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Retinal Cell Biology  |   September 2012
SEMA4A Mutations Lead to Susceptibility to Light Irradiation, Oxidative Stress, and ER Stress in Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Kazuhiro Tsuruma
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and the
  • Yuhei Nishimura
    Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinfomatics, Mie University Graduate School of Medicine, Tsu, Japan.
  • Seiya Kishi
    Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinfomatics, Mie University Graduate School of Medicine, Tsu, Japan.
  • Masamitsu Shimazawa
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and the
  • Toshio Tanaka
    Department of Molecular and Cellular Pharmacology, Pharmacogenomics and Pharmacoinfomatics, Mie University Graduate School of Medicine, Tsu, Japan.
  • Hideaki Hara
    From the Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; and the
  • Corresponding author: Hideaki Hara, Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, 1-25-4 Daigaku-nishi, Gifu 501-1196, Japan; hidehara@gifu-pu.ac.jp
Investigative Ophthalmology & Visual Science September 2012, Vol.53, 6729-6737. doi:10.1167/iovs.11-9378
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      Kazuhiro Tsuruma, Yuhei Nishimura, Seiya Kishi, Masamitsu Shimazawa, Toshio Tanaka, Hideaki Hara; SEMA4A Mutations Lead to Susceptibility to Light Irradiation, Oxidative Stress, and ER Stress in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(10):6729-6737. doi: 10.1167/iovs.11-9378.

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

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Abstract

Purpose.: RP is a retinal degeneration disorder that is caused by mutations of various genes, including semaphorin-4A (SEMA4A). A number of retinal diseases, including RP, are associated with light exposure, oxidative stress, and endoplasmic reticulum (ER) stress. In this study, we investigated whether mutant SEMA4A causes retinal dysfunction via light exposure, oxidative stress, and ER stress.

Methods.: Mutant SEMA4A (D345H or F350C) was overexpressed in a human retinal epithelium cell line ARPE19. Intracellular localization of mutant SEMA4A was investigated using confocal laser scanning microscopy. The ARPE-19 cells were also irradiated with white light, and expression of 78 kDa glucose-regulated protein (GRP78), a marker of ER stress, and phagocytosis were measured. The cells were treated with an ER stress inducer, tunicamycin, or an oxidative stressor, H2O2, and cell death was measured. Human SEMA4A mutants were expressed in zebrafish embryos with tunicamycin and mRNA of DNA damage-inducible transcript 3 (ddit3) was measured as an ER stress marker.

Results.: Mutant SEMA4A was localized in the ER, whereas wild type (WT) SEMA4A was observed in cell membranes. The expression of GRP78 was increased by mutant SEMA4A following light irradiation, and phagocytosis was suppressed in mutant SEMA4A-transfected cells. Mutant SEMA4A induced susceptibility to ER stress and oxidative stress. In zebrafish, human mutant SEMA4A increased ddit3 mRNA compared with WT under the ER stress condition.

Conclusions.: Our results suggest that mutations in SEMA4A may cause susceptibility to light exposure, oxidative stress, and ER stress, which may be involved in the progression and pathology of RP.

