The Expression of Rab8, Ezrin, Radixin and Moesin in the Ciliary Body of Cynomolgus Monkeys

Purpose The purpose of this study was to determine what proteins are present in the ciliary body (CB). To accomplish this, we conducted a proteomic analysis of the CB of cynomolgus monkeys. We also determined the location of the proteins in CB by immunohistology. Methods The eyes of euthanized cynomolgus monkeys were enucleated, and the CB, were isolated from the eyes. Proteins were extracted from the CB and determined by liquid chromatography-mass spectrometry. Separated CB epithelial cells were cultured, and the proteins expressed in the CB were determined by Western blotting. The location of these proteins in the CB was determined by immunohistochemical staining. We also investigated whether adding dexamethasone to the culture medium changed protein expression by the epithelial cells. Results Proteomic analysis of the CBs showed that 813 proteins were expressed in the epithelium and stroma. These proteins included the small guanosine triphosphate-binding protein Rab8 and the ezrin/radixin/moesin (ERM) family. Tissue and immunohistological staining confirmed the colocalization of these proteins in non-pigmented CB epithelium. Western blotting of cultured CB epithelial cell lysates showed a tendency that adding dexamethasone changed Rab8 protein expression levels. Conclusions Proteomic analysis of CBs identified several proteins involved in the transport and secretion of proteins. These proteins may be involved in the production of aqueous humor and protein secretion by the CB.


Introduction
Glaucoma is a major cause of blindness worldwide: Approximately 60 million people have glaucoma, and approximately 8 million of them are blind in both eyes 1,2) .The disease is characterized by a progressive loss of retinal ganglion cells, resulting in constriction of the visual fields 2,3) .Elevated intraocular pressure (IOP) enhances the progression of the disease processes, and a reduction of IOP can slow or block the progression of glaucoma.The IOP can also affect aqueous humor dynamics 4) .Other components that influence IOP are the trabecular meshwork and Schlemm's canal, which are involved in the outflow of aqueous humor, and the ciliary body (CB), which is involved in aqueous humor production.
Besides aqueous humor formation, the CB is involved in accommodation and anterior chamber-associated immune deviation.The CB, especially the non-pigmented ciliary epithelium, is also involved in the synthesis and secretion of various proteins found in the aqueous humor that are believed to be involved in controlling IOP [4][5][6] .
Proteins secreted from the CB can affect the trabecular meshwork cells.The glycoproteins in aqueous humor have been shown to be secreted from the CB epithelium 7) , and molecules in the aqueous humor, e.g., collagenases, can affect the trabecular meshwork morphology 8,9) .However, a comprehensive description of the proteins present in the aqueous humor has not been published.
Recent advances in proteomics technology have made it possible to perform comprehensive investigations of the proteins in body fluids and tissues.In ophthalmology, proteomics studies have analyzed the proteins in the cells of the trabecular meshwork, aqueous humor, retina, retinal pigment epithelium, and retinal drusen [10][11][12][13][14][15][16][17] .
To date, no study has comprehensively determined the proteins expressed in the CB.In addition, the many steps involved in the synthesis and secretion of aqueous humor have not been determined.Therefore, the purpose of this study was to determine the proteins present in the CB.To accomplish this, we conducted a proteomic analysis of the CB of cynomolgus monkeys and thereby focused on the small guanosine triphosphate (GTP)-binding protein Rab8, which has been shown to be involved in vesicle transport and protein localization in small intestinal epithelial cells.Rab8 is also involved in the morphology of the microvilli in small intestinal epithelial 18) and is associated with optineurin 19,20) , a glaucoma gene.

Preparation of cynomolgus monkey eyes
All experimental procedures were approved by the Animal Welfare and Animal Care Committee of the Tsukuba Primate Research Center (TRPC) and the Experimental Animal Committee of the National Tokyo Medical Center.The facilities for housing the monkeys are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC Inter-national).Monkeys are routinely examined for physical and ophthalmic conditions by veterinarians and ophthalmologists, and all experiments on monkeys are conducted in accordance with The Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Eyes were obtained in a collaborative study to make effective use of all the monkey tissues for different research programs.Eyes were enucleated immediately after the monkeys were euthanized, and the CBs were isolated from the eyes within 4 hours after death.

Protein extraction from CBs
The tissues from the CBs of a healthy male monkey were homogenized and sonified in lysis buffer (50 mM Tris-HCL, 2 mM EDTA, 0.5% Triton-X, 2% SDS).After centrifugation for 10 minutes at 10 000 rpm (9,300g), the supernatant was collected.The protein concentration in the supernatant was determined with the RC DC protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions.Twenty micrograms of the protein sample were combined with an equal volume of 2 × Laemmli buffer and heated for 5 minutes at 100 °C.Then, all samples were stored at -20 °C until use.

