April 2015
Volume 56, Issue 4
Free
Retinal Cell Biology  |   April 2015
TUDCA Promotes Phagocytosis by Retinal Pigment Epithelium via MerTK Activation
Author Affiliations & Notes
  • Hiromi Murase
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Kazuhiro Tsuruma
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Masamitsu Shimazawa
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Hideaki Hara
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Correspondence: Hideaki Hara, Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan; hidehara@gifu-pu.ac.jp
  • Kazuhiro Tsuruma, Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan; tsuruma@gifu-pu.ac.jp
Investigative Ophthalmology & Visual Science April 2015, Vol.56, 2511-2518. doi:10.1167/iovs.14-15962
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      Hiromi Murase, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hideaki Hara; TUDCA Promotes Phagocytosis by Retinal Pigment Epithelium via MerTK Activation. Invest. Ophthalmol. Vis. Sci. 2015;56(4):2511-2518. doi: 10.1167/iovs.14-15962.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: Renewal and elimination of the aged photoreceptor outer segment (POS) by RPE cells is a daily rhythmic process that is important for long-term vision. Phagocytic dysfunction results in photoreceptor cell death. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, is known to show neuroprotective effects in stroke, neurological diseases, and retinal degeneration models. In this study, we investigated the effects of TUDCA on retinal phagocytosis.

Methods.: We used pHrodo-succinimidyl ester (SE), a pH-sensitive fluorescent dye, to label the POS for monitoring phagocytosis. After ingestion, the intensity of pHrodo fluorescence increases because of the pH changes inside the liposome. An RPE cell line, ARPE-19, and primary human RPE cells were used to investigate the hydrogen peroxide (H2O2)–induced disruption of phagocytosis in the pH-sensitive fluorescence POS phagocytosis assay. Additionally, we examined whether TUDCA could promote phagocytic function.

Results.: The intensity of pHrodo light emission increased in a time-dependent manner. Tauroursodeoxycholic acid enhanced phagocytosis of POS and protected against H2O2-induced phagocytic dysfunction. It also promoted phagocytic function via activation of Mer tyrosine kinase receptor (MerTK), which is known to have a key role in the physiological renewal of POS.

Conclusions.: These results suggest that TUDCA activates MerTK, which is important for phagocytosis of POS. Tauroursodeoxycholic acid may represent a new therapeutic option for the treatment of retinal diseases.