Introduction
RP is known as a group of hereditary retinal diseases that are characterized by progressive degeneration of photoreceptors. RP is one of the primary causes of visual disability or blindness, and a worldwide prevalence of RP is approximately 1 in 5000. 1 RP patients typically lose night vision in puberty and peripheral vision in young adulthood. In later life, central vision is damaged due to progressive photoreceptor degeneration, which leads to blindness in some cases. At present, no effective therapy exists for RP. 
Endoplasmic reticulum (ER) stress is caused by various biochemical and physiological stimuli, including genetic alteration, which result in the accumulation of unfolded proteins in the ER lumen. As a response to ER stress, cells activate a self-protective system termed the unfolded protein response, which includes increased expression of molecular chaperones such as 78 kDa glucose-regulated protein (GRP78) and GRP94, translational attenuation, and ER-associated degradation. 2 However, when excessive stress is caused in the ER, the unfolded protein response ultimately activates an apoptotic pathway that involves a CCAAT/enhancer-binding protein homologous protein (CHOP). 3  
In the case of RP, rods first degenerate due to gene mutations, followed by a gradual death of cones. The disease can be inherited as an autosomal dominant, autosomal recessive, or X-linked trait. The etiology underlying most forms of RP are mutations associated with photopigment metabolism. 4 Some genes are known to have critical roles in visual function such as those for rhodopsin, 5 phosphodiesterase 6, 6,7 and retinal pigment epithelium-specific 65 kDa protein (RPE65). 8 Some genes affected in RP are expressed in photoreceptors as well as in other retinal cells, such as the retinal pigment epithelium (RPE), and in tissues outside the eye. 4 Recently, mutant rhodopsin has been reported to be associated with ER stress. 9 Carbonic anhydrase 4, one of the RP genes expressed in various tissues, has also been reported to show a relationship with ER stress. 10 Moreover, ER stress is induced by oxidative stress, known as an exacerbating factor of RP, in RPE cells. 11 Although ER stress may have a common role in the pathology and progression of RP, the association between most other genes and ER stress remains unclear. 
The toxic effects of accumulated photo-oxidative products are prevented by a daily renewal process that photoreceptors undergo, wherein approximately 10% of their volume is shed and, subsequently, phagocytosed by adjacent RPE cells. The mutation of a receptor tyrosine kinase gene, which is found in RP patients, has been shown to result in phagocytotic dysfunction in RPE cells and, subsequent, retinal degeneration. 12 This finding suggests that disruption of phagocytosis can lead to the development of RP. 
The Semaphorin 4A (SEMA4A) protein, a member of semaphorin family, 13 is expressed in many tissues, and is known to enhance T-cell activation. 14 In the retina, SEMA4A is expressed in the RPE and the retinal ganglion cell layer but not in the outer nuclear layer, which contains photoreceptor cells. 15 SEMA4A plays an important role in retinal degeneration 15 and some mutations of the SEMA4A gene have been found in RP patients. 16 SEMA4A has a transmembrane domain and putative glycosylation sites, which implicate its maturation in the ER. 
In the present study, we investigated the influence of mutant SEMA4A on RPE cells and we also investigated the association between SEMA4A and various stresses, including ER stress, in vitro using a human RPE (HRPE) cell line and in vivo in zebrafish. 
Materials and Methods
Materials
A plasmid containing a Myc-tagged human SEMA4A gene (pReceiver-M9-SEMA4A) was obtained from GeneCopoeia (Germantown, MD). Drugs and sources were as follows: H2O2 (Wako, Osaka, Japan), tunicamycin (Wako), Hoechst 33342 (Invitrogen, Carlsbad, CA), propidium iodide (PI; Invitrogen). 
Cell Culture
The ARPE-19 line, a transformed cell line for HRPE cells, was obtained from American Type Culture Collection (Manassas, VA). The cells were maintained in Dulbecco's modified Eagle's medium (DMEM)/F-12 (Wako) 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 ARPE-19 cells were passaged by trypsinization every 3 to 4 days. 
Construction of Mutant SEMA4A
The wild type (WT) SEMA4A gene was mutated using a PrimeSTAR Mutagenesis Basal Kit (TaKaRa, Otsu, Japan) with the following primers (aspartic acid to histidine in position 345; D345H, phenylalanine to cysteine in position 350; F350C, and arginine to glutamine in position 713; R713Q), according to the manufacturer's protocol: 
  1.  
    D345H sense; 5′-CTCTTGCACATTGAACGTGTCTTTAAG-3′,
  2.  
    D345H antisense; 5′-TTCAATGTGCAAGAGAGAGAAGGCACA-3′,
  3.  
    F350C sense; 5′-CGTGTCTGTAAGGGGAAATACAAAGAG-3′,
  4.  
    F350C antisense; 5′-CCCCTTACAGACACGTTCAATGTCCAA-3′,
  5.  
    R713Q sense; 5′-CGGGCTCAGGGCAAGGTTCAGGGCTGT-3′,
  6.  
    R713Q antisense; 5′-CTTGCCCTGAGCCCGGAGTGCTCTCAA-3′.
Each mutation was confirmed by DNA sequencing. ARPE-19 cells were transfected with each plasmid using Lipofectamine 2000 (Invitrogen). 
Immunostaining
The cells were seeded on a chamber slide (Nunc, Rochester, NY) and transfected with plasmids. Twenty-four hours after transfection, the cells were washed by PBS and fixed with 4% paraformaldehyde. Cells were incubated with anti-myc antibody (Cell Signaling Technology, Danvers, MA), anti-pan cadherin antibody (Abcam, Cambridge, UK) and anti-GRP78 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by Alexa Fluor 488- and Alexa Fluor 548-conjugated secondary antibodies (Invitrogen), respectively. Nuclei were stained by Hoechst 33342. Stained cells were observed using a confocal laser scanning microscope (FV10i-DOC; Olympus, Tokyo, Japan). 
Immunoblot Analysis
Cells were washed with PBS, harvested, and lysed in RIPA buffer (Sigma-Aldrich, St. Louis, MO) with protease inhibitor cocktail and phosphatase inhibitor cocktail 1 and 2 (Sigma-Aldrich). Lysates were boiled for 5 minutes in SDS sample buffer (Wako). Equal amounts of protein were subjected to 5% to 20% SDS-PAGE gradient gels and then transferred to polyvinylidene difluoride membranes. After blocking with Blocking One-P (Nakarai Tesque, Kyoto, Japan) for 30 minutes, membranes were incubated with the primary antibody (anti-Myc [Cell Signaling Technology], anti-β-actin, [Sigma-Aldrich], and anti-GRP78 [BD Transduction Laboratories, Lexington, KY]). Subsequently, the membranes were incubated with the secondary antibody (HRP-conjugated goat anti-mouse immunoglobulin G [IgG] [Pierce Biotechnology, Rockford, IL]). The immunoreactive bands were visualized using Super Signal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology) and then measured using LAS 4000 UV mini (Fujifilm, Tokyo, Japan). 
Cell Death Assay