Gel digestion and liquid chromatography-mass spectrometry analyses
Twenty micrograms of protein sample was separated on 12.5% acrylamide SDS-PAGE gel.The gel was stained with Colloidal Coomassie Blue (Invitrogen, Carlsbad, CA, USA) and cut into 15 equal pieces of approximately 1 mm 3 .The pieces were washed twice with 50mM ammonium bicarbonate/50% acetonitrile, and after destaining, the gel pieces were rinsed with distilled water and incubated with acetonitrile for 20 minutes.The supernatant was discarded, and the gel pieces were completely dried before incubation with 10mM DTT in 100mM ammonium bicarbonate for 45 minutes at 56 °C.The supernatant was discarded, and the pieces were incubated in the dark with 55mM iodoacetamide in 100mM ammonium bicarbonate for 30 minutes at room temperature.Then, the supernatant was discarded, and the gels were washed three times.Finally, the gel pieces were completely dried before tryptic digestion in sequencing grade trypsin solution (12.5 μg/μL; Thermo Fisher Scientific Inc, Rockford, IL, USA) in 50mM ammonium bicarbonate.The digestion was performed at 37 °C overnight, and then the extraction step was performed once with 25mM ammonium bicarbonate, twice with 5% formic acid, and then once with distilled water.The extracted peptides were pooled and dried.After re-suspending in 40 μL of aqueous 0.01% trifluoroacetic acid/2% acetonitrile, the samples were analyzed by liquid chromatography-mass spectrometry (LCQ Deca XP plus, Thermo Fisher Scientific Inc).

Proteomic analysis of monkey CB
Database searches were performed with the assistance of Bio Works 3.3.1,a protein search program.The UniProt-SwissProt database was initially used by querying the entire theoretical peptide masses provided in the public domain by the Swiss Institute of Bioinformatics.The number of registrations in the UniProt-SwissProt database is about 17 000 for humans and about 14 000 for mice.The data analyses of the monkey CB were aimed at cynomolgus monkeys, humans, orangutans, chimpanzees, rhesus macaques, and lowland gorillas.

Immunohistochemistry of the CB
Enucleated eyes from normal cynomolgus monkeys were fixed in 10% neutralized and buffered formaldehyde solution at 4 °C overnight and then dehydrated.The eyes were embedded in paraffin and serially sectioned at 4 μm thickness.After deparaffinization and rehydration, the specimens were prepared for antigen retrieval by warming in hot water in Target Retrieval Solution (Dako, Glostrup, Denmark) for 30 minutes at 100 °C.The sections were then blocked with phosphate-buffered saline (PBS) containing 10% BSA for one hour and then incubated overnight with primary antibodies (Abs) that were the same as those used for the Western blotting.The slides were washed in PBS and, for nuclear staining, were incubated with Alexa 488 or Alexa 568 (1:500 dilution; Invitrogen) and 4',6'diamidino-2-phenylindole (DAPI) for one hour at room temperature.The stained tissues were examined with a confocal fluorescence laser microscope (Radiance 2000, Bio-Rad Laboratories).Control slides were prepared by a similar process, but the primary Abs were omitted.

Primary culture of ciliary epithelial cells from cynomolgus monkey
The ciliary epithelial cells isolated from cynomolgus monkey eyes were suspended in DMEM (GIBCO, Carlsbad, CA, USA) containing penicillin-streptomycin (100 U/mL final concentration; Invitrogen) and 10% FBS.Cells were grown to confluence at 37 °C in 5% CO 2 .Then, cells were cultured with 100 nM of the glucocorticoid dexamethasone (DEX), 500 nM of timolol malate, or 1 μM of acetazolamide (Sigma-Aldrich, St Louis, MO, USA) for five days.

