; 274:19347-51.
64. Junghans D, Hack I, Frotscher M, Taylor V, Kemler R. Beta-catenin-mediated cell-adhesion is vital for embryonic forebrain development. Dev Dyn 2005; 233:528-39.
65. Erez N, Zamir E, Gour BJ, Blaschuk OW, Geiger B. Induction of apoptosis in cultured endothelial cells by a cadherin antagonist peptide: involvement of fibroblast growth factor receptor-mediated signalling. Exp Cell Res 2004; 294:366-78.
66. Andley UP, Song Z, Wawrousek EF, Fleming TP, Bassnett S. Differential protective activity of alpha A- and alphaB-crystallin in lens epithelial cells. J Biol Chem 2000; 275:36823-31.
67. Andley UP, Patel HC, Xi JH. The R116C mutation in alpha A-crystallin diminishes its protective ability against stress-induced lens epithelial cell apoptosis. J Biol Chem 2002; 277:10178-86.
68. Liu JP, Schlosser R, Ma WY, Dong Z, Feng H, Lui L, Huang XQ, Liu Y, Li DW. Human alphaA- and alphaB-crystallins prevent UVA-induced apoptosis through regulation of PKCalpha, RAF/MEK/ERK and AKT signaling pathways. Exp Eye Res 2004; 79:393-403.
69. Calautti E, Li J, Saoncella S, Brissette JL, Goetinck PF. Phosphoinositide 3-kinase signaling to Akt promotes keratinocyte differentiation versus death. J Biol Chem 2005; 280:32856-65.
70. Weber GF, Menko AS. Phosphatidylinositol 3-kinase is necessary for lens fiber cell differentiation and survival. Invest Ophthalmol Vis Sci 2006; 47:4490-9.
71. Lyu J, Kim JA, Chung SK, Kim KS, Joo CK. Alteration of cadherin in dexamethasone-induced cataract organ-cultured rat lens. Invest Ophthalmol Vis Sci 2003; 44:2034-40.