Studies of Optineurin, a Glaucoma Gene
2006; Elsevier BV; Volume: 169; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.060400
ISSN1525-2191
AutoresBum‐Chan Park, Xiang Shen, Mishan Samaraweera, Jianbo Yue,
Tópico(s)Cellular transport and secretion
ResumoOptineurin (OPTN) has recently been linked to glaucoma, a major cause of blindness worldwide. Mutations in OPTN such as Glu50→Lys (E50K) have been reported in patients, particularly those with normal pressure glaucoma. Here, we show that the endogenous OPTN was not secreted in two ocular cell types, human trabecular meshwork and retinal pigment epithelial cells. It localized instead in the cytoplasm in a diffuse pattern without a distinct association with the Golgi apparatus. When overexpressed, however, wild-type OPTN-green fluorescent protein (GFP) formed foci especially around the Golgi, colocalizing partially with the common endocytic pathway marker transferrin receptor in both cell types. Fragmentation of the Golgi was also observed. On nocodazole treatment, the OPTN foci were dispersed into the cytoplasm. Overexpression of mutant OPTNE50K-GFP resulted in a greater number (P < 0.0055) and size of the foci, compared with the wild type, and the Golgi alteration was potentiated. Cell loss observed in OPTN-expressing cultures was also more pronounced in OPTNE50K-GFP compared with that of wild-type OPTN-GFP counterparts (P < 0.01). This study highlights a possible role of OPTN in vesicle trafficking and Golgi integrity. It also provides in-sights into the possible mechanisms why E50K would exhibit a propensity toward the development of glaucoma. Optineurin (OPTN) has recently been linked to glaucoma, a major cause of blindness worldwide. Mutations in OPTN such as Glu50→Lys (E50K) have been reported in patients, particularly those with normal pressure glaucoma. Here, we show that the endogenous OPTN was not secreted in two ocular cell types, human trabecular meshwork and retinal pigment epithelial cells. It localized instead in the cytoplasm in a diffuse pattern without a distinct association with the Golgi apparatus. When overexpressed, however, wild-type OPTN-green fluorescent protein (GFP) formed foci especially around the Golgi, colocalizing partially with the common endocytic pathway marker transferrin receptor in both cell types. Fragmentation of the Golgi was also observed. On nocodazole treatment, the OPTN foci were dispersed into the cytoplasm. Overexpression of mutant OPTNE50K-GFP resulted in a greater number (P < 0.0055) and size of the foci, compared with the wild type, and the Golgi alteration was potentiated. Cell loss observed in OPTN-expressing cultures was also more pronounced in OPTNE50K-GFP compared with that of wild-type OPTN-GFP counterparts (P < 0.01). This study highlights a possible role of OPTN in vesicle trafficking and Golgi integrity. It also provides in-sights into the possible mechanisms why E50K would exhibit a propensity toward the development of glaucoma. Glaucoma, a major cause of blindness worldwide, is a group of diseases characterized by a progressive loss of retinal ganglion cells and their axons. Primary open angle glaucoma (POAG), the most common form of the disease, is age-related and is frequently associated with elevated intraocular pressure (IOP). The IOP is controlled by a balance between the production and outflow of the aqueous humor in the anterior chamber. The trabecular meshwork (TM), a specialized eye tissue neighboring the cornea, is the major site for regulation of the aqueous humor outflow.1Bill A The drainage of aqueous humor.Invest Ophthalmol Vis Sci. 1975; 14: 1-3Google Scholar A subset of POAG that accounts for approximately 30% of the POAG cases2Hoyng PFJ Kitazawa Y Medical treatment of normal tension glaucoma.Surv Ophthalmol. 