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Age-related susceptibility to apoptosis in human retinal pigment epithelial cells is triggered by disruption of p53-Mdm2 association.
Bhattacharya S, Chaum E, Johnson DA, Johnson LR
(2012) Invest Ophthalmol Vis Sci 53: 8350-66
MeSH Terms: Acetylation, Adult, Aged, Aged, 80 and over, Aging, Apoptosis, Apoptosis Regulatory Proteins, Benzamides, Blotting, Western, Caspase 3, Cell Proliferation, Cells, Cultured, DNA Fragmentation, Disease Susceptibility, Enzyme-Linked Immunosorbent Assay, Fluorescent Antibody Technique, Indirect, Humans, Imidazoles, In Situ Nick-End Labeling, Middle Aged, Naphthols, Phosphorylation, Piperazines, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-mdm2, RNA, Small Interfering, Retinal Pigment Epithelium, Sirtuin 1, Sirtuin 2, Tumor Suppressor Protein p53
Show Abstract · Added June 11, 2018
PURPOSE - Relatively little is known about the contribution of p53/Mdm2 pathway in apoptosis of retinal pigment epithelial (RPE) cells or its possible link to dysfunction of aging RPE or to related blinding disorders such as age-related macular degeneration (AMD).
METHODS - Age-associated changes in p53 activation were evaluated in primary RPE cultures from human donor eyes of various ages. Apoptosis was evaluated by activation of caspases and DNA fragmentation. Gene-specific small interfering RNA was used to knock down expression of p53.
RESULTS - We observed that the basal rate of p53-dependent apoptosis increased in an age-dependent manner in human RPE. The age-dependent increase in apoptosis was linked to alterations in several aspects of the p53 pathway. p53 phosphorylation Ser15 was increased through the stimulation of ATM-Ser1981. p53 acetylation Lys379 was increased through the inhibition of SIRT1/2. These two posttranslational modifications of p53 blocked the sequestration of p53 by Mdm2, thus resulting in an increase in free p53 and of p53 stimulation of apoptosis through increased expression of PUMA (p53 upregulated modulator of apoptosis) and activation of caspase-3. Aged RPE also had reduced expression of antiapoptotic Bcl-2, which contributed to the increase in apoptosis. Of particular interest in these studies was that pharmacologic treatments to block p53 phosphorylation, acetylation, or expression were able to protect RPE cells from apoptosis.
CONCLUSIONS - Our studies suggest that aging in the RPE leads to alterations of specific checkpoints in the apoptotic pathway, which may represent important molecular targets for the treatment of RPE-related aging disorders such as AMD.
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MeSH Terms
Monoclonal antibodies to DIPA: a novel binding partner of p120-catenin isoform 1.
Markham NO, Cooper T, Goff M, Gribben EM, Carnahan RH, Reynolds AB
(2012) Hybridoma (Larchmt) 31: 246-54
MeSH Terms: Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Antibodies, Monoclonal, Murine-Derived, Antibody Specificity, Blotting, Western, Catenins, Cell Line, Dogs, Fluorescent Antibody Technique, Indirect, Gene Knockdown Techniques, Humans, Immunoglobulin G, Immunoprecipitation, Mice, Molecular Sequence Data, Protein Isoforms, RNA Interference, Rats, Repressor Proteins
Show Abstract · Added December 12, 2013
The coiled-coil domain-containing delta-interacting protein A (DIPA) is a transcription factor implicated in developmental regulation. DIPA is the first protein discovered to selectively interact with the p120-catenin (p120) isoform 1, an alternatively spliced form of p120 expressed preferentially in mesenchymal cells. Although a small fraction of p120 can be observed in the nucleus under certain circumstances, the vast majority of it associates with classical cadherins at adherens junctions. We observed for the first time that a discrete fraction of DIPA exists at cell-cell junctions, in addition to its predominantly nuclear localization. Thus, the p120-DIPA interaction may regulate cell signaling and/or transcriptional events, as has been described previously for β-catenin and the LEF/TCF transcription factor family. To facilitate further study of DIPA and to determine the physiological relevance of its interaction with p120, we have generated and characterized a panel of five DIPA-specific monoclonal antibodies (MAbs) that function in immunoblotting, immunoprecipitation, and immunofluorescence assays.