ARPE-19 cells, 24 hours after transfection, were used for cell death assays based on induction by tunicamycin or H2O2. The cells were immersed in DMEM supplemented with 0.1% or 1% FBS. Tunicamycin (2 μg/mL) or H2O2 (200 μM) was added to these cultures. After 24 hours, cell mortality was measured using a single cell digital imaging-based method that detected fluorescent staining of nuclei. At the end of the culture period, Hoechst 33342 (λex = 360 nm, λem > 490 nm) and PI (λex = 535 nm, λem > 617 nm) were added to the culture medium for 15 minutes at final concentrations of 8.1 μM 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. PI is a membrane impermeable dye that is generally excluded from viable cells. Images were collected using an Olympus IX70 inverted epifluorescence microscope (Olympus, Tokyo, Japan). The total number of cells was counted in a blind manner (by KT) and calculated the percent of PI-positive cells, as described in a previous report. 17  
Exposure of ARPE-19 Cells to White Light
ARPE-19 cells, 24 hours after transfection, were exposed to light irradiation. The entire medium was then replaced with fresh medium containing 1% FBS. The cells were exposed to 2500 lux (lx) of white fluorescent light (Nikon, Tokyo, Japan) for 24 hours or 48 hours at 37°C in a humidified atmosphere of 95% air and 5% CO2. The luminance was measured with a light meter (LM-332; ASONE, Osaka, Japan). 
Phagocytosis Assays
The transfected cells were confirmed to have reached confluence by light microscopy (Olympus) and the entire medium was then replaced with fresh medium containing 1% FBS. The cells were exposed to 2500 lx of white fluorescent light (Nikon) for 48 hours at 37°C. After 48 hours of incubation, 1.4 μL latex beads diluted with 450 μL medium were added to the wells (at a final concentration of 1 × 106 beads/wells in a final volume of 500 μL) and incubated for 4 hours. Subsequently, the cells were washed five times with PBS to remove extracellular latex beads and then exposed to 4% paraformaldehyde (PFA; Wako) for 10 minutes. The cells were washed again with PBS to remove PFA, and Hoechst 33342 was added to the culture medium for 15 minutes, at final concentrations of 8.1 μM, for nuclear staining. Images were collected using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan). We counted the total number of cells and the number of intracellular latex beads and calculated the percentage of intracellular latex beads relative to the total number of cells, as previously described. 17  
Injection of SEMA4A mRNA in Zebrafish
Zebrafish embryos were raised at 28.5°C and staged according to Kimmel et al. 18 The open reading frame (ORF) of human SEMA4A (WT, D345H, or F350C) was cloned in the pCS2P+ vector (Addgene, Cambridge, MA) using EcoRI and XbaI sites. The ORF of enhanced green fluorescent protein (EGFP) was cloned in the pCS2P+ using ClaI and StuI sites The sequences were confirmed by DNA sequencing (FASMAC, Kanagawa, Japan). PCR was performed using SP6 and T3 primers and the cloned pCD2P+ vector as a template. The PCR product was purified using QIAquick PCR purification kit (QIAGEN, Valencia, CA). By using the mMESSAGE mMACHINE SP6 Kit (Life Technologies, Grand Island, NY), mRNA for SEMA4A or EGFP was transcribed from the PCR product and purified and concentrated using LiCl precipitation. The RNA concentration was determined using NanoDrop (Thermo Scientific, Wilmington, DE). Zebrafish were bred and maintained according to the methods described by Westerfield. 19 Briefly, zebrafish were raised at 28.5 ± 0.5°C with a 14 hours/10 hours light/dark cycle. Embryos were cultured in egg water. We prepared three different mRNA solutions: (1) mRNA solution for EGFP only containing 300 ng/μL EGFP mRNA, (2) mRNA solution for SEMA4A-WT and EGFP containing 200 ng/μL EGFP mRNA and 100 ng/μL SEMA4A-WT mRNA, and (3) mRNA solution for SEMA4A-mutants and EGFP containing 200 ng/μL EGFP mRNA, 50 ng/μL SEMA4A- D345H mRNA, and 50 ng/μL SEMA4A- F350C mRNA. Injection of the mRNA solution was made in the two to four cell stages of the zebrafish embryo and delivered approximately 1 to 2 nl. At 6 hours post injection, only zebrafish embryo expressing EGFP were collected and treated with or without 0.1 μM tunicamycin (Sigma-Aldrich) for 20 hours. After the treatment, only zebrafish embryo expressing EGFP and showing no apparent external malformation were collected and used for further experiments. The investigation conformed to the ethical guidelines established by the Institutional Animal Care and Use Committee at Mie University, and was in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
qPCR Using Zebrafish
Total RNA was extracted from whole zebrafish embryo using RNAqueous Micro Kit (Life Technologies) according to the manufacturer's protocol. The RNA concentration was determined using NanoDrop (Thermo Scientific) and used to generate cDNAs using an iScript Select cDNA Synthesis Kit (Bio-Rad, Hercules, CA). qPCR was done using an ABI Prism 7300 (Life Technologies, Carlsbad, CA) with SYBR Green Realtime PCR Master Mix Plus (Toyobo, Osaka, Japan). The thermal cycling condition comprised an initial step at 95°C for 1 minute followed by 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 45 seconds. We measured the expression of ddit3 (alias name is CHOP), a well known biomarker for ER stress. The data were normalized by the quantity of eukaryotic translation elongation factor 1 alpha 1 (eef1a1). This allowed us to account for any variability in the initial template concentration as well as the conversion efficiency of the reverse transcription reaction. The primer sequences were 5′-CAGCTGAACAATGGTTAACATGA-3′ and 5′-AATCAAGTTTGAATGTGAGTTGTTG-3′ for ddit3, and 5′-CTTTCTGTTACCTGGCAAAGG-3′ and 5′-ACATCGTGGTTATTGGCCAC-3′for eef1a1. 
Statistical Analysis
Data are presented as the means ± SEM. Statistical comparisons were made using Student's t-test and one-way ANOVA followed by Dunnett's or Bonferroni multiple comparison test (using STAT VIEW version 5.0 [SAS Institute, Cary, NC]). P value less than 0.05 was considered to indicate statistical significance. 
Results
Altered Cellular Localization of Mutant SEMA4A
Previously, Abid et al. 16 identified three mutations in the SEMA4A gene from RP patients. D345H and F350C were identified as heterozygous mutations, and both mutations are required for RP onset. The other was a R713Q mutation, which is an autosomal dominant mode of inheritance. First, we investigated the localization of SEMA4A in ARPE-19 cells. WT and mutant SEMA4A were expressed at the same level (Fig. 1A). Transiently transfected-WT SEMA4A was observed in cell membranes merged with pan cadherin, a plasma membrane marker (Fig. 1B), whereas the mutant SEMA4As, D345H, and F350C, showed altered intracellular localization. Although a part of the D345H mutant was also observed in cell membranes, SEMA4A mutants were merged with GRP78, an ER molecular chaperone. On the other hand, R713Q mutant was observed in cell membrane as well as WT (Fig. 1B). 
Figure 1. 
 
