FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1993506411> ?p ?o ?g. }

• W1993506411 endingPage "1301" @default.
• W1993506411 startingPage "1291" @default.
• W1993506411 abstract "Prion diseases are associated with the conformational conversion of the host-encoded cellular prion protein into an abnormal pathogenic isoform. Reduction in prion protein levels has potential as a therapeutic approach in treating these diseases. Key targets for this goal are factors that affect the regulation of the prion protein gene. Recent in vivo and in vitro studies have suggested a role for prion protein in copper homeostasis. Copper can also induce prion gene expression in rat neurons. However, the mechanism involved in this regulation remains to be determined. We hypothesized that transcription factors SP1 and metal transcription factor-1 (MTF-1) may be involved in copper-mediated regulation of human prion gene. To test the hypothesis, we utilized human fibroblasts that are deleted or overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. Menkes deletion fibroblasts have high intracellular copper, whereas Menkes overexpressed fibroblasts have severely depleted intracellular copper. We have utilized this system previously to demonstrate copper-dependent regulation of the Alzheimer amyloid precursor protein. Here we demonstrate that copper depletion in MNK overexpressed fibroblasts decreases cellular prion protein and PRNP gene levels. Conversely, expression of transcription factors SP1 and/or MTF-1 significantly increases prion protein levels and up-regulates prion gene expression in copper-replete MNK deletion cells. Furthermore, siRNA “knockdown” of SP1 or MTF-1 in MNK deletion cells decreases prion protein levels and down-regulates prion gene expression. These data support a novel mechanism whereby SP1 and MTF-1 act as copper-sensing transcriptional activators to regulate human prion gene expression and further support a role for the prion protein to function in copper homeostasis. Expression of the prion protein is a vital component for the propagation of prion diseases; thus SP1 and MTF-1 represent new targets in the development of key therapeutics toward modulating the expression of the cellular prion protein and ultimately the prevention of prion disease. Prion diseases are associated with the conformational conversion of the host-encoded cellular prion protein into an abnormal pathogenic isoform. Reduction in prion protein levels has potential as a therapeutic approach in treating these diseases. Key targets for this goal are factors that affect the regulation of the prion protein gene. Recent in vivo and in vitro studies have suggested a role for prion protein in copper homeostasis. Copper can also induce prion gene expression in rat neurons. However, the mechanism involved in this regulation remains to be determined. We hypothesized that transcription factors SP1 and metal transcription factor-1 (MTF-1) may be involved in copper-mediated regulation of human prion gene. To test the hypothesis, we utilized human fibroblasts that are deleted or overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. Menkes deletion fibroblasts have high intracellular copper, whereas Menkes overexpressed fibroblasts have severely depleted intracellular copper. We have utilized this system previously to demonstrate copper-dependent regulation of the Alzheimer amyloid precursor protein. Here we demonstrate that copper depletion in MNK overexpressed fibroblasts decreases cellular prion protein and PRNP gene levels. Conversely, expression of transcription factors SP1 and/or MTF-1 significantly increases prion protein levels and up-regulates prion gene expression in copper-replete MNK deletion cells. Furthermore, siRNA “knockdown” of SP1 or MTF-1 in MNK deletion cells decreases prion protein levels and down-regulates prion gene expression. These data support a