Copper Depletion Down-regulates Expression of the Alzheimer's Disease Amyloid-β Precursor Protein Gene
2004; Elsevier BV; Volume: 279; Issue: 19 Linguagem: Inglês
10.1074/jbc.m400805200
ISSN1083-351X
AutoresShayne A. Bellingham, Debomoy K. Lahiri, Bryan Maloney, Sharon La Fontaine, Gerd Multhaup, James Camakaris,
Tópico(s)Aluminum toxicity and tolerance in plants and animals
ResumoAlzheimer's disease is characterized by the accumulation of amyloid-β peptide, which is cleaved from the amyloid-β precursor protein (APP). Reduction in levels of the potentially toxic amyloid-β has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-β in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease. Alzheimer's disease is characterized by the accumulation of amyloid-β peptide, which is cleaved from the amyloid-β precursor protein (APP). Reduction in levels of the potentially toxic amyloid-β has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-β in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease. A number of neurodegenerative disorders including Alzheimer's disease (AD), 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid-β peptide; APP, amyloid-β precursor protein; CuRE, copper response element; MRE, metal response element; Ace/AmtRE, ACE1/AMT1 response element; CAT, chloramphenicol acetyltransferase; β-Gal, β-galactosidase; BME, Eagle's basal medium; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance. 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid-β peptide; APP, amyloid-β precursor protein; CuRE, copper response element; MRE, metal response element; Ace/AmtRE, ACE1/AMT1 response element; CAT, chloramphenicol acetyltransferase; β-Gal, β-galactosidase; BME, Eagle's basal medium; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; ANOVA, analysis of variance. Parkinson's disease, amyotrophic lateral sclerosis, and prion disease have been closely linked to disturbances in copper homeostasis in the central nervous system and the brain (1Strausak D. Mercer J.F. Dieter H.H. Stremmel W. Multhaup G. Brain Res. Bull. 2001; 55: 175-185Crossref PubMed Scopus (393) Google Scholar, 2Sayre L.M. Perry G. Atwood C.S. Smith M.A. Cell. Mol. Biol. 2000; 46: 731-741PubMed Google Scholar). AD is the most common progressive neurodegenerative disorder in elderly people and is characterized by neuronal loss with the accumulation of senile plaques and neurofibrillary tangles. The major proteinaceous component of senile plaques is a 39-42-amino acid peptide, termed amyloid-β peptide (Aβ) (3Masters C.L. Multhaup G. Simms G. Pottgiesser J. Martins R.N. Beyreuther K. EMBO J. 1985; 4: 2757-2763Crossref PubMed Scopus (796) Google Scholar), that is proteolytically cleaved from the larger amyloid-β precursor protein (APP) (4Kang J. Lemaire H.G. Unterbeck A. Salbaum J.M. Masters C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3930) Google Scholar). An apparent relationship between copper homeostasis, APP, and AD has been suggested (1Strausak D. Mercer J.F. Dieter H.H. Stremmel W. Multhaup G. Brain Res. Bull. 2001; 55: 175-185Crossref PubMed Scopus (393) Google Scholar). APP has a specific type II copper-binding site that binds copper with a Kd of 10 μm (5Multhaup G. Schlicksupp A. Hesse L. Beher D. Ruppert T. Masters C.L. Beyreuther K. Science. 1996; 271: 1406-1409Crossref PubMed Scopus (586) Google Scholar) and can modulate copper-induced toxicity and oxidative stress in primary mouse neuronal cultures (6White A.R. Multhaup G. Maher F. Bellingham S. Camakaris J. Zheng H. Bush A.I. Beyreuther K. Masters C.L. Cappai R. J. Neurosci. 1999; 19: 9170-9179Crossref PubMed Google Scholar). APP knockout mice have increased copper levels in the brain (7White A.R. Reyes R. Mercer J.F. Camakaris J. Zheng H. Bush A.I. Multhaup G. Beyreuther K. Masters C.L. Cappai R. Brain Res. 1999; 842: 439-444Crossref PubMed Scopus (256) Google Scholar), whereas conversely, transgenic overexpression of APP and Aβ in mice correlates with reduced brain copper levels (8Maynard C.J. Cappai R. Volitakis I. Cherny R.A. White A.R. Beyreuther K. Masters C.L. Bush A.I. Li Q.X. J. Biol. Chem. 2002; 277: 44670-44676Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar). In a cellular system overexpressing APP, increasing copper