Cellular localization of WT and mutant SEMA4A. (A) Transiently transfected WT SEMA4A and mutants were detected by immunoblotting. (B) Cellular localization of SEMA4A was altered by its mutations. Anti–pan-cadherin and anti-GRP78 antibody were used as a plasma membrane marker and an endoplasmic reticulum marker, respectively. Hoechst 33342 was used as a nuclear marker in merged images. The horizontal scale bar equals 10 μm.
Figure 1. 
 
Cellular localization of WT and mutant SEMA4A. (A) Transiently transfected WT SEMA4A and mutants were detected by immunoblotting. (B) Cellular localization of SEMA4A was altered by its mutations. Anti–pan-cadherin and anti-GRP78 antibody were used as a plasma membrane marker and an endoplasmic reticulum marker, respectively. Hoechst 33342 was used as a nuclear marker in merged images. The horizontal scale bar equals 10 μm.
Expression of GRP78 in SEMA4A Mutants Increased by White Light Irradiation
SEMA4A mutant proteins were observed in the ER, and SEMA4A has putative glycosylation sites. Therefore, mutant SEMA4A appeared to be associated with ER stress. The possibility that mutant SEMA4A caused ER stress, was examined by measuring the expression of GRP78, an ER stress marker, by immunoblotting analysis (Fig. 2A). A 24 hour white light irradiation did not increase in the expression of GRP78 in control EGFP, WT, or D345H transfected cells (Fig. 2B). On the other hand, GRP78 was increased in the cells expressing F350C mutant under the white light irradiation; however cell death was not observed in any transfectants (data not shown). Moreover, co-expression of D345H and F350C mutants, which assume the pathogenesis of RP, did not cause an additive or a synergistic effect in the increase of GRP78. 
Figure 2. 
 
GRP78 protein expression in light-irradiated SEMA4A F350C transfected cells. (A) ARPE-19 cells were transiently transfected with each plasmid and exposed to fluorescent light (2500 lx) for 24 hours. The cells were analyzed by immunoblotting. (B) Quantification of GRP78 expression was corrected by β-actin expression. Data are expressed as the mean ± SEM (n = 6). *P less than 0.05 versus the WT exposed to light group.
Figure 2. 
 
