Retinoid X receptor activation is essential for docosahexaenoic acid protection of retina photoreceptors
2013; Elsevier BV; Volume: 54; Issue: 8 Linguagem: Inglês
10.1194/jlr.m039040
ISSN1539-7262
AutoresOlga Lorena German, Sandra Monaco, Daniela L. Agnolazza, Nora P. Rotstein, Luis E. Politi,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoWe have established that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, promotes survival of rat retina photoreceptors during early development in vitro and upon oxidative stress by activating the ERK/MAPK signaling pathway. Here we have investigated whether DHA turns on this pathway through activation of retinoid X receptors (RXRs) or by inducing tyrosine kinase (Trk) receptor activation. We also evaluated whether DHA release from phospholipids was required for its protective effect. Addition of RXR antagonists (HX531, PA452) to rat retinal neuronal cultures inhibited DHA protection during early development in vitro and upon oxidative stress induced with Paraquat or H2O2. In contrast, the Trk inhibitor K252a did not affect DHA prevention of photoreceptor apoptosis. These results imply that activation of RXRs was required for DHA protection whereas Trk receptors were not involved in this protection. Pretreatment with 4-bromoenol lactone, a phospholipase A2 inhibitor, blocked DHA prevention of oxidative stress-induced apoptosis of photoreceptors. It is noteworthy that RXR agonists (HX630, PA024) also rescued photoreceptors from H2O2-induced apoptosis. These results provide the first evidence that activation of RXRs prevents photoreceptor apoptosis and suggest that DHA is first released from phospholipids and then activates RXRs to promote the survival of photoreceptors. We have established that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid in the retina, promotes survival of rat retina photoreceptors during early development in vitro and upon oxidative stress by activating the ERK/MAPK signaling pathway. Here we have investigated whether DHA turns on this pathway through activation of retinoid X receptors (RXRs) or by inducing tyrosine kinase (Trk) receptor activation. We also evaluated whether DHA release from phospholipids was required for its protective effect. Addition of RXR antagonists (HX531, PA452) to rat retinal neuronal cultures inhibited DHA protection during early development in vitro and upon oxidative stress induced with Paraquat or H2O2. In contrast, the Trk inhibitor K252a did not affect DHA prevention of photoreceptor apoptosis. These results imply that activation of RXRs was required for DHA protection whereas Trk receptors were not involved in this protection. Pretreatment with 4-bromoenol lactone, a phospholipase A2 inhibitor, blocked DHA prevention of oxidative stress-induced apoptosis of photoreceptors. It is noteworthy that RXR agonists (HX630, PA024) also rescued photoreceptors from H2O2-induced apoptosis. These results provide the first evidence that activation of RXRs prevents photoreceptor apoptosis and suggest that DHA is first released from phospholipids and then activates RXRs to promote the survival of photoreceptors. Photoreceptor cell death, which leads to vision loss, occurs in several retinal neurodegenerative diseases, such as retinitis pigmentosa and age-related macular degeneration. Photoreceptor loss in these pathologies occurs by apoptosis and oxidative stress, and lack of trophic factors is implicated in the activation of the apoptotic mechanisms (1Portera-Cailliau C. Sung C.H. Nathans J. Adler R. Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa.Proc. Natl. Acad. Sci. USA. 1994; 91: 974-978Crossref PubMed Scopus (579) Google Scholar). Identifying the trophic factors and intracellular pathways activated to prevent this apoptosis is crucial for designing procedures to rescue photoreceptors in these diseases. Previous research from our laboratory has demonstrated that docosahexaenoic acid (DHA), the major n-3 polyunsaturated fatty acid (PUFA) in the retina, acts as a trophic factor for photoreceptor cells. DHA promotes differentiation and postpones apoptosis of photoreceptors, which otherwise occurs in the absence of trophic factors during their early development in culture (2Rotstein N.P. Aveldano M.I. Barrantes F.J. Politi L.E. Docosahexaenoic acid is required for the survival of rat retinal photoreceptors in vitro.J. Neurochem. 