concentration can modulate APP processing, stimulating levels of cell-bound and secreted forms of APP with reduced production of Aβ (9Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (158) Google Scholar). The importance of copper in AD pathology has also been demonstrated by the ability of Aβ to bind copper with a high affinity (10Huang X. Cuajungco M.P. Atwood C.S. Hartshorn M.A. Tyndall J.D. Hanson G.R. Stokes K.C. Leopold M. Multhaup G. Goldstein L.E. Scarpa R.C. Saunders A.J. Lim J. Moir R.D. Glabe C. Bowden E.F. Masters C.L. Fairlie D.P. Tanzi R.E. Bush A.I. J. Biol. Chem. 1999; 274: 37111-37116Abstract Full Text Full Text PDF PubMed Scopus (725) Google Scholar), promoting amyloid plaque aggregation and neurotoxicity (11Atwood C.S. Scarpa R.C. Huang X. Moir R.D. Jones W.D. Fairlie D.P. Tanzi R.E. Bush A.I. J. Neurochem. 2000; 75: 1219-1233Crossref PubMed Scopus (573) Google Scholar). Conversely, treatment with a copper-zinc chelator can disaggregate Aβ both in vitro and in transgenic mouse models in vivo (12Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1313) Google Scholar). The APP gene is expressed in all major tissues but predominantly in the brain, where expression is primarily in neurons (13Neve R.L. Finch E.A. Dawes L.R. Neuron. 1988; 1: 669-677Abstract Full Text PDF PubMed Scopus (313) Google Scholar). Although there is both developmental and cell-type specific regulation of APP, expression in the adult is mostly ubiquitous (14Salbaum J.M. Ruddle F.H. J. Exp. Zool. 1994; 269: 116-127Crossref PubMed Scopus (48) Google Scholar). The regulation of the APP gene as a pathogenic factor for AD has received considerable attention. Down's syndrome patients, who have an extra copy of the APP gene, invariably exhibit early onset AD-like pathology (15Holtzman D.M. Bayney R.M. Li Y.W. Khosrovi H. Berger C.N. Epstein C.J. Mobley W.C. EMBO J. 1992; 11: 619-627Crossref PubMed Scopus (59) Google Scholar). Overexpression of APP in certain areas of the brain in AD patients also suggest that the regulation of APP might be an important factor in the neuropathology of AD (16Johnson S.A. McNeill T. Cordell B. Finch C.E. Science. 1990; 248: 854-857Crossref PubMed Scopus (226) Google Scholar). These observations illustrate the importance of elucidating the mechanisms of APP gene regulation in the development of AD. The human APP promoter closely resembles that of a typical housekeeping gene and contains the consensus sequences for the binding of several transcription factors (17Salbaum J.M. Weidemann A. Lemaire H.G. Masters C.L. Beyreuther K. EMBO J. 1988; 7: 2807-2813Crossref PubMed Scopus (248) Google Scholar, 18Theuns J. Van Broeckhoven C. Hum. Mol. Genet. 2000; 9: 2383-2394Crossref PubMed Scopus (65) Google Scholar). Nerve growth factor, interleukin-1, retinoic acid, and various transcription factors are among several cellular mediators that cause an increase in APP mRNA levels in neuronal and nonneuronal cells (19Lahiri D.K. Song W. Ge Y.W. Brain Res. Mol. Brain Res. 2000; 77: 185-198Crossref PubMed Scopus (18) Google Scholar). In contrast, thyroid hormone and interferon-γ have been reported to down-regulate APP expression (20Belandia B. Latasa M.J. Villa A. Pascual A. J. Biol. Chem. 1998; 273: 30366-30371Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 21Ringheim G.E. Szcepanik A.M. Burgher K.L. Petko W. Heroux J.A. Cavalieri F. Biochem. Biophys. Res. Commun. 1996; 224: 246-251Crossref PubMed Scopus (20) Google Scholar). Recently, an iron-responsive element located in the 5′-untranslated region has been implicated in the regulation of APP expression (22Rogers J.T. Randall J.D. Cahill C.M. Eder P.S. Huang X. Gunshin H. Leiter L. McPhee J. Sarang S.S. Utsuki T. Greig N.H. Lahiri D.K. Tanzi R.E. Bush A.I. Giordano T. Gullans S.R. J. Biol. Chem. 2002; 277: 45518-45528Abstract Full Text Full Text PDF PubMed Scopus (453) Google Scholar). We hypothesized that metals and in particular copper might alter APP gene expression. To investigate the role of copper in APP gene regulation, we utilized a novel approach involving cultured human fibroblasts overexpressing the Menkes protein (MNK; encoded by ATP7A), a major mammalian copper translocating P-type ATPase involved in copper efflux (23Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (532) Google Scholar, 24Camakaris J. Petris M.J. Bailey L. Shen P. Lockhart P. Glover T.W. Barcroft C. Patton J. Mercer J.F. Hum. Mol. Genet. 