novel mechanism whereby SP1 and MTF-1 act as copper-sensing transcriptional activators to regulate human prion gene expression and further support a role for the prion protein to function in copper homeostasis. Expression of the prion protein is a vital component for the propagation of prion diseases; thus SP1 and MTF-1 represent new targets in the development of key therapeutics toward modulating the expression of the cellular prion protein and ultimately the prevention of prion disease. Prion diseases, traditionally known as transmissible spongiform encephalopathies, are invariably fatal, transmissible neurodegenerative disorders that include Creutzfeldt-Jakob disease and kuru in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle. According to the protein-only model of prion propagation, these diseases are associated with the conformational conversion of the host-encoded cellular prion protein (PrPC) into an abnormal pathogenic isoform (PrPSc) (1Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4014) Google Scholar). PrPC and PrPSc both have the same primary sequence and are encoded for by a single gene, PRNP (2Basler K. Oesch B. Scott M. Westaway D. Walchli M. Groth D.F. McKinley M.P. Prusiner S.B. Weissmann C. Cell. 1986; 46: 417-428Abstract Full Text PDF PubMed Scopus (634) Google Scholar). PrPC expression is an absolute requirement for prion infection, because mice in which PRNP has been ablated are completely resistant to infection when inoculated with prions (3Bueler H. Aguzzi A. Sailer A. Greiner R.A. Autenried P. Aguet M. Weissmann C. Cell. 1993; 73: 1339-1347Abstract Full Text PDF PubMed Scopus (1791) Google Scholar), and this protective phenotype can be inhibited when transgenes expressing PrPC are reintroduced (4Asante E.A. Li Y.G. Gowland I. Jefferys J.G. Collinge J. Neurosci. Lett. 2004; 360: 33-36Crossref PubMed Scopus (13) Google Scholar). PrPC is a cell surface glycoprotein anchored at the plasma membrane by a glycosylphosphatidylinositol anchor (5Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar). Expression is most abundant in the central nervous system, primarily in neuronal (6Kretzschmar H.A. Prusiner S.B. Stowring L.E. DeArmond S.J. Am. J. Pathol. 1986; 122: 1-5PubMed Google Scholar) and glial cells (7Moser M. Colello R.J. Pott U. Oesch B. Neuron. 1995; 14: 509-517Abstract Full Text PDF PubMed Scopus (269) Google Scholar). PrPC is also expressed in many non-neuronal tissues, including blood lymphocytes, gastroepithelial cells, heart, kidney, and muscle (8Horiuchi M. Yamazaki N. Ikeda T. Ishiguro N. Shinagawa M. J. Gen. Virol. 1995; 76: 2583-2587Crossref PubMed Scopus (174) Google Scholar, 9Fournier J.G. Escaig-Haye F. Billette de Villemeur T. Robain O. Lasmezas C.I. Deslys J.P. Dormont D. Brown P. Cell Tissue Res. 1998; 292: 77-84Crossref PubMed Scopus (68) Google Scholar). Although this widespread expression pattern has suggested potential functional roles for PrPC in a variety of cellular mechanisms (10Martins V.R. Linden R. Prado M.A. Walz R. Sakamoto A.C. Izquierdo I. Brentani R.R. FEBS Lett. 2002; 512: 25-28Crossref PubMed Scopus (120) Google Scholar), the cellular functions of PrPC are still poorly understood. Several studies have demonstrated that a major function of PrPC relates to the maintenance of intracellular copper homeostasis. PrPC contains two distinct copper-binding domains. The primary copper-binding domain is located in the N-terminal region between residues 60-91 (11Wadsworth J.D. Hill A.F. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (276) Google Scholar), consists of four to six octapeptide repeats of the sequence Pro-His-Gly-Gly-Gly-Trp-Gly-Gln (12Hornshaw M.P. McDermott J.R. Candy J.M. Biochem. Biophys. Res. Commun. 1995; 207: 621-629Crossref PubMed Scopus (321) Google Scholar, 13Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar, 14Miura T. Hori-i A. Mototani H. Takeuchi H. Biochemistry. 