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1.
Am J Hum Genet ; 96(2): 266-74, 2015 Feb 05.
Article in English | MEDLINE | ID: mdl-25620203

ABSTRACT

Singleton-Merten syndrome (SMS) is an autosomal-dominant multi-system disorder characterized by dental dysplasia, aortic calcification, skeletal abnormalities, glaucoma, psoriasis, and other conditions. Despite an apparent autosomal-dominant pattern of inheritance, the genetic background of SMS and information about its phenotypic heterogeneity remain unknown. Recently, we found a family affected by glaucoma, aortic calcification, and skeletal abnormalities. Unlike subjects with classic SMS, affected individuals showed normal dentition, suggesting atypical SMS. To identify genetic causes of the disease, we performed exome sequencing in this family and identified a variant (c.1118A>C [p.Glu373Ala]) of DDX58, whose protein product is also known as RIG-I. Further analysis of DDX58 in 100 individuals with congenital glaucoma identified another variant (c.803G>T [p.Cys268Phe]) in a family who harbored neither dental anomalies nor aortic calcification but who suffered from glaucoma and skeletal abnormalities. Cys268 and Glu373 residues of DDX58 belong to ATP-binding motifs I and II, respectively, and these residues are predicted to be located closer to the ADP and RNA molecules than other nonpathogenic missense variants by protein structure analysis. Functional assays revealed that DDX58 alterations confer constitutive activation and thus lead to increased interferon (IFN) activity and IFN-stimulated gene expression. In addition, when we transduced primary human trabecular meshwork cells with c.803G>T (p.Cys268Phe) and c.1118A>C (p.Glu373Ala) mutants, cytopathic effects and a significant decrease in cell number were observed. Taken together, our results demonstrate that DDX58 mutations cause atypical SMS manifesting with variable expression of glaucoma, aortic calcification, and skeletal abnormalities without dental anomalies.


Subject(s)
Aortic Diseases/genetics , DEAD-box RNA Helicases/genetics , Dental Enamel Hypoplasia/genetics , Glaucoma/genetics , Metacarpus/abnormalities , Models, Molecular , Muscular Diseases/genetics , Odontodysplasia/genetics , Osteoporosis/genetics , Vascular Calcification/genetics , Adult , Aortic Diseases/pathology , Base Sequence , Cells, Cultured , Child, Preschool , DEAD Box Protein 58 , DEAD-box RNA Helicases/chemistry , Dental Enamel Hypoplasia/pathology , Exome/genetics , Female , Genes, Dominant/genetics , Humans , Male , Metacarpus/pathology , Molecular Sequence Data , Muscular Diseases/pathology , Musculoskeletal Abnormalities/diagnostic imaging , Musculoskeletal Abnormalities/genetics , Mutation, Missense/genetics , Odontodysplasia/diagnostic imaging , Odontodysplasia/pathology , Osteoporosis/pathology , Pedigree , Polymorphism, Single Nucleotide/genetics , Radiography , Receptors, Immunologic , Sequence Analysis, DNA , Vascular Calcification/pathology
2.
Muscle Nerve ; 45(5): 713-20, 2012 May.
Article in English | MEDLINE | ID: mdl-22499099

ABSTRACT

INTRODUCTION: We investigated the distribution of myosin heavy chain (MyHC) isoforms along the length of the global and orbital layers of human extraocular muscles (EOMs). METHODS: Whole muscle tissue extracts of human EOMs were cross-sectioned consecutively and separated into orbital and global layers. The extracts from these layers were subjected to electrophoretic analysis, followed by quantification with scanning densitometry. RESULTS: MyHC isoforms displayed different distributions along the lengths of EOMs. In the orbital and global layers of all EOMs except for the superior oblique muscle, MyHCeom was enriched in the central regions. MyHCIIa and MyHCI were most abundant in the proximal and distal ends. CONCLUSIONS: A variation in MyHC isoform expression was apparent along the lengths of human EOMs. These results provide a basis for understanding the molecular mechanisms underlying the functional diversity of EOMs.


Subject(s)
Myosin Heavy Chains/metabolism , Oculomotor Muscles/metabolism , Adult , Female , Humans , Longitudinal Studies , Male , Middle Aged , Oculomotor Muscles/anatomy & histology , Protein Isoforms/metabolism
3.
Mol Vis ; 15: 545-56, 2009.
Article in English | MEDLINE | ID: mdl-19287508

ABSTRACT

PURPOSE: Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein. METHODS: Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry. RESULTS: Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER. CONCLUSIONS: Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.