Phagocytosis of the shed photoreceptor outer segments (POS) by the RPE is essential for retinal function. Retinal pigment epithelium cells are the most active phagocytes in the body, because each RPE cell disposes off several thousand shed membranous disks per day.1 Failure to phagocytize POS by the RPE cells causes retinal dystrophy in rodents and humans.2 For phagocytosis, the RPE uses phagocyte cell-surface receptors, such as the lipid scavenger receptor CD36, integrin adhesion receptor αvβ5, focal adhesion kinase (FAK), and Mer tyrosine kinase receptor (MerTK).35 In RPE phagocytosis, CD36 and MerTK participate in the engulfment step of the phagocytosis process, while αvβ5 integrin promotes POS recognition or binding and leads to a downstream cytoplasmic signaling cascade in the RPE.6,7 Mer tyrosine kinase receptor and αvβ5 interact via the FAK, which in turn may functionally interact with MerTK during the POS binding phase when both proteins still reside at the apical plasma membrane of the RPE.8 
The RPE cell dysfunction has been implicated in the pathogenesis of age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Age-related macular degeneration is a degenerative disease that causes severe visual impairment in one-fourth of the population over 65 years of age in the developed world.9 The causes of this disease include genetic and environmental factors, such as oxidative stress, light exposure, smoking, and low eye pigmentation.10 Lipofuscin accumulates in the RPE with age, and has been associated with AMD.11 Retinitis pigmentosa is one of the major causes of visual handicaps, blindness, and loss of peripheral vision followed by progressive loss of central vision that has affected 1.5 million people globally.12 This photoreceptor degeneration begins with the loss of rods, generally preceding the loss of cones. The rods are more susceptible to oxidative damage in the retina, which is exposed directly to the effects of light.13 When the rods die, the oxygen content in the retina rises, causing oxidative stress and secondary degeneration of the remaining cone cells.14 Many antioxidants, such as vitamin A, lutein, docosahexaenoic acid, and omega-3 fatty acids, have been characterized as potential candidates that may delay progression in vivo and in vitro.1520 Moreover, mutations in MerTK, found in patients with RP, result in phagocytic dysfunction in RPE cells and subsequent retinal degeneration.2124 Our prior findings showed that white light irradiation leads to the phagocytic dysfunction of RPE cells.25,26 
Tauroursodeoxycholic acid (TUDCA) is present in human bile and has been used in Chinese medicine for a long time for a variety of biliary and liver diseases. It is known to enhance phagocytosis in cultured rat Küpffer cells.27 Recently, it has been shown to have antiapoptotic properties in the retina. Systemic administration of TUDCA can protect photoreceptors from cell death after retinal detachment.28 Tauroursodeoxycholic acid is known to protect cones, rods, as well as presynaptic and postsynaptic elements in an animal model for autosomal dominant RP, the P23H rat,29 and to preserve vision in the retinal degeneration 10 (rd10) mouse, which is a model for RP.30 
Despite several reports on the characterization of aged phagocytes, the factors promoting RPE phagocytosis are yet to be appreciated. In this study, we examined the effect of TUDCA as a promoter of RPE phagocytosis using a pH-sensitive dye. We also tested whether TUDCA protected the RPE from oxidative stress-induced phagocytic dysfunction in vitro. 
Materials and Methods
Cell Culture
The ARPE-19 line was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin. Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. The cells were passaged by trypsinization every 3 to 4 days. 
Primary human RPE (hRPE) cells were obtained from Lonza (Walkersville, MD, USA). The cells were maintained in Retinal Pigment Epithelial Basal Medium (Lonza) containing 2% FBS, L-glutamine (Lonza), GA-1000 (Lonza), Growth factor (FGF-B; Lonza) according to the manufacturer's protocol. Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. The cells were passaged by Trypsin/EDTA (Lonza), Trypsin Neutralizing Solution (Lonza), HEPES Buffered Saline Solution (Lonza) every 3 to 4 days. 
Isolation of POS From Porcine Eyes
All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Retinas from freshly obtained porcine eyes were shaken gently in a solution containing 115 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 10 mM HEPES/KOH pH 7.5, and 1 mM dithiothreitol (DTT; POS buffer) containing 1.5 M sucrose (0.8–1.0 mL/retina).31 The suspension was centrifuged for 7 minutes at 7510g to sediment large pieces of the sample. The supernatant was filtered using a 0.22-μm syringe filter (BD Falcon, Franklin Lakes, NJ, USA). The filtrate was diluted with POS buffer 1:1, and centrifuged again. The pellet (crude POS) was resuspended in the POS buffer containing 0.6 M sucrose (5 mL per retina) and layered on a linear continuous sucrose gradient (0.6–1.5 M sucrose in POS buffer). After centrifugation (90 minutes; 103,500g in RP55T rotor; Hitachi Co. Ltd. Tokyo, Japan), the POS layer were collected and diluted 1:3 with balanced salt solution (BSS; 10 mM HEPES, 137 mM NaCl, 5.36 mM KCl, 0.81 mM MgSO4, 1.27 mM CaCl2, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, pH 7.4-7.5).6 The POSs were pelleted by centrifugation for 7 minutes at 7510g and stored in darkness at −80°C. 
Fluorescence Labeled-POS
Unlabeled POSs (10 mg) were resuspended to 5 mL of the BSS to make at a final concentration of 2 mg/mL. Resuspend POS was labeled with pHrodo (dilute 1 mg plus 150 μL dimethyl sulfoxide [DMSO]; Thermo Fisher Scientific K.K., Waltham, MA, USA) or FITC Isomer I (dilute 10 mg plus 1 mL DMSO; Thermo Fisher Scientific K.K.). They were rotated 1 hour in the dark. Then, they were concentrated by centrifugation at 4000g using the Amicon Ultra-15 molecular weight cutoff: 3,000 (Millipore, Billerica, MA, USA) for 6 hours at 4°C. 
Phagocytosis Assays