1999; 38: 11560-11569Crossref PubMed Scopus (247) Google Scholar), and binds copper ions with between femto- and nanomolar affinities (15Thompsett A.R. Abdelraheim S.R. Daniels M. Brown D.R. J. Biol. Chem. 2005; 280: 42750-42758Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). The secondary copper-binding domain is located between residues 91 and 111 just outside of the octapeptide repeat region (16Hasnain S.S. Murphy L.M. Strange R.W. Grossmann J.G. Clarke A.R. Jackson G.S. Collinge J. J. Mol. Biol. 2001; 311: 467-473Crossref PubMed Scopus (125) Google Scholar, 17Cereghetti G.M. Schweiger A. Glockshuber R. Van Doorslaer S. Biophys. J. 2001; 81: 516-525Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 18Jobling M.F. Huang X. Stewart L.R. Barnham K.J. Curtain C. Volitakis I. Perugini M. White A.R. Cherny R.A. Masters C.L. Barrow C.J. Collins S.J. Bush A.I. Cappai R. Biochemistry. 2001; 40: 8073-8084Crossref PubMed Scopus (249) Google Scholar). This domain is coordinated by two histidine residues, His96 and His111, and is suggested to have a much lower affinity for copper than the primary copper-binding domain (15Thompsett A.R. Abdelraheim S.R. Daniels M. Brown D.R. J. Biol. Chem. 2005; 280: 42750-42758Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 19Treiber A. Schneiter R. Hausler S. Stieger B. Drug Metab. Dispos. 2007; 35: 1400-1407Crossref PubMed Scopus (260) Google Scholar). These copper-binding regions may have functional significance, because they are very highly conserved among a wide variety of mammalian species (11Wadsworth J.D. Hill A.F. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (276) Google Scholar), and insertions of one or more octapeptide repeat units are associated with familial forms of prion disease in humans (20Duchen L.W. Poulter M. Harding A.E. Brain. 1993; 116: 555-567Crossref PubMed Scopus (60) Google Scholar, 21Perera W.S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Copper binding to the octapeptide repeat region induces the endocytosis of PrPC from the cell surface in a reversible manner, which suggests PrPC may act as a recycling receptor for cellular uptake or efflux of copper (22Pauly P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (539) Google Scholar, 23Brown L.R. Harris D.A. J. Neurochem. 2003; 87: 353-363Crossref PubMed Scopus (105) Google Scholar). Furthermore, it has been shown in PRNP null mice that copper levels in cerebellar cells are significantly reduced compared with cells from age- and sex-matched controls (13Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar), suggesting that PrPC is an important component for maintaining brain copper homeostasis. In addition, the octapeptide region of human PrPC reduces Cu2+ to Cu+ in vitro, which is an important step required for cellular copper uptake (24Ruiz F.H. Silva E. Inestrosa N.C. Biochem. Biophys. Res. Commun. 2000; 269: 491-495Crossref PubMed Scopus (75) Google Scholar, 25Opazo C. Barria M.I. Ruiz F.H. Inestrosa N.C. Biometals. 2003; 16: 91-98Crossref PubMed Scopus (76) Google Scholar). It has also been reported that copper can regulate the expression of the rat prion gene promoter in neurons via putative metal response element (MRE) 3The abbreviations used are: MRE, metal response element; APP, amyloid precursor protein; MNK, Menkes protein; MTF-1, metal transcription factor-1; MLS, MRE-like sequence; siRNA, small interfering RNA; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; ANOVA, analysis of variance.3The abbreviations used are: MRE, metal response element; APP, amyloid precursor protein; MNK, Menkes protein; MTF-1, metal transcription factor-1; MLS, MRE-like sequence; siRNA, small interfering RNA; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription; ANOVA, analysis of variance. DNA sequences (26Varela-Nallar L. Toledo E.M. Larrondo L.F. Cabral A.L. Martins V.R. Inestrosa N.C. Am. J. Physiol. 