2002; 47: S116-S124Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar is called normal tension or normal pressure glaucoma (NPG). In this condition, the glaucomatous damage occurs with IOPs within the normal limits, although the progression of the damage is still believed to be IOP dependent.3Anderson DR Collaborative normal tension glaucoma study.Curr Opin Ophthalmol. 2003; 14: 86-90Crossref PubMed Scopus (229) Google Scholar Genetic studies have established that POAG is genetically heterogeneous and is caused by several susceptibility genes4Wiggs JL Allingham RR Hossain A Kern J Auguste J DelBono EA Broomer B Graham FL Hauser M Pericak-Vance M Haines JL Genome-wide scan for adult onset primary open angle glaucoma.Hum Mol Genet. 2000; 9: 1109-1117Crossref PubMed Scopus (115) Google Scholar, 5Fan BJ Wang DY Lam DS Pang CP Gene mapping for primary open angle glaucoma.Clin Biochem. 2006; 39: 249-258Crossref PubMed Scopus (146) Google Scholar and perhaps also environmental factors.6Wang N Chintala SK Fini ME Schuman JS Activation of a tissue-specific stress response in the aqueous outflow pathway of the eye defines the glaucoma disease phenotype.Nat Med. 2001; 7: 304-309Crossref PubMed Scopus (227) Google Scholar To date, a total of 12 chromosomal loci, designated as GLC1A to GLC1L, have been mapped for POAG.4Wiggs JL Allingham RR Hossain A Kern J Auguste J DelBono EA Broomer B Graham FL Hauser M Pericak-Vance M Haines JL Genome-wide scan for adult onset primary open angle glaucoma.Hum Mol Genet. 2000; 9: 1109-1117Crossref PubMed Scopus (115) Google Scholar, 5Fan BJ Wang DY Lam DS Pang CP Gene mapping for primary open angle glaucoma.Clin Biochem. 2006; 39: 249-258Crossref PubMed Scopus (146) Google Scholar Three genes, myocilin, optineurin (OPTN), and WDR36, have been identified, respectively, as the GLC1A gene on 1q23-q25,7Sheffield VC Stone EM Alward WL Drack AV Johnson AT Streb LM Nichols BE Genetic linkage of familial open angle glaucoma to chromosome 1q21–q31.Nat Genet. 1993; 4: 47-50Crossref PubMed Scopus (404) Google Scholar, 8Stone EM Fingert JH Alward WL Nguyen TD Polansky JR Sunden LF Nishimura D Clark AF Nystuen A Nichols BE Mackey DA Ritch R Kalenak JW Craven ER Sheffield VC Identification of a gene that causes primary open angle glaucoma.Science. 1997; 275: 668-670Crossref PubMed Scopus (1238) Google Scholar GLC1E gene on 10p15-p14,9Sarfarazi M Child A Stoilova D Brice G Desai T Trifan OC Poinoosawmy D Crick RP Localization of the fourth locus (GLC1E) for adult-onset primary open-angle glaucoma to the 10p15–p14 region.Am J Hum Genet. 1998; 62: 641-652Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar and GLC1G gene on 5q22.1.11Monemi S Spaeth G DaSilva A Popinchalk S Ilitchev E Liebmann J Ritch R Heon E Crick RP Child A Sarfarazi M Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1.Hum Mol Genet. 2005; 14: 725-733Crossref PubMed Scopus (385) Google Scholar Among them, OPTN is linked particularly to NPG cases.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar, 12Sarfarazi M Rezaie T Optineurin in primary open angle glaucoma.Ophthalmol Clin North Am. 2003; 16: 529-541Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Mutations, including Glu50→Lys (E50K), Met98→Lys (M98K), and Arg45→Gln (R545Q), in OPTN have been found in 16.7% of families with hereditary POAG. Approximately 80% of those families had the most prevalent E50K mutation. Of the E50K-affected subjects, 18% had high and the remaining had normal IOP values.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar, 12Sarfarazi M Rezaie T Optineurin in primary open angle glaucoma.Ophthalmol Clin North Am. 2003; 16: 529-541Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The human OPTN gene contains three noncoding exons in the 5′-untranslated region and 13 exons that code for a 577-amino acid protein.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar Alternative splicing at the 5′-untranslated region generates at least three different isoforms, but all have the same open reading frame.