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1.
Exp Eye Res ; 144: 73-80, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26142952

ABSTRACT

Optineurin is a cytosolic protein encoded by the OPTN gene. Mutations of OPTN are associated with normal tension glaucoma and amyotrophic lateral sclerosis. Autophagy is an intracellular degradation system that delivers cytoplasmic components to the lysosomes. It plays a wide variety of physiological and pathophysiological roles. The optineurin protein is a selective autophagy receptor (or adaptor), containing an ubiquitin binding domain with the ability to bind polyubiquitinated cargoes and bring them to autophagosomes via its microtubule-associated protein 1 light chain 3-interacting domain. It is involved in xenophagy, mitophagy, aggrephagy, and tumor suppression. Optineurin can also mediate the removal of protein aggregates through an ubiquitin-independent mechanism. This protein in addition can induce autophagy upon overexpression or mutation. When overexpressed or mutated, the optineurin protein also serves as a substrate for autophagic degradation. In the present review, the multiple connections of optineurin to autophagy are highlighted.


Subject(s)
Autophagy/genetics , Low Tension Glaucoma/genetics , Transcription Factor TFIIIA/genetics , Cell Cycle Proteins , Humans , Membrane Transport Proteins
2.
Protein Pept Lett ; 22(11): 1025-30, 2015.
Article in English | MEDLINE | ID: mdl-26343062

ABSTRACT

We previously reported that neostatin, a proteolytic fragment of collagen XVIII that includes endostatin, inhibits basic fibroblast growth factor-induced corneal angiogenesis and lymphangiogenesis. In experiments to determine which fragments in neostatin are responsible for binding to VEGF receptors (VEGFRs), we previously showed that a 28- mer sequence at the C-terminal of endostatin, known as endostatin peptide 9, preferentially binds VEGFR3-Fc over VEGFR1-Fc and VEGFR2-Fc. In the present study, we show that a different endostatin fragment, endostatin peptide 4 (26 mers long), also selectively binds VEGFR3-Fc and not VEGFR1-Fc or VEGFR2-Fc. From surface plasmon resonance data, the KD and Chi² (RU²) values for endostatin peptide 4 binding to VEGFR3-Fc are 5.72 × 10⁻8 M and 0.354, respectively. In conclusion, endostatin peptides 4 and 9 may be responsible for endostatin binding to VEGFR3-Fc, and this improved understanding of endostatin peptide binding to VEGFR3-Fc may support the development of therapeutics targeting lymphangiogenic processes.


Subject(s)
Endostatins/chemistry , Endostatins/metabolism , Vascular Endothelial Growth Factor Receptor-3/chemistry , Vascular Endothelial Growth Factor Receptor-3/metabolism , Amino Acid Sequence , Collagen Type XVIII , Molecular Sequence Data , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism
3.
BMC Cell Biol ; 16: 14, 2015 May 06.
Article in English | MEDLINE | ID: mdl-25943884

ABSTRACT

BACKGROUND: Optineurin is a gene associated with normal tension glaucoma and amyotrophic lateral sclerosis. It has been reported previously that in cultured RGC5 cells, the turnover of endogenous optineurin involves mainly the ubiquitin-proteasome pathway (UPP). When optineurin is upregulated or mutated, the UPP function is compromised as evidenced by a decreased proteasome ß5 subunit (PSMB5) level and autophagy is induced for clearance of the optineurin protein. RESULTS: Adeno-associated type 2 viral (AAV2) vectors for green fluorescence protein (GFP) only, GFP-tagged wild-type and Glu50Lys (E50K) mutated optineurin were intravitreally injected into rats for expression in retinal ganglion cells (RGCs). Following intravitreal injections, eyes that received optineurin vectors exhibited retinal thinning, as well as RGC and axonal loss compared to GFP controls. By immunostaining and Western blotting, the level of PSMB5 and autophagic substrate degradation marker p62 was reduced, and the level of autophagic marker microtubule associated protein 1 light chain 3 (LC3) was enhanced. The UPP impairment and autophagy induction evidently occurred in vivo as in vitro. The optineurin level, RGC and axonal counts, and apoptosis in AAV2-E50K-GFP-injected rat eyes were averted to closer to normal limits after treatment with rapamycin, an autophagic enhancer. CONCLUSIONS: The UPP function was reduced and autophagy was induced when wild-type and E50K optineurin was overexpressed in rat eyes. This study validates the in vitro findings, confirming that UPP impairment and autophagy induction also occur in vivo. In addition, rapamycin is demonstrated to clear the accumulated mutant optineurin. This agent may potentially be useful for rescuing of the adverse optineurin phenotypes in vivo.