2006; 290: C271-C281Crossref PubMed Scopus (58) Google Scholar). However, the precise transcriptional machinery involved in this regulation has not been determined. Because copper homeostasis from yeast to mammals is regulated by several cellular mechanisms including copper-dependent transcriptional regulation (27Rutherford J.C. Bird A.J. Eukaryot. Cell. 2004; 3: 1-13Crossref PubMed Scopus (207) Google Scholar, 28Balamurugan K. Schaffner W. Biochim. Biophys. Acta. 2006; 1763: 737-746Crossref PubMed Scopus (176) Google Scholar) and the PRNP promoter region is highly conserved among several species (29Mahal S.P. Asante E.A. Antoniou M. Collinge J. Gene (Amst.). 2001; 268: 105-114Crossref PubMed Scopus (41) Google Scholar), we propose that copper regulation of the human PRNP gene is controlled by metal-regulated or metal-responsive transcription factors via putative MRE sequences. The human PRNP promoter region contains a number of putative transcription factor-binding sites, including the transcriptional activator SP1, AP1, AP2, and a CCAAT box (29Mahal S.P. Asante E.A. Antoniou M. Collinge J. Gene (Amst.). 2001; 268: 105-114Crossref PubMed Scopus (41) Google Scholar). The active promoter region has been determined to be within a 273-bp region, -148 to +125, relative to the cap start site (29Mahal S.P. Asante E.A. Antoniou M. Collinge J. Gene (Amst.). 2001; 268: 105-114Crossref PubMed Scopus (41) Google Scholar, 30Puckett C. Concannon P. Casey C. Hood L. Am. J. Hum. Genet. 1991; 49: 320-329PubMed Google Scholar). Metal-responsive transcription factor-1 (MTF-1) can bind to MRE sequences to regulate genes encoding metallothioneins (31Stuart G.W. Searle P.F. Chen H.Y. Brinster R.L. Palmiter R.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7318-7322Crossref PubMed Scopus (243) Google Scholar, 32Culotta V.C. Hamer D.H. Mol. Cell. Biol. 1989; 9: 1376-1380Crossref PubMed Scopus (159) Google Scholar, 33Westin G. Schaffner W. EMBO J. 1988; 7: 3763-3770Crossref PubMed Scopus (225) Google Scholar, 34Radtke F. Heuchel R. Georgiev O. Hergersberg M. Gariglio M. Dembic Z. Schaffner W. EMBO J. 1993; 12: 1355-1362Crossref PubMed Scopus (329) Google Scholar), a family of conserved metal detoxification proteins (35Vasak M. Hasler D.W. Curr. Opin. Chem. Biol. 2000; 4: 177-183Crossref PubMed Scopus (368) Google Scholar). In Drosophila, dMTF-1 can also regulate the expression of copper detoxification metallothioneins (36Balamurugan K. Egli D. Selvaraj A. Zhang B. Georgiev O. Schaffner W. Biol. Chem. 2004; 385: 597-603Crossref PubMed Scopus (46) Google Scholar) and paradoxically control the expression of copper import and export proteins DmCtr1b (37Selvaraj A. Balamurugan K. Yepiskoposyan H. Zhou H. Egli D. Georgiev O. Thiele D.J. Schaffner W. Genes Dev. 2005; 19: 891-896Crossref PubMed Scopus (120) Google Scholar) and DmATP7 (38Burke R. Commons E. Camakaris J. Int. J. Biochem. Cell Biol. 2008; 40: 1850-1860Crossref PubMed Scopus (44) Google Scholar), respectively. SP1, a general activator of transcription, can also bind to putative MRE sequences, possibly in a negative-regulatory manner in competition with MTF-1 to regulate gene expression (39Ogra Y. Suzuki K. Gong P. Otsuka F. Koizumi S. J. Biol. Chem. 2001; 276: 16534-16539Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The contribution of these transcription factors, SP1 and MTF-1, in copper regulation of the human PRNP gene has not yet been determined. To investigate the role of SP1 and MTF-1 in copper-dependent regulation of the human PRNP gene, we utilized a novel human cell culture model system that has previously been used to demonstrate copper-dependent regulation of the Alzheimer amyloid precursor protein (APP) gene (40Bellingham S.A. Lahiri D.K. Maloney B. La Fontaine S. Multhaup G. Camakaris J. J. Biol. Chem. 2004; 279: 20378-20386Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). This