GRP78 protein expression in light-irradiated SEMA4A F350C transfected cells. (A) ARPE-19 cells were transiently transfected with each plasmid and exposed to fluorescent light (2500 lx) for 24 hours. The cells were analyzed by immunoblotting. (B) Quantification of GRP78 expression was corrected by β-actin expression. Data are expressed as the mean ± SEM (n = 6). *P less than 0.05 versus the WT exposed to light group.
Suppression of Phagocytosis by White Light Irradiation in SEMA4A Mutants
We investigated the effect of mutant SEMA4A on RPE function by measuring phagocytosis in ARPE-19 cells. Phagocytosis is one of the major functions of RPE to maintain the photoreceptor and reduce the oxidative stress. Previously, we have reported that the uptake of latex beads (i.e., phagocytosis) into ARPE-19 cells was reduced by irradiation with white light. 17 In SEMA4A mutants, phagocytosis of fluorescent-labeled latex beads was reduced after 48 hours of light irradiation. The SEMA4A F350C mutant showed strong suppression of phagocytosis compared with WT SEMA4A following light irradiation (Fig. 3). In contrast, the D345H mutation had no effect on phagocytosis. When maintained in dark conditions, none of the mutations showed any effect on phagocytosis when compared with a mock transfectant. An additive or a synergistic effect by co-expression of D345H and F350C was not observed in decrease of phagocytosis. 
Figure 3. 
 
Phagocytosis in light-irradiated SEMA4A F350C transfected cells. ARPE-19 cells were transiently transfected with each plasmid and exposed to fluorescent light (2500 lx) for 48 hours. Phagocytosis was measured as described in the Materials and Methods section. Data are expressed as the mean ± SEM (n = 4). ## P less than 0.01 versus the WT without light exposure group, **P less than 0.01 versus the WT with light exposure group.
Figure 3. 
 
Phagocytosis in light-irradiated SEMA4A F350C transfected cells. ARPE-19 cells were transiently transfected with each plasmid and exposed to fluorescent light (2500 lx) for 48 hours. Phagocytosis was measured as described in the Materials and Methods section. Data are expressed as the mean ± SEM (n = 4). ## P less than 0.01 versus the WT without light exposure group, **P less than 0.01 versus the WT with light exposure group.
Susceptibility of SEMA4A Mutants to ER Stress and Oxidative Stress
Light irradiation to retina induces oxidative stress, 20 and inflammatory cytokines, 21 which lead to the ER stress in RPE cells. 11,22 The RPE is also constantly subjected to oxidative stress as a result of exposure to arterial blood. We examined whether mutant SEMA4A alters the sensitivity of H2O2 and tunicamycin, an ER stress inducer. Representative photographs of Hoechst 33342 and PI staining are shown in Figure 4A. Hoechst 33342 stains all cells (live and dead cells), whereas PI stains only dead cells. WT SEMA4A showed no differences when compared with an EGFP transfectant with respect to tunicamycin-induced cell death (Fig. 4B). In contrast, the D345H mutation increased tunicamycin-induced cell death, whereas the F350C or R713Q transfectants showed no effect. Transfection with D345H increased H2O2-induced cell death compared with WT, while transfection with F350C slightly, but not significantly, increased it (Fig. 4C). 
Figure 4. 
 
Cell death induced by endoplasmic reticulum stress and oxidative stress in SEMA4A mutants transfected cells. (A) Typical images of nuclear staining. ARPE-19 cells were transiently transfected with each plasmid and tunicamycin (2 μg/mL) was added for 24 hours. (B) Quantification of tunicamycin-induced cell death was calculated as described in the Materials and Methods section. (C) ARPE-19 cells were transiently transfected with each plasmid and H2O2 (200 μM) was added for 24 hours. Data are expressed as the mean ± SEM (n = 6). ## P less than 0.01 versus the WT without tunicamycin group, **P less than 0.01 versus the WT with tunicamycin group.
Figure 4. 
 