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar Sequence analysis indicates that OPTN is a protein containing multiple coiled coil domains, at least one leucine zipper (amino acids 143 to 164), and a carboxyl-terminal zinc finger.13Li Y Kang J Horwitz MS Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor α-inducible cellular protein containing leucine zipper domains.Mol Cell Biol. 1998; 18: 1601-1610Crossref PubMed Scopus (176) Google Scholar, 14Hattula K Peranen J FIP-2, a coiled-coil protein, links Huntington to Rab8 and modulates cellular morphogenesis.Curr Biol. 2000; 10: 1603-1606Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 15Schwamborn K Weil R Courtois G Whiteside ST Israel A Phorbol esters and cytokines regulate the expression of the NEMO-related-protein, a molecule involved in a NF-κB-independent pathway.J Biol Chem. 2000; 275: 22780-22789Crossref PubMed Scopus (96) Google Scholar OPTN was also identified previously as FIP-2 (14.7-interacting protein-2) and NRP (nuclear factor-κB essential modulator-related protein).13Li Y Kang J Horwitz MS Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor α-inducible cellular protein containing leucine zipper domains.Mol Cell Biol. 1998; 18: 1601-1610Crossref PubMed Scopus (176) Google Scholar, 15Schwamborn K Weil R Courtois G Whiteside ST Israel A Phorbol esters and cytokines regulate the expression of the NEMO-related-protein, a molecule involved in a NF-κB-independent pathway.J Biol Chem. 2000; 275: 22780-22789Crossref PubMed Scopus (96) Google Scholar It was suggested that OPTN might have a role in TNF-α-induced apoptosis,13Li Y Kang J Horwitz MS Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor α-inducible cellular protein containing leucine zipper domains.Mol Cell Biol. 1998; 18: 1601-1610Crossref PubMed Scopus (176) Google Scholar although this has not been well documented. OPTN is expressed in many tissues, such as the heart, brain, liver, skeletal muscle, kidney, pancreas, and the eye.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar, 13Li Y Kang J Horwitz MS Interaction of an adenovirus E3 14.7-kilodalton protein with a novel tumor necrosis factor α-inducible cellular protein containing leucine zipper domains.Mol Cell Biol. 1998; 18: 1601-1610Crossref PubMed Scopus (176) Google Scholar Previous studies suggested that OPTN might be associated with the Golgi apparatus.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar, 15Schwamborn K Weil R Courtois G Whiteside ST Israel A Phorbol esters and cytokines regulate the expression of the NEMO-related-protein, a molecule involved in a NF-κB-independent pathway.J Biol Chem. 2000; 275: 22780-22789Crossref PubMed Scopus (96) Google Scholar, 16Stroissnigg H Repitz M Miloloza A Linhartova I Beug H Wiche G Propst F FIP-2, an IκB-kinase-γ-related protein, is associated with the Golgi apparatus and translocates to the marginal band during chicken erythroblast differentiation.Exp Cell Res. 2002; 278: 133-145Crossref PubMed Scopus (19) Google Scholar, 17Sahlender DA Roberts RC Arden SD Spudich G Taylor MJ Luzio JP Kendrick-Jones J Buss F Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis.J Cell Biol. 2005; 169: 285-295Crossref PubMed Scopus (325) Google Scholar OPTN has been shown to interact with Rab8, huntingtin, and myosin VI.14Hattula K Peranen J FIP-2, a coiled-coil protein, links Huntington to Rab8 and modulates cellular morphogenesis.Curr Biol. 2000; 10: 1603-1606Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 17Sahlender DA Roberts RC Arden SD Spudich G Taylor MJ Luzio JP Kendrick-Jones J Buss F Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis.J Cell Biol. 2005; 169: 285-295Crossref PubMed Scopus (325) Google Scholar Rab8 regulates membrane trafficking and is known to promote changes in cell shape by reorganizing actin and microtubules.18Huber LA Pimplikar S Parton RG Virta H Zerial M Simons K Rab8, a small GTPase involved in vesicular traffic between the TGN and the basolateral plasma membrane.J Cell Biol. 1993; 123: 35-45Crossref PubMed Scopus (380) Google Scholar, 19Peranen J Auvinen P Virta H Wepf R Simons K Rab8 promotes polarized membrane transport through reorganization of actin and microtubules in fibroblasts.J Cell Biol. 1996; 135: 153-167Crossref PubMed Scopus (214) Google Scholar Huntingtin has been localized to endocytic and secretory membrane organelles.20Velier J Kim M Schwarz C Kim TW Sapp E Chase K Aronin N DiFiglia M Wild-type and mutant huntingtins function in vesicle trafficking in the secretory and endocytic pathways.Exp Neurol. 