approach involves cultured human fibroblasts overexpressing the Menkes protein (MNK; encoded by ATP7A), a major mammalian copper translocating P-type ATPase involved in copper efflux (41Petris M.J. Mercer J.F. Culvenor J.G. Lockhart P. Gleeson P.A. Camakaris J. EMBO J. 1996; 15: 6084-6095Crossref PubMed Scopus (527) Google Scholar, 42Camakaris 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 (155) Google Scholar, 43La 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 caused by the lack of active copper efflux, whereas cells transfected and hence overexpressing MNK have markedly reduced copper levels (40Bellingham S.A. Lahiri D.K. Maloney B. La Fontaine S. Multhaup G. Camakaris J. J. Biol. Chem. 2004; 279: 20378-20386Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 43La 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, 44Du T. La Fontaine S.L. Abdo M. Bellingham S.A. Greenough M. Volitakis I. Cherny R.A. Bush A.I. Hudson P.J. Camakaris J. Mercer J.F. Crouch P.J. Masters C.L. Perreau V.M. White A.R. Proteomics. 2008; 8: 1819-1831Crossref PubMed Scopus (7) Google Scholar, 45Cater M.A. McInnes K.T. Li Q.X. Volitakis I. La Fontaine S. Mercer J.F. Bush A.I. Biochem. J. 2008; 412: 141-152Crossref PubMed Scopus (71) Google Scholar). Here we report that transcription factors SP1 and MTF-1 increase both cellular prion protein levels and PRNP gene expression under copper-replete conditions. Conversely, reducing levels of SP1 and MTF-1 using siRNA “knockdown” decreases both cellular prion protein and PRNP gene expression under copper-replete conditions. In addition, depletion of intracellular copper results in complete reduction of PrPC protein and PRNP gene levels. Overall, these data suggest that SP1 and MTF-1 are required for copper-mediated regulation of the PRNP gene and supports the increasing evidence that the cellular prion protein is involved in copper homeostasis. Cell Lines—Establishment and characterization of human skin fibroblast cells, MNK(Del), MNK(v/o), and MNK(++) has been reported previously (43La 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). Briefly, primary fibroblast cells from a classical Menkes disease patient were immortalized by the SV40 gene transfer to derive Me32aT22/2L (43La 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) and designated MNK(Del). The 4.6-kb cDNA encoding the human MNK protein was cloned into a mammalian expression vector and transfected into Me32aT22/2L, and MNK-expressing clone A12-H9 was designated MNK(++) (43La 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 Me32aT22/2L to derive Me32aT22/2L(pCMB77) and designated MNK(v/o) (43La 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), was used as a control. Cell Culture Conditions—MNK(Del) and normal human fibroblast cells (43La 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) were maintained in Eagle's basal medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen), 2 mm l-glutamine, 20 mm HEPES. The MNK(v/o) and MNK(++) cell lines were maintained in 10% Eagle's basal medium prepared as above with the addition of 500 μg/ml geneticin (Invitrogen). All of the cells were incubated at 37 °C in a 5% CO2 atmosphere. Cellular Metal Analysis—Copper, zinc, and iron levels were determined in MNK(Del), MNK(v/o), MNK(++), and normal human fibroblasts according to established procedures (40Bellingham S.A. Lahiri D.K. Maloney B. La Fontaine S. Multhaup G. Camakaris J. J. Biol. Chem. 2004; 279: 20378-20386Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Preparation of Expression Constructs—Full-length True-Clone™ cDNA expression constructs (Origene) for SP1 (SC116396) and MTF-1 (SC101137) were prepared for transfection using a plasmid DNA maxi kit (Promega) according to the manufacturer's instructions. Transient Transfection and Lysate Preparation—The cells were seeded at ∼1 × 105 cells/well 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. Transfection mixture was prepared containing 4 μg of pSP1 and/or 4 μg of pMTF-1/well. 24 h post-transfection, the medium was changed to either basal medium or 100 μm CuCl2, 50 μm ZnCl2, or 50 μm FeCl3 supplemented medium. 