Oxidative Stress Modulates Complement Factor H Expression in Retinal Pigmented Epithelial Cells by Acetylation of FOXO3

Age-related macular degeneration (AMD), the leading cause of severe vision loss in the elderly, is a complex disease that results from genetic modifications that increase susceptibility to environmental exposures. Smoking, a major source of oxidative stress, increases the incidence and severity of AMD, and antioxidants slow progression, suggesting that oxidative stress plays a major role. Polymorphisms in the complement factor H (CFH) gene that reduce activity of CFH increase the risk of AMD. In this study we demonstrate an interaction between these two risk factors, because oxidative stress reduces the ability of an inflammatory cytokine, interferon-γ, to increase CFH expression in retinal pigmented epithelial cells. The interferon-γ-induced increase in CFH is mediated by transcriptional activation by STAT1, and its suppression by oxidative stress is mediated by acetylation of FOXO3, which enhances FOXO3 binding to the CFH promoter, reduces its binding to STAT1, inhibits STAT1 interaction with the CFH promoter, and reduces expression of CFH. Expression of SIRT1, a mammalian homolog of NAD-dependent protein deacetylase sir2, attenuated FOXO3 recruitment to the CFH regulatory region and reversed the H2O2-induced repression of CFH gene expression. These data suggest an important interaction between environmental exposure and genetic susceptibility in the pathogenesis of AMD and, by elucidating molecular signaling involved in the interaction, provide potential targets for therapeutic intervention. Age-related macular degeneration (AMD), the leading cause of severe vision loss in the elderly, is a complex disease that results from genetic modifications that increase susceptibility to environmental exposures. Smoking, a major source of oxidative stress, increases the incidence and severity of AMD, and antioxidants slow progression, suggesting that oxidative stress plays a major role. Polymorphisms in the complement factor H (CFH) gene that reduce activity of CFH increase the risk of AMD. In this study we demonstrate an interaction between these two risk factors, because oxidative stress reduces the ability of an inflammatory cytokine, interferon-γ, to increase CFH expression in retinal pigmented epithelial cells. The interferon-γ-induced increase in CFH is mediated by transcriptional activation by STAT1, and its suppression by oxidative stress is mediated by acetylation of FOXO3, which enhances FOXO3 binding to the CFH promoter, reduces its binding to STAT1, inhibits STAT1 interaction with the CFH promoter, and reduces expression of CFH. Expression of SIRT1, a mammalian homolog of NAD-dependent protein deacetylase sir2, attenuated FOXO3 recruitment to the CFH regulatory region and reversed the H2O2-induced repression of CFH gene expression. These data suggest an important interaction between environmental exposure and genetic susceptibility in the pathogenesis of AMD and, by elucidating molecular signaling involved in the interaction, provide potential targets for therapeutic intervention. Age-related macular degeneration (AMD) 2The abbreviations used are: AMD, age-related macular degeneration; CFH, complement factor H; sir2, silent information regulator 2; IFN-γ, interferon-γ; RPE cells, retinal pigmented epithelial cells; STAT, signal transducers and activators of transcription; ChIP, chromatin immunoprecipitation; ANOVA, analysis of variance; JNK, Jun-N-terminal kinase. 