1998; 152: 34-40Crossref PubMed Scopus (247) Google Scholar Myosin VI is a multifunctional motor protein that moves toward the minus end of actin filaments and is found in a number of intracellular compartments, including endocytic vesicles, the Golgi, and secretory vesicles.21Buss F Spudich G Kendrick-Jones J Myosin VI: cellular functions and motor properties.Annu Rev Cell Dev Biol. 2004; 20: 649-676Crossref PubMed Scopus (145) Google Scholar Cellular and molecular biological studies of OPTN on eye tissues or cells have been limited. Immunolabeling for OPTN did locate this protein in the TM, cornea, nonpigmented ciliary epithelium, iris, and retina, especially the retinal pigment epithelium (RPE).22Rezaie T Sarfarazi M Molecular cloning, genomic structure, and protein characterization of mouse optineurin.Genomics. 2005; 85: 131-138Crossref PubMed Scopus (44) Google Scholar, 23Rezaie T Waitzman DM Seeman JL Kaufman PL Sarfarazi M Molecular cloning and expression profiling of optineurin in the rhesus monkey.Invest Ophthalmol Vis Sci. 2005; 46: 2404-2410Crossref PubMed Scopus (22) Google Scholar OPTN was also reported to be in the aqueous humor, suggesting that it may be a secretory protein.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar Nevertheless, the exact properties and the role of OPTN in ocular cells and the pathogenic mechanisms of mutations such as E50K remain undefined. In the present study, we examined the localization of OPTN in two ocular cell types, namely, normal human TM and RPE cells. We also investigated the consequences from overexpression of wild-type OPTN and OPTN containing mutation E50K. Our study implicated a role of OPTN in vesicle trafficking and Golgi integrity in TM and RPE cells. It also unveiled possible mechanisms why E50K mutation may lead to pathology. These findings are of clinical relevance in understanding of the development of glaucoma. Normal human TM tissues excised from donor eyes (Illinois Eye Bank, Chicago, IL) were cultured on Falcon Primaria flasks in complete medium containing Eagle's minimum essential medium (Sigma, St. Louis, MO), 10% fetal bovine serum, 5% calf serum, essential and nonessential amino acids, and antibiotics. Donors were 14, 19, 21, 24, 26, 44, and 49 years old. When TM cells reached confluence, they were trypsinized and subcultured. Second- to fourth-passage cells were used in this study. RPE (ARPE-19) cells obtained from American Type Culture Collection (Manassas, VA) were grown and maintained in complete medium. Freshly confluent TM cells were equilibrated in basal medium (Eagle's minimum essential medium with 2 mg/ml bovine serum albumin, 0.1 mg/ml lima bean trypsin inhibitor, 1 μg/ml insulin, and 0.1 μg/ml transferrin) for 1 hour. Cells were then incubated with fresh basal medium, and the medium was collected for two sequential 30-minute periods. The medium was replaced with fresh basal medium containing 1 mmol/L BaCl2 for another two sequential 30-minute periods.24El Meskini R Mains RE Eipper BA Cell type-specific metabolism of peptidylglycine α-amidating monooxygenase in anterior pituitary.Endocrinology. 