The ARPE-19 cells were seeded at 1.5 × 104 cells per well in 96-well plates and confirmed that the cells were 100% confluent, and then incubated for 4 days. The morphology of cells were checked using a microscope (Olympus, Tokyo, Japan), and the medium was replaced with fresh medium containing 1% FBS. Tauroursodeoxycholic acid (Wako) was added, followed by hydrogen peroxide (H2O2) 1 hour later, at final concentrations of 30 to 300 μM. After 1 hour of incubation, 1 × 107 POS/well were added and incubated for 6 hours. Subsequently, the cells were washed five times with 1% FBS DMEM/F-12 to remove extracellular POS. Images were taken using BZ-9000 Biorevo all-in-one fluorescence microscope (Keyence, Osaka, Japan), and quantified using an image processing software (ImageJ ver. 1.43 h; National Institutes of Health [NIH], Bethesda, MD, USA). pHrodo succinimidyl ester (SE) is a pH-sensitive fluorescent dye (pHrodo-SE). It is a lipophilic molecule that diffuses passively into the cells.32 The SE group anchors the dye by covalently binding to the amino groups on the intracellular macromolecules. In a neutral pH condition, the light emission by pHrodo-SE is almost undetectable.33 The postphagocytic fusion of the lysosomes reduces the pH within the phagolysosome due to the acidic environment of lysosomes.34 This phenomenon can be detected using pHrodo-SE that emits fluorescence in the red range at an increasing intensity with decreasing environmental pH. 
The hRPE cells were seeded at 1.5 × 104 cells per well in 96-well plates and confirmed that the cells were 100% confluent, and then incubated for 7 days. The morphology of cells was checked using a microscope (Olympus), and the medium was replaced with Retinal Pigment Epithelial Basal Medium without FBS. Tauroursodeoxycholic acid (Wako) was added, followed by H2O2 1 hour later, at a final concentration of 100 μM. After 1 hour of incubation, 1 × 107 POS (labeled with pHrodo or FITC Isomer I)/well were added and incubated for 6 hours. Subsequently, the cells were washed five times with Retinal Pigment Epithelial Basal Medium without FBS to remove extracellular POS. Images were taken using BZ-9000 Biorevo all-in-one fluorescence microscope, and quantified using an image processing software (ImageJ ver. 1.43 h). 
Cell Death Assay
The ARPE-19 cells were seeded at 1.5 × 104 cells per well in 96-well plates. After incubating for 24 hours, the entire medium was then replaced with fresh medium without FBS. Next, the cells were exposed to H2O2 (100 μM) for 6 hours at 37°C. Nuclear staining assays were carried out at the end of the H2O2 treatment. Hoechst 33342 (λex = 360 nm, λem > 490 nm) and propidium iodide (PI; λex = 535 nm, λem > 617 nm) were added to the culture medium for 15 minutes at a final concentrations of 8.1 and 1.5 μM, respectively. Hoechst 33342 freely enters living cells and then stains the nuclei of viable cells, as well as those that have suffered apoptosis or necrosis. Propidium iodide is a membrane-impermeable dye that generally is excluded from viable cells. Images were collected using an Olympus IX70 inverted epifluorescence microscope (Olympus). The total number of cells was counted and the percentage of PI-positive cells calculated. 
Radical Scavenging-Capacity Assay
The ARPE-19 cells were seeded at 1.5 × 104 cells per well in 96-well plates. After incubating for 4 days, cells were washed with 1% FBS DMEM/F-12. After 1 hour of pretreatment with TUDCA or N-acetylcysteine, 10 μM 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA; the radical probe; Thermo Fisher Scientific K.K.) was added to the cells. After 20 minutes, the cell culture medium was replaced with fresh 1% FBS DMEM/F12 in the absence of TUDCA or N-acetylcysteine to remove any extracellular CM-H2DCFDA (inactive for reactive oxygen species [ROS]). The radical probe was converted to 2′, 7′-dichlorodihydrofluorescein diacetate (DCFH, active for ROS) by the action of intracellular esterase. To generate the ROS, we added 100 μM of H2O2 as the radical probe loading medium. Fluorescence was measured after H2O2 exposure for various time points using a SkanIt RE for Varioskan Flash 2.4 (a microplate reader; Thermo Fisher Scientific K.K.) at excitation/emission wavelengths of 485/535 nm. Radical integrals were calculated by integrating the area under the CM-H2DCFDA fluorescence intensity curve after a 20-minute treatment with ROS-generating compounds. 
Western Blot Analysis
The ARPE-19 and hRPE cells were supplemented with a 1% protease inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO, USA), 1% phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich Corp.), and sample buffer (Wako). The cells were washed with PBS, harvested, and lysed in RIPA buffer (Sigma-Aldrich Corp.) supplemented with a protease inhibitor cocktail and phosphatase inhibitor cocktails 2 and 3. Lysates were centrifuged at 12,000g for 15 minutes at 4°C. Protein concentrations were measured by comparing with known concentrations of BSA, using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). Subsequently, the samples were boiled in the sample buffer for 5 minutes. The samples were subjected to a 5% to 20% gradient SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). The following primary antibodies were used for immunoblotting: anti-MerTK (phospho Y749 + Y 753 + Y754; ab14921) rabbit polyclonal (1:500; Abcam, Cambridge, UK); anti-MerTK (ab137673) rabbit polyclonal; anti-phospho-FAK (Tyr397) rabbit polyclonal (1:1000; Cell Signaling Technology, Danvers, MA, USA); anti-FAK (C-903, sc-932) rabbit polyclonal (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin mouse monoclonal antibody (1:5000; Sigma-Aldrich Corp.). An HRP-conjugated goat anti-rabbit antibody (1:2000; Thermo Fisher Scientific K.K.) and goat anti-mouse antibody (1:2000; Thermo Fisher Scientific K.K.) were used as secondary antibodies. Immunoreactive bands were visualized using Immunostar-LD (Wako) and LAS-4000 luminescent image analyzer (Fuji Film Co., Ltd., Tokyo, Japan). 
Statistical Analysis
Data are presented as the mean ± SEM. Statistical comparisons were made using Student's t-test and 1-way ANOVA followed by Dunnett's test (using STAT VIEW version 5.0; SAS Institute, Cary, NC, USA). A P value < 0.05 was considered statistically significant. 
Results
The Intensity of pHrodo Light Emission From Labeled POS in ARPE-19 Cells
The intensity of pH-sensitive fluorescence was measured using fluorescence micrographs (Fig. 1A). The intensity of pHrodo emission increased in a time-dependent manner (Figs. 1Aa–1Ad). Representative phase contrast images show the shape of ARPE-19 cells at 24 hours (Fig. 1Ae). Representative merged micrograph shows pHrodo-SE-labeled POS with ARPE-19 (Fig. 1Af). Fluorescence intensity significantly increased from 3 hours after POS addition compared to 0 hours (Fig. 1B). 
Figure 1
 