2The abbreviations used are: AMD, age-related macular degeneration; CFH, complement factor H; sir2, silent information regulator 2; IFN-γ, interferon-γ; RPE cells, retinal pigmented epithelial cells; STAT, signal transducers and activators of transcription; ChIP, chromatin immunoprecipitation; ANOVA, analysis of variance; JNK, Jun-N-terminal kinase. is the most prevalent cause of severe vision loss in patients over the age of 60 in developed countries (1Klein R. Klein B.E. Linton K.L. Ophthalmology. 1992; 99: 933-943Abstract Full Text PDF PubMed Scopus (1600) Google Scholar, 2Mitchell P. Smith W. Attebo K. Wang J.J. Ophthalmology. 1995; 102: 1450-1460Abstract Full Text PDF PubMed Scopus (931) Google Scholar). It is a complex group of diseases in which genetic susceptibility combined with environmental exposures result in a disease phenotype that consists of deposits (drusen) along Bruch's membrane, atrophy of photoreceptors and retinal pigmented epithelial (RPE) cells (geographic atrophy), and enhanced risk of choroidal neovascularization. Several genetic susceptibility loci for AMD have been identified, but some variants in and around the complement factor H (CFH) gene are associated with particularly high risk, and others are associated with reduced risk (3Klein R.J. Zeiss C. Chew E.Y. Tsai J.Y. Sackler R.S. Haynes C. Henning A.K. SanGiovanni J.P. Mane S.M. Mayne S.T. Bracken M.B. Ferris F.L. Ott J. Barnstable C. Hoh J. Science. 2005; 308: 385-389Crossref PubMed Scopus (3434) Google Scholar, 4Edwards A.O. Ritter R.R. Abel K.J. Manning A. Panhuysen C. Farrer L.A. Science. 2005; 308: 421-424Crossref PubMed Scopus (2042) Google Scholar, 5Haines J.L. Hauser M.A. Schmidt S. Scott W.K. Olson L.M. Gallins P. Spencer K.L. Kwan S.Y. Noureddine M. Gilbert J.R. Schnetz-Boutaud N. Agarwal A. Postel E.A. Pericak-Vance M.A. Science. 2005; 308: 419-421Crossref PubMed Scopus (2033) Google Scholar, 6Rivera A. Fisher S.A. Fritsche L.G. Keilhauer C.N. Lichtner P. Meitinger T. Weber B.H. Hum. Mol. Genet. 2005; 14: 3227-3236Crossref PubMed Scopus (654) Google Scholar, 7Li M. Atmaca-Sonmez P. Othman M. Branham K.E.H. Khanna R. Wade M.S. Yun L. Liang L. Zereparsi S. Swaroop A. Abecasis G.R. Nat. Genet. 2006; 38: 1049-1054Crossref PubMed Scopus (286) Google Scholar). A polymorphism encoding the sequence variation Y402H is associated with increased risk (3Klein R.J. Zeiss C. Chew E.Y. Tsai J.Y. Sackler R.S. Haynes C. Henning A.K. SanGiovanni J.P. Mane S.M. Mayne S.T. Bracken M.B. Ferris F.L. Ott J. Barnstable C. Hoh J. Science. 2005; 308: 385-389Crossref PubMed Scopus (3434) Google Scholar, 4Edwards A.O. Ritter R.R. Abel K.J. Manning A. Panhuysen C. Farrer L.A. Science. 2005; 308: 421-424Crossref PubMed Scopus (2042) Google Scholar, 5Haines J.L. Hauser M.A. Schmidt S. Scott W.K. Olson L.M. Gallins P. Spencer K.L. Kwan S.Y. Noureddine M. Gilbert J.R. Schnetz-Boutaud N. Agarwal A. Postel E.A. Pericak-Vance M.A. Science. 2005; 308: 419-421Crossref PubMed Scopus (2033) Google Scholar, 6Rivera A. Fisher S.A. Fritsche L.G. Keilhauer C.N. Lichtner P. Meitinger T. Weber B.H. Hum. Mol. Genet. 2005; 14: 3227-3236Crossref PubMed Scopus (654) Google Scholar), and in vitro studies have shown that CFH His-402 has reduced binding to C-reactive protein, heparin, and RPE cells, which reduces its complement control activity (8Skerka C. Lauer N. Weinberger A.A.W.A. Keilhauer C.N. Suhnel J. Smith R. Schlotzer-Schrehardt U. Fritsche L. Heinen S. Hartmann A. Weber B.H.F. Zipfel P.F. Mol. Immunol. 2007; 44: 3398-3406Crossref PubMed Scopus (158) Google Scholar, 9Laine M. Jarva H. Seitsonen S. Haapasalo K. Lehtinen M.J. Lindeman N. Anderson D.H. Johnson P.T. Jarvela I. Jokiranta T.S. Hageman G.S. Immonen I. Meri S. J. Immunol. 