Subject(s)
Autophagy , Transcription Factor TFIIIA/metabolism , Amino Acid Substitution , Animals , Cell Line , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Intraocular Pressure/drug effects , Male , Microtubule-Associated Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Rats , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/metabolism , Sirolimus/pharmacology , Transcription Factor TFIIIA/genetics
4.
Surv Ophthalmol ; 60(6): 524-35, 2015.
Article in English | MEDLINE | ID: mdl-25980779

ABSTRACT

Angiogenesis may pose a clinical challenge in glaucoma, for example, during the wound healing phase after glaucoma filtration surgery and in the severe secondary glaucoma called neovascular glaucoma (NVG). Upregulation of vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, occurs in eyes that have undergone glaucoma filtration surgery, as well as those with NVG. This has led investigation of the ability of anti-vascular endothelial growth factor therapy to improve outcomes, and we examine the findings with respect to the safety and efficacy of anti-vascular endothelial growth factor agents, mainly bevacizumab and ranibizumab, in eyes that have undergone glaucoma filtration surgery or have NVG. Combining conventional therapies-such as antimetabolites after filtration surgery and panretinal photocoagulation in NVG-and anti-vascular endothelial growth factor drugs may produce a synergetic effect, although further studies are required to evaluate the long-term efficacy of combination treatments.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Filtering Surgery/adverse effects , Glaucoma, Neovascular/etiology , Iris/blood supply , Neovascularization, Pathologic/etiology , Antimetabolites/therapeutic use , Bevacizumab/therapeutic use , Combined Modality Therapy , Glaucoma, Neovascular/drug therapy , Glaucoma, Neovascular/metabolism , Humans , Laser Coagulation , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Ranibizumab/therapeutic use , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism
5.
Springerplus ; 3: 99, 2014.
Article in English | MEDLINE | ID: mdl-24683533

ABSTRACT

Optineurin is a gene associated with normal tension glaucoma (NTG) and amyotrophic lateral sclerosis (ALS). Foci formation and functional consequences including Golgi fragmentation, impairment of vesicle trafficking and apoptosis were observed previously upon overexpression and/or mutation of optineurin. In the current study, a total of 15 GFP tagged constructs that included NTG (E50K and 2 bp-AG insertion), ALS (exon 5 deletion, R96L, Q398X, and E478G) and non-disease (L157A and D474N) associated mutants and a series of deletion fragments were cloned into mammalian expression vectors and transfected into RGC5 and/or Neuro2A cells to evaluate whether their expression confer the optineurin phenotypes. The cells were monitored for foci formation and stained by immunofluorescence with anti-GM130 to analyze the Golgi integrity. Transferrin uptake experiments were performed to evaluate the protein trafficking process and apoptosis was assessed with the active caspase 3/7 detection kit. We demonstrated that cells expressing E50K and R96L optineurin exhibited all of the optineurin phenotypes. Q398X mutant did not induce foci formation, but triggered Golgi fragmentation, impairment of transferrin uptake and increase in apoptosis. The 2 bp-AG insertion mutant had a nuclear localization, compromised the transferrin uptake and strongly induced apoptosis. The foci formation, which might not predict the rest of the phenotypes, appeared to require both the leucine zipper and ubiquitin binding domains of the optineurin sequence. Interactions of optineurin with proteins including Rab8, myosin VI, huntingtin and transferrin receptor might directly determine whether the Golgi and protein trafficking phenotypes would be manifested. Examination of mutants and deletion fragments located at various sites of optineurin gene provide clues as to what regions of the gene may play a critical role in the development of pathologic consequences.

6.
PLoS One ; 9(4): e92845, 2014.
Article in English | MEDLINE | ID: mdl-24732711

ABSTRACT

BACKGROUND: Myocilin (MYOC) is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Pro370Leu (P370L) and Gln368stop (Q368X) have been identified in patients. In the present study, we investigated the processing of myocilin in human trabecular meshwork (TM) cells as well as in inducible, stable RGC5 cell lines. METHODOLOGY/PRINCIPAL FINDINGS: The turnover and photoactivation experiments revealed that the endogenous myocilin in human trabecular meshwork (TM) cells was a short-lived protein. It was found that the endogenous myocilin level in TM cells was increased by treatment of lysosomal and proteasomal inhibitors, but not by autophagic inhibitor. Multiple bands immunoreactive to anti-ubiquitin were seen in the myocilin pull down, indicating that myocilin was ubiquitinated. In inducible cell lines, the turnover rate of overexpressed wild-type and mutant P370L and Q368X myocilin-GFP fusion proteins was much prolonged. The proteasome function was compromised and autophagy was induced. A decreased PSMB5 level and an increased level of autophagic marker, LC3, were demonstrated. CONCLUSIONS/SIGNIFICANCE: The current study provided evidence that in normal homeostatic situation, the turnover of endogenous myocilin involves ubiquitin-proteasome and lysosomal pathways. When myocilin was upregulated or mutated, the ubiquitin-proteasome function is compromised and autophagy is induced. Knowledge of the degradation pathways acting on myocilin can help in design of novel therapeutic strategies for myocilin-related glaucoma.