Fluorescence microscopic detection of phagocytosis of pHrodo-SE–labeled photoreceptor outer segment. (AaAd) Representative fluorescence micrographs showing pHrodo-SE–labeled POS engulfed by ARPE-19. (Ae) Representative phase contrast images showing shape of ARPE-19 cells at 24 hours. (Af) Representative merged micrograph showing pHrodo-SE–labeled POS with ARPE-19. (B) Measurements of the pH sensitive fluorescence POS. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control (Dunnett's test).
Figure 1
 
Fluorescence microscopic detection of phagocytosis of pHrodo-SE–labeled photoreceptor outer segment. (AaAd) Representative fluorescence micrographs showing pHrodo-SE–labeled POS engulfed by ARPE-19. (Ae) Representative phase contrast images showing shape of ARPE-19 cells at 24 hours. (Af) Representative merged micrograph showing pHrodo-SE–labeled POS with ARPE-19. (B) Measurements of the pH sensitive fluorescence POS. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control (Dunnett's test).
H2O2 Decreased the Phagocytic Function in ARPE-19 Cells
To investigate the effect of oxidative stress on phagocytic dysfunction in this system, H2O2 was used at concentrations of 30 to 300 μM. H2O2 induced the reduction of phagocytic activity in ARPE-19 cells. The fluorescence intensity significantly decreased after H2O2 treatment in a concentration-dependent manner (Figs. 2A, 2B). In contrast, pretreatment with N-acetylcysteine at a concentration of 1 mM inhibited the phagocytic dysfunction by the treatment with 100 μM of H2O2 (Fig. 2C). This concentration did not affect the cell viability (Fig. 2D). 
Figure 2
 
H2O2-induced reduction of phagocytic activity in ARPE-19 cells. (A, B) The reduction of phagocytic activity was stimulated with 30, 100, 300 μM H2O2, and fluorescence was measured at 6 hours. (A) Representative fluorescence micrographs showing pHrodo fluorescence upon addition at 6 hours after H2O2. (B) The H2O2 treatment decreased the phagocytic activity in a concentration-dependent manner. (C) N-acetylcysteine was used as a typical antioxidant. Pretreatment with N-acetylcysteine (1 mM) inhibited H2O2 100 μM-induced phagocytic dysfunction. (D) Cell death assay was performed using Hoechst 33342 and propidium iodide. H2O2 (100 μM) did not induce cell death. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control, ##P < 0.01 versus H2O2 (Student's t-test).
Figure 2
 
H2O2-induced reduction of phagocytic activity in ARPE-19 cells. (A, B) The reduction of phagocytic activity was stimulated with 30, 100, 300 μM H2O2, and fluorescence was measured at 6 hours. (A) Representative fluorescence micrographs showing pHrodo fluorescence upon addition at 6 hours after H2O2. (B) The H2O2 treatment decreased the phagocytic activity in a concentration-dependent manner. (C) N-acetylcysteine was used as a typical antioxidant. Pretreatment with N-acetylcysteine (1 mM) inhibited H2O2 100 μM-induced phagocytic dysfunction. (D) Cell death assay was performed using Hoechst 33342 and propidium iodide. H2O2 (100 μM) did not induce cell death. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control, ##P < 0.01 versus H2O2 (Student's t-test).
TUDCA Increased the Phagocytosis and Inhibited the Phagocytic Dysfunction Induced by H2O2 Treatment in RPE Cells
The effect of TUDCA on H2O2-induced reduction of phagocytosis was investigated using pHrodo-SE–labeled POS assay. H2O2 significantly decreased phagocytosis and TUDCA significantly inhibited this reduction at concentrations of 10 and 100 μM in ARPE-19 cells (Fig. 3A) and primary hRPE cells (Fig. 3B). Furthermore, we investigated whether TUDCA promoted phagocytosis in the H2O2 nontreated statement. Tauroursodeoxycholic acid at a concentration of 0.1 and 1 μM promoted phagocytosis in ARPE-19 cells (Fig. 3C). Since TUDCA inhibited the phagocytic dysfunction induced by H2O2 treatment, we examined the radical scavenging activity of TUDCA. We used radical scavenging-capacity assays using ROS-sensitive probes, CM-H2DCFDA. N-acetylcysteine, at a concentration of 1 mM, significantly scavenged the H2O2 radicals. However, TUDCA did not affect the free radical generation in ARPE-19 cells (Fig. 3D). To clarify the phases affected by TUDCA, FITC Isomer I and pHrodo were used. FITC Isomer I detects all phases of POS, while pHrodo detects the POS which have been digested within the RPE lysosome. Tauroursodeoxycholic acid significantly increased the fluorescence of FITC Isomer I and that of pHrodo after POS addition in hRPE cells (Figs. 3E, 3F). 
Figure 3
 