2007; 178: 3831-3836Crossref PubMed Scopus (198) Google Scholar). In addition, detailed analysis of single nucleotide polymorphisms in and around the CFH gene has identified variants, some in noncoding regions, with high risk (7Li M. Atmaca-Sonmez P. Othman M. Branham K.E.H. Khanna R. Wade M.S. Yun L. Liang L. Zereparsi S. Swaroop A. Abecasis G.R. Nat. Genet. 2006; 38: 1049-1054Crossref PubMed Scopus (286) Google Scholar). No single polymorphism could account for the contribution of CFH, and instead, haplotypes associated with increased or decreased risk were identified. The authors hypothesized that these haplotypes might modulate risk of AMD not by disrupting protein function but, rather, by altering expression. Therefore, either reducing the amount of CFH or reducing its complement-modulating activity may increase the risk of AMD. High risk and protective variants have also been identified in other genes that code for proteins involved in the regulation of complement, complement factor B, and complement component 2 (10Gold B. Merriam J.E. Zernant J. Hancox L.S. Taiber A.J. Gehrs K. Cramer K. Neel J. Bergeron J. Barile G.R. Smith R.T. Hageman G.S. Dean M. Allikmets R. Nat. Genet. 2006; 38: 458-462Crossref PubMed Scopus (919) Google Scholar). This supports the hypothesis that dysregulation of complement activity contributes to the development of AMD. The strongest environmental risk factor for AMD is cigarette smoking, which is associated with substantially higher incidences of geographic atrophy and choroidal neovascularization (11Smith W. Assink J. Klein R. Mitchell P. Klaver C.C. Klein B.E. Hofman A. Jensen S. Wang J.J. de Jong P.T. Ophthalmology. 2001; 108: 697-704Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar, 12Khan J.C. Thurlby D.A. Shahid H. Clayton D.G. Yates J.R. Bradley M. Moore A.T. Bird A.C. Br. J. Ophthalmol. 2006; 90: 75-80Crossref PubMed Scopus (292) Google Scholar). The mechanism by which smoking increases the incidence and severity of AMD is uncertain, but cigarette smoke is known to contain numerous oxidants (13Smith C.J. Hansch C. Food Chem. Toxicol. 2000; 38: 637-646Crossref PubMed Scopus (187) Google Scholar). Exposure of mice to cigarette smoke or hydroquinone, an oxidant known to be present in cigarette smoke, resulted in sub-RPE deposits and diffuse thickening of Bruch's membrane (14Espinosa-Heidmann D.G. Suner I.J. Catanuto P. Hernandez E.P. Marin-Castano M.E. Cousins S.W. Investig. Ophthalmol. Vis. Sci. 2006; 47: 729-737Crossref PubMed Scopus (153) Google Scholar), suggesting that oxidative stress is capable of promoting these phenotypic characteristics of AMD. Oxidative stress has also been implicated in AMD by the Age-Related Eye Disease Study, which showed that antioxidants and zinc reduce the risk of individuals with large drusen progressing to advanced AMD, defined as choroidal neovascularization or severe geographic atrophy involving the center of the fovea (15The Age-related Eye Disease Study Research GroupArch. Ophthalmol. 2001; 119: 1417-1436Crossref PubMed Scopus (2521) Google Scholar). Also, proteomic analysis has demonstrated that drusen contain numerous proteins with adducts that are commonly caused by oxidative damage (16Crabb J.W. Miyagi M. Gu X. Shadrach K. West K. Sakaguchi H. Kamei M. Hasan A. Yan L. Rayborn M.E. Salomon R.G. Hollyfield J.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14682-14687Crossref PubMed Scopus (961) Google Scholar). Thus, genetic studies suggest that the level of expression of CFH may play a role in the pathogenesis of AMD and several other lines of evidence implicate oxidative stress. In this study we sought to test the hypothesis that oxidative stress influences the expression of CFH. Reagents, Cells, and Transfections—Interferon-γ (IFN-γ) was obtained from Abcam (Cambridge, MA). The inhibitors PD98059, LY294002, p38 inhibitor, and PP2 were purchased from Calbiochem and used at concentrations of 50, 20, 10, and 5 μm, respectively. These concentrations were found to be effective in previous studies (17Ceryak S. Zingarello C. O'Brien T. Patierno S.R. Mol. Cell. Biochem. 