Protein expression of lysate from blotting of CB
Cultured monkey ciliary epithelial cells were lysed in TNE buffer containing 50mM Tris, 137mM NaCL, 1mM EDTA, 1% TritonX-100, and protease inhibitors (Complete EDTA-free; Roche, Basel, Switzerland).After homogenization and centrifugation for 15 minutes at 14 000 rpm, the supernatant was collected.The protein concentration was determined with the RC-DC protein assay kit (Bio-Rad) according to the manufacturer's instructions.Ten micrograms of proteins from the monkey ciliary epithelium cells lysates were diluted in an equal volume of 2×Laemmli buffer and heated for 5 minutes at 100 °C.Samples were separated by 7.5% SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membrane.Membranes were blocked in PBS containing 0.05% tween20 (PBS-T) and 5% non-fat dry milk for one hour and probed overnight at 4 °C with one of the following primary Abs: rabbit anti-Rab8 Ab (Sigma-Aldrich), goat anti-Ezrin Ab (Sigma-Aldrich), goat anti-Radixin Ab (Sigma-Aldrich), goat anti-Moesin Ab (Sigma), and mouse anti-Actin Ab (Millipore, Billerica, MA, USA).The specific signals were detected with horseradish peroxidase (HRP)conjugated donkey Ab to goat IgG or rabbit IgG as secondary Abs.The signals were made visible by chemiluminescence reactions and examined with a ChemiDoc XRS plus (Bio-Rad).

Proteomic analysis of monkey CB
The proteomic analysis of proteins extracted from the stroma and epithelium of the monkey CB identified a total of 813 proteins.Detailed information on these proteins is shown as supplemental data.These proteins were classified by a biological process registered in the Gene Ontology database that uses pathway tool software (MetaCore, Gene GO, Infocom; Figure 1).The proteins identified in the CB are involved in protein transport, localization, and secretion (Table 1).

Immunohistochemical analyses
The results of proteomic analysis of monkey CB included the Rab8 protein (No.170), which is a small GTP-binding protein that is related to the glaucoma gene optineurin, and is known to be involved in protein localization and transportation in the small intestine and can affect the morphology of microvilli 18) .The ezrin (No.70)/radixin (No.160)/ moesin (No.108), ERM family, which are core proteins in the microvilli that also act as linker   proteins between actin filaments and the cell membranes.Rab8 and moesin have also been reported to interact in the photoreceptor cells 21) .It is reasonable to assume that Rab8 and ERM family interact also in CB, and are involved in protein secretion.Double immunostaining with anti-Rab8 Ab and anti-ERM family Abs showed expression of Rab8, ezrin, radixin, and moesin in the CB epithelium and especially in the non-pigmented epithelium.A co-expression of Rab8 and the ERM family proteins was also detected in the ciliary epithelium (Figure 2A, 2B, 2C) and primary cultured ciliary epithelial cells (Figure 2D).

Drug effects on ciliary epithelium cells in vitro
The secondary glaucoma caused by glucocorticoids is both clinically and morphologically similar to primary open angle glaucoma.When trabecular meshwork cells are exposed to DEX, the extracellular matrix increases and the trabecular meshwork thickens, and resulting in IOP elevation.It is known that glucocorticoids affect IOP.However, the relationship between CB function and glucocorticoids remains unclear.We investigated whether Rab8 interacts the mechanism of IOP elevation by glucocorticoids stimulation.Light microscopy did not show any morphological changes in the morphology and protein expression of cultured cynomolgus monkey non-pigmented ciliary epithelial cells with different concentrations of DEX(data not shown).Western blotting of epithelial cell lysates showed a tendency for the addition of DEX to decrease the expression of Rab8 protein and this effect seemed dose-dependent, though this is not significant(Figure 3A).The experiments repeated twice and the results were similar.In addition, when cells were cultured in the same way and timolol maleate or acetazolamide was added, no changes were observed in the expression of Rab8(-Figure 3B, 3C).