2000; 141: 3020-3034Crossref PubMed Scopus (26) Google Scholar The media collected were centrifuged to remove nonadherent cells. Protease inhibitor cocktail (Roche, Indianapolis, IN) was added, and the media were concentrated by Centricon YM-10 (Millipore, Bedford, MA). Cells were harvested in CelLytic-M cell lysis reagent (Sigma) containing protease inhibitor cocktail. Aliquots of each media sample and total cell lysate were analyzed by Western blotting. In brief, proteins were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and electroblotted for 1 hour onto Protran BA83 nitrocellulose membrane (Whatman, Sanford, ME). After blocking for 1 hour with 5% nonfat dry milk in 20 mmol/L Tris buffer (pH 7.4) containing 150 mmol/L NaCl and 0.1% Tween 20, the membrane was incubated for 1 hour with either anti-OPTN (1:2000; Cayman Chemical, Ann Arbor, MI) or anti-myocilin25Hardy KM Hoffman EA Gonzalez P McKay BS Stamer WD Extracellular trafficking of myocilin in human trabecular meshwork cells.J Biol Chem. 2005; 280: 28917-28926Crossref PubMed Scopus (83) Google Scholar (1:5000; a gift from Dr. Daniel Stamer, University of Arizona, Tucson, AZ). According to the manufacturer's specification, the anti-OPTN antibody, raised in rabbits against a peptide corresponding to amino acid 115 to 130 sequence, recognizes human OPTN with a molecular size of 74 kd. The blot was further incubated for 1 hour with horseradish peroxidase-conjugated secondary antibody (1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA), and protein bands were detected using SuperSignal Substrate (Pierce, Rockford, IL). For repeated probing, the blot was stripped for 1 hour at room temperature with ImmunoPure IgG Elution buffer (Pierce). TM and RPE cells (5000 cells/well) plated onto Lab-Tek eight-well CC2 glass chamber slides (Nalge Nunc, Rochester, NY) were fixed in 4% paraformaldehyde for 15 minutes, washed with 100 mmol/L glycine in phosphate-buffered saline, and permeabilized in 0.2% Triton X-100 for 4 minutes. After 30 minutes of blocking with 3% bovine serum albumin, the cells were incubated for 1 hour at room temperature with anti-OPTN (1:200), anti-GM130 (1:100; BD Biosciences, San Jose, CA), or anti-golgin97 (1:200; Invitrogen, Carlsbad, CA). They were further incubated for 45 minutes with fluorescein isothiocyanate- or Cy3-conjugated secondary antibody (1:200; Jackson ImmunoResearch Laboratories) and mounted in Vectashield with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA). Photography was performed using a 63× oil objective on an Axioscope (Carl Zeiss MicroImaging, Thornwood, NY) with the aid of Metamorph software (Molecular Devices, Downingtown, PA). In some experiments, confocal microscopic analysis was performed on a Leica SP2 confocal system (Leica Microsystems, Bannockburn, IL) with a 63× oil objective using sequential scanning to minimize the bleed through.26North AJ Seeing is believing? A beginners' guide to practical pitfalls in imaging acquisition.J Cell Biol. 2006; 172: 9-18Crossref PubMed Scopus (183) Google Scholar The extent of colocalization was quantified using the ImageJ software by generating contour maps of each image by overlaying the green [wild-type OPTN (OPTNWT)-green fluorescent protein (GFP)] and red (Golgi markers) channels. Total number of green foci and that overlapped with the red staining around the Golgi areas were counted.27Aivazian D Serrano RL Pfeffer S TIP47 is a key effector for Rab9 localization.J Cell Biol. 2006; 173: 917-926Crossref PubMed Scopus (79) Google Scholar TM and RPE (5 × 106) cells were harvested in homogenization buffer (0.25 mol/L sucrose, 10 mmol/L HEPES, pH 7.4, and 1 mmol/L ethylenediamine tetraacetic acid). The cells were broken by repeated strokes in a Dounce homogenizer. Cell debris and nuclei were pelleted by centrifugation at 1000 × g for 10 minutes. The supernatant was overlaid onto the top of the discontinuous gradient, which consisted of 30, 25, 20, 15, and 10% iodixanol solution (OptiPrep; Accurate Chemical & Scientific Corp., Westbury, NY). After centrifugation at 4°C in a Beckman SW rotor at 100,000 × g for 3 hours, 17 fractions were collected from top to bottom. One-sixth of each fraction was loaded on SDS-PAGE gels and immunoblotted with anti-optineurin, anti-GM130 (1:500), and