2004; 255: 139-149Crossref PubMed Scopus (35) Google Scholar, 18Hayashi H. Matsuzaki O. Muramatsu S. Tsuchiya Y. Harada T. Suzuki Y. Sugano S. Matsuda A. Nishida E. J. Biol. Chem. 2005; 281: 1332-1337Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 19Lee K.W. Jung J.W. Kang K.S. Lee H.J. Ann. N. Y. Acad. Sci. 2004; 1030: 258-263Crossref PubMed Scopus (15) Google Scholar, 20Lee M.C. Kim J.Y. Koh W.S. J. Cell. Biochem. 2004; 93: 629-638Crossref PubMed Scopus (20) Google Scholar). Goat polyclonal anti-human CFH antibody was obtained from Calbiochem. Anti-phospho-Akt, anti-phospho-STAT1, anti-FOXO3, anti-STAT1, anti-Akt, and anti-β-actin antibodies were obtained from Cell Signaling (Beverly, MA). Anti-acetyl-lysine (clone 4G12) was obtained from Upstate Biotechnologies (Lake Placid, NY). Human RPE cells (ARPE-19 cells) (21Dunn K.C. Aotaki-Keen A.E. Putkey F.R. Hjelmeland L.M. Exp. Eye Res. 1996; 62: 155-169Crossref PubMed Scopus (1027) Google Scholar) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. For transfection, ARPE-19 cells were grown in six-well plates and transfected with Lipofectamine™ LTX (Invitrogen) according to the manufacturer’s instructions. DNA Constructs—The CFH promoter-luciferase reporter constructs were generated by PCR amplification using purified genomic DNA as template and the primers listed in Table 1. The PCR products were inserted into the HindIII and XhoI sites of pGL3 basic vector (Promega, Madison, WI). The plasmids FOXO3.WT, FOXO3.TM, FOXO3.TMΔDB, SIRT1.WT, and SIRT1.H363Y were provided by Dr. Michael Greenberg (Department of Neurobiology, Center for Blood Research Institute for Biomedical Research, Harvard Medical School, Boston, MA). The FOXO1-GFP construct was provided by Dr. William Sellers (Department of Medical Oncology, Dana Farber Cancer Institute, Boston, MA). STAT1α.Y701F and STAT1α.WT plasmids were provided by Dr. Jim Darnell (The Rockefeller University, New York, NY).TABLE 1Primers used for PCR amplificationsGeneRegionPrimersCFH-553 to +1075′-AAACTCGAGTCAGCATTTCAATTTGTTGATTTTTGGATT-3′5′-AAAAAGCTTGGATCTTTTAAGAGGACATTTACCAGCTAA-3′CFH-420 to +1075′-AAACTCGAGCCAAATTCATCAAGCACTGCATTCTTGGCA-3′5′-AAAAAGCTTGGATCTTTTAAGAGGACATTTACCAGCTAA-3′CFH-553 to -625′-AAACTCGAGTCAGCATTTCAATTTGTTGATTTTTGGATT-3′5′-AAAAAGCTTAAGCCACAAGCCCAGAAATGCCAGAAGTT-3′CFH-33 to +1075′-AAACTCGAGGTTTCTGATAGGCGGAGCATCTAGTT-3′5′-AAAAAGCTTGGATCTTTTAAGAGGACATTTACCAGCTAA-3′CFH+1118 to +13295′-TTGCACACAAGATGGATGGT-3′5′-GGATGCATCTGGGAGTAGGA-3′Ribosomal protein (large P0)+19 to +1275′-CGACCTGGAAGTCCAACTAC-3′5′-ATCTGCTGCATCTGCTTG-3′ Open table in a new tab Immunoblots—ARPE-19 cells were seeded in 6-well plates (105 cells/well), and after various treatments they were lysed in pre-warmed Laemmli buffer (Bio-Rad). For each sample the same amount of total protein was added to a well of a 10% acrylamide gel and resolved by SDS-PAGE. The separated proteins were transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences). Nonspecific binding was blocked by incubation with 5% nonfat milk at room temperature for 2 h before overnight incubation with primary antibody at 4 °C. A 1:500 dilution of anti-CFH and a 1:1000 dilution of all other primary antibodies was used. The proteins were detected by SuperSignal West Pico substrate solution using horseradish peroxidase-linked anti-rabbit IgG (Pierce). Blots were quantified by densitometry using a Bio-Rad Molecular Imager FX and Quantity One software. Quantitative Real Time Reverse Transcription-PCR—The cells were harvested