The effects of TUDCA on RPE phagocytosis. (A) The ARPE-19 cells were incubated with 100 μM H2O2 for 6 hours. Measurements of the pHrodo fluorescence in the H2O2 treated statement. (B) The hRPE cells were incubated with 100 μM H2O2 for 6 hours. Measurements of the pHrodo fluorescence in the H2O2 treated statement. (C) In ARPE-19 cells, pHrodo fluorescence was measured in the H2O2 nontreated statement. (D) Measurements of ROS production. Intracellular ROS levels were determined by measuring the fluorescence of CM-H2DCFDA (at excitation 488 nm/emission 525 nm) after H2O2 addition at 6 hours in ARPE-19 cells. N-acetylcysteine was used as a typical antioxidant. (E) In hRPE cells, FITC Isomer I fluorescence was measured. The TUDCA (1 mM) was treated 1 hour before POS addition. (F) In hRPE cells, pHrodo fluorescence was measured. The TUDCA (1 mM) was treated 1 hour before POS addition. Data are shown as mean ± SEM ([AE], n = 6; [F], n = 12). #P < 0.05, ##P < 0.01 versus control (Student's t-test [A, B, DF] and Dunnett's test [C]). **P < 0.01 versus H2O2 (Dunnett's test).
Figure 3
 
The effects of TUDCA on RPE phagocytosis. (A) The ARPE-19 cells were incubated with 100 μM H2O2 for 6 hours. Measurements of the pHrodo fluorescence in the H2O2 treated statement. (B) The hRPE cells were incubated with 100 μM H2O2 for 6 hours. Measurements of the pHrodo fluorescence in the H2O2 treated statement. (C) In ARPE-19 cells, pHrodo fluorescence was measured in the H2O2 nontreated statement. (D) Measurements of ROS production. Intracellular ROS levels were determined by measuring the fluorescence of CM-H2DCFDA (at excitation 488 nm/emission 525 nm) after H2O2 addition at 6 hours in ARPE-19 cells. N-acetylcysteine was used as a typical antioxidant. (E) In hRPE cells, FITC Isomer I fluorescence was measured. The TUDCA (1 mM) was treated 1 hour before POS addition. (F) In hRPE cells, pHrodo fluorescence was measured. The TUDCA (1 mM) was treated 1 hour before POS addition. Data are shown as mean ± SEM ([AE], n = 6; [F], n = 12). #P < 0.05, ##P < 0.01 versus control (Student's t-test [A, B, DF] and Dunnett's test [C]). **P < 0.01 versus H2O2 (Dunnett's test).
TUDCA Promoted Phagocytic Function by Activating MerTK and FAK in the H2O2 Treated Statement
To investigate whether TUDCA might affect the phagocytosis pathway in RPE cells, we focused on the phosphorylation of MerTK and FAK, which are initially activated by phagocytosis in RPE.8 In ARPE-19 cells, TUDCA significantly increased the phosphorylation of MerTK in a concentration-dependent manner (Figs. 4A, 4B). Treatment with H2O2 significantly reduced the phosphorylation of MerTK and FAK, and TUDCA inhibited these decrease in ARPE-19 cells (Figs. 4A–D). Furthermore, in hRPE cells, TUDCA increased the phosphorylation of MerTK in normal and oxidative stress conditions (Figs. 4E, 4F). Tauroursodeoxycholic acid is known as a chemical chaperon, which reduces endoplasmic reticulum (ER) stress.35 We assessed the effect of TUDCA on glucose-regulated protein-78 (GRP-78), an ER stress marker, expression induced by ER stress. However, TUDCA did not affect the expression of GRP-78 in ARPE-19 cells (Supplementary Fig. S1). 
Figure 4
 
Effects of TUDCA on MerTK and FAK phosphorylation. (A, B) Pretreatment with vehicle (lanes 1 and 5) or with TUDCA (lane 24, 68) was followed by 6 hours of additional incubation with 100 μM H2O2 (lane 58) in ARPE-19 cells. (C, D) Pretreatment with vehicle (lanes 1 and 2) or with TUDCA (1 mM, lanes 3 and 4) was followed by 6 hours of additional incubation with 100 μM H2O2 (lanes 2 and 3) in ARPE-19 cells. (E, F) Pretreatment with vehicle (lanes 1 and 3) or with TUDCA (1 mM, lanes 2 and 4) was followed by 6 hours of additional incubation with 100 μM H2O2 (lanes 3 and 4) in hRPE cells. Representative band images show (A) MerTK expression in ARPE-19 cells, (C) FAK expression in ARPE-19 cells, and (E) MerTK expression in hRPE cells. (B, F) Expression of p-MerTK was quantified by densitometry and corrected by reference to β-actin. (D) Expression of p-FAK was quantified by densitometry and corrected with reference to t-FAK. Data are shown as mean ± SEM ([B, D], n = 4; [F], n = 7). #P < 0.05, ##P < 0.01 versus control (Dunnett's test [B] and Student's t-test [D, F]), *P < 0.05, **P < 0.01 versus H2O2 (Dunnett's test [B] and Student's t-test [D, F]). C, control; V, vehicle.
Figure 4
 