Subject(s)
Cytoskeletal Proteins/metabolism , Endoplasmic Reticulum/metabolism , Extracellular Space/metabolism , Eye Proteins/metabolism , Glycoproteins/metabolism , Cell Line , Cell Proliferation , Cytoskeletal Proteins/chemistry , Endoplasmic Reticulum Chaperone BiP , Eye Proteins/chemistry , Female , Glaucoma, Open-Angle/metabolism , Glycoproteins/chemistry , Green Fluorescent Proteins , Heat-Shock Proteins/biosynthesis , Humans , Molecular Chaperones/biosynthesis , Oligopeptides , Peptides , Protein Folding , Recombinant Fusion Proteins , Secretory Pathway , Time Factors , Trabecular Meshwork/metabolism , Up-Regulation
4.
Invest Ophthalmol Vis Sci ; 43(12): 3680-5, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12454036

ABSTRACT

PURPOSE: To investigate the subcellular localizations and potential cytotoxicities of wild-type and truncated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: Full-length wild-type myocilin, truncated myocilin, and stromelysin were expressed as green fluorescence (GFP) or DsRed fusion proteins in TM cells by using adenoviral vectors, and their secretory properties and cytopathic effects were evaluated by Western blot analysis and cell proliferation assay, respectively. To determine the subcellular localizations of myocilins, the cellular organelles of the infected TM cells were stained with organelle-specific antibodies or fluorescent indicators and examined under a confocal microscope. RESULTS: Wild-type myocilin was expressed mainly in the perinuclear region of TM cells and was localized preferentially in endoplasmic reticulum (ER), but not in actin, microtubules, or mitochondria. Truncated myocilin was also localized in ER, and its expression was found to be potentially toxic to TM cells, leading to deformed cellular morphology and diminished cell proliferation, but it had no effect on the secretion of stromelysin. The truncated myocilin was also found to be colocalized with and appeared to aggregate with wild-type myocilin when the proteins were coexpressed. CONCLUSIONS: TM cells participating in the turnover of trabecular extracellular matrix (ECM) components are important in regulating aqueous outflow. The truncated myocilin, colocalized and coaggregated with wild-type myocilin, is believed to cause a dysfunction of the cells, resulting in alterations in structural compartmentalization of trabecular ECM and obstruction of aqueous outflow.


Subject(s)
Eye Proteins/metabolism , Eye Proteins/toxicity , Glycoproteins/metabolism , Glycoproteins/toxicity , Trabecular Meshwork/metabolism , Adenoviridae/genetics , Blotting, Western , Cell Division , Cells, Cultured , Cytoskeletal Proteins , Endoplasmic Reticulum/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/metabolism , Eye Proteins/genetics , Gene Expression , Genetic Vectors , Glycoproteins/genetics , Green Fluorescent Proteins , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Microscopy, Confocal , Recombinant Fusion Proteins/metabolism , Subcellular Fractions , Trabecular Meshwork/drug effects , Trabecular Meshwork/pathology
5.
J Ocul Pharmacol Ther ; 30(10): 847-53, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25188901

ABSTRACT

PURPOSE: The study investigated the effect of intravitreally administered tanibirumab, a fully human monoclonal antibody against vascular endothelial growth factor receptor 2, in a rat model of laser-induced choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation on day 0 in the eyes of Brown Norway rats. Intravitreal injection of tanibirumab or phosphate-buffered saline (PBS) was done on day 0 (prevention arm) or day 7 (treatment arm). Seven days after injection, the eyes were enucleated and retinal pigment epithelium-choroid-sclera flat mounts were prepared. Areas of CNV were determined in the flat mounts using tetramethylrhodamine isothiocyanate Bandeiraea simplicifolia (BS) isolectin labeling and intravenously administered fluorescein isothiocyanate-dextran and quantified using an image analysis program. RESULTS: In the prevention arm, the mean area of CNV measured by BS isolectin labeling was reduced by 28.2% and 53.9% in tanibirumab-treated eyes (20 and 60 µg, respectively) compared with PBS-treated control eyes on day 7 (P=0.038 and P<0.001, respectively). In the treatment arm, the mean area of CNV measured by BS isolectin labeling was reduced by 28.7% and 46.0% in tanibirumab-treated eyes (20 and 60 µg, respectively) compared with PBS-treated control eyes on day 14 (P=0.048 and P<0.001, respectively). CONCLUSIONS: Intravitreally administered tanibirumab partially suppressed the formation of new CNV and partially regressed preformed laser-induced CNV in the rat model. Tanibirumab may be a feasible treatment for CNV associated with age-related macular degeneration or other causes.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Choroidal Neovascularization/drug therapy , Animals , Antibodies, Monoclonal, Humanized , Choroidal Neovascularization/etiology , Choroidal Neovascularization/pathology , Disease Models, Animal , Humans , Intravitreal Injections , Laser Coagulation , Plant Lectins/chemistry , Random Allocation , Rats , Staining and Labeling/methods , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors
6.
Korean J Ophthalmol ; 27(1): 48-51, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23372380