after various 24-h treatments, and total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA) and then incubated with rDNase I (DNA-free™ kit, Ambion, Austin, TX) to remove any traces of contaminating DNA. Reverse transcription was performed at 50 °C for 1 h using 2 μg of total RNA, 1 μl of Superscript ™ III reverse transcriptase, 1 μl of 50 μm oligo(dT)20, 1 μl of 10 mm dNTP, 4 μl of 5× first-strand buffer, 1 μl of 0.1 m dithiothreitol, and 1 μl of RNAseOUT (Invitrogen). Each 20-μl PCR reaction mixture was prepared using the LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Diagnostics). The +1118 to +1329 CFH primers (Table 1) were designed to hybridize in two different exons or at exon/intron boundaries to prevent amplification of any remaining genomic DNA. The amplification conditions were denaturation at 95 °C for 15 s, annealing at 55 °C for 5 s, and extension at 72 °C for 12 s for a total of 40 cycles. Under optimized conditions there was a single melting curve and no primer-dimer formation. The copy number for each mRNA was determined using a standard curve generated with external standards of known copy number. A fragment of the large P0 subunit of human ribosomal protein (primers, Table 1) was amplified to provide an internal control. Luciferase Assay—ARPE-19 cells were plated in 6-well plates, and transfections were done at 60–70% confluence. CFH promoter-luciferase reporter constructs in combination with Renilla luciferase control vector (pRL-SV40 vector, Promega) were transfected along with relevant FOXO3 constructs into ARPE-19 cells using Lipofectamine™ LTX (Invitrogen). Twenty-four hours after transfection, cells maintained in the medium containing serum were treated with IFN-γ and H2O2 for 5 h and lysed with passive lysis buffer (Promega). For inhibitor studies, cells were pretreated with LY294002 for 30 min before the addition of IFN-γ and H2O2. Luciferase activity was measured using the dual-luciferase reporter assay system with a luminometer according to the manufacturer’s instructions. Firefly luciferase activities were normalized to Renilla luciferase activities for transfection efficiency. Immunoprecipitation—ARPE-19 cells were plated (107/10-cm dish), and the following day at 80–90% confluence they were treated with IFN-γ and H2O2 for 1 h. Cells were washed with phosphate-buffered saline and lysed in 1 ml of lysis buffer (50 mm Tris, pH 8.0, 50 mm KCl, 10 mm EDTA, 1% Nonidet P-40) containing deacetylase inhibitors (10 mm nicotinamide and 1 μm Trichostatin A), protease inhibitors (Roche Diagnostics mixture tablet), and phosphatase inhibitors (20 mm NaF and 1 mm orthovanadate). The lysates were immunoprecipitated with indicated antibodies for 5 h followed by the addition of protein G-Sepharose (Sigma) for overnight incubation. The beads were washed three times with lysis buffer and once with phosphate-buffered saline. The immunoprecipitated proteins were released from the beads by boiling in Laemmli buffer (Bio-Rad) for 5 min and subsequently analyzed by immunoblotting with appropriate antibodies. Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were conducted as described by Nelson et al. (22Nelson J.D. Denisenko O. Bomsztyk K. Nat. Protoc. 