Discussion
Proteomic analyses of the CB of cynomolgus monkeys detected many proteins, some of which are involved in protein transport and secretion.In the CB, the difference of protein characteristics was found between ciliary epithelium and stroma.Structure proteins were major component in stroma, and catalytic and metabolic proteins were more expressed in epithelium than stroma.Diffusion, ultrafiltration, and active secretion are physiological processes that participate in the production of aqueous humor, especially active secretion 4) .Blood flows through the CB, and aqueous humor is produced from the blood plasma by the non-pigmented ciliary epithelium.The proteins in the aqueous humor differ both quantitatively and qualitatively from those in the plasma 4,22,23) , indicating that the CB must be involved in the secretion of proteins into the aqueous humor.In comparison of CB with aqueous humor, Chowdhury et al reported that 355 proteins were identified from human aqueous humor by narrow liquid chromatography electrospray ionization tandem mass spectrometry, and most of the proteins had catalytic, enzymatic, and structural properties 23) .Catalytic and metabolic proteins are almost 30% each of top 50 distribution by function by Gene Ontology in CB, and approximately 20% each in aqueous humor.Structure proteins take up around 35% in CB, whereas 9.9% in aqueous humor 23) , though simple comparison is not possible according to different way of classification.
Myocilin, a glaucoma gene, is found in the aqueous humor, and the trabecular meshwork cells are known to secrete exosomes into the aqueous humor 24) .The CB appears to secrete proteins into the aqueous humor in the same way.
Rab8, one of the proteins identified by our proteomic analysis, is a small GTP-binding protein that is related to optineurin, a glaucoma gene [18][19][20] . I is known to be involved in protein localization and transportation in the small intestine and can affect the morphology of microvilli 18) .Although no reports have described the role that Rab8 plays in the CB, it is quite reasonable to assume that it is involved in protein secretion.
Our proteomic analysis also identified the ERM family proteins, which are core proteins of the microvilli and are found directly beneath the cell membranes.They act as linker proteins and connect actin filaments to the cell membranes 25,26) .Rab8 and moesin have been reported to interact to regulate the transport of rhodopsin in photoreceptor cells 21) .Both proteins appear to be related to protein transport and to be active in the CB.
We double immunostained for Rab8, ezrin, radixin, and moesin in cynomolgus monkey CB tissues and also in cultured non-pigmented ciliary epithelial cells.Confocal microscopy showed a co-expression of Rab8 and the ERM family in the non-pigmented epithelium of the CB, suggesting that these proteins are involved in the function of the non-pigmented epithelium of the CB.
The secondary glaucoma caused by glucocorticoids is both clinically and morphologically similar to primary open angle glaucoma.Thus, glucocorticoids have been used in both in vivo and in vitro glaucoma research [27][28][29][30][31] . Wen trabecular mesh- work cells are exposed to DEX, the extracellular matrix increases and the trabecular meshwork thickens, resulting in an elevation of IOP 31) .It is also widely known that glucocorticoids affect IOP. Howver, the relationship between CB function and glucocorticoids remains unclear.
We cultured non-pigmented ciliary epithelial cells and found a tendency for adding DEX to reduce the level of Rab8 expression in these cells.Although drugs such as beta-blockers and acetazolamide can alter the production of aqueous humor, we did not observe any changes in the level of Rab8 expression when timolol maleate or acetazolamide was added to cultured CB epithelial cells.We suggest that these proteins influence the function and morphology of the trabecular meshwork.They may also affect the aqueous humor outflow route and thus the IOP.Determining these functions would be relevant for understanding the mechanism of IOP control and glaucoma pathogenesis.
Although Rab8 might be involved in functions in the CB besides the secretion of aqueous humor, the finding that Rab8 expression in the CB was affected by DEX stimulation indicates that Rab8 may have some effect on IOP dynamics.To obtain more information on this relationship, analyses of the CB epithelial cell secretome and secreted exosomes and experiments involving co-culture models with trabecular meshwork cells are required.
In conclusion, Rab8 probably plays some role in the secretion of aqueous humor in the CB of cynomolgus monkeys and co-exists with ERM family molecules.Its expression is dependent on stimulation by glucocorticoids.Further studies will be needed to investigate the exact role of Rab8 in the CB.

Figure 1
Figure 1 Classification according to the Gene Ontology databaseThe pathway tool software (MetaCore,Gene GO,Infocom) was used to classify all of the identified proteins by the biological process registered in the Gene Ontology database.Upper bar, proteins identified mainly in the ciliary epithelium; lower bar, proteins identified mainly in the ciliary stroma

Figure 2
Figure 2 Immunohistochemistry of normal cynomolgus monkey ciliary body, and immunofluorescence staining of cultured monkey ciliary body cells Paraffin sections of cynomolgus monkey ciliary body (CB) were labeled with antibodies specific to Rab8 (A-C), ezrin (A), radixin (B), and moesin (C).Monkey CB cells were stained with Rab8 and ezrin (D).Co-expression of Rab8 and the ezrin/radixin/moesin (ERM) family can be seen at the ciliary non-pigmented epithelium cells (arrow).

Figure 3
Figure 3 Effect of dexamethasone on the expression ofRab8 in monkey ciliary epithelial cells A: Western blot analysis with an antibody against Rab8 showed a tendency of decrease in the expression of Rab8 in monkey ciliary epithelial cells after exposure to dexamethasone (Dex) for 5 days.Western blot analysis with an antibody against actin was the control.The relative amount of Rab8/ actin was quantified.B: Western blot analysis of ciliary epithelial cells after exposure to timolol maleate for five days.No change was seen in the expression of Rab8 proteins.C: Western blot analysis of ciliary epithelial cells after exposure to acetazolamide for five days.No change was seen in the expression of Rab8 proteins.

Table 1
Principal proteins of cynomolgus monkey ciliary body