anti-golgin97 (1:2000). The open reading frame (ORF) without stop codon of OPTN was amplified by polymerase chain reaction (PCR) using pcDNA3-NRP (a gift from Dr. Robert Weil, Institute Pasteur, Paris, France) as template and primers 5′-GGCGAATTCCCACCATGTCCCATCAACCTCTCAGC-3′ and 5′-GGCGGATCCCGAATGATGCAATCCATCACGTG-3′. To generate the Rab8 ORF, cDNAs synthesized from total RNA of human TM cells by Superscript II cDNA synthesis kit (Invitrogen) were used as template, and PCR was performed with primers 5′-GGCGAATTCTATGGCGAAGACCTACGATTA-3′ and 5′-GGCGGATCCTCACAGAAGAACACATCGGA-3′. The resulting PCR products of OPTN and Rab8 were digested with EcoRI and BamHI and cloned in frame into pEGFP-N1 and pDsRed-Monomer-C1 (BD Biosciences), respectively, at the same restriction sites to yield constructs containing wild-type OPTN and GFP, pOPTNWT-GFP, and containing DsRedM and wild-type Rab8, pDsRedM-Rab8WT, respectively. All mutant constructs were generated by QuikChange II Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). The sense primer sequences used for mutagenesis were 5′-GAAAGAGCTCCTGACCAAGAACCACCAGCTGAAAG-3′ for pOPTNE50K-GFP and 5′-GACACAGCCGGTCTGGAACGGTTTCGG-3′ for pDsRedM-Rab8Q67L. The antisense primer sequences were the reverse. To construct untagged OPTN, PCR amplification of the OPTN ORF with stop codon was performed using pcDNA3-NRP as template and primers 5′-CCACCATGTCCCATCAACCTCTCAGC (sense) and 5′-TACTAAATGATGCAATCCATCACGTG-3′ (antisense). The antisense primer was also used to amplify FLAG-tagged OPTN along with a sense primer, 5′-CCACCATGGATTACAAGGATGACGACGATAAGATGTCCCATCAACCTCTCAGC-3′. The amplified PCR products were then inserted into pTarget (Promega, Madison, WI), yielding pTarget-OPTN and pTarget-FLAG-OPTN. Sequencing was followed to verify all of the constructs. Transient transfection was performed using FuGENE6 reagent (Roche) per the manufacturer's instruction with minor modifications. In brief, cells were plated at 60 to 70% confluency overnight. Cells were washed with Dulbecco's modified Eagle's minimum essential medium (DMEM; Invitrogen) 1 hour before the transfection and transferred to DMEM containing 2.5% fetal bovine serum. FuGENE6 reagent in DMEM was mixed with plasmid DNA at a 3:1 ratio and added to the cells for indicated time periods. Two micrograms of plasmid DNA per milliliter of the final transfection mixture was used unless otherwise noted. The transfection efficiency was estimated from the number of GFP-expressing green cells versus total number of cells under a Zeiss fluorescence/phase contrast microscope. TM and RPE cells in six-well plates were transfected with pEGFP-N1 (control) or pOPTNWT-GFP for 16 hours. The media were collected and concentrated. The cells were lysed for 15 minutes in CelLytic-M cell lysis reagent containing protease inhibitor cocktail. Cell debris was removed, and the protein content was determined by bi- cinchoninic acid protein assay kit (Pierce). Protein extracts (10 or 20 μg) and concentrated media samples were resolved on SDS-PAGE and immunoblotted with anti-OPTN, anti-GFP (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:5000; Trevigen, Gaithersburg, MD). TM cells plated on eight-well glass chamber slides were transfected with pOPTNWT-GFP, untagged pTarget-OPTNWT, or pTarget-FLAG-OPTNWT for 16 hours. Cells were fixed for immunofluorescence staining with anti-OPTN, anti-GM130, anti-golgin97, anti-myosin VI (1:100; Sigma), anti-FLAG (1:500; Sigma), or anti-transferrin receptor (1:100; Invitrogen). For colocalization with Rab8, cells were co-transfected with pOPTNWT-GFP and pDsRedM-Rab8Q67L (constitutively active Rab8). The OPTN foci distribution was examined after treatment of transfected cells for 30 minutes with vehicle [dimethylsulfoxide (DMSO)], brefeldin A (BFA; 5 μg/ml), nocodazole (10 μg/ml), or cytochalasin D (CytoD; 1 μmol/L). TM and RPE cells were transfected with pEGFP-N1, pOPTNWT-GFP, or pOPTNE50K-GFP for 16 hours and stained with