2006; 1: 179-185Crossref PubMed Scopus (594) Google Scholar) with minor modifications. ARPE-19 cells upon reaching 80–90% confluence were treated with IFN-γ and H2O2 for 1 h in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and then cross-linked with 1.42% formaldehyde at room temperature for 15 min followed by the addition of glycine to a final concentration of 125 mm and incubated for 5 min. After 2 washes with cold phosphate-buffered saline (PBS), cells were scraped in 1 ml of PBS and collected by centrifugation. The cell pellet was lysed in 1 ml of IP buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 0.5% Nonidet P-40, and 1% Triton X-100) containing deacetylase inhibitors (10 mm nicotinamide and 1 μm Trichostatin A), protease inhibitors (Roche Diagnostics mixture tablet), and phosphatase inhibitors (20 mm NaF and 1 mm orthovanadate). The resulting nuclear pellet was washed once, resuspended in IP buffer, and sonicated for three rounds of 15 pulses each at 50% power output and 90% duty cycle (Branson Sonifer 250, VWR, West Chester, PA). DNA shearing was controlled by extracting the DNA from sheared chromatin and running on an agarose gel to ensure that that DNA fragment size was 300–1000 bp. The sonicated lysate was cleared by centrifugation, and 20 μl was retained as an input control. Two hundred μl of cleared lysate was used for immunoprecipitation with or without the addition of antibodies (5 μg of each antibody per immunoprecipitation reaction) in an ultrasonic water bath for 15 min at 4 °C. The immune complexes were recovered with protein G-Sepharose (Sigma) and washed five times with cold IP buffer without any inhibitors. DNA was precipitated from input control samples by the addition of 100% ethanol. The immunoprecipitated DNA and input DNA were extracted by incubation with 100 μl of 10% Chelex (Bio-Rad) directly with precipitated beads or input DNA pellet and boiled for 10 min to reverse the cross-link. The DNA was purified by removing the Chelex slurry by centrifugation. PCR was performed with the purified DNA, according to instructions, with a High Fidelity PCR kit (Roche Diagnostics) using the following primers: forward, 5′-AAA CTC GAG CCA AAT TCA TCA AGC ACT GCA TTC TTG GCA-3′; reverse, 5′-AAA AAG CTT GGA TCT TTT AAG AGG ACA TTT ACC AGC TAA-3′. Analysis of PCR products was performed on a standard 1% agarose gel. Site-specific Mutagenesis—Point mutations in the FOXO3 binding site were generated by site-specific mutagenesis by overlap PCR extension. The two smaller PCR products were generated using primer A forward, 5′-AAA CTC GAG CCA AAT TCA TCA AGC ACT GCA TTC TTG GCA-3′, and reverse, 5′-TAT CAG AAA CTT TTG CAA AAG CAA TAA AAA ATC AAC CAC A-3′, and primer B forward, 5′-TGT GGT TGA TTT TTT ATT GCT TTT GCA AAA GTT TCT GAT A-3′, and reverse, 5′-AAA AAG CTT GGA TCT TTT AAG AGG ACA TTT ACC AGC TAA-3′. Statistical Analyses—Data are expressed as the mean (±S.D.), and statistical comparisons were done by analysis of variance (ANOVA) with Dunnett’s correction for multiple comparisons using SAS version 9 (SAS Institute Inc., Cary, NC) with p < 0.05 considered statistically significant. Oxidative Stress Reduces IFN-γ-induced Up-regulation of CFH—The function of CFH is to down-regulate the alternative complement pathway to minimize bystander damage (for review, see Ref. 23Zipfel P.F. Semin. Thromb. Hemostasis. 2001; 27: 191-199Crossref PubMed Scopus (66) Google Scholar). The expression of CFH is increased by the inflammatory cytokine, IFN-γ (24Ward H.M. Higgs N.M. Blackmore T.K. Sadlon T.A. Gordon D.L. Immunol. Cell Biol. 1997; 75: 508-510Crossref PubMed Scopus (13) Google Scholar). The liver is the major source of CFH, but the RPE is a local source of production in the eye (25Mandal M.N. Ayyagari R. Investig. Ophthalmol. Vis. Sci. 2006; 47: 4091-4097Crossref PubMed Scopus (66) Google Scholar). Incubation of cultured human RPE cells with IFN-γ increased expression of CFH with a maximal effect achieved with 10 ng/ml (Fig. 1a). Exposure of RPE cells to 0.5 or 0.75 mm H2O2 reduced the IFN-γ-induced stimulation of CFH production, with 0.75 mm causing a greater reduction. Serum promotes cell survival and provides some degree of protection from noxious stimuli, but