<strong>ARVO</strong> 2013 Annual Meeting Abstracts by Scientific Section/Group - <strong>Biochemistry</strong>/<strong>Molecular</strong> <strong>Biology</strong>5-aza-dc -treated cells compared to controls and qPCR demonstrateda concomitant increase in RHO expression. In 5-aza-dc-treated HEK293 cells, addition of CRX and NRL greatly increased Gluc activityfrom the reporter plasmid and increased expression of endogenousRHO. Additional chromatin remodeling drugs, including HDACinhibitors, are currently being examined for possible synergisticeffects in the regulation of RHO gene expression.Conclusions: Our results show that DNA hypomethylation by 5-azadccan induce RHO expression from low-expressing cells.Endogenous RHO expression can be further increased by the additionof transcription factors required during normal gene expression. Ourresults suggest that DNA methylation plays an important role in thesuppression of RHO gene expression and is not a passive marker ofRHO silencing. The ability to manipulate the expression of retinaspecificgenes using epigenetic modifiers currently in therapeutic useenables further study into the role that these mechanisms may play inboth normal eye development and disease.Commercial Relationships: Jin Song, None; Tomohiro Masuda,None; Donald J. Zack, Alcon (C), Merck (F), Allergan (C);Shannath L. Merbs, NoneSupport: RPB unrestricted fundsProgram Number: 1600 Poster Board Number: D0027Presentation Time: 8:30 AM - 10:15 AMMicroRNA 184 regulates Ezrin expression and potentiallyimpacts Ezrin-dependent functions in human Retinal PigmentEpitheliumNishantha Gunawardena, Nady Golestaneh, Maria Kokkinaki, MiaGunawan. Georgetown University, Washington, DC.Purpose: The purpose of this study is to investigate the role ofmicroRNA 184 (miR-184) in regulating RPE functions. It is knownthat miR-184 plays a key role in neurological development and ishighly expressed in mice corneal epithelium and in human fetal RPE,however, its role in RPE is largely unknown.Methods: Proteomic analysis of RPE transfected with hairpins formiR-184 was performed and the data were analyzed to identify thegenes that were highly affected by the down-regulation of miR-184.Since RPE express miR-184, Hela cells were used to analyze thebinding of miR-184 to the 3’UTR of the targeted genes. Luciferaseassays were performed after transfection of Hela cells with bothplasmids for EZR 3’UTR in pEZX-MT01 vector and miR-184 codingDNA sequence in pEZX-MR04. Real Time PCR was used torelatively quantify the mRNA expression levels of the targeted gene.Western blot was used to measure the protein levels of ezrin in miR-184 transfected cells. RNAi technology was used to verify the effectof EZR down-regulation in RPE function. Phagocytosis assay andultrastructural analysis using electron microscopy were performed.Results: Our proteomic data showed that EZR is a potential target formiR-184.Ezrin is an actin-binding cytoplasmic peripheral membrane proteinthat functions as a protein-tyrosine kinase substrate in the apicalmicrovilli and as an intermediate between the plasma membrane andthe actin cytoskeleton. Luciferase assay revealed that miR-184 bindsto the 3’UTR of EZR and directly down-regulates EZR geneexpression. In addition, real time PCR revealed that EZR geneexpression is inhibited by miR-184. Western blot confirmed thedown-regulation of ezrin protein by miR-184. Using EZR siRNA, weshowed that RPE morphology and phagocytosis function wereaffected by ezrin down-regulation.Conclusions: Our results suggest that miR-184 affects actindepolymerization and in turn impacts membrane trafficking and RPEfunctions through down-regulation of EZR. In addition, since ezrin isexpressed in the apical region of polarized RPE, its regulation bymiR-184 might be crucial for RPE cell polarity and related biologicalfunctions. Further research is required to delineate the posttranslationalregulation of EZR mRNA by miR-184 and its possiblerole in RPE related diseases such as Age-related MacularDegeneration.Commercial Relationships: Nishantha Gunawardena, None;Nady Golestaneh, None; Maria Kokkinaki, None; Mia Gunawan,NoneSupport: Not applicable.Program Number: 1601 Poster Board Number: D0028Presentation Time: 8:30 AM - 10:15 AMThe role of Rgcs1 gene Spink2 in Autophagy and ganglion cellsusceptibility to optic nerve damageMargaret Maes 1, 2 , Joel Dietz 1 , Cassandra Schlamp 1 , Robert W.Nickells 1 . 