2 Communities
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20 MeSH Terms
Optimum length and flexibility of reovirus attachment protein σ1 are required for efficient viral infection.
Bokiej M, Ogden KM, Ikizler M, Reiter DM, Stehle T, Dermody TS
(2012) J Virol 86: 10270-80
MeSH Terms: Animals, Capsid, Capsid Proteins, Carbohydrates, Cell Adhesion Molecules, Cell Line, Cell Membrane, Fibroblasts, Fluorescent Antibody Technique, Indirect, Gene Deletion, Gene Expression Regulation, Viral, Genome, Viral, HeLa Cells, Humans, Mice, Mutation, Protein Binding, Protein Folding, Receptors, Cell Surface, Reoviridae, Virus Replication
Show Abstract · Added December 10, 2013
Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.
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21 MeSH Terms
Tight junction-associated signaling pathways modulate cell proliferation in uveal melanoma.
Jayagopal A, Yang JL, Haselton FR, Chang MS
(2011) Invest Ophthalmol Vis Sci 52: 588-93
MeSH Terms: Blotting, Western, CCAAT-Enhancer-Binding Proteins, Cell Adhesion Molecules, Cell Cycle, Cell Line, Tumor, Cell Proliferation, Flow Cytometry, Fluorescent Antibody Technique, Indirect, Genetic Vectors, Heat-Shock Proteins, Humans, Melanoma, Membrane Proteins, Muscle Proteins, Occludin, Phosphoproteins, Signal Transduction, Tight Junctions, Transcription Factors, Transfection, Uveal Neoplasms, Zonula Occludens-1 Protein
Show Abstract · Added May 27, 2014
PURPOSE - To investigate the role of tight junction (TJ)-associated signaling pathways in the proliferation of uveal melanoma.
METHODS - Human uveal melanoma cell lines overexpressing the TJ molecule blood vessel epicardial substance (Bves) were generated. The effects of Bves overexpression on TJ protein expression, cell proliferation, and cell cycle distribution were quantified. In addition, localization and transcription activity of the TJ-associated protein ZO-1-associated nucleic acid binding protein (ZONAB) were evaluated using immunofluorescence and bioluminescence reporter assays to study the involvement of Bves signaling in cell proliferation-associated pathways.
RESULTS - Bves overexpression in uveal melanoma cell lines resulted in increased expression of the TJ proteins occludin and ZO-1, reduced cell proliferation, and increased sequestration of ZONAB at TJs and reduced ZONAB transcriptional activity.
CONCLUSIONS - TJ proteins are present in uveal melanoma, and TJ-associated signaling pathways modulate cell signaling pathways relevant to proliferation in uveal melanoma.
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22 MeSH Terms
Ectopic Mitf in the embryonic chick retina by co-transfection of β-catenin and Otx2.
Westenskow PD, McKean JB, Kubo F, Nakagawa S, Fuhrmann S
(2010) Invest Ophthalmol Vis Sci 51: 5328-35
MeSH Terms: Animals, Cell Count, Cell Differentiation, Cells, Cultured, Chick Embryo, Electroporation, Fluorescent Antibody Technique, Indirect, Gene Expression Regulation, Developmental, Microphthalmia-Associated Transcription Factor, Microscopy, Confocal, Otx Transcription Factors, Plasmids, Retina, Retinal Neurons, Retinal Pigment Epithelium, Transcriptional Activation, Transfection, Wnt Proteins, beta Catenin
Show Abstract · Added November 19, 2015
PURPOSE - Development of the retinal pigment epithelium (RPE) is controlled by intrinsic and extrinsic regulators including orthodenticle homeobox 2 (Otx2) and the Wnt/β-catenin pathway, respectively. Otx2 and β-catenin are necessary for the expression of the RPE key regulator microphthalmia-associated transcription factor (Mitf); however, neither factor is sufficient to promote Mitf expression in vivo. The study was conducted to determine whether Otx2 and β-catenin act in a combinatorial manner and tested whether co-expression in the presumptive chick retina induces ectopic Mitf expression.