Effects of TUDCA on MerTK and FAK phosphorylation. (A, B) Pretreatment with vehicle (lanes 1 and 5) or with TUDCA (lane 24, 68) was followed by 6 hours of additional incubation with 100 μM H2O2 (lane 58) in ARPE-19 cells. (C, D) Pretreatment with vehicle (lanes 1 and 2) or with TUDCA (1 mM, lanes 3 and 4) was followed by 6 hours of additional incubation with 100 μM H2O2 (lanes 2 and 3) in ARPE-19 cells. (E, F) Pretreatment with vehicle (lanes 1 and 3) or with TUDCA (1 mM, lanes 2 and 4) was followed by 6 hours of additional incubation with 100 μM H2O2 (lanes 3 and 4) in hRPE cells. Representative band images show (A) MerTK expression in ARPE-19 cells, (C) FAK expression in ARPE-19 cells, and (E) MerTK expression in hRPE cells. (B, F) Expression of p-MerTK was quantified by densitometry and corrected by reference to β-actin. (D) Expression of p-FAK was quantified by densitometry and corrected with reference to t-FAK. Data are shown as mean ± SEM ([B, D], n = 4; [F], n = 7). #P < 0.05, ##P < 0.01 versus control (Dunnett's test [B] and Student's t-test [D, F]), *P < 0.05, **P < 0.01 versus H2O2 (Dunnett's test [B] and Student's t-test [D, F]). C, control; V, vehicle.
Discussion
Prompt and complete phagocytic clearance of the shed POS by the underlying RPE is important for lifelong visual function.36 In this manuscript, we report the assessment of phagocytosis by using a pH-sensitive dye.37 The pHrodo-SE–labeled assay might help find new therapeutic agents for the treatment of retinal diseases, which are related to phagocytosis. The results of hRPE were similar to those of ARPE-19 (Figs. 3B, 4E, 4F). Therefore, our method using ARPE-19 cells would be useful to validate a potential new therapeutic approach for retinal degenerative diseases. 
FITC Isomer I-tagged POS detects all phases of phagocytosis; that is, recognition, engulfment, and lysosomal digestion, while pHrodo detects the lysosomal digestion phase. The fluorescence of FITC Isomer I increased to a plateau at 1 hour, whereas that of pHrodo increased in a time-dependent manner. These results indicated that most of POS were in the phases of recognition and/or engulfment, and the lysosomal digestion phase has a little effect to the fluorescence of FITC Isomer I. Tauroursodeoxycholic acid significantly increased the fluorescence of FITC Isomer I and that of pHrodo after POS addition (Figs. 3E, 3F). If TUDCA promotes the digestion without the increase of recognition/engulfment, only the fluorescence of pHrodo is supposed to increase particularly. However, TUDCA increases not only the fluorescence of pHrodo, but also that of FITC Isomer I. Thus, it would be suggested that TUDCA affects the phase of recognition/engulfment and TUDCA may have a positive impact on the phase of digestion collaterally. It could be strengthened by our experiment of Figure 4 (TUDCA increases the level of MerTK phosphorylation, which is important for POS engulfment). 
Bear bile has been used in Chinese medicine for ophthalmic and hepatic indications for over 3000 years. To our knowledge, this is the first study to determine the effects of TUDCA against retinal phagocytic dysfunction. The systemic administration of TUDCA 500 mg/kg attenuates the retinal degeneration in P23H rats38 and rd10 mice.39 It is suggested that TUDCA exerts its ability in the retina. 
Oxidative stress has a critical role in photoreceptor death, and we have shown that ROS reduction is neuroprotective in the photoreceptor cells.40,41 Furthermore, oxidative stress could reduce phagocytic efficiency by affecting receptor distribution.42 However, TUDCA did not have a radical scavenging activity in our assay. This is surprising in light of recent reports on the ability of TUDCA to reduce the oxidative stress–induced ROS production.43 Tauroursodeoxycholic acid is known as a chemical chaperone and is widely used in ER stress.35,44,45 It prevents the ER stress-associated rapid cone degeneration.46 Previously, we revealed that mutant semaphorin-4A, which is implicated in RP, caused ER stress and phagocytic dysfunction under the light-irradiated condition in ARPE-19 cells.26 However, TUDCA did not reduce the increase of ER stress marker (Supplementary Fig. S1). It could be thought that treatment with TUDCA improved phagocytosis without reduction in ER stress. Hence, TUDCA might affect phagocytosis through an independent pathway of ER stress. The functional protection against H2O2 by TUDCA was supported by the activation of MerTK. Mer tyrosine kinase receptor has a critical role in the physiological renewal of POS.47 It triggers photoreceptor POS ingestion by the RPE. Mutations in MerTK impair the phagocytic activity of the RPE cells and lead to accumulation of POS debris in the interphotoreceptor space, ultimately resulting in retinal degeneration. It has been reported that photoreceptors underwent apoptotic cell death in the MerTK−/− mice. Consistent with this finding, the photoreceptor degeneration seen in the Royal College of Surgeons (RCS) rat was associated with a loss of function deletion in the rat MerTK gene.24,48 Nearly all MerTK mutations identified in human patients are associated with early onset retinal dystrophy.5 In the present study, TUDCA increased the level of MerTK phosphorylation. However, the mechanism by which TUDCA promotes MerTK phosphorylation is unclear. In H2O2 nontreated condition, TUDCA increased pHrodo fluorescence at 0.1 μM and more (Fig. 3C), although TUDCA significantly increased MerTK phosphorylation at 1000 μM (Figs. 4A, 4B). These results suggest that TUDCA may not directly act to promote the phagocytosis via MerTK phosphorylation in the normal condition. Integrin, which is associated with the recognition of POS, is activated by TUDCA,49 and that TUDCA activates FAK and Src via integrins, which mediate downstream signaling toward mitogen-activated protein kinase in the rat liver.50 Focal adhesion kinase signaling in response to integrin-dependent POS binding by RPE is upstream of receptor MerTK phosphorylation.8 Therefore, TUDCA may promote the phosphorylation of MerTK via integrin activation, and further studies for clarifying the effects of TUDCA on RPE phagocytosis are required. 
In the present study, we highlighted the challenges involved in deciphering the molecular mechanisms of TUDCA. In conclusion, systemic administration of TUDCA could prevent vision deficits in various retinal disorders associated with photoreceptor loss. We also propose new research approaches, and envisage future strategies to prevent phagocytic dysfunction. 
Acknowledgments
The authors thank Yoshiki Kuse and Tomoyo Imamura for technical support. 
Supported by Gifu Pharmaceutical University. The authors alone are responsible for the content and writing of the paper. 
Disclosure: H. Murase, None; K. Tsuruma, None; M. Shimazawa, None; H. Hara, None 
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Figure 1
 