either anti-GM130 or anti-golgin97. Images were taken using a 63× oil objective as described above. The number of foci formed was counted in at least 60 transfected cells manually and through the use of Metamorph computer software. The integrity of the Golgi apparatus in the same set of transfectants was determined. The judgment of a broken Golgi complex was based on the presence of disconnected, small and round GM130-immunolabeled Golgi membranes dispersed in the cell.28Liazoghli D Perreault S Micheva KD Desjardins M Leclerc N Fragmentation of the Golgi apparatus induced by the overexpression of wild-type and mutant human tau forms in neurons.Am J Pathol. 2005; 166: 1499-1514Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Percentage of transfected cells that displayed broken or fragmented Golgi and the degree of Golgi fragmentation were analyzed manually and through the Metamorph software. Significance of the data was determined by Student's t-tests. Three sets of independent experiments were analyzed. RPE cells on glass chamber slides were transiently transfected to express GFP alone, OPTNWT-GFP, or OPTNE50K-GFP. Five to six 10× fields, each containing at least 50 transfected cells, were selected. Images in the selected areas were captured after washing to remove nonsurviving cells at 24-hour intervals for 4 days. The number of remaining cells was counted using the Metamorph software. Three sets of independent experiments were performed. The significance of the data was determined by Student's t-tests. OPTN was reported to be in the aqueous humor, suggesting that it might be secreted by cells in the anterior chamber such as TM and nonpigmented ciliary epithelial cells.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar However, we did not detect any OPTN protein by Western blotting even on overloading or overexposure (data not shown) in culture media concentrated from either human TM or RPE cultures. To carefully address this issue, secretion assay was performed using BaCl2, a secretagogue known to induce protein secretion.24El Meskini R Mains RE Eipper BA Cell type-specific metabolism of peptidylglycine α-amidating monooxygenase in anterior pituitary.Endocrinology. 2000; 141: 3020-3034Crossref PubMed Scopus (26) Google Scholar TM cells were incubated in basal medium for two sequential 30-minute periods followed by the challenge of BaCl2 for another two sequential 30-minute periods. The media collected along with total cell lysate were analyzed by immunoblotting (Figure 1). Myocilin, a protein well documented to be secreted in TM cells,29Jacobson N Andrews M Shepard AR Nishimura D Searby C Fingert JH Hageman G Mullins R Davidson BL Kwon YH Alward WL Stone EM Clark AF Sheffield VC Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor.Hum Mol Genet. 2001; 10: 117-125Crossref PubMed Scopus (244) Google Scholar was used as a positive control. As expected, myocilin was found in the basal media, and the secretion was enhanced by BaCl2. By contrast, OPTN was not discerned in any of the media samples (Figure 1, top, lanes 1 to 4) under the same conditions. Meanwhile, abundant OPTN was present in the total cell lysate (Figure 1, top, lane 5). OPTN has previously been suggested to localize to the Golgi apparatus in nonocular or transformed cell types.10Rezaie T Child A Hitchings R Brice G Miller L Coca-Prados M Heon E Krupin T Ritch R Kreutzer D Crick PP Sarfarazi M Adult-onset primary open-angle glaucoma caused by mutations in optineurin.Science. 2002; 295: 1077-1079Crossref PubMed Scopus (903) Google Scholar, 15Schwamborn K Weil R Courtois G Whiteside ST Israel A Phorbol esters and cytokines regulate the expression of the NEMO-related-protein, a molecule involved in a NF-κB-independent pathway.J Biol Chem. 2000; 275: 22780-22789Crossref PubMed Scopus (96) Google Scholar, 16Stroissnigg H Repitz M Miloloza A Linhartova I Beug H Wiche G Propst F FIP-2,
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