ABSTRACT

PURPOSE: To assess whether the expression of heat shock protein 72 (Hsp72) protects rat retinal ganglion cells (RGC-5) from apoptotic cell death. METHODS: Hsp72 expression in RGC-5 cells transduced with replication-deficient recombinant adenovirus was analyzed by Western blot analysis and immunofluorescence. The effect of Hsp72 expression on etoposide-induced apoptotic cell death was examined by microscopic analysis and confirmed by cell proliferation assay. RESULTS: Western blot analysis and immunofluorescence clearly showed adenovirus-mediated Hsp72 expression in RGC-5 cells. Treatment with etoposide resulted in the death of a proportion of the cells by apoptosis. However, this apoptotic cell death was significantly reduced in cells expressing Hsp72, with the reduction in cell death correlating to the level of Hsp72 expression. CONCLUSIONS: Over-expression of Hsp72 alone is sufficient to rescue neuronal cells from apoptotic cell death, suggesting that fine-tuning its expression may be an effective neuroprotective approach in retinal degenerative disease.


Subject(s)
Cell Death/genetics , DNA/genetics , Gene Expression Regulation , HSP72 Heat-Shock Proteins/genetics , Retinal Degeneration/genetics , Retinal Ganglion Cells/metabolism , Animals , Blotting, Western , Cell Survival , Cells, Cultured , Disease Models, Animal , Etoposide/toxicity , HSP72 Heat-Shock Proteins/biosynthesis , Immunohistochemistry , Rats , Retinal Degeneration/metabolism , Retinal Degeneration/pathology , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/pathology
7.
Vision Res ; 51(9): 1033-8, 2011 May 11.
Article in English | MEDLINE | ID: mdl-21334360

ABSTRACT

To understand the regulatory mechanisms governing glucocorticoid-mediated MYOC induction in human trabecular meshwork (HTM) cells, the expression and degradation of MYOC mRNA were quantified in HTM cells by Northern blot analysis, and the transcriptional activity of constructs containing variable lengths of putative MYOC promoters was assessed by luciferase reporter assay. Here, we confirmed that MYOC is a delayed secondary glucocorticoid-responsive gene by demonstrating that its transcription was not initiated immediately by the addition of dexamethasone (DEX) and was completely inhibited by treatment with cycloheximide. In addition, we demonstrated that MYOC mRNA is degraded very slowly, with approximately half persisting for at least 4 days, suggesting that its mRNA is intrinsically quite stable. Promoter analysis of up to 5271 base pairs upstream of MYOC revealed that luciferase induction by DEX was increased by 280 ± 34% in HTM cells. Moreover, DEX induction required the region between base pairs -2548 and -1541. However, the putative regulatory element exhibited little activity in other cell lines, including TM-5, 293A, SH-SY5Y, and human retinal pigment epithelium (RPE) cells. To our knowledge, this study provides the first evidence for the presence of a cis-acting region for secondary glucocorticoid responsiveness in the 5'-flanking sequences of MYOC. It will be a major step towards understanding the expression pattern of MYOC in HTM cells and TM tissue.


Subject(s)
Cytoskeletal Proteins/metabolism , Dexamethasone/pharmacology , Eye Proteins/metabolism , Glucocorticoids/pharmacology , Glycoproteins/metabolism , Trabecular Meshwork/metabolism , Blotting, Northern , Cells, Cultured , Cycloheximide/pharmacology , Cytoskeletal Proteins/genetics , Eye Proteins/genetics , Glycoproteins/genetics , Humans , Protein Synthesis Inhibitors/pharmacology , RNA, Messenger/metabolism , Trabecular Meshwork/drug effects
8.
Br J Ophthalmol ; 94(5): 639-42, 2010 May.
Article in English | MEDLINE | ID: mdl-20447966