48 h post-transfection the cells were washed with ice-cold PBS several times, lysed, and extracted for total protein or total RNA using the PARIS™ kit (Ambion) according to the manufacturer's instructions. The protein concentrations were measured using a BCA protein assay (Pierce). Total RNA was analyzed for quality and quantity by standard spectrometry procedures. Antibodies—Anti-MNK polyclonal antibody raised to the MNK N-terminal region (42Camakaris 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 (155) Google Scholar) was diluted 1:2500. Anti-APP (WO2) monoclonal antibody raised to the amyloid-β region (46Ida N. Hartmann T. Pantel J. Schroder J. Zerfass R. Forstl H. Sandbrink R. Masters C.L. Beyreuther K. J. Biol. Chem. 1996; 271: 22908-22914Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar) was diluted 1:10000. Anti-PrP (3F4) monoclonal antibody raised to human PrPC region was diluted 1:10000 for Western blot analysis, whereas Anti-PrP (ICSM-18; D-Gen) was diluted at 1:250 for immunofluorescence. Anti-MTF-1 (clone ab55522) and anti-SP1 (clone ab58199) monoclonal antibodies (Abcam) were diluted at 1:500. Anti-GAPDH monoclonal antibody, clone 6C5 (Ambion) was diluted at 1:20000. Anti-mouse horseradish peroxidase secondary antibody (GE Healthcare) was diluted at 1:15000. Confocal Microscopy—The cells were seeded onto glass coverslips and grown for 48 h prior to being fixed in 3.2% paraformaldehyde/PBS and permeabilized in 0.1% Triton X-100/PBS. The coverslips were then placed in blocking solution containing 10% goat serum (Invitrogen)/2% bovine serum albumin in PBS at 4 °C overnight. PrPC was detected with primary antibody ICSM-18 and secondary antibody Alexa-488 conjugated to goat anti-mouse (Molecular Probes) diluted at 1:250 and 1:500 in blocking solution, respectively. Nuclear staining was performed with 4′,6′-diamino-2-phenylindole (Sigma) diluted at 1:1000 and co-incubated with the secondary antibody. Coverslips were mounted on glass slides in DABCO (Sigma) and scanned using a Leica DMIRE2 confocal microscope under identical exposure conditions. Western Immunoblot Analysis—Protein extracts were fractionated on NuPage™ Bis-Tris (4-12%) gradient acrylamide gel (Invitrogen) and electroblotted to nitrocellulose filters or polyvinylidene difluoride filters. Detection of protein was performed using an ECL chemiluminescence kit (GE Healthcare), according to the manufacturer's instructions. Quantitative Real Time RT-PCR—1 μg of total RNA extracted from cell lysates was converted to cDNA using a high capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions. Real time RT-PCR samples were then prepared using Taqman® gene expression master mix and human-specific Taqman® gene expression assays (Applied Biosystems) for PRNP (Hs00175591_m1) with endogenous controls for human GAPDH (Hs99999905_m1) and RPLP0 (Hs99999902_m1) according to the manufacturer's instructions. Real Time RT-PCR samples were then run on a RotorGene 3000 (Corbett Research). The data were analyzed and quantified using the DeltaDelta CT method (47Pfaffl M.W. Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (24586) Google Scholar). siRNA—Silencer® predesigned siRNA for SP1 (SP#1, identification code 116547; SP#2, identification code 1165476; SP#3, identification code 143158), MTF-1 (MTF1#1, identification code 3247; MTF1#2, identification code 3337; MTF1#3, identification code 107900), and negative control 1 siRNA (NEG#1) was purchased from Ambion. siRNA transfection reagent siPORT NeoFx was purchased from Applied Biosystems. siRNA Knockdown—The cells were post-seeded at ∼1 × 105 cells/6 wells and either mock transfected or transfected with siPORT NeoFx transfection reagent and negative control #1 siRNA or Silencer® predesigned siRNAs for SP1 and MTF-1 at a final concentration of 30 nm using the reverse transfection method according to the manufacturer's instructions. 