Subject(s)
Cytoskeletal Proteins/metabolism , Eye Proteins/metabolism , Glycoproteins/metabolism , Protein Processing, Post-Translational , Trabecular Meshwork/cytology , Trabecular Meshwork/metabolism , Adult , Autophagy/radiation effects , Cell Line , Cytoskeletal Proteins/antagonists & inhibitors , Eye Proteins/antagonists & inhibitors , Glycoproteins/antagonists & inhibitors , Green Fluorescent Proteins/metabolism , Humans , Light , Microtubule-Associated Proteins/metabolism , Mutation/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Processing, Post-Translational/radiation effects , Recombinant Fusion Proteins/metabolism , Trabecular Meshwork/radiation effects , Ubiquitination/radiation effects , Young Adult , beta Catenin/metabolism
7.
J Ocul Pharmacol Ther ; 30(2-3): 291-9, 2014.
Article in English | MEDLINE | ID: mdl-24611521

ABSTRACT

PURPOSE: The purpose of this study was to distinguish differences in gene expression between cells cultured from the juxtacanalicular trabecular meshwork (JCTM) and those from Schlemm's canal (SC), to gain clues to differences between those cell types, and to add to our baseline knowledge of gene expression differences in these cell types for later comparison between cells from nonprimary open-angle glaucoma (POAG) and POAG outflow tissues. METHODS: A set of JCTM and SC cells was cultured from each of 2 donor eyes by an explant method, grown to passage 3, and frozen in liquid nitrogen. The cells were thawed, total RNA was extracted, and the probes made from total RNAs were hybridized to MICROMAX human cDNA microarray slides in 2 separate trials. Differentially expressed genes were analyzed using PubMed, Prosite, and IPA software, and the expression of several of the genes including intercellular adhesion molecule-1 (ICAM-1), tenascin, and ß-spectrin was assessed by immunofluorescence. RESULTS: Schlemm's canal cells differentially expressed ICAM-1, spectrin, complement, fibulin-1, and several genes consistent with an endothelial origin in both arrays, while the JCTM cells more often overexpressed genes consistent with contractile, matrix function, and neural character. At the same time, many genes highly expressed in the first array were not highly overexpressed in the second. One highly overexpressed gene in the JCTM in both arrays, that for heparan sulfate 3-O-sulfotransferase-1 precursor, is thought to be somewhat unique, and could affect the glycosaminoglycan functionality in the extracellular matrix (ECM). CONCLUSIONS: We found generally good agreement between the 2 array trials, but some contradictions as well. Many of the genes overexpressed in each cell type had been described in earlier work, but several were new. Tables of genes, grouped by cellular function, and the complete datasets are provided for the development of new hypotheses.


Subject(s)
Gene Expression Regulation , Sclera/cytology , Trabecular Meshwork/cytology , Adolescent , Aged , Cells, Cultured , Glaucoma, Open-Angle/genetics , Glaucoma, Open-Angle/pathology , Humans , Oligonucleotide Array Sequence Analysis , RNA/metabolism
8.
Invest Ophthalmol Vis Sci ; 54(12): 7424-30, 2013 Nov 13.
Article in English | MEDLINE | ID: mdl-24106124

ABSTRACT

PURPOSE: Corneal stromal scarring partly involves the production of corneal myofibroblasts. The purpose of this study was to examine the effects of rapamycin (an inhibitor of the mammalian target of rapamycin [mTOR] pathway) on myofibroblast formation in vitro and in-vivo. METHODS: Human corneal fibroblasts were grown in culture and transformed into myofibroblasts using TGF-ß (2 ng/mL). The phosphorylation (activation) of the mTOR pathway was examined by immunoblotting. Cell proliferation with and without rapamycin was examined by thiazolyl blue tetrazolium bromide (MTT) assay and Ki67 staining. The expression of the myofibroblast differentiation marker smooth muscle actin (SMA) was examined by immunostaining and immunoblotting. The functional effects of rapamycin were measured using a gel contraction assay. For in vivo studies, 140 µm laser ablation was performed on rabbit corneas followed by subconjunctival rapamycin or vehicle. Corneal haze development was graded at 4 weeks, while the expression of myofibroblast markers was examined by immunostaining and immunoblotting. RESULTS: The TGF-ß activated the mTOR pathway with peak phosphorylation at 2 to 4 hours. Treatment of corneal fibroblasts with rapamycin reduced their proliferation by 46% compared to control. Rapamycin significantly inhibited TGF-ß-induced expression of myofibroblast markers (17.2% SMA positive cells with rapamycin compared to 69.0% in control). Rapamycin also significantly inhibited TGF-ß-induced collagen gel contraction. In the rabbit eyes treated with rapamycin, corneal haze development was significantly less compared to controls (0.75 ± 0.4 vs. 2.17 ± 0.7). CONCLUSIONS: Rapamycin appears to inhibit proliferation and differentiation of corneal myofibroblasts and, thus, may provide an effective therapeutic measure for preventing corneal scarring.