ABSTRACT

BACKGROUND/AIM To determine if overexpression of the glaucoma gene MYOC is involved in the development of open-angle glaucoma (OAG) and if its promoter variants are associated with glaucoma in the Korean population. METHODS Human trabecular meshwork cells were cultured in the presence of ophthalmic steroids such as fluorometholone, fluorometholone acetate, dexamethasone, prednisolone acetate and rimexolone. The cells were cultured at a hydrostatic pressure of 32 mm Hg above atmospheric pressure and induction of MYOC was evaluated by northern blot analysis. Genomic DNA was extracted from blood samples obtained from 74 normal controls and 168 unrelated Korean patients with OAG, including primary OAG, normal tension glaucoma and steroid-induced glaucoma. A 461 base pair (bp) DNA fragment of the MYOC promoter region was amplified using PCR and its genotype was analysed by directly sequencing the product. RESULTS The potencies of steroid eye drops in MYOC induction in vitro was the same regardless of their potential for elevating intraocular pressure in vivo. Hydrostatic pressure had no effect on MYOC induction. A dinucleotide repeat polymorphism and three single nucleotide polymorphisms were identified, but no obvious differences in the genotype distribution and allele frequency of the variants between the control group and any type of OAG were observed. CONCLUSION Our data suggest that MYOC overexpression is not a cause or an effect of intraocular pressure elevation and that MYOC itself is not associated with OAG.


Subject(s)
Cytoskeletal Proteins/genetics , Eye Proteins/genetics , Glaucoma, Open-Angle/genetics , Glycoproteins/genetics , Cells, Cultured , Cytoskeletal Proteins/metabolism , Eye Proteins/metabolism , Gene Expression Regulation/drug effects , Gene Frequency , Genetic Predisposition to Disease , Glucocorticoids/pharmacology , Glycoproteins/metabolism , Humans , Hydrostatic Pressure , Polymorphism, Single Nucleotide , Trabecular Meshwork/cytology , Trabecular Meshwork/drug effects , Trabecular Meshwork/metabolism
9.
Biochem Biophys Res Commun ; 336(4): 1201-6, 2005 Nov 04.
Article in English | MEDLINE | ID: mdl-16198165

ABSTRACT

Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG.


Subject(s)
Cytoskeletal Proteins/metabolism , Eye Proteins/metabolism , Glaucoma, Open-Angle/metabolism , Glycoproteins/metabolism , Membrane Proteins/metabolism , Cell Line , Cytoskeletal Proteins/genetics , Eye Proteins/genetics , Gene Library , Glaucoma, Open-Angle/genetics , Glycoproteins/genetics , Humans , Immunoprecipitation , Mutation , Protein Binding , Protein Structure, Tertiary , Two-Hybrid System Techniques
10.
Biochem Biophys Res Commun ; 312(3): 592-600, 2003 Dec 19.
Article in English | MEDLINE | ID: mdl-14680806

ABSTRACT

MYOC encoding a 55kDa secretory glycoprotein named myocilin is closely linked to primary open-angle glaucoma (POAG). To understand a role played by MYOC in glaucoma, we examined the cellular fate of various mutant myocilins that were adenovirally expressed in human trabecular meshwork cells. Most myocilins with mutations such as G364V, Q368X, K423E, Y437H, and I477N were intrinsically stable, and appeared to have interactions with wild-type myocilin but not with stromelysin and thereby selectively inhibited the secretion of the former protein. The myocilins expressed were identified to be concentrated into fine punctate aggregates in endoplasmic reticulum, but never developed into the formation of aggresomes. In endoplasmic reticulum, the accumulation of the myocilins resulted in the upregulation of 78kDa glucose-regulated protein and protein disulfide isomerase. In addition, the expression of the myocilins led to deformed cellular morphology and diminished cell proliferation, an effect postulated to result in the dysfunction of trabecular cells that could be a cause of glaucoma. Therefore, our results support the statement that gain of function rather than haploinsufficiency is a critical mechanism for POAG in individuals with mutations on MYOC.


Subject(s)
Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/ultrastructure , Eye Proteins/metabolism , Glycoproteins/metabolism , Stress, Physiological/metabolism , Trabecular Meshwork/growth & development , Trabecular Meshwork/metabolism , Cells, Cultured , Cytoskeletal Proteins , Eye Proteins/chemistry , Eye Proteins/genetics , Glaucoma, Open-Angle/metabolism , Glaucoma, Open-Angle/pathology , Glycoproteins/chemistry , Glycoproteins/genetics , Humans , Mutagenesis, Site-Directed , Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Stress, Physiological/pathology , Structure-Activity Relationship , Tissue Distribution , Trabecular Meshwork/pathology
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