1995; 4: 2117-2123Crossref PubMed Scopus (156) Google Scholar, 25La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Cells lacking the MNK protein show high intracellular copper levels because of the lack of active copper efflux, whereas cells transfected and overexpressing MNK have markedly reduced copper levels (25La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). We report that depletion of intracellular copper results in significant reduction of APP gene expression. In addition, APP promoter analysis suggests that putative metal regulatory elements may be involved in mediating the response to copper depletion to regulate APP gene expression. Overall, these data suggest that copper is a required co-factor for the basal regulation of the APP gene and support the increasing evidence that APP is involved in copper homeostasis as a copper detoxification/efflux protein. Cell Lines—Human skin fibroblast cells, Me32a, were isolated from a classical Menkes disease patient (26Mercer J.F. Livingston J. Hall B. Paynter J.A. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemieniak D. Glower T.W. Nat. Genet. 1993; 3: 20-25Crossref PubMed Scopus (626) Google Scholar). The patient carried a 4-bp deletion that resulted in a frameshift mutation and premature stop codon of the MNK gene, whose expression was undetectable by Western and Northern analysis (25La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 26Mercer J.F. Livingston J. Hall B. Paynter J.A. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemieniak D. Glower T.W. Nat. Genet. 1993; 3: 20-25Crossref PubMed Scopus (626) Google Scholar, 27Ambrosini L. Mercer J.F. Hum. Mol. Genet. 1999; 8: 1547-1555Crossref PubMed Scopus (60) Google Scholar). Me32a cells were immortalized by SV40 gene transfer to derive Me32aT22/2L and designated MNK deletion (25La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The 4.6-kb cDNA encoding the human MNK protein was cloned into a mammalian expression vector and transfected into Me32aT22/2L (25La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Two independently derived clones that expressed MNK were isolated, MNK A12-H9 and MNK C3-C1, and designated MNK transfected A and MNK transfected B (25La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The empty mammalian expression vector, pCMB77, was transfected alone into MNK deletion to derive Me32aT22/2L(pCMB77) and designated vector only (25La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The SV40-immortalized human fibroblast cell line, normal human (GM2069), and human neuroblastoma cell line, human neuron (SY5Y), were used as controls. Culture Conditions—The cell lines MNK deletion and normal human were maintained in basal medium, which consisted of Eagle's basal medium (BME; Thermo Trace) (24Camakaris J. Petris M.J. Bailey L. Shen P. Lockhart P. Glover T.W. Barcroft C. Patton J. Mercer J.F. Hum. Mol. Genet. 1995; 4: 2117-2123Crossref PubMed Scopus (156) Google Scholar). The BME was supplemented with 10% fetal calf serum (Thermo Trace), 2 mm l-glutamine, 0.2 mm proline, 20 mm HEPES, and 0.2% (w/v) sodium bicarbonate. Vector only, MNK transfected A, and MNK transfected B cell lines were maintained in 10% BME as above with the additional supplement of 400 μg/ml Geneticin (Invitrogen). The cell line human neuron was maintained in basal medium, which consisted of RPMI 1640 (Thermo Trace). The RPMI 1640 was supplemented with 20% fetal calf serum, 0.8 mm l-glutamine, 0.4 mm sodium pyruvate, 0.02 mm uridine, 16 mm HEPES, and 0.05% (w/v) sodium bicarbonate. The basal copper concentrations of 10% BME and 20% RPMI 1640 were 0.78 (0.05 μg/ml) and 1.02 μm (0.07 μg/ml), respectively, as measured by atomic absorption spectroscopy using a Perkin Elmer 5000 Atomic Absorption Spectrophotometer. The cells were incubated at 37 °C. Bioinformatics—The human APP gene promoter sequence (GenBank™ accession number D87875) and rhesus monkey APP gene promoter sequence (GenBank™ accession number AF067971) were searched for copper response element (28Labbe S. Zhu Z. Thiele