Cell death induced by endoplasmic reticulum stress and oxidative stress in SEMA4A mutants transfected cells. (A) Typical images of nuclear staining. ARPE-19 cells were transiently transfected with each plasmid and tunicamycin (2 μg/mL) was added for 24 hours. (B) Quantification of tunicamycin-induced cell death was calculated as described in the Materials and Methods section. (C) ARPE-19 cells were transiently transfected with each plasmid and H2O2 (200 μM) was added for 24 hours. Data are expressed as the mean ± SEM (n = 6). ## P less than 0.01 versus the WT without tunicamycin group, **P less than 0.01 versus the WT with tunicamycin group.
Mutant SEMA4A Increased Susceptibility to ER Stress In Vivo
To examine whether mutant SEMA4A would increase susceptibility to ER stress in vivo as well as in vitro, we injected mRNA encoding human WT SEMA4A or SEMA4A mutants (D345H and F350C) with mRNA encoding EGFP. For control, we injected only mRNA encoding EGFP. The total concentration of mRNA in each group was 300 ng/ml. Following the mRNA injection, only embryo showing fluorescence of EGFP and normal morphology at the developmental stage were used for qPCR experiment to assess the expression level of ddit3 (CHOP). It has been shown that the expression of ddit3 is induced in ER stress response. As shown in Figure 5, there was no significant difference in the expression level of ddit3 among the three groups without tunicamycin treatment. However, among the three groups with tunicamycin treatment, the expression level of ddit3 in zebrafish injected with SEMA4A mutants was significantly higher than that in zebrafish injected with WT SEMA4A or EGFP only. These results suggest that SEMA4A mutants (mixture of D345H and F350C) may not cause ER stress by itself, but they can increase susceptibility to ER stress in vivo as well as ARPE-19 cells. Moreover, partially knock down of SEMA4A by morpholinos caused increases of X-box binding protein 1 (XBP1) and ddit3 (See Supplementary Material and Supplementary Fig. S1). 
Figure 5. 
 
ddit3 mRNA expression in tunicamysin-treated SEMA4A, D345H, and F350C expressing zebrafish. Zebrafish embryos were injected with EGFP only, the mixture of WT SEMA4A and EGFP or the mixture of SEMA4A mutants (D345H and F350C) and EGFP. After 6 hours post injection, the embryos were treated with or without 0.1 μM tunicamycin for 20 hours. Total RNA was extracted from the whole embryo and qPCR analysis was performed to examine the expression level of ddit3 and eef1a. In zebrafish treated with tunicamycin, the relative expression level of ddit3 to eef1a in zebrafish injected with SEMA4A mutants was significantly higher than those in zebrafish injected with WT SEMA4A or EGFP only. Results represent means ± SEM of each group. P value was calculated by Bonferroni-Dunn multiple comparison test (n = 3–7 for zebrafish without tunicamycin treatment, n = 9–14 for zebrafish with tunicamycin treatment). *P less than 0.01.
Figure 5. 
 