1996; 66: 1851-1859Crossref PubMed Scopus (101) Google Scholar, 3Rotstein N.P. Aveldano M.I. Barrantes F.J. Roccamo A.M. Politi L.E. Apoptosis of retinal photoreceptors during development in vitro: protective effect of docosahexaenoic acid.J. Neurochem. 1997; 69: 504-513Crossref PubMed Scopus (108) Google Scholar–4Politi L.E. Rotstein N.P. Carri N.G. Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid.Invest. Ophthalmol. Vis. Sci. 2001; 42: 3008-3015PubMed Google Scholar); it also effectively prevents photoreceptor apoptosis due to oxidative stress induced by Paraquat (PQ) (5Rotstein N.P. 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In this work we investigated the mechanisms involved in DHA protection upstream ERK/MAPK in different experimental models of retinal degeneration in vitro, either induced by oxidative damage or by trophic factor deprivation during early development in vitro. Our results support that RXR activation is essential for DHA protection of photoreceptors and that its release and subsequent action as free DHA is required for this protection. In addition, our data suggest that activation of RXRs has an intrinsically neuroprotective effect, promoting photoreceptor survival independently of the agonist involved in their activation. One to two day old albino Wistar rats bred in our own colony were used in all the experiments. All procedures concerning animal use were carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and following the guidelines of the Institutional Committee for the Care of Laboratory Animals from the Universidad Nacional del Sur (Argentina). Plastic 35 mm diameter culture dishes and multi-chambered slides (NUNC) were from Inter Med (Naperville, IL). Dulbecco's modified Eagle's medium (DMEM) (GIBCO) was from Life Technologies (Grand Island, NY). Trypsin, trypsin inhibitor, transferrin, hydrocortisone, putrescine, insulin, polyornithine, selenium, gentamycin, 4,6-diamidino-2-phenylindole (DAPI), fluorescein-conjugated secondary antibodies, paraformaldehyde, PQ, bovine serum albumin (BSA), and monoclonal anti-syntaxin clone HPC-1 syntaxin were from Sigma (St. Louis, MO). Monoclonal antibodies for pan-RXR (sc-774) and secondary antibody, goat anti-mouse IgG-HRP, and goat anti-rabbit IgG-HRP were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary antibodies, Cy2-conjugated-goat anti-mouse, and Cy5-conjugated-goat anti-rabbit were from Molecular Probes (Eugene, OR). Monoclonal Rho4D2 was a generous gift from Dr. R. Molday (University of South Columbia). DHA was from Nu-Chek (Elysian, MN). MitoTracker (CMXRos) was from Molecular Probes. Terminal deoxynucleotidyl transferase (TdT), recombinant, 5-bromo-2′deoxyuridine 5′-triphosphate (BrdUTP), and TdT buffer were from Molecular Probes and Invitrogen (Argentina), respectively. H2O2 was from Merck (Argentina). 4-Bromoenol lactone (BEL) was from Santa Cruz Biotechnology, Inc. and generously provided by Dr. Gabriela Salvador (Universidad Nacional del Sur, Argentina). RXR noncommercial pan-agonists (HX630 and PA024) and pan-antagonists (HX531 and PA452) were generous gifts from Dr. Kagechika (Tokyo Medical and Dental University, Japan). All other reagents used were analytical grade. Pure retinal cultures were obtained following procedures previously established (2Rotstein N.P. Aveldano M.I. Barrantes F.J. Politi L.E. Docosahexaenoic acid is required for the survival of rat retinal photoreceptors in vitro.J. Neurochem. 1996; 66: 1851-1859Crossref PubMed Scopus (101) Google Scholar, 3Rotstein N.P. Aveldano M.I. Barrantes F.J. Roccamo A.M. Politi L.E. Apoptosis of retinal photoreceptors during development in vitro: protective effect of docosahexaenoic acid.J. Neurochem. 1997; 69: 504-513Crossref PubMed Scopus (108) Google Scholar, 36Politi L.E. Bouzat C. de los Santos E.B. Barrantes F.J. 