METHODS - The sufficiency of Wnt/β-catenin activation and/or Otx2 expression to induce RPE-specific gene expression was examined in chick optic vesicle explant cultures or in the presumptive neural retina using in ovo-electroporation. Luciferase assays were used to examine the transactivation potentials of Otx2 and β-catenin on the Mitf-D enhancer and autoregulation of the Mitf-D and Otx2T0 enhancers.
RESULTS - In optic vesicles explant cultures, RPE-specific gene expression was activated by lithium chloride, a Wnt/β-catenin agonist. However, in vivo, Mitf was induced only in the presumptive retina if both β-catenin and Otx2 are co-expressed. Furthermore, both Mitf and Otx2 can autoregulate their own enhancers in vitro.
CONCLUSIONS - The present study provides evidence that β-catenin and Otx2 are sufficient, at least in part, to convert retinal progenitor cells into presumptive RPE cells expressing Mitf. Otx2 may act as a competence factor that allows RPE specification in concert with additional RPE-promoting factors such as β-catenin.
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19 MeSH Terms
Inhibition of RhoA signaling with increased Bves in trabecular meshwork cells.
Russ PK, Kupperman AI, Presley SH, Haselton FR, Chang MS
(2010) Invest Ophthalmol Vis Sci 51: 223-30
MeSH Terms: Avian Proteins, Blotting, Western, Cell Adhesion Molecules, Cell Line, Electric Impedance, Fluorescein, Fluorescent Antibody Technique, Indirect, Humans, Membrane Proteins, Microfilament Proteins, Muscle Proteins, Myosin Light Chains, Occludin, Permeability, Phosphoproteins, Phosphorylation, Signal Transduction, Tight Junctions, Trabecular Meshwork, Transfection, Zonula Occludens-1 Protein, rhoA GTP-Binding Protein
Show Abstract · Added May 27, 2014
PURPOSE - Blood vessel epicardial substance (Bves) is a novel adhesion molecule that regulates tight junction (TJ) formation. TJs also modulate RhoA signaling, which has been implicated in outflow regulation. Given that Bves has been reported in multiple ocular tissues, the authors hypothesize that Bves plays a role in the regulation of RhoA signaling in trabecular meshwork (TM) cells.
METHODS - Human TM cell lines NTM-5 and NTM-5 transfected to overexpress Bves (NTM-w) were evaluated for TJ formation, and levels of occludin, cingulin, and ZO-1 protein were compared. Assays of TJ function were carried out using diffusion of sodium fluorescein and transcellular electrical resistance (TER). Levels of activated RhoA were measured using FRET probes, and phosphorylation of myosin light chain (MLC-p), a downstream target of RhoA, was assessed by Western blot analysis.
RESULTS - Overexpression of Bves led to increased TJ formation in NTM-5 cells. Increased TJ formation was confirmed by increased occludin, cingulin, and ZO-1 protein. Functionally, NTM-w cells showed decreased permeability and increased TER compared with NTM-5 cells, consistent with increased TJ formation. NTM-w cells also exhibited decreased levels of active RhoA and lower levels of MLC-p than did NTM-5 cells. These findings support a TJ role in RhoA signaling.
CONCLUSIONS - Increased Bves in TM cells leads to increased TJ formation with decreased RhoA activation and decreased MLC-p. This is the first report of a regulatory pathway upstream of RhoA in TM cells. In TM tissue, RhoA has been implicated in outflow regulation; thus, Bves may be a key regulatory molecule in aqueous outflow.
