1Department of Ophthalmology, Gunma University School of Medicine; and 2Second Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, Japan
Submitted 1 March 2004 ; accepted in final form 13 December 2004
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ABSTRACT |
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angiogenesis; transcription factors; retinoid
The molecular mechanisms underlying the intraocular neovascularization have been the focus of intensive research. VEGF is an angiogenic peptide that is increased greatly in response to hypoxia in retinal cells (25). To date, it has been well established that hypoxia-mediated increase in VEGF expression plays a major role in development of many retinal diseases such as retinopathy of prematurity and diabetic retinopathy (19, 27). However, the molecular mechanisms underlying VEGF induction independent of hypoxia remain unclear. Thus the identification of the mediators that are capable of inducing VEGF expression may provide new insight into the mechanism of hypoxia-independent ocular disease. In this regard, our study, which indicates that light exposure increases VEGF expression in retinal cells, seems to provide a clue to an important clinical relevance.
Light exposure initiates a cascade leading to the transmission of a visual signal through the phototransduction pathway. During this process, 11-cis retinal is converted to the all-trans retinal, which is then reduced to all-trans retinol by reduced nicotinamide adenine dinucleotide phosphate and photoreceptor retinol dehydrogenase within the rod outer segment (12). Rhodopsin undergoes conformational change by which it catalytically activates a G protein and transmits a visual signal (3). These events could be blocked by guanosine 5'-[-thio]diphosphate (GDP
S) (18a), and many biological effects of pertussis toxin are the result of a toxin-catalyzed transfer of an ADP ribose moiety from NAD to the
-subunits of signal-transducing G protein (43). All-trans retinal subsequently dissociates from opsin, leaving the chromophore pocket empty (3). Recently, one report (28) has shown that exposure of retinal tissue to light induces retinoic acid (RA) synthesis. Our previous studies have shown that treatment of Y79 cells, a cultured human retinoblastoma cell line, with all-trans RA increases the VEGF expression (1). These lines of evidence led us to hypothesize that light exposure increases VEGF expression in photoreceptor cells.
One of the major findings in this study is that a cis element involving light-mediated increase in VEGF expression is localized at 89 and 68 of VEGF promoter, whose sequence matches Sp1 binding sites. In fact, the results of electrophoretic mobility shift assays (EMSAs) showed that Sp1 protein binds to this site and light stimulation increases the binding of nuclear factors to the Sp1 site. We also showed that light increased Sp1 protein levels but not Sp1 mRNA levels, thus suggesting that light has a potential to enhance the stability of Sp1 protein. Previous studies done by us and others support the idea that Sp1 plays a role in mediating the inducible expression of various genes, such as superoxide dismutase (37), glucose activation of the carboxylase (9), plasminogen activator inhibitor-1 (6), and VEGF genes (31). In addition, a role of Sp1 has been described in PMA-induced expression of the WAF1/CIP1 (4) gene and the platelet thromboxane receptor gene (8).
In this study, we determined the effects of light on the VEGF gene expression in human retinoblastoma cell line Y79 and showed that the exposure of Y79 cells to visible light induces VEGF transcription via RA-mediated activation of RA receptor- (RAR
). These data suggest a novel and important role of light for the induction of VEGF gene in ocular tissues.
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MATERIALS AND METHODS |
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Fluorescent light exposure.
A fluorescent lamp (Neoball Z 40W EFA8EL, Toshiba) provided visible light for culture dishes in a 37°C incubator in 5% CO2. According to the data sheet provided by the manufacturer, the wavelengths of this illumination range from 400 to 740 nm with the major peaks at 550 and 620 nm, and with the minor peaks at 410, 440, 500, 600, 640, and 730 nm. The fluorescent lamp was adjusted 50 cm above culture dishes. The wavelengths of this illumination range from 400 to 740 nm. A control dish was shaded with aluminum foil. The length of exposure time and illumination intensity are indicated in the figure legends. Illumination light intensity was measured with the use of a SUN ILLUMIND SLX-1330 (Sanyo, Tokyo, Japan), and medium temperature was taken with a measuring kit (model 950, Testo, Yokohama, Japan) as stated in the figure legends.
Plasmid constructions.
We obtained the VEGF promoter/pGL3 from Dr. J. A. Abraham, which contained DNA fragment from 1,180 to +338 of the human VEGF gene fused to luciferase reporter plasmid (41). Plasmids 480 and 89LUC were made by subcloning the BglII and SmaI insert from 1180LUC into the corresponding site of pGL3 (Promega, Madison, WI). The RA response element, RAR dominant-negative/pCMX, was a generous gift from Dr. A. Kakizuka (32) (Osaka Bioscience Institute).