Fluorescence microscopic detection of phagocytosis of pHrodo-SE–labeled photoreceptor outer segment. (AaAd) Representative fluorescence micrographs showing pHrodo-SE–labeled POS engulfed by ARPE-19. (Ae) Representative phase contrast images showing shape of ARPE-19 cells at 24 hours. (Af) Representative merged micrograph showing pHrodo-SE–labeled POS with ARPE-19. (B) Measurements of the pH sensitive fluorescence POS. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control (Dunnett's test).
Figure 1
 
Fluorescence microscopic detection of phagocytosis of pHrodo-SE–labeled photoreceptor outer segment. (AaAd) Representative fluorescence micrographs showing pHrodo-SE–labeled POS engulfed by ARPE-19. (Ae) Representative phase contrast images showing shape of ARPE-19 cells at 24 hours. (Af) Representative merged micrograph showing pHrodo-SE–labeled POS with ARPE-19. (B) Measurements of the pH sensitive fluorescence POS. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control (Dunnett's test).
Figure 2
 
H2O2-induced reduction of phagocytic activity in ARPE-19 cells. (A, B) The reduction of phagocytic activity was stimulated with 30, 100, 300 μM H2O2, and fluorescence was measured at 6 hours. (A) Representative fluorescence micrographs showing pHrodo fluorescence upon addition at 6 hours after H2O2. (B) The H2O2 treatment decreased the phagocytic activity in a concentration-dependent manner. (C) N-acetylcysteine was used as a typical antioxidant. Pretreatment with N-acetylcysteine (1 mM) inhibited H2O2 100 μM-induced phagocytic dysfunction. (D) Cell death assay was performed using Hoechst 33342 and propidium iodide. H2O2 (100 μM) did not induce cell death. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control, ##P < 0.01 versus H2O2 (Student's t-test).
Figure 2
 
H2O2-induced reduction of phagocytic activity in ARPE-19 cells. (A, B) The reduction of phagocytic activity was stimulated with 30, 100, 300 μM H2O2, and fluorescence was measured at 6 hours. (A) Representative fluorescence micrographs showing pHrodo fluorescence upon addition at 6 hours after H2O2. (B) The H2O2 treatment decreased the phagocytic activity in a concentration-dependent manner. (C) N-acetylcysteine was used as a typical antioxidant. Pretreatment with N-acetylcysteine (1 mM) inhibited H2O2 100 μM-induced phagocytic dysfunction. (D) Cell death assay was performed using Hoechst 33342 and propidium iodide. H2O2 (100 μM) did not induce cell death. Data are shown as mean ± SEM (n = 6). **P < 0.01 versus control, ##P < 0.01 versus H2O2 (Student's t-test).
Figure 3
 