ddit3 mRNA expression in tunicamysin-treated SEMA4A, D345H, and F350C expressing zebrafish. Zebrafish embryos were injected with EGFP only, the mixture of WT SEMA4A and EGFP or the mixture of SEMA4A mutants (D345H and F350C) and EGFP. After 6 hours post injection, the embryos were treated with or without 0.1 μM tunicamycin for 20 hours. Total RNA was extracted from the whole embryo and qPCR analysis was performed to examine the expression level of ddit3 and eef1a. In zebrafish treated with tunicamycin, the relative expression level of ddit3 to eef1a in zebrafish injected with SEMA4A mutants was significantly higher than those in zebrafish injected with WT SEMA4A or EGFP only. Results represent means ± SEM of each group. P value was calculated by Bonferroni-Dunn multiple comparison test (n = 3–7 for zebrafish without tunicamycin treatment, n = 9–14 for zebrafish with tunicamycin treatment). *P less than 0.01.
Discussion
In this study, SEMA4A mutations have been revealed the susceptibility to various stresses. Although both D345H and F350C mutations caused changes in the intracellular localization of SEMA4A from the cell membrane to the ER in normal state, SEMA4A mutants do not cause by itself ER stress and cell dysfunction. 
Residues at positions 345 and 350 are included in the semaphorin domain, a conserved protein domain of approximately 500 amino acids found in the extracellular region of all members of the semaphorin protein family, whose key function is neuronal axon guidance. 23 Recently, a three-dimensional structure of human SEMA4D and its binding domain to plexin-B1, a receptor for SEMA4D, have been confirmed. 24 The residues at positions 345 and 350 of SEMA4A are included in α-helix and exist at surface of protein, 24 and seem to be important for protein interactions. Thus, these mutants may lead to be anchored in ER by abnormal protein interactions or these residues may be important during the maturation of SEMA4A. Recently, SEMA4A has been reported to bind prosaponin and rab11/FIP2, which are associated with endosomal sorting between the cell surface and endoplasmic reticulum and important in oxidative stress. 25 These interacting proteins may be associated with vulnerabilities to light, ER, and oxidative stress. Although the observed effect on cell dysfunction could be due to a general effect of SEMA4A accumulation in the ER, other factor(s) such as melanin (which is synthesized in the RPE and important to reduce light-induced toxicity) and its synthase (also found in the ER) may be involved in converting light stress to ER stress, and may be altered by the F350C mutation. Differences of D345H and F350C in susceptibility to each stress may be attributed to the affinity to such interacting proteins. 
The R713Q mutant showed no protein localization changes or increased vulnerability to stimuli. The R713Q mutant also did not show GRP78 induction or phagocytotic dysfunction (data not shown). These results suggest that the R713Q mutant may cause RP by a different mechanism from that invoked by D345H/F350C heterozygous mutations. Actually, R713Q mutation is an autosomal dominant inheritance, whereas either D345H or F350C mutation does not cause RP. The residue at position 713 is located on the intracellular domain 13,23 ; thus, R713Q mutation may obtain the abnormal function, disturbing interactions with intracellular proteins, and further experiments will be required. 
Phagocytosis is one of the most important functions of the RPE and disruption of phagocytosis can lead to the development of RP. RPE can phagocytose photo-oxidative products, thereby, reducing damaging effects of oxidative stress on photoreceptors and on the RPE itself. Phagocytosis decreases in vitro in response to light irradiation. 26 In the present study, a 48 hour light irradiation treatment decreased phagocytosis in ARPE-19 cells, as reported previously, 17 and the F350C mutation suppressed the amount of phagocytosis compared with the WT (Fig. 4). This result suggests that the F350C mutant may cause oxidative stress in the retina of RP patients via phagocytotic dysfunction. In contrast, the D345H mutant did not show a similar phagocytotic dysfunction, but did show an increased vulnerability to H2O2 stress. Moreover, the vulnerability to ER stress caused by the D345H mutation. Together with these results, F350C may have some roles in initiation of RPE dysfunction, while D345H may enhance and accelerate damage to RPE, resulting ultimately in photoreceptor cell death. The coexistence of each mutation is required for RP, in agreement with a previous report. 16  
We investigated the potential association between ER stress and phagocytosis using the chemical chaperones, 4-phenylbutyrate and tauroursodeoxycholic acid, which are known to reduce ER stress. 27,28 However, these chemical chaperones did not reduce the GRP78 expression induced by tunicamycin or light irradiation in F350C mutant ARPE-19 cells (data not shown). Understanding the mechanism of light irradiation-induced ER stress and phagocytotic dysfunction in the presence of F350C mutation will require further experiments. 
In vivo studies also revealed that SEMA4A mutants lead to the susceptibility to ER stress. The results of intracellular localization (Fig. 1B) and SEMA4A knockdown (See Supplementary Material and Supplementary Fig. S1) suggest that SEMA4A mutants may cause the loss of function by their accumulation in ER, which leads to the susceptibility to ER stress in vivo. Knockout of SEMA4A leads to protein accumulation in ER of ARPE-19 cells,25 therefore, SEMA4A may be important in maintaining the ER environment. Although SEMA4A mutants could exert the abnormality in tunicamycin-treated condition (Figs. 4B, 5), endogenous SEMA4A may counteract the abnormal function of SEMA4A mutants. SEMA4A responds to oxidative stress in retina25; therefore, SEMA4A mutants may also lead to the susceptibility to oxidative stress in vivo. 
In conclusion, our findings suggest that SEMA4A mutations cause the susceptibility to light exposure, oxidative stress, and ER stress. The SEMA4A mutations may elevate ER stress in the retina exposed to light irradiation. One of the precipitating factors of RP may in fact be ER stress, acting in conjunction with light irradiation stress and oxidative stress. 
Supplementary Materials
Acknowledgments
The authors thank Reiko Kawase for her skillful assistance in the experiments using zebrafish. 
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Footnotes
 Supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan for Grants-in-Aid for Scientific Research (B) (20592082).
Footnotes
 Disclosure: K. Tsuruma, None; Y. Nishimura, None; S. Kishi, None; M. Shimazawa, None; T. Tanaka, None; H. Hara, None
Figure 1. 
 
Cellular localization of WT and mutant SEMA4A. (A) Transiently transfected WT SEMA4A and mutants were detected by immunoblotting. (B) Cellular localization of SEMA4A was altered by its mutations. Anti–pan-cadherin and anti-GRP78 antibody were used as a plasma membrane marker and an endoplasmic reticulum marker, respectively. Hoechst 33342 was used as a nuclear marker in merged images. The horizontal scale bar equals 10 μm.
Figure 1. 
 
Cellular localization of WT and mutant SEMA4A. (A) Transiently transfected WT SEMA4A and mutants were detected by immunoblotting. (B) Cellular localization of SEMA4A was altered by its mutations. Anti–pan-cadherin and anti-GRP78 antibody were used as a plasma membrane marker and an endoplasmic reticulum marker, respectively. Hoechst 33342 was used as a nuclear marker in merged images. The horizontal scale bar equals 10 μm.
Figure 2. 
 