48 h post-transfection total protein and RNA were extracted and analyzed as described above. Bioinformatics—The human PRNP gene promoter sequence (GenBank™ accession number AJ289875) was analyzed for the presence of metal response element (48Koizumi 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 TESS: Transcription Element Search Software (49Schug J. Overton G.C. TESS: Transcriptional Element Search Software on the WWW. Computational Biology and Informatics, Laboratory School of Medicine, University of Pennsylvania1997Google Scholar). In addition, promoters were searched for MRE-like sequences (MLS), with no more than one base mismatch from the last three MRE consensus residues (5′-TGCRCNC-3′). Statistical Analysis—The results were expressed as the means ± S.E. Statistical analysis involving two groups was performed by unpaired t test, whereas one-way analysis of variance (ANOVA) followed by Bonferroni's multiple comparison of mean's post-test was performed to compare more than two groups using Prism 4 for Macintosh (GraphPad Software Inc.). Statistically significant was defined as p < 0.05. The Human PRNP Gene Promoter Contains a Number of Putative Metal Regulatory Sequences—The human PRNP gene promoter (GenBank™ accession code AJ289875) was analyzed for the presence of MREs and MRE-like sequences found in the mammalian copper detoxification gene, metallothionein (MT) promoter (48Koizumi S. Suzuki K. Ogra Y. Yamada H. Otsuka F. Eur. J. Biochem. 1999; 259: 635-642Crossref PubMed Scopus (101) Google Scholar). Utilizing this search criteria, we identified three consensus MTF-1-binding sites, several potential MRE-like sequences (Fig. 1). In addition, multiple SP1-binding sites in the human PRNP promoter were also identified (Fig. 1). Of interest, the 273-bp (-148 to +125) active promoter region (29Mahal S.P. Asante E.A. Antoniou M. Collinge J. Gene (Amst.). 2001; 268: 105-114Crossref PubMed Scopus (41) Google Scholar) contained tandem MTF-1- and SP1-binding sites immediately prior to the suggested 5′-untranslated region cap start site (30Puckett C. Concannon P. Casey C. Hood L. Am. J. Hum. Genet. 1991; 49: 320-329PubMed Google Scholar). These observations supported our hypothesis that transcription factors SP1 and MTF-1 may mediate copper regulation of the human PRNP gene. MNK Fibroblasts Have Altered Cellular Copper Levels—MNK encodes a P-type copper-transporting ATPase (50Mercer J.F. Livingston J. Hall B. Paynter J.A. Begy C. Chandrasekharappa S. Lockhart P. Grimes A. Bhave M. Siemieniak D. Glover T.H. Nat. Genet. 1993; 3: 20-25Crossref PubMed Scopus (621) Google Scholar, 51Vulpe C. Levinson B. Whitney S. Packman S. Gitschier J. Nat. Genet. 1993; 3: 7-13Crossref PubMed Scopus (1204) Google Scholar). Mutations in the MNK gene, ATP7A, lead to Menkes disease in humans associated with increased intracellular copper in cultured cells from Menkes patients (52Camakaris J. Danks D.M. Ackland L. Cartwright E. Borger P. Cotton R.G. Biochem. Genet. 1980; 18: 117-131Crossref PubMed Scopus (82) Google Scholar), whereas overexpression of MNK results in copper resistance and reduced intracellular copper (42Camakaris 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 (155) Google Scholar). Immortalized human fibroblasts isolated from a Menkes disease patient (43La Fontaine S.L. Firth S.D. Cam" @default.
• W1993506411 created "2016-06-24" @default.
• W1993506411 creator A5012082589 @default.
• W1993506411 creator A5019296552 @default.
• W1993506411 creator A5033891416 @default.
• W1993506411 creator A5047385288 @default.
• W1993506411 creator A5061389893 @default.
• W1993506411 date "2009-01-01" @default.