D.J. J. Biol. Chem. 1997; 272: 15951-15958Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) (CuRE consensus sequence, 5′-WWWTTTGCKCR-3′), ACE1/AMT1 response element (29Thiele D.J. Mol. Cell. Biol. 1988; 8: 2745-2752Crossref PubMed Scopus (240) Google Scholar) (Ace/AmtRE consensus sequence, 5′-THNNGCTG-3′), and metal response element (30Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (101) Google Scholar) (MRE consensus sequence, 5′-TGCRCNC-3′) consensus sequences using Transcription Element Search Software (31Schug J. Overton G.C. Computational Biology and Informatics. Laboratory School of Medicine, University of Pennsylvania, Philadelphia, PA1997Google Scholar). In addition, the promoters were searched for ACE1/AMT1-like sequences, with no more than one base mismatch to the first two ACE1/AMT1 consensus residues (5′-THNNGCTG-3′), and MRE-like sequences, with no more than one base mismatch from the last three MRE consensus residues (5′-TGCRCNC-3′). Antibodies—Anti-MNK polyclonal antibodies raised to the MNK N-terminal region (24Camakaris J. Petris M.J. Bailey L. Shen P. Lockhart P. Glover T.W. Barcroft C. Patton J. Mercer J.F. Hum. Mol. Genet. 1995; 4: 2117-2123Crossref PubMed Scopus (156) Google Scholar) were diluted 1:2500 for use in Western blot analysis. Anti-APP (WO2) monoclonal antibodies raised to the amyloid-β region (32Ida N. Hartmann T. Pantel J. Schroder J. Zerfass R. Forstl H. Sand-brink R. Masters C.L. Beyreuther K. J. Biol. Chem. 1996; 271: 22908-22914Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar) were diluted 1:1000 for use in immunofluorescence studies and 1:10000 for use in Western blot analysis. Steady-state Measurement of 64Cu Accumulation—The cells were grown to near confluency in basal growth medium. The medium was replaced with BME supplemented with 2% fetal calf serum containing 5-10 μCi/ml 64Cu (as CuCl2; ARI, Lucas Heights, Australia). After incubation at 37 °C for 16, 24, and 36 h, the cells were immediately harvested to obtain the total 64Cu accumulated at the end of the time period (four 35-mm Petri-dishes containing 1 × 106 cells/time point). All of the cells were washed twice in ice-cold serum-free medium to remove any nonspecifically bound 64Cu and harvested by dissolution in 1.5% SDS, 2 mm EDTA. The cell lysates were scraped and collected. 64Cu was measured in cell lysates using an LKB-Wallac Ultragamma counter. The protein concentration was estimated in each cell lysate using a Bio-Rad protein assay (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). Total Cellular Measurement of Copper, Zinc, and Iron—The cells were grown for 4 days in basal growth medium as described above. The cells were harvested as previously described (34Camakaris J. Danks D.M. Ackland L. Cartwright E. Borger P. Cotton R.G. Biochem. Genet. 1980; 18: 117-131Crossref PubMed Scopus (84) Google Scholar), and copper, zinc, and iron were analyzed using a PerkinElmer Life Sciences 5000 Atomic Absorption Spectrophotometer. Indirect Immunofluorescence—The cells were seeded in basal medium onto 13-mm round coverslips 48 h prior to immunofluorescence. The coverslips were washed in ice-cold phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 15 min, washed several times with PBS, permeabilized with 0.1% Triton X-100 in PBS for 5 min, washed several times in PBS, and then blocked overnight in PBS containing 1% bovine serum albumin. All of the antibody incubations were for 1 h in 1% bovine serum albumin/PBS. Monoclonal APP antiserum was visualized using Alexa 488™-conjugated to goat anti-mouse IgG antibodies (Molecular Probes). The coverslips were washed overnight in PBS then mounted using 100 mg/ml DABCO (Sigma) in 90% glycerol. Confocal microscopy was carried out using an Olympus BX60 microscope with a 60× PlanApo lens with a 1.40 N/A. This microscope was connected to an "Optiscan F900e" laser-scanning unit with a krypton/argon ion laser with excitation wavelengths of 488 and 568 nm. Protein Extraction and Western Immunoblot Analysis—The cell lines were sonicated in 62.5 mm Tris-HCl pH 6.8 buffer containing 2% SDS, 10 mm dithiothreitol, 1 mm EDTA, and COMPLETE™ EDTA-free protease inhibitor mixture (Roche Applied Science). 