H2O2 had the same suppressive effect on IFN-γ-induced stimulation of CFH production in serum-containing media (Fig. 1b) as seen in serum-free media (Fig. 1a). This effect of H2O2 is due to oxidative stress because it is abrogated in a dose-dependent manner by preincubation with the antioxidant N-acetylcysteine (Fig. 1, c and d). Consistent with this conclusion, H2O2 and two other causes of oxidative stress, paraquat and FeSO4, at concentrations previously shown not to reduce viability over this time frame (26Lu L. Hackett S.F. Mincey A. Lai H. Campochiaro P.A. J. Cell. Physiol. 2006; 206: 119-125Crossref PubMed Scopus (103) Google Scholar), significantly reduced CFH mRNA in RPE cells (Fig. 1e). This could be due to destabilization of the mRNA or reduction in its production. To distinguish between these possibilities we examined the effect of oxidative stress on CFH promoter activity using a dual luciferase reporter assay. The IFN-γ-induced stimulation of CFH promoter activity was significantly reduced by H2O2-induced oxidative stress (Fig. 1f). STAT1 Mediates IFN-γ-induced Stimulation of CFH Promoter Activity—STAT1 is a transcription factor that mediates many effects of IFN-γ (27Dunn G.P. Koebel C.M. Schreiber R.D. Nat. Rev. Immunol. 2006; 6: 836-848Crossref PubMed Scopus (1128) Google Scholar). To determine if STAT1 mediates the IFN-γ-induced effects in RPE cells, transfections were done to generate cells with increased levels of STAT1 or dominant-negative STAT1. Cells overexpressing STAT1 showed enhanced IFN-γ-induced stimulation of p3xSTAT1-luc artificial promoter activity, and this was significantly decreased in cells expressing dominant-negative STAT1 (Fig. 2a). Western blots confirmed that, in the presence of IFN-γ, RPE cells expressing dominant-negative STAT1 had substantially less CFH than cells expressing wild type STAT1 (Fig. 2b). This suggests that STAT1 plays an important role in IFN-γ-induced up-regulation of CFH in RPE cells. Oxidative Stress Stimulates Phosphorylation of 46-kDa Jun-N-terminal Kinase (JNK) and Translocation of FOXO Proteins into the Nucleus of RPE Cells—IFN-γ stimulates phosphorylation of STAT1, which allows it to enter the nucleus and stimulate transcription. We postulated that oxidative stress reduces phosphorylation of STAT1 and thereby prevents IFN-γ from increasing levels of CFH in RPE cells. To test this hypothesis, we performed Western blots with an antibody that specifically recognizes phosphorylated STAT1. IFN-γ increased pSTAT1 in RPE cells, and this was not reduced by co-incubation with H2O2; instead the IFN-γ-induced increase was further increased by H2O2 (Fig. 3a). Phosphorylation is an important component of several signaling pathways. Small molecule kinase inhibitors were used to determine if activation of Src, phosphatidylinositol 3-kinase, or mitogen-activated protein kinase was involved in oxidative stress-mediated down-regulation of CFH expression. In contrast to the mitogen-activated protein kinase inhibitors PD98059 (17Ceryak S. Zingarello C. O'Brien T. Patierno S.R. Mol. Cell. Biochem. 2004; 255: 139-149Crossref PubMed Scopus (35) Google Scholar) and p38 inhibitor (19Lee K.W. Jung J.W. Kang K.S. Lee H.J. Ann. N. Y. Acad. Sci. 2004; 1030: 258-263Crossref PubMed Scopus (15) Google Scholar) and the Src inhibitor PP2 (20Lee M.C. Kim J.Y. Koh W.S. J. Cell. Biochem. 