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22 MeSH Terms
Confocal microscopy reveals zones of membrane remodeling in the outer cortex of the human lens.
Lim JC, Walker KL, Sherwin T, Schey KL, Donaldson PJ
(2009) Invest Ophthalmol Vis Sci 50: 4304-10
MeSH Terms: Adolescent, Aged, Aquaporins, Cell Differentiation, Cell Membrane, Coloring Agents, Epithelial Cells, Eye Proteins, Fluorescent Antibody Technique, Indirect, Humans, Immunohistochemistry, Lens Cortex, Crystalline, Microscopy, Confocal, Microtomy, Middle Aged, Tissue Fixation, Wheat Germ Agglutinins
Show Abstract · Added May 27, 2014
PURPOSE - To optimize fixation, sectioning, and immunolabeling protocols to map the morphology of the human lens with confocal microscopy.
METHODS - Transparent human lenses were fixed in 0.75% paraformaldehyde for 24 hours, cut in half, and fixed for another 24 hours. Lenses were cryoprotected, sectioned, and labeled with wheat germ agglutinin, aquaporin-0 antibodies, Hoechst, or toluidine blue. Before fixation, some lenses were incubated in an extracellular marker dye, Texas Red-dextran. Labeled sections were imaged with a confocal microscope. Overlapping images were tiled together to form a continuous image montage of fiber cell morphology from the periphery to the lens center.
RESULTS - Fiber cell morphologies were identical with those previously described by electron microscopy and allowed immunohistochemistry to be performed for a representative membrane protein, aquaporin-0. Sectioning protocols enabled the epithelium and outer cortex to be retained, leading to the identification of two unique morphologic zones. In the first zone, an age-independent compaction of nucleated fiber cells and the initiation of extensive membrane remodeling occur. In the second zone, fiber cells retain their interdigitations but lose their nuclei, exhibit a distorted shape, and are less compressed. These zones are followed by the adult nucleus, which is marked by extensive compaction and a restriction of the extracellular space to the diffusion of Texas Red-dextran.
CONCLUSIONS - The authors have developed sectioning and imaging protocols to capture differentiation-dependent changes in fiber cell morphology and protein expression throughout the human lens. Results reveal that differentiating fiber cells undergo extensive membrane remodeling before their internalization into the adult nucleus.
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17 MeSH Terms
Rlbp1 promoter drives robust Müller glial GFP expression in transgenic mice.
Vázquez-Chona FR, Clark AM, Levine EM
(2009) Invest Ophthalmol Vis Sci 50: 3996-4003
MeSH Terms: Animals, Carrier Proteins, Female, Fluorescent Antibody Technique, Indirect, Gene Expression Regulation, Genotype, Green Fluorescent Proteins, Male, Mice, Mice, Inbred C57BL, Mice, Inbred CBA, Mice, Transgenic, Microscopy, Confocal, Neuroglia, Promoter Regions, Genetic, Retinal Degeneration, Retinal Neurons, Retinaldehyde
Show Abstract · Added November 2, 2015
PURPOSE - Müller glia are essential for maintaining retinal homeostasis and exhibit neuroprotective and deleterious responses during retinal degeneration. Having the ability to visualize and genetically manipulate Müller glia in vivo will facilitate a better understanding of how these cells contribute to these processes. The goal of this study was to determine whether regulatory elements of the retinaldehyde binding protein 1 (Rlbp1; formerly Cralbp) gene can drive robust Müller glial gene expression in vivo.
METHODS - Transgenic mice were generated by pronuclear injection of a construct carrying a 3-kilobase (kb) region of the Rlbp1 gene and 5'-flanking sequences linked to the enhanced green fluorescent protein (GFP) cDNA. GFP expression was analyzed by immunohistology in regions of the central nervous system in which RLBP1 protein is expressed, in retinas from wild-type and retinal degeneration 1 (rd1) mice, and during retinal development.