Subject(s)
Cicatrix/prevention & control , Corneal Opacity/surgery , Corneal Stroma/pathology , Myofibroblasts/pathology , Photorefractive Keratectomy , Sirolimus/pharmacology , Animals , Blotting, Western , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Cicatrix/metabolism , Cicatrix/pathology , Corneal Opacity/pathology , Corneal Stroma/metabolism , Corneal Stroma/surgery , Disease Models, Animal , Female , Humans , Immunosuppressive Agents/pharmacology , Myofibroblasts/drug effects , Myofibroblasts/metabolism , Rabbits , Transforming Growth Factor beta/metabolism
9.
Mol Pharm ; 10(8): 3023-3032, 2013 Aug 05.
Article in English | MEDLINE | ID: mdl-23795867

ABSTRACT

Accumulation of extracellular matrix (ECM) materials in the trabecular meshwork (TM) is believed to be a contributing factor to intraocular pressure (IOP) elevation, a risk factor/cause of primary open angle glaucoma, a major blinding disease. Matrix metalloproteinase-3 (MMP-3) is one of the proteinases that can effectively degrade ECM elements such as fibronectin, and MMP-3 delivery to the TM represents a promising approach for IOP reduction and treatment of glaucoma. In this study, we tested the feasibility of using polymeric microparticles to achieve a slow and sustained release of active MMP-3 to cultured human TM cells. ß-Casein, with molecular weight (24 kDa) and hydrophobicity similar to those of the active MMP-3 fragment (19.2 kDa), was first employed as a model for initial testing. ß-casein was encapsulated into poly(lactic-co-glycolic acid) (PLGA) microparticles using a double emulsion procedure at an encapsulation efficiency of approximately 45%. The PLGA microparticles were chosen given their biocompatibility and the proven capacity of sustained release of encapsulated molecules. The release test conducted in the culture medium showed a slow and sustained release of the protein over 20 days without a significant initial burst release. Active MMP-3 was subsequently encapsulated into PLGA microparticles with an encapsulation efficiency of approximately 50%. A biofunctional assay utilizing human TM cells was set up in which the reduction of fibronectin was used as an indicator of enzyme activity. It was observed that fibronectin staining was markedly reduced by the medium collected from MMP-3-microparticle-treated cultures compared to that from blank- and ß-casein-microparticle controls, which was validated using a direct MMP-3 activity assay. The controlled release of MMP-3 from the microparticles resulted in sustained degradation of fibronectin up to 10 days. This proof-of-concept undertaking represents the first study on the controlled and sustained release of active MMP-3 to TM cells via encapsulation into PLGA microparticles as a potential treatment of glaucoma.


Subject(s)
Lactic Acid/chemistry , Matrix Metalloproteinase 3/metabolism , Polyglycolic Acid/chemistry , Trabecular Meshwork/cytology , Glaucoma/metabolism , Humans , In Vitro Techniques , Matrix Metalloproteinase 3/chemistry , Matrix Metalloproteinase 3/pharmacokinetics , Microscopy, Electron, Scanning , Particle Size , Polylactic Acid-Polyglycolic Acid Copolymer , Trabecular Meshwork/metabolism
10.
Ophthalmic Genet ; 34(3): 167-73, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23289809

ABSTRACT

PURPOSE: To document a novel NYX gene mutation in a patient with X-linked complete congenital stationary night blindness and to describe this patient's electroretinogram (ERG) characteristics. METHODS: ERGs were recorded from a 17-year-old male with a previously unreported NYX mutation (819G > A) that results in a missense codon change (Trp237Ter). ERGs were recorded in response to brief-flash stimuli, 6.33-Hz sawtooth flicker, and sinusoidal flicker ranging from 6.33-100 Hz. The omitted stimulus response (OSR) of the flicker ERG, which is thought to be generated within the ON-pathway, was also assessed. RESULTS: The patient's single-flash responses were consistent with previously documented NYX ERG characteristics, including a high-luminance flash response that was electronegative under dark-adapted conditions and a square-like a-wave followed by an abnormally shaped positive potential under light-adapted conditions, both of which are consistent with an ON-pathway deficit. Further evidence for an ON-pathway deficit included: (1) ERGs to rapid-on sawtooth flicker in which b-wave amplitude was reduced more than a-wave amplitude, and (2) responses to sinusoidal flicker that lacked the normal amplitude minimum and phase inflection near 12 Hz, ERG characteristics that are like those of patients with other NYX mutations. Novel findings included a pronounced amplitude attenuation for sinusoidal flicker at frequencies above approximately 50 Hz and an absent OSR, suggesting ON-pathway dysfunction at high frequencies. CONCLUSION: The substantial loss of ERG amplitude and apparent ON-pathway dysfunction at high temporal frequencies distinguish this patient with a Trp237Ter NYX mutation from those with other previously reported NYX mutations.