GRP78 protein expression in light-irradiated SEMA4A F350C transfected cells. (A) ARPE-19 cells were transiently transfected with each plasmid and exposed to fluorescent light (2500 lx) for 24 hours. The cells were analyzed by immunoblotting. (B) Quantification of GRP78 expression was corrected by β-actin expression. Data are expressed as the mean ± SEM (n = 6). *P less than 0.05 versus the WT exposed to light group.
Figure 2. 
 
GRP78 protein expression in light-irradiated SEMA4A F350C transfected cells. (A) ARPE-19 cells were transiently transfected with each plasmid and exposed to fluorescent light (2500 lx) for 24 hours. The cells were analyzed by immunoblotting. (B) Quantification of GRP78 expression was corrected by β-actin expression. Data are expressed as the mean ± SEM (n = 6). *P less than 0.05 versus the WT exposed to light group.
Figure 3. 
 
Phagocytosis in light-irradiated SEMA4A F350C transfected cells. ARPE-19 cells were transiently transfected with each plasmid and exposed to fluorescent light (2500 lx) for 48 hours. Phagocytosis was measured as described in the Materials and Methods section. Data are expressed as the mean ± SEM (n = 4). ## P less than 0.01 versus the WT without light exposure group, **P less than 0.01 versus the WT with light exposure group.
Figure 3. 
 
Phagocytosis in light-irradiated SEMA4A F350C transfected cells. ARPE-19 cells were transiently transfected with each plasmid and exposed to fluorescent light (2500 lx) for 48 hours. Phagocytosis was measured as described in the Materials and Methods section. Data are expressed as the mean ± SEM (n = 4). ## P less than 0.01 versus the WT without light exposure group, **P less than 0.01 versus the WT with light exposure group.
Figure 4. 
 
Cell death induced by endoplasmic reticulum stress and oxidative stress in SEMA4A mutants transfected cells. (A) Typical images of nuclear staining. ARPE-19 cells were transiently transfected with each plasmid and tunicamycin (2 μg/mL) was added for 24 hours. (B) Quantification of tunicamycin-induced cell death was calculated as described in the Materials and Methods section. (C) ARPE-19 cells were transiently transfected with each plasmid and H2O2 (200 μM) was added for 24 hours. Data are expressed as the mean ± SEM (n = 6). ## P less than 0.01 versus the WT without tunicamycin group, **P less than 0.01 versus the WT with tunicamycin group.
Figure 4. 
 
Cell death induced by endoplasmic reticulum stress and oxidative stress in SEMA4A mutants transfected cells. (A) Typical images of nuclear staining. ARPE-19 cells were transiently transfected with each plasmid and tunicamycin (2 μg/mL) was added for 24 hours. (B) Quantification of tunicamycin-induced cell death was calculated as described in the Materials and Methods section. (C) ARPE-19 cells were transiently transfected with each plasmid and H2O2 (200 μM) was added for 24 hours. Data are expressed as the mean ± SEM (n = 6). ## P less than 0.01 versus the WT without tunicamycin group, **P less than 0.01 versus the WT with tunicamycin group.
Figure 5. 
 
ddit3 mRNA expression in tunicamysin-treated SEMA4A, D345H, and F350C expressing zebrafish. Zebrafish embryos were injected with EGFP only, the mixture of WT SEMA4A and EGFP or the mixture of SEMA4A mutants (D345H and F350C) and EGFP. After 6 hours post injection, the embryos were treated with or without 0.1 μM tunicamycin for 20 hours. Total RNA was extracted from the whole embryo and qPCR analysis was performed to examine the expression level of ddit3 and eef1a. In zebrafish treated with tunicamycin, the relative expression level of ddit3 to eef1a in zebrafish injected with SEMA4A mutants was significantly higher than those in zebrafish injected with WT SEMA4A or EGFP only. Results represent means ± SEM of each group. P value was calculated by Bonferroni-Dunn multiple comparison test (n = 3–7 for zebrafish without tunicamycin treatment, n = 9–14 for zebrafish with tunicamycin treatment). *P less than 0.01.
Figure 5. 
 
ddit3 mRNA expression in tunicamysin-treated SEMA4A, D345H, and F350C expressing zebrafish. Zebrafish embryos were injected with EGFP only, the mixture of WT SEMA4A and EGFP or the mixture of SEMA4A mutants (D345H and F350C) and EGFP. After 6 hours post injection, the embryos were treated with or without 0.1 μM tunicamycin for 20 hours. Total RNA was extracted from the whole embryo and qPCR analysis was performed to examine the expression level of ddit3 and eef1a. In zebrafish treated with tunicamycin, the relative expression level of ddit3 to eef1a in zebrafish injected with SEMA4A mutants was significantly higher than those in zebrafish injected with WT SEMA4A or EGFP only. Results represent means ± SEM of each group. P value was calculated by Bonferroni-Dunn multiple comparison test (n = 3–7 for zebrafish without tunicamycin treatment, n = 9–14 for zebrafish with tunicamycin treatment). *P less than 0.01.
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