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Neurons were then incubated for 24 h before fixation. Treatment of the cultures with H2O2 was done essentially according to Chucair et al. (41Chucair A.J. Rotstein N.P. Sangiovanni J.P. During A. Chew E.Y. Politi L.E. Lutein and zeaxanthin protect photoreceptors from apoptosis induced by oxidative stress: relation with docosahexaenoic acid.Invest. Ophthalmol. Vis. Sci. 2007; 48: 5168-5177Crossref PubMed Scopus (149) Google Scholar), with slight modifications. Briefly, cells were treated at day 3 in culture with 10 μM H2O2 for 30 min at 36°C; the medium was then removed, replaced with fresh neuronal medium, and the cultures were returned to the incubator for 5.5 h before fixation. Cultures were treated with two different RXR pan-antagonists, HX531 and PA452 (42Ebisawa M. Umemiya H. Ohta K. Fukasawa H. Kawachi E. Christoffel G. Gronemeyer H. Tsuji M. Hashimoto Y. Shudo K. et al.Retinoid X receptor-antagonistic diazepinylbenzoic acids.Chem. Pharm. Bull. 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A 1 μM concentration of each RXR pan-antagonist was used in further experiments, because a lower HX531 concentration showed little effect on apoptosis and higher HX531 and PA542 concentrations were toxic to neurons. Cultures were treated with either of two different RXR pan-agonists, HX630 and PA024 (44Suzuki K. Takahashi K. Nishimaki-Mogami T. Kagechika H. Yamamoto M. Itabe H. Docosahexaenoic acid induces adipose differentiation-related protein through activation of retinoid x receptor in human choriocarcinoma BeWo cells.Biol. Pharm. Bull. 2009; 32: 1177-1182Crossref PubMed Scopus (27) Google Scholar, 45Umemiya H. Fukasawa H. Ebisawa M. Eyrolles L. Kawachi E. Eisenmann G. Gronemeyer H. Hashimoto Y. Shudo K. Kagechika H. Regulation of retinoidal actions by diazepinylbenzoic acids. Retinoid synergists which activate the RXR-RAR heterodimers.J. Med. Chem. 1997; 40: 4222-4234Crossref PubMed Scopus (182) Google Scholar–46Ohta K. Kawachi E. Inoue N. Fukasawa H. Hashimoto Y. Itai A. 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Day 1 retinal neurons were supplemented with BSA or DHA for 24 h; culture medium was then replaced by fresh medium and at day 3 and cells were incubated with BEL (5 μM) for 30 min and finally either treated or not treated with H2O2 for 6 h. Cultures were fixed for at least 1 h with 2% paraformaldehyde in PBS, followed by permeation with Triton X-100 (0.1%) for 15 min. After blocking with PBS (5% BSA), samples were incubated with the appropriate primary antibody prepared in PBS (3% BSA). After washing with PBS, the samples were incubated with secondary Cy2-conjugated-goat anti-mouse or Cy5-conjugated-goat anti-rabbit antibody. To evaluate mitochondrial function, cultures were incubated for 30 min before fixing with the fluorescent probe MitoTracker (0.1 μg/ml). The samples were examined using a Leica TCS SP2 AOBS confocal laser microscope. Neuronal cell types were identified with specific monoclonal antibodies as described above. Cultures were analyzed by phase contrast and epifluorescence microscopy using a Nikon Eclipse E600 microscope with a C-C phase contrast turret condenser and a Y-FL Epi-Fluorescence attachment and a laser scanning confocal microscope (Leica DMIRE2) with a 63× water objective; images were collected and processed with LCS software (Leica) and Photoshop 8.0 (Adobe Systems, San Jose, CA). Proteins (20 μg) dissolved in 4× Laemmli sample buffer were seeded in SDS polyacrylamide gels (10% acrylamide) and electrotransferred to PVDF membranes. After blocking with 5% nonfat milk in TBST buffer (50 mM Tris pH 7.2–7.4, 200 mM NaCl, 0.1% Tween-20), the membranes were incubated overnight with a pan-RXR antibody in TBST plus 3% nonfat milk. After washing, membranes were incubated with a horse radish peroxidase-conjugated secondary antibody in TBST plus 3% nonfat milk. Finally, the blots were developed by ECL with the use of Kodak BioMax Light film and digitalized with a GS-700 imaging densitometer (Bio-Rad, Hercules, CA). ARPE-19 cells were used as a positive control for RXR expression. Cell death was determined by quantifying cells labeled with propidium iodide (PI), 0.5 μg/ml in culture, after 30 min incubation (51Jordán J. Galindo M.F. Prehn J.H. Weichselbaum R.R. Beckett M. Ghadge G.D. Roos R.P. Le
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