Cell culture and transfection. Y79 retinoblastoma and retinal pigment epithelium cells were obtained from the American Type Culture Collection and cultured in RPMI 1640 supplemented with 10% FBS and antibiotics at 37°C in 5% CO2. Transfection into Y79 cells was performed with a modified calcium phosphate coprecipitation technique as previously described (14). The cells were transfected with 1 µg of reporter plasmid. Twenty-four hours after transfection, the cells were washed twice with phosphate-buffered saline and treated with or without light (700 lux) stimulation for 12 h. After 24 h of incubation, the cells were harvested for luciferase assay. Luciferase activity was measured with the use of a luminometer (Lumat model LB9501, Berthold, Bad Wildbad, Germany) and was normalized to cellular protein concentration. Each transfection was repeated, and means ± SE are presented.
EMSAs and supershift assays. Nuclear extracts from Y79 cells were prepared as previously described (14). The sequences of double-stranded oligonucleotides used as probes or competitors in EMSAs were as follows, with the mutations of wild-type sequences in boldface: VEGF-89/-67, 5'-CCCGGGGCGGGCCGGGGGCGGGG-3'; VEGF-89/-67(Sp1m), 5'-CCCGGGAAGGGCCGGGGAAGGGG-3'; Sp1, 5'-ATTCGATCGGGGCGGGGCGAGC-3'; cAMP response element binding protein (CREB), 5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3'; and AP-2, 5'-GATCGAACTGACCGCCCGCGGCCCGT-3'.
Binding reactions, EMSAs, and supershift assays were performed as previously described (14).
Northern and Western blot analyses. A 642-bp fragment of human VEGF cDNA sequence and 2.0-kb fragment of the rat Sp1 cDNA sequence were used as a probe for Northern blot analyses. Nuclear extracts from vehicle- or light-treated Y79 cells were directly subjected to immunoblotting for Sp1. Western blot analyses were performed essentially as previously described (14). Sp1 was visualized by using an affinity-purified rabbit polyclonal antibody and a horseradish peroxidase-linked anti-rabbit IgG secondary antibody (Amersham).
ELISA for VEGF. The concentration of VEGF produced was measured using a commercially available ELISA kit (Immuno-Biological Laboratories, Fujioka, Japan), as stated in the Fig. 2 legend. The culture supernatants were collected after stimulation for 48 h, and the absorbency was measured at 450 nm. VEGF production was normalized to the volume of the medium and cell number.
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RESULTS |
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Specific inhibitors for G protein, pertussis toxin, and GDPS, repress light-induced VEGF expression.
To define the signaling pathways responsible for the effects of light stimulation on the transcription of VEGF gene, we tested the effects on light stimulation of a set of different protein kinase inhibitors, including SB-203580 (inhibitor of p38 mitogen-activated protein kinase) (38), wortmannin (phosphatidylinositol 3-kinase inhibitor) (40), PD-98059 (mitogen-activated protein kinase inhibitor) (38), and PP1 (Src family kinase inhibitor) (33), as well as pertussis toxin and GDP
S of G protein inhibitor. Northern blot analyses showed that PP1 partly inhibited and GDP
S and pertussis toxin completely abolished the induction of light-mediated increase in VEGF mRNA expression (Fig. 3). These results suggest that light induces the VEGF gene expression through phototransduction, given that the transduction of light into a neural signal in rod and cone receptor goes through signaling by G protein.
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We then attempted to map the regions that confer both basal and light-induced activities on the VEGF promoter. Light significantly increased the promoter activity of both 480Luc and 89Luc (Fig. 4B). Luciferase activity of 89Luc was significantly higher than that of promoterless construct pGL3, suggesting that the sequences downstream of 89 contain elements required for both basal and light-induced expression of the VEGF promoter. A response of the VEGF promoter activity to light appears to be promoter specific because -actin promoter was totally unresponsive (data not shown). These results indicate that a sequence between 89 and +338 is necessary for light-induced VEGF gene transcription as well as for basal expression.