Subject(s)
Eye Diseases, Hereditary/genetics , Eye Diseases, Hereditary/physiopathology , Genetic Diseases, X-Linked/genetics , Genetic Diseases, X-Linked/physiopathology , Mutation, Missense , Myopia/genetics , Myopia/physiopathology , Night Blindness/genetics , Night Blindness/physiopathology , Polymorphism, Single Nucleotide , Proteoglycans/genetics , Retina/physiopathology , Adolescent , Adult , Dark Adaptation , Electroretinography , Exons/genetics , Female , Humans , Male , Middle Aged , Photic Stimulation , Polymerase Chain Reaction , Young Adult
11.
PLoS One ; 7(10): e47307, 2012.
Article in English | MEDLINE | ID: mdl-23082156

ABSTRACT

BACKGROUND: Myocilin is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Gln368stop (Q368X) and Pro370Leu (P370L) have been identified in patients. The exact role of myocilin and its functional association with glaucoma are still unclear. In the present study, we established tetracycline-inducible (Tet-on) wild type and mutant myocilin-green fluorescence protein (GFP) expressing RGC5 stable cell lines and studied the changes in cell migration and barrier function upon induction. METHODOLOGY/PRINCIPAL FINDINGS: After several rounds of selection, clones that displayed low, moderate, or high expression of wild type, Q368X or P370L myocilin-GFP upon doxycycline (Dox) induction were obtained. The levels of wild type and mutant myocilin-GFP in various clones were confirmed by Western blotting. Compared to non-induced controls, the cell migration was retarded, the actin stress fibers were fewer and shorter, and the trypsinization time needed for cells to round up was reduced when wild type or mutant myocilin was expressed. The barrier function was in addition aberrant following induced expression of wild type, Q368X or P370L myocilin. Immunoblotting further showed that tight junction protein occludin was downregulated in induced cells. CONCLUSIONS/SIGNIFICANCE: Tet-on inducible, stable RGC5 cell lines were established. These cell lines, expressing wild type or mutant (Q368X or P370L) myocilin-GFP upon Dox induction, are valuable in facilitating studies such as proteomics, as well as functional and pathogenesis investigations of disease-associated myocilin mutants. The barrier function was found impaired and the migration of cells was hindered with induced expression of wild type and mutant myocilin in RGC5 cell lines. The reduction in barrier function might be related to the declined level of occludin. The retarded cell migration was consistent with demonstrated myocilin phenotypes including the loss of actin stress fibers, lowered RhoA activities and compromised cell-matrix adhesiveness.


Subject(s)
Cell Culture Techniques/methods , Cytoskeletal Proteins/metabolism , Eye Proteins/metabolism , Glycoproteins/metabolism , Green Fluorescent Proteins/metabolism , Mutant Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Actins/metabolism , Animals , Blotting, Western , Cell Line , Cell Movement/drug effects , Doxycycline/pharmacology , Humans , Plasmids/genetics , Rats , Staining and Labeling , Stress Fibers/drug effects , Stress Fibers/metabolism , Tetracycline/pharmacology , Trypsin/metabolism , rhoA GTP-Binding Protein/metabolism
12.
PLoS One ; 7(9): e44902, 2012.
Article in English | MEDLINE | ID: mdl-23028669

ABSTRACT

BACKGROUND: Myocilin is a gene linked to the most prevalent form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The Pro³7° to Leu (P370L) mutation of myocilin is associated with severe glaucoma phenotypes and Gln³68 stop (Q368X) is the most common myocilin mutation reported. Myocilin, upon overexpression, has been shown to induce phenotypes that include a loss of actin stress fibers, an increase in the cAMP level and protein kinase A (PKA) activity, as well as a reduction in the RhoA activity. We examined herein whether Wnt signaling pathway is involved in the myocilin phenotypes and whether P370L and Q368X mutants also display biological effects similar to those of the wild type myocilin. METHODOLOGY/PRINCIPAL FINDINGS: Wild type myocilin, when transfected into cultured human TM cells, induced a loss of actin stress fibers as judged by phalloidin staining. Such a loss was averted by treatment of secreted Frizzled-related protein 1 (sFRP1), an inhibitor of Wnt signaling. Consistent with the notion that Wnt pathway mediates the myocilin phenotype, Wnt activation was demonstrated by TOP/FOP-Flash reporter assays. Treatment of human TM cells of a Wnt activator, SB216763, as well as transfection of myocilin P370L and Q368X mutants all resulted in actin stress fiber loss, PKA activation and RhoA inactivation. The PKA elevation was obviated by the sFRP1 treatment, indicating that Wnt signaling was upstream that of PKA. CONCLUSIONS/SIGNIFICANCE: The present study demonstrated that following forced expression of wild type myocilin, Wnt was activated, triggering in turn other myocilin-related alterations. P370L and Q368X mutations induced similar phenotypes, suggesting one possible mechanism how the mutants may lead to TM cell damage and pathology.