RESULTS - Three transgenic lines were generated, and the one with the strongest and most consistent GFP expression was characterized further. Müller glia displayed robust GFP expression at all postnatal developmental stages and in the rd1 retina. Onset of expression occurred by birth in retinal progenitor cells.
CONCLUSIONS - Regulatory elements in a restricted region of the Rlbp1 gene are sufficient to drive GFP expression in vivo. This transgenic line provides robust GFP expression that can be used to visualize retinal progenitor cells during postnatal development and Müller glia during their differentiation and in the healthy or degenerating adult retina.
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18 MeSH Terms
A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding.
Lan H, Liu Y, Bell MI, Gurevich VV, Neve KA
(2009) Mol Pharmacol 75: 113-23
MeSH Terms: Animals, Arrestin, Cell Line, Cyclic AMP, Dopamine, Extracellular Signal-Regulated MAP Kinases, Fluorescent Antibody Technique, Indirect, GTP-Binding Proteins, Glutathione Transferase, Horseradish Peroxidase, Humans, Kidney, Mutation, Protein Binding, Radioligand Assay, Rats, Receptors, Dopamine D2, Recombinant Fusion Proteins, Signal Transduction, Sulpiride, Time Factors, Transfection
Show Abstract · Added December 10, 2013
Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.
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22 MeSH Terms
A nonautonomous role for retinal frizzled-5 in regulating hyaloid vitreous vasculature development.
Zhang J, Fuhrmann S, Vetter ML
(2008) Invest Ophthalmol Vis Sci 49: 5561-7
MeSH Terms: Animals, Apoptosis, Cell Proliferation, Female, Fluorescent Antibody Technique, Indirect, Frizzled Receptors, Gene Expression Regulation, Developmental, Gene Silencing, Genotype, In Situ Hybridization, In Situ Nick-End Labeling, Integrases, Male, Mice, Mice, Knockout, Mice, Transgenic, Persistent Hyperplastic Primary Vitreous, Polymerase Chain Reaction, Receptors, G-Protein-Coupled, Retina, Retinal Dysplasia, Retinal Ganglion Cells, Vitreous Body
Show Abstract · Added November 19, 2015
PURPOSE - Frizzled-5 (Fzd5) is expressed in the developing retina of multiple species and appears to play species-specific roles during eye development. The present study analyzed the effects of tissue-specific deletion of Fzd5 on mammalian eye development.
METHODS - To generate Fzd5 conditional knockout (CKO) mice, Fzd5(+/-) mice carrying the Six3-Cre transgene were crossed with Fzd5(LoxP/LoxP) mice. To determine which cell lineages in the eye had Cre recombinase activity, Six3-Cre transgenic mice were crossed with ROSA-26 reporter mice, and lacZ activity was assayed. Histologic analysis, immunofluorescence, and TUNEL labeling were performed from embryonic day (E)12.5 to postnatal stages to analyze vascularization, cell proliferation, retinal organization, and apoptosis.
RESULTS - On conditional disruption of Fzd5 specifically in the retina, but not in vitreous hyaloid vasculature (VHV), an abnormal accumulation consisting of pericytes and endothelial cells was observed in the vitreous as early as E12.5. The abundant retrolental cells persisted into postnatal stages and appeared as a pigmented intravitreal mass. In Fzd5 CKO mice there was failure of normal apoptosis of the VHV, and cells in the persistent VHV were maintained in the cell cycle up to postnatal day 23. Moreover, morphogenesis of the retina adjacent to the vasculature was disrupted, leading to retinal folds, detachment, and abnormal lamination. This phenotype is similar to that of human eye disease persistent hyperplastic primary vitreous (PHPV).
CONCLUSIONS - Selective loss of Fzd5 in the retina results in PHPV and retinal defects through an apparently cell-nonautonomous effect, revealing a potential requirement for retina-derived signals in regulating the development of the VHV.
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23 MeSH Terms