Sp1 sites play a critical role in light-mediated VEGF expression. We have previously shown that a sequence between 89 and +338 contains two functional Sp1 sites at 85 and 74 and a consensus AP-2 site at 80 located upstream of the transcription start site. To test whether Sp1 sites serve as the light-regulatory element, 89(Sp1m)Luc, a plasmid that contains mutations within the two Sp1 binding sites, was transiently transfected into Y79 cells. The disruption of both Sp1 sites markedly impaired the responsiveness to light stimulation (Fig. 4B). Such a loss of responsiveness of 89(Sp1m)Luc to light was not due to the disruption of the essential elements for the basal transcription, because the luciferase activity of 89(Sp1m)Luc was significantly higher than that of pGL3. These results indicate that the activation of VEGF promoter in response to light depends on the integrity of at least one of the two Sp1 sites.
Identification of nuclear factors binding to light-responsive elements in human VEGF promoter. To examine the ability of these sites to interact with Sp1 or related factors, EMSAs were performed using nuclear extracts prepared from either untreated or light-treated Y79 cells and the 32P-labeled double-stranded oligonucleotide probe containing the sequence between 89 and 67. As shown in Fig. 5A, the 32P-labeled VEGF 89/67 probe gave rise to two specific DNA:protein complexes (C1 and C2). The intensity of C1 complex was enhanced by light. By contrast, the C2 complex was not affected (Fig. 5A). Both C1 and C2 complexes were proved to be sequence specific because formation of these complexes was competed by wild type but not by a mutated version of the probe sequence (Fig. 5B). These complexes could also be competed by consensus Sp1 sequence but not by CREB and AP-2 binding sequence (Fig. 5B). To verify that C1 and C2 complexes contain Sp1- or Sp1-related proteins, we carried out supershift assays using Sp1- or Sp3-antisera (Fig. 5C). The addition of an Sp1 antibody resulted in a supershift of a complex C1, indicating that Sp1 is a principal DNA binding component of this complex. An Sp3 antibody completely supershifted complex C2. The addition of the CREB and AP-2 antibodies had no effect on complex formation. These results provide the evidence that Sp1 and Sp3 but not AP-2 or CREB bind to the VEGF 89/67 probe, and light increased the binding of Sp1 to this sequence.
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DISCUSSION |
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In addition to the pleiotropic effects of RA, such as the regulation of cellular growth and differentiation of the retinal tissue (16, 18, 29, 42), most of the earlier studies on RA in the retinal tissues focused on its role in visual cycles. Among them, a significant observation was that light exposure induces RA synthesis in retinal tissue in vivo (28). Along a similar line, we have recently reported that atRA induces the VEGF gene expression in Y79 cells (1). Thus we tested whether Y79 cells afford a model to analyze the molecular events accounting for some forms of light-induced retinopathy. Here, we specified the role of RA in light-induced VEGF expression by testing the effects of RAR antagonist on VEGF induction. LE-135, a RAR
antagonist (17), potently inhibited the light-mediated VEGF induction. We also showed that cotransfection of dominant negative mutant of RAR
attenuates the light-induced VEGF promoter activity. Thus these data suggest that atRA-activated RAR
mediate the light-induced VEGF expression in Y79 cells.
In view of the mechanisms of photoreceptor cell death via apoptosis that characterize retinal dystrophies, previous studies (10, 39) have shown that light exposure creates the condition of oxidative stress leading to oxidative damage. Although oxidative events are important for a variety of biological processes, such as signal transduction and gene expression, we should emphasize that light-induced VEGF expression is not mainly due to the oxidative stress, because an inhibitor for G protein and antagonist of RAR inhibited the response. In this regard, we propose that light serves as a regulator of gene expression via ligand-activated nuclear receptors.
We have identified RAR as a mediator of the light-induced VEGF expression. In mice disrupted with RAR
, the eyes are extremely small with gross morphological defects in choroid, sclera, and retinal dysplasia (13). Furthermore, previous reports (36) indicated that RA produces rod photoreceptor-selective apoptosis in developing mammalian retina. Future studies on the light-induced VEGF expression using the conditional knockout of RAR
loci will uncover the role of RAR
in the mature retina.
In conclusion, we found that light induces the VEGF gene expression through Sp1-binding sites of the human VEGF promoter. In addition, light increases the levels of Sp1 protein and enhances its binding activity to Sp1 sites within the VEGF promoter. Given that light induces RA synthesis in retinal tissue in vivo, RA may be one of the critical mediators for neovascularization through induction of VEGF expression. Therefore, our findings raise the possibility that pharmacological intervention that inhibits the signals elicited by light or RA may be effective in treating VEGF-mediated retinopathies.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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