2004; 93: 629-638Crossref PubMed Scopus (20) Google Scholar), which had minimal effect, the phosphatidylinositol 3-kinase inhibitor LY294002 (18Hayashi H. Matsuzaki O. Muramatsu S. Tsuchiya Y. Harada T. Suzuki Y. Sugano S. Matsuda A. Nishida E. J. Biol. Chem. 2005; 281: 1332-1337Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) caused substantial reduction of IFN-γ-induced expression of CFH (Fig. 3b). Treatment with LY294002 also caused significant decrease in CFH promoter activity, and the addition of H2O2 caused a further reduction suggesting an additive effect (Fig. 3c). LY294002 is a specific inhibitor for the phosphatidylinositol 3-kinase-Akt signaling pathway. Members of the FOXO family of forkhead transcription factors are key downstream targets of the phosphatidylinositol 3-kinase-Akt pathway (28del Peso L. Gonzalez V.M. Hernandez R. Barr F.G. Nunez G. Oncogene. 1999; 18: 7328-7333Crossref PubMed Scopus (109) Google Scholar, 29Zheng W-H. Kar S. Quirion R. J. Biol. Chem. 2000; 275: 39152-39158Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). In mammals, four highly conserved FOXO family members have been identified and implicated in many cellular processes such as cell cycle arrest, apoptosis, DNA repair, and detoxification of reactive oxygen species (29Zheng W-H. Kar S. Quirion R. J. Biol. Chem. 2000; 275: 39152-39158Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The effect of LY294002 on CFH expression caused us to investigate for involvement of FOXO proteins. Regulation of FOXO proteins occurs in part through subcellular localization; enhanced activity of the phosphatidylinositol 3-kinase-Akt pathway results in phosphorylation of FOXO proteins causing nuclear exclusion and altered gene expression (30Matsuzaki H. Ichino A. Hayashi T. Yamamoto T. Kikkawa U. J. Biochem. (Tokyo). 2005; 138: 485-491Crossref PubMed Scopus (36) Google Scholar). We tested whether oxidative stress could override the sequestration of FOXO by Akt. RPE cells transfected with a FOXO1-GFP fusion protein showed diffuse fluorescence throughout their cytoplasm in the presence of 10% serum (Fig. 3d). After treatment with IFN-γ, a fraction of the fluorescently labeled FOXO1 entered the nucleus. However, in response to oxidative stress, nuclear translocation was essentially complete. Because we observed that IFN-γ did not inhibit activation of Akt (Fig. 3a), we postulated that the partial translocation of FOXO1 to the nucleus by IFN-γ was not mediated by changes in the phosphorylation state of Akt. In some tissues oxidative stress has been shown to increase nuclear localization and FOXO transcriptional activity through activation of JNK (31Essers M.A. Weijzen S. de Vries-Smits A.M. Saarloos I. de Ruiter N.D. Bos J.L. Burgering B.M. EMBO J. 2004; 23: 4802-4812Crossref PubMed Scopus (608) Google Scholar). Both Akt and JNK phosphorylate FOXO proteins but do so at different sites. Akt, which is activated by growth factors, phosphorylates FOXO proteins at sites that promote their export from the nucleus, whereas JNK, which is activated by oxidative stress or other causes of stress, phosphorylates FOXO proteins at sites that promote translocation to the nucleus, and when both Akt and JNK are activated, the nuclear localization effect of JNK predominates, which explains why oxidative stress overrides the effect of Akt (31Essers M.A. Weijzen S. de Vries-Smits A.M. Saarloos I. de Ruiter N.D. Bos J.L. Burgering B.M. EMBO J. 2004; 23: 4802-4812Crossref PubMed Scopus (608) Google Scholar, 32Sunayama J. Tsuruta F. Masuyama N. Gotoh Y. J. Cell Biol. 2005; 170: 295-304Crossref PubMed Scopus (277) Google Scholar). This appears to be the case in RPE cells because incubation with H2O2 caused activation of JNK in the presence and absence of IFN-γ or serum (Fig. 3e), and as we noted previously, expression of CFH was reduced by H2O2 in the presence or absence of serum (Fig. 1b), consistent with the notion that oxidative stress overrides the effect of Akt on FOXO proteins. FOXO3 Mediates the H2O2-induced Reduction in CFH Expression in RPE Cells—Examination