Subject(s)
Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Eye Proteins/genetics , Eye Proteins/metabolism , Glycoproteins/genetics , Glycoproteins/metabolism , Mutation , Trabecular Meshwork/cytology , Wnt Proteins/metabolism , Actins/metabolism , Adult , Caco-2 Cells , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Focal Adhesions/drug effects , Focal Adhesions/metabolism , Humans , Indoles/pharmacology , Intracellular Signaling Peptides and Proteins , Maleimides/pharmacology , Middle Aged , Phenotype , Proteins/pharmacology , Signal Transduction/drug effects , Trabecular Meshwork/drug effects , Trabecular Meshwork/metabolism , Transfection , Young Adult
13.
Invest Ophthalmol Vis Sci ; 53(12): 7476-83, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-23049092

ABSTRACT

PURPOSE: To determine the role of Notch signaling in corneal epithelial migration and wound healing. METHODS: Immunolocalization of Notch1 was performed during epithelial wound healing in vivo in mouse corneal epithelial debridement wounds and in vitro in primary human corneal epithelial cells following a linear scratch wound. The effects of Notch inhibition, using the γ-secretase inhibitor N-(N-[3,5-difluorophenacetyl]-l-alanyl)-S-phenylglycine t-butyl ester (DAPT) or following stable transfection with Notch1-short hairpin RNA (shRNA), was evaluated in a scratch assay and transwell migration assay. Likewise, in vitro adhesion, proliferation and the actin cytoskeleton was examined. The DAPT effect was also evaluated in vivo in a mouse model of corneal epithelial wound healing. RESULTS: The expression of Notch1 was reduced at the leading edge of a healing corneal epithelium both in vivo and in vitro. Notch inhibition using DAPT and using Notch1-shRNA both enhanced in vitro migration in scratch and transwell migration assays. Consistent with this increased migratory behavior, Notch inhibited cells demonstrated decreased cell-matrix adhesion and enhanced lamellipodia formation. Notch inhibition by DAPT was also found to accelerate corneal epithelial wound closure in an in vivo murine model without affecting proliferation. CONCLUSIONS: The results highlight the role of Notch in regulating corneal epithelial migration and wound healing. In particular, Notch signaling appears to decrease in the early stages of wound healing which contributes to cytoskeletal changes with subsequent augmentation of migratory behavior.


Subject(s)
Corneal Diseases/metabolism , Epithelium, Corneal/metabolism , Eye Injuries/metabolism , Gene Expression Regulation , RNA/genetics , Receptor, Notch1/genetics , Wound Healing/genetics , Animals , Cell Movement , Cells, Cultured , Corneal Diseases/genetics , Corneal Diseases/pathology , Epithelium, Corneal/injuries , Epithelium, Corneal/pathology , Eye Injuries/pathology , Humans , Mice , Phosphorylation , Receptor, Notch1/biosynthesis , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction
14.
Int Rev Cell Mol Biol ; 294: 223-58, 2012.
Article in English | MEDLINE | ID: mdl-22364875

ABSTRACT

Optineurin is a gene linked to glaucoma, amyotrophic lateral sclerosis, other neurodegenerative diseases, and Paget's disease of bone. This review describes the characteristics of optineurin and summarizes the cellular and molecular biology investigations conducted so far on optineurin. Data from a number of laboratories indicate that optineurin is a cytosolic protein containing 577 amino acid residues. Interacting with proteins such as myosin VI, Rab8, huntingtin, transferrin receptor, and TANK-binding kinase 1, optineurin is involved in basic cellular functions including protein trafficking, maintenance of the Golgi apparatus, as well as NF-κB pathway, antiviral, and antibacteria signaling. Mutation or alteration of homeostasis of optineurin (such as overexpression or knockdown) results in adverse consequences in the cells, leading to the development of neurodegenerative diseases including glaucoma.


Subject(s)
Transcription Factor TFIIIA/chemistry , Transcription Factor TFIIIA/physiology , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Cell Cycle Proteins , Golgi Apparatus/chemistry , Golgi Apparatus/metabolism , Humans , Membrane Transport Proteins , Protein Transport/physiology , Signal Transduction/genetics , Signal Transduction/physiology , Transcription Factor TFIIIA/genetics
15.
J Biol Chem ; 286(5): 3618-29, 2011 Feb 04.
Article in English | MEDLINE | ID: mdl-21059646