1 Ophthalmology and Visual Sciences, University ofWisconsin-Madison, Madison, WI; 2 Cellular and <strong>Molecular</strong>Pathology Graduate Program, University of Wisconsin-Madison,Madison, WI.Purpose: Rgcs1 is a QTL that influences ganglion cell death afteroptic nerve crush. Spink2, a gene of interest in this locus, isupregulated in mouse retina after crush. SPINK 2 is a serine proteaseinhibitor Kazal type 2. Previous studies suggest that these inhibitorsfunction as suppressors of autophagic flux (AF). High levels ofSPINK 2 may therefore alter the AF in RGCs, a process that has beenshown to increase their susceptibility to optic nerve damage.Methods: Resistant (DBA/2J) and susceptible (Balb/c) mice wereused to perform optic nerve crush. All crush studies compare thecrushed retina to the control (non-crushed) retina. Changes intranscript abundance of autophagy markers Beclin 1, Atg5, Atg7 andPik3c3 were examined 7 days post-crush using qPCR. Activatedcaspase 3/7 was measured using a cell death assay with D407 cells inthe presence or absence of SPINK 2 and/or staurosporine. Spinningdisc microscopy was used to image GFP-LC3 labeledautophagosomes in rapamycin-treated D407 RPE cells in thepresence or absence of SPINK 2. Fusion of the autophagosome andlysosome was analyzed using tfLC3 (GFP-RFP-LC3) in the presenceor absence of Rapamycin and/or SPINK 2. Quantification andkinetics of live imaging LC3 experiments were analyzed using Imarissoftware.Results: Transcript levels of autophagy markers were upregulatedpost-crush, and were significantly higher in resistant DBA/2J mice.D407 cells expressing Spink2 exhibited an increase in caspaseactivity in response to staurosporine, relative to control GFPtransfected cells. Rapamycin caused a significant increase in LC3containing vesicles within 30 minutes in cells with and withoutSPINK 2. D407 cells expressing Spink2 accumulated significantlymore vesicles. These remained small even 4 hours after rapamycintreatment, suggesting impaired fusion between autophagosomes andlysosomes. Further experiments directly examining this fusion eventare underway and results will be reported.Conclusions: SPINK 2 increases cell susceptibility to apoptoticstimuli, possibly by impairing the AF response. This provides arationale for the difference in ganglion cell susceptibility associatedwith the Rgcs1 locus.Commercial Relationships: Margaret Maes, None; Joel Dietz,None; Cassandra Schlamp, None; Robert W. Nickells, NoneSupport: NEI R01EY018869, P30 EY016665, and Research toPrevent BlindnessProgram Number: 1602 Poster Board Number: D0029Presentation Time: 8:30 AM - 10:15 AM©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the <strong>ARVO</strong> Office at arvo@arvo.org.
<strong>ARVO</strong> 2013 Annual Meeting Abstracts by Scientific Section/Group - <strong>Biochemistry</strong>/<strong>Molecular</strong> <strong>Biology</strong>mTor is Involved in the Dexamethasone Induced Expression ofβ3 Integrin in Human Trabecular Meshwork CellsJennifer A. Faralli 1 , Debjani Gagen 1 , Donna M. Peters 1, 2 .1 Pathology and Laboratory Medicine, University of Wisconsin,Madison, WI; 2 Ophthalmology and Visual Sciences, University ofWisconsin, Madison, WI.Purpose: αvβ3 integrin signaling is involved in the formation ofcross-linked actin networks (CLANs), a structure proposed to play arole in steroid induced glaucoma. The purpose of this study was todetermine the effect of dexamethasone (DEX) treatment on theexpression of β3 integrin in human trabecular meshwork (HTM)cells.Methods: Post-confluent HTM cells were treated with either 500nMDEX or 0.1% EtOH for 0-6 days. Western blot analysis and FACSwere used to determine changes in protein expression. Changes inmRNA expression were determined using qPCR in the absence orpresence of 5μg/ml actinomycin D or 25μg/ml cycloheximide. Theglucocorticoid inhibitor RU486 was used to determine if changes inmRNA levels were due to direct glucocorticoid receptor activation.The immunosuppressant rapamycin was used to determine if mTORwas involved. FKBP5 (positive control), and β1integrin subunit(housekeeping gene) were used as controls.Results: Protein expression of the β3 integrin subunit increasedstarting after 2 days of DEX treatment and remained high as long asDEX was present and for at least 14 days following the removal ofDEX. FACS analysis showed that DEX increased β3 integrinactivation at the cell surface and it remained activated for at least 7days after removal of DEX. By qPCR, DEX treatment of HTM cellsinduced a 6.2-fold increase (p
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