50 μg of protein extracts were fractionated on NuPage™ Bis-Tris (4-12%) gradient acrylamide gel (Invitrogen) and electroblotted overnight to nitrocellulose filters. Detection of MNK and APP protein was performed using an ECL Plus chemiluminescence kit (Amersham Biosciences) according to the manufacturer's instructions. The protein concentrations were measured using a Bio-Rad protein assay (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar), and MultiMark® (Invitrogen) protein molecular weight standards were loaded. Purified human APP695 isoform ectodomain was loaded as a control (35Henry A. Masters C.L. Beyreuther K. Cappai R. Protein Expression Purif. 1997; 10: 283-291Crossref PubMed Scopus (49) Google Scholar). Total RNA Extraction and Northern Blot Analysis—Total RNA was extracted from cell lines using a Total RNAeasy kit (Qiagen). 5-μg total RNA samples were denatured in formamide, formaldehyde, MOPS, and ethidium bromide at 65 °C for 15 min, cooled on ice, and electrophoresed on a 1.0% agarose-formaldehyde gel. The gel was blotted on Hybond-N+ nitrocellulose filter and immobilized by 50 mm sodium hydroxide. Each filter was prehybridized in hybridization buffer containing 50× Denhardt's reagent, sheared Salmon sperm DNA, 0.5 m EDTA, 1 m Tris, 20% SDS, and 1 m NaPO4 for at least 2 h. The filter was hybridized with a [α-32P]dATP-labeled cDNA probe using the Prime-a-Gene labeling System (Promega). After hybridization, the filters were washed to a stringency of 0.01% SDS, 0.1× SSC at 60 °C. APP cDNA probe corresponded to 3-kb fragment generated from human APP cDNA. Equal loading of samples was verified by rehybridizing the filter with the "housekeeping gene cDNA probe" rat GAPDH. The filter were exposed for 2 h to a PhosphorScreen (Molecular Dynamics) and scanned with a Typhoon 8600 PhosphorImager (Molecular Dynamics). Densitometer quantification was done with ImageQuant (Molecular Dynamics) software for both APP and GAPDH Northern analysis. Preparation of Plasmid Constructs—The promoter constructs pRSV-βGal, pBLCAT3, pβBIV, pβPB, and pβHB were as previously described (36Song W. Lahiri D.K. Gene (Amst.). 1998; 217: 151-164Crossref PubMed Scopus (29) Google Scholar, 37Song W. Lahiri D.K. Gene (Amst.). 1998; 217: 165-176Crossref PubMed Scopus (36) Google Scholar). The plasmids were prepared for transfection using an EndoFree® plasmid maxi kit (Qiagen) according to the manufacturer's instructions. Transient Transfection and Lysate Preparation—The cells were seeded in 6-well plates 24 h prior to transfection in basal medium. All of the cells were transfected with FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. The transfection mixture was prepared containing 0.5 μg of pRSV-βGal and either 3 μg of pBLCAT3, pβBIV, pβPB, or pβHB/well. Prior to transfection, the medium was changed to basal medium (0.78 μm copper), copper-supplemented medium (50, 100, 250, or 500 μm Cu) or copper-chelator diamsar-treated (38Bingham M.J. Sargeson A.M. McArdle H.J. Am. J. Physiol. 1997; 272: G1400-G1407PubMed Google Scholar) medium (5, 10, or 50 μm). 48 h post-transfection the cells were washed with ice-cold PBS several times, lysed in reporter gene lysis buffer (Roche Applied Science), and collected by microcentrifuge centrifugation (16,000 × g) to remove any cellular debris. The protein concentration was measured in each cell extract using a BioRad protein assay (33Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (214455) Google Scholar). β-Gal Assay—β-Gal activity was used to standardize transfection efficiency for subsequent CAT ELISAs. The cell extracts from transfected cells were analyzed for their β-Gal activity using a β-Gal enzyme assay system according to the manufacturer's instructions (Promega). Briefly, 5 μg of protein (an amount within the linear range of the assay) was assayed for 30 min at 37 °C in 2× Assay Buffer before the reaction was stopped with the addition of 1 m sodium carbonate. The absorbance was measured on a Bio-Rad 2550 EIA plate reader at 420 nm. Chloramphenicol Acetyltransferase Promoter Assay—The cell extracts from transfected cells were analyzed for their CAT activity using a colorimetric ELISA according to the manufacturer's instructions (Roche Applied Science). Briefly, 50 μg of protein (an amount within the linear