ABSTRACT

Optineurin is a gene linked to amyotrophic lateral sclerosis, Paget disease of bone, and glaucoma, a major blinding disease. Mutations such as E50K were identified in glaucoma patients. We investigated herein the involvement of ubiquitin-proteasome pathway (UPP) and autophagy, two major routes for protein clearance, in processing of optineurin in a retinal ganglion cell model line RGC5 and neuronal PC12 cells. It was found that the endogenous optineurin level in neuronal cells was increased by treatment of proteasomal inhibitor but not by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pulldown, indicating that optineurin was ubiquitinated. In cells overexpressing wild type and E50K optineurin, the level of the proteasome regulatory ß5 subunit (PSMB5, indicative of proteasome activity) was reduced, whereas that for autophagy marker microtubule-associated protein 1 light chain 3 was enhanced compared with controls. Autophagosome formation was detected by electron microscopy. The foci formed after optineurin transfection were increased upon treatment of an autophagic inhibitor but were decreased by treatment of an inducer, rapamycin. Moreover, the level of optineurin-triggered apoptosis was reduced by rapamycin. This study thus provides compelling evidence that in a normal homeostatic situation, the turnover of endogenous optineurin involves mainly UPP. When optineurin is up-regulated or mutated, the UPP function is compromised, and autophagy comes into play. A decreased PSMB5 level and an induced autophagy were also demonstrated in vivo in retinal ganglion cells of E50K transgenic mice, validating and making relevant the in vitro findings.


Subject(s)
Autophagy , Neurons/metabolism , Proteasome Endopeptidase Complex/metabolism , Transcription Factor TFIIIA/metabolism , Ubiquitination , Animals , Apoptosis , Cell Cycle Proteins , Cell Line , Eye Proteins , Humans , Membrane Transport Proteins , Mice , Mice, Transgenic , Neurons/cytology , PC12 Cells , Rats , Retina
16.
Invest Ophthalmol Vis Sci ; 51(9): 4549-56, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20805571

ABSTRACT

PURPOSE: To evaluate the role of crumbs genes and related epithelial polarity loci in the vertebrate cornea. METHODS: The authors used histologic analysis and electron microscopy to evaluate the corneas of zebrafish mutant for a crumbs locus oko meduzy (ome) and in mutants of four other loci, nagie oko (nok), heart and soul (has), mosaic eyes (moe), and ncad (formerly glass onion), that function in the same or related genetic pathways. In parallel, they performed an evaluation of corneas in human carriers of a crumbs gene, CRB1, and mutations using topography and biomicroscopy. The expression of the CRB1 gene in the normal human cornea was examined by polymerase chain reaction (PCR) and immunohistochemical staining. RESULTS: The corneas of zebrafish mutants display severe abnormalities of the epithelial and stromal layers. The epithelial cells do not properly adhere to each other, and fluid-filled spaces form between them. In addition, the layering of the corneal stroma is poorly formed or absent. The corneas of human carriers of CRB1 mutations display shape deviations compared with what has been observed in normal individuals. A PCR product of the correct size was obtained from normal human corneal samples. Sequence analyses confirmed its identity to be the human CRB1 gene. Immunohistochemical staining using anti-CRB1 yielded positive brown deposits in the human cornea. CONCLUSIONS: crumbs genes play a role in the differentiation of the vertebrate cornea. Corneal defects associated with crumbs gene mutations are very severe in the zebrafish model and, in comparison, appear clinically less pronounced in the human eye.


Subject(s)
Epithelium, Corneal/abnormalities , Epithelium, Corneal/physiology , Eye Proteins/genetics , Gene Expression Regulation, Developmental , Membrane Proteins/genetics , Nerve Tissue Proteins/genetics , Zebrafish Proteins/genetics , Animals , Cell Polarity/physiology , Epithelial Cells/pathology , Epithelial Cells/physiology , Epithelial Cells/ultrastructure , Epithelium, Corneal/pathology , Eye Proteins/metabolism , Female , Heterozygote , Humans , Immunohistochemistry , Male , Membrane Proteins/metabolism , Microscopy, Electron , Nerve Tissue Proteins/metabolism , Pedigree , Polymerase Chain Reaction , Severity of Illness Index , Stromal Cells/pathology , Stromal Cells/physiology , Stromal Cells/ultrastructure , Vertebrates , Zebrafish , Zebrafish Proteins/metabolism
17.
PLoS One ; 5(7): e11547, 2010 Jul 12.
Article in English | MEDLINE | ID: mdl-20634958

ABSTRACT

BACKGROUND: Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu(50) to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis. METHODOLOGY/PRINCIPAL FINDINGS: Human retinal pigment epithelial (RPE) and rat RGC5 cells transfected to overexpress wild type optineurin were incubated with Texas Red-Tf to evaluate Tf uptake. Granular structures or dots referred to as foci formed in perinuclear regions after transfection. An impairment of the Tf uptake was in addition observed in transfected cells. Compared to overexpression of the wild type, E50K mutation yielded an increased foci formation and a more pronounced defect in Tf uptake. Co-transfection with TfR, but not Rab8 or myosin VI, construct rescued the optineurin inhibitory effect, suggesting that TfR was the factor involved in the trafficking phenotype. Forced expression of both wild type and E50K optineurin rendered TfR to colocalize with the foci. Surface biotinylation experiments showed that the surface level of TfR was also reduced, leading presumably to an impeded Tf uptake. A non-consequential Leu(157) to Ala (L157A) mutation that displayed much reduced foci formation and TfR binding had normal TfR distribution, normal surface TfR level and normal Tf internalization. CONCLUSIONS/SIGNIFICANCE: The present study demonstrates that overexpression of wild type optineurin results in impairment of the Tf uptake in RPE and RGC5 cells. The phenotype is related to the optineurin interaction with TfR. Our results further indicate that E50K induces more dramatic effects than the wild type optineurin, and is thus a gain-of-function mutation. The defective protein trafficking may be one of the underlying bases why glaucoma pathology develops in patients with E50K mutation.