The effects of TUDCA on RPE phagocytosis. (A) The ARPE-19 cells were incubated with 100 μM H2O2 for 6 hours. Measurements of the pHrodo fluorescence in the H2O2 treated statement. (B) The hRPE cells were incubated with 100 μM H2O2 for 6 hours. Measurements of the pHrodo fluorescence in the H2O2 treated statement. (C) In ARPE-19 cells, pHrodo fluorescence was measured in the H2O2 nontreated statement. (D) Measurements of ROS production. Intracellular ROS levels were determined by measuring the fluorescence of CM-H2DCFDA (at excitation 488 nm/emission 525 nm) after H2O2 addition at 6 hours in ARPE-19 cells. N-acetylcysteine was used as a typical antioxidant. (E) In hRPE cells, FITC Isomer I fluorescence was measured. The TUDCA (1 mM) was treated 1 hour before POS addition. (F) In hRPE cells, pHrodo fluorescence was measured. The TUDCA (1 mM) was treated 1 hour before POS addition. Data are shown as mean ± SEM ([AE], n = 6; [F], n = 12). #P < 0.05, ##P < 0.01 versus control (Student's t-test [A, B, DF] and Dunnett's test [C]). **P < 0.01 versus H2O2 (Dunnett's test).
Figure 3
 
The effects of TUDCA on RPE phagocytosis. (A) The ARPE-19 cells were incubated with 100 μM H2O2 for 6 hours. Measurements of the pHrodo fluorescence in the H2O2 treated statement. (B) The hRPE cells were incubated with 100 μM H2O2 for 6 hours. Measurements of the pHrodo fluorescence in the H2O2 treated statement. (C) In ARPE-19 cells, pHrodo fluorescence was measured in the H2O2 nontreated statement. (D) Measurements of ROS production. Intracellular ROS levels were determined by measuring the fluorescence of CM-H2DCFDA (at excitation 488 nm/emission 525 nm) after H2O2 addition at 6 hours in ARPE-19 cells. N-acetylcysteine was used as a typical antioxidant. (E) In hRPE cells, FITC Isomer I fluorescence was measured. The TUDCA (1 mM) was treated 1 hour before POS addition. (F) In hRPE cells, pHrodo fluorescence was measured. The TUDCA (1 mM) was treated 1 hour before POS addition. Data are shown as mean ± SEM ([AE], n = 6; [F], n = 12). #P < 0.05, ##P < 0.01 versus control (Student's t-test [A, B, DF] and Dunnett's test [C]). **P < 0.01 versus H2O2 (Dunnett's test).
Figure 4
 
Effects of TUDCA on MerTK and FAK phosphorylation. (A, B) Pretreatment with vehicle (lanes 1 and 5) or with TUDCA (lane 24, 68) was followed by 6 hours of additional incubation with 100 μM H2O2 (lane 58) in ARPE-19 cells. (C, D) Pretreatment with vehicle (lanes 1 and 2) or with TUDCA (1 mM, lanes 3 and 4) was followed by 6 hours of additional incubation with 100 μM H2O2 (lanes 2 and 3) in ARPE-19 cells. (E, F) Pretreatment with vehicle (lanes 1 and 3) or with TUDCA (1 mM, lanes 2 and 4) was followed by 6 hours of additional incubation with 100 μM H2O2 (lanes 3 and 4) in hRPE cells. Representative band images show (A) MerTK expression in ARPE-19 cells, (C) FAK expression in ARPE-19 cells, and (E) MerTK expression in hRPE cells. (B, F) Expression of p-MerTK was quantified by densitometry and corrected by reference to β-actin. (D) Expression of p-FAK was quantified by densitometry and corrected with reference to t-FAK. Data are shown as mean ± SEM ([B, D], n = 4; [F], n = 7). #P < 0.05, ##P < 0.01 versus control (Dunnett's test [B] and Student's t-test [D, F]), *P < 0.05, **P < 0.01 versus H2O2 (Dunnett's test [B] and Student's t-test [D, F]). C, control; V, vehicle.
Figure 4
 
Effects of TUDCA on MerTK and FAK phosphorylation. (A, B) Pretreatment with vehicle (lanes 1 and 5) or with TUDCA (lane 24, 68) was followed by 6 hours of additional incubation with 100 μM H2O2 (lane 58) in ARPE-19 cells. (C, D) Pretreatment with vehicle (lanes 1 and 2) or with TUDCA (1 mM, lanes 3 and 4) was followed by 6 hours of additional incubation with 100 μM H2O2 (lanes 2 and 3) in ARPE-19 cells. (E, F) Pretreatment with vehicle (lanes 1 and 3) or with TUDCA (1 mM, lanes 2 and 4) was followed by 6 hours of additional incubation with 100 μM H2O2 (lanes 3 and 4) in hRPE cells. Representative band images show (A) MerTK expression in ARPE-19 cells, (C) FAK expression in ARPE-19 cells, and (E) MerTK expression in hRPE cells. (B, F) Expression of p-MerTK was quantified by densitometry and corrected by reference to β-actin. (D) Expression of p-FAK was quantified by densitometry and corrected with reference to t-FAK. Data are shown as mean ± SEM ([B, D], n = 4; [F], n = 7). #P < 0.05, ##P < 0.01 versus control (Dunnett's test [B] and Student's t-test [D, F]), *P < 0.05, **P < 0.01 versus H2O2 (Dunnett's test [B] and Student's t-test [D, F]). C, control; V, vehicle.
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