range of the assay) was placed on anti-CAT coated microtiter plate modules and allowed to bind for 1 h at 37 °C. The plates were washed thoroughly after each step. Next, a digoxigenin-labeled anti-CAT antibody was added to the samples and incubated for 1 h at 37 °C. A subsequent antibody, anti-digoxigenin conjugated to peroxidase, was placed in the wells for 1 h at 37 °C. Finally, peroxidase substrate, ABTS (Roche Applied Science), was added, and the absorbance was measured at 405 nm using an ELISA plate reader. CAT activity was determined from the ratio of pg CAT/milliunit of β-Gal/μg of protein. Statistical Analysis—One-way ANOVA of more than two means followed by Bonferroni's multiple comparison of mean's post-test was performed for Northern analysis, 64Cu accumulation, cellular metal determination, and promoter assays using GraphPad Prism3 for Macintosh (GraphPad Software Inc.). Statistically significant was defined as p < 0.05. Intracellular Copper Levels, but Not Zinc or Iron, Are Severely Decreased in MNK transfected Fibroblasts—Immortalized human fibroblasts isolated from a Menkes disease patient (Table I), herein referred to as MNK deletion cells, and MNK deletion cells stably transfected with and hence overexpressing the MNK efflux protein (Table I), herein referred to as MNK transfected cells, represented powerful tools to investigate the hypothesis that copper levels can modulate APP gene expression. This is due to MNK deletion cells having high intracellular copper, whereas MNK transfected cells have low intracellular copper (25La Fontaine S.L. Firth S.D. Camakaris J. Englezou A. Theophilos M.B. Petris M.J. Howie M. Lockhart P.J. Greenough M. Brooks H. Reddel R.R. Mercer J.F. J. Biol. Chem. 1998; 273: 31375-31380Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). To confirm that MNK deletion and MNK transfected fibroblast cell lines (Fig. 1A) have dramatically altered copper levels, we investigated cells for intracellular levels of copper as well as zinc and iron.Table IDescription of fibroblast cell lines and their MNK expressionNameCell lineDescriptionaFor descriptions, refer to "Experimental Procedures" for further information.MNKMNK deletionMe32aT22/2LImmortalized human fibroblast isolated from classical Menkes disease patientNone detectedVector onlypCMB77Me32aT22/2L stably transfected with empty vectorNone detectedMNK transfected AMNK A12-H9Me32aT22/2L stably transfected with MNK expression vectorOverexpressedMNK transfected BMNK C3-C1Me32aT22/2L stably transfected with MNK expression vectorOverexpressedNormal humanGM2069Immortalized normal human fibroblastWild typea For descriptions, refer to "Experimental Procedures" for further information. Open table in a new tab Intracellular copper levels were measured using two independent approaches. The first was to analyze cells for copper accumulation using the Cu radioisotope, 64Cu, under steady-state conditions. 64Cu analysis showed MNK deletion and vector only control cells maintained copper levels significantly higher than normal human fibroblast cells with an average increase of 125% (Fig. 1B). MNK transfected cells maintained copper levels significantly lower than normal human fibroblast cells with an average decrease of 82% (Fig. 1B). MNK transfected cells also maintained copper levels significantly lower than MNK deletion cells with an average decrease of 92% (Fig. 1B). The second method of analysis examined the total amount of copper present in cell pellets utilizing atomic absorption spectroscopy. This analysis showed MNK deletion and vector only control cells contained significantly elevated copper levels compared with normal human fibroblast cells with an average increase of 65% (Fig. 1C). MNK transfected cells contained significantly reduced copper levels, with an average decrease of 79%, compared with normal human fibroblast cells (Fig. 1C). MNK transfected cells also contained significantly reduced copper levels compared with MNK deletion cells with an average decrease of 87% (Fig. 1C). There was no significant difference in the zinc and iron content of these fibroblast cell lines (Fig. 1, D and E). Overall, the cellular metal analysis demonstrated that by over
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