Subject(s)
Mutation , Transcription Factor TFIIIA/genetics , Transcription Factor TFIIIA/metabolism , Animals , Cell Cycle Proteins , Cell Line , Endocytosis/genetics , Endocytosis/physiology , Humans , Immunoblotting , Membrane Transport Proteins , Microscopy, Fluorescence , Protein Transport/genetics , Protein Transport/physiology , RNA, Small Interfering , Rats , Receptors, Transferrin/genetics , Receptors, Transferrin/metabolism , Transferrin/metabolism
18.
PLoS One ; 5(2): e9168, 2010 Feb 11.
Article in English | MEDLINE | ID: mdl-20161783

ABSTRACT

BACKGROUND: Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin. METHODOLOGY/PRINCIPAL FINDINGS: Lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation or membrane protein extraction. The phosphorylation status was evaluated after immunoprecipitation. It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. RGC5 and human retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with molecules including Rab8, myosin VI, and transferrin receptor to assemble into supermolecular complexes. When overexpressed, optineurin-green fluorescence protein (GFP) fusion protein formed punctate structures termed "foci" in the perinuclear region. Treatment of nocadazole resulted in dispersion of the optineurin foci. In addition, tetracycline-regulated optineurin-GFPs expressing RGC5 stable cell lines were established for the first time. CONCLUSIONS/SIGNIFICANCE: The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology. The inducible optineurin-GFP-expressing cell lines are also anticipated to facilitate in-depth studies of optineurin. Furthermore, the demonstrations that optineurin is an aggregation-prone protein and that the foci formation is microtubule-dependent bear similarities to features documented in neurodegenerative diseases, supporting a neurodegenerative paradigm for glaucoma.


Subject(s)
Low Tension Glaucoma/metabolism , Protein Processing, Post-Translational , Transcription Factor TFIIIA/metabolism , Animals , Blotting, Western , Cell Cycle Proteins , Cell Line , Glycosylation , Golgi Apparatus/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Low Tension Glaucoma/genetics , Membrane Transport Proteins , Microscopy, Confocal , Myosin Heavy Chains/metabolism , Phosphorylation , Protein Binding , Protein Multimerization , Rats , Receptors, Transferrin/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/metabolism , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/metabolism , Transcription Factor TFIIIA/chemistry , Transcription Factor TFIIIA/genetics , Transfection
19.
AIDS Res Hum Retroviruses ; 20(3): 327-35, 2004 Mar.
Article in English | MEDLINE | ID: mdl-15117456

ABSTRACT

Mannose-binding lectin (MBL), a microbe-recognition protein in serum, binds to high mannose glycans on HIV-1 gp120 and has been reported to neutralize the cell line-adapted strain HIV(IIIB). Because HIV primary isolates (PI) are generally more resistant to neutralization by antibodies and considering that PI are produced in primary cells that could alter the number of high mannose glycans on HIV relative to cell lines, we assessed the ability to MBL to neutralize HIV PI. MBL at concentrations up to 50 microg/ml mediated relatively little neutralization (<20%) of HIV PI infection of peripheral blood mononuclear cells (PBMCs). MBL-neutralizing activity was slightly higher for cell line-adapted HIV infection of the H9 T cell line (up to 64% at 50 microg/ml). However, this effect was specific for H9 cells since MBL did not neutralize cell line-adapted virus infection of PBMCs, HIV PI infection of the GHOST cell line, or VSV pseudotyped with HIV gp160 from cell line-derived virus or PI. In contrast to its low activity in neutralization assays, MBL efficiently bound infectious HIV PI and opsonized HIV PI for uptake by monocytic cells. These results show that both PI and cell line-adapted HIV, despite binding of MBL, are relatively resistant to neutralization by levels of MBL normally present in serum. However, binding and opsonization of HIV by MBL may alter virus trafficking and viral-antigen presentation during HIV infection.


Subject(s)
HIV-1/metabolism , Mannose-Binding Lectin/metabolism , Cell Line , Cells, Cultured , HIV Infections/virology , HIV-1/isolation & purification , Humans , Leukocytes, Mononuclear/virology , Mannose-Binding Lectin/genetics , Neutralization Tests , Opsonin Proteins , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , T-Lymphocytes/virology
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