• W1993506411 modified "2023-10-11" @default.
• W1993506411 title "Regulation of Prion Gene Expression by Transcription Factors SP1 and Metal Transcription Factor-1" @default.
• W1993506411 cites W1502443218 @default.
• W1993506411 cites W1511938293 @default.
• W1993506411 cites W1575039155 @default.
• W1993506411 cites W1601881612 @default.
• W1993506411 cites W1611407165 @default.
• W1993506411 cites W1704732331 @default.
• W1993506411 cites W192250034 @default.
• W1993506411 cites W1971022916 @default.
• W1993506411 cites W1974939001 @default.
• W1993506411 cites W1975536870 @default.
• W1993506411 cites W1979857580 @default.
• W1993506411 cites W1986291674 @default.
• W1993506411 cites W1993651887 @default.
• W1993506411 cites W2004846702 @default.
• W1993506411 cites W2005555304 @default.
• W1993506411 cites W2010388034 @default.
• W1993506411 cites W2013288672 @default.
• W1993506411 cites W2013679718 @default.
• W1993506411 cites W2019968489 @default.
• W1993506411 cites W2023163708 @default.
• W1993506411 cites W2023918463 @default.
• W1993506411 cites W2025174245 @default.
• W1993506411 cites W2027128963 @default.
• W1993506411 cites W2028345570 @default.
• W1993506411 cites W2042978244 @default.
• W1993506411 cites W2043251937 @default.
• W1993506411 cites W2044257400 @default.
• W1993506411 cites W2044627978 @default.
• W1993506411 cites W2046972756 @default.
• W1993506411 cites W2050498090 @default.
• W1993506411 cites W2051432193 @default.
• W1993506411 cites W2051564837 @default.
• W1993506411 cites W2051968416 @default.
• W1993506411 cites W2052706468 @default.
• W1993506411 cites W2052987925 @default.
• W1993506411 cites W2063767041 @default.
• W1993506411 cites W2070045579 @default.
• W1993506411 cites W2071474630 @default.
• W1993506411 cites W2082105991 @default.
• W1993506411 cites W2082872027 @default.
• W1993506411 cites W2083050608 @default.
• W1993506411 cites W2092341724 @default.
• W1993506411 cites W2095193325 @default.
• W1993506411 cites W2098407672 @default.
• W1993506411 cites W2106044548 @default.
• W1993506411 cites W2108244474 @default.
• W1993506411 cites W2118296350 @default.
• W1993506411 cites W2118539817 @default.
• W1993506411 cites W2125542198 @default.
• W1993506411 cites W2128389187 @default.
• W1993506411 cites W2131773446 @default.
• W1993506411 cites W2132661882 @default.
• W1993506411 cites W2133019124 @default.
• W1993506411 cites W2133778193 @default.
• W1993506411 cites W2138038687 @default.
• W1993506411 cites W2147276565 @default.
• W1993506411 cites W2149884263 @default.
• W1993506411 cites W2150612343 @default.
• W1993506411 cites W2152058429 @default.
• W1993506411 cites W2152735279 @default.
• W1993506411 cites W2154852310 @default.
• W1993506411 cites W2159294156 @default.
• W1993506411 cites W2162594375 @default.
• W1993506411 cites W2231807690 @default.
• W1993506411 doi "https://doi.org/10.1074/jbc.m804755200" @default.
• W1993506411 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18990686" @default.
• W1993506411 hasPublicationYear "2009" @default.
• W1993506411 type Work @default.
• W1993506411 sameAs 1993506411 @default.
• W1993506411 citedByCount "62" @default.
• W1993506411 countsByYear W19935064112012 @default.
• W1993506411 countsByYear W19935064112013 @default.
• W1993506411 countsByYear W19935064112014 @default.
• W1993506411 countsByYear W19935064112015 @default.
• W1993506411 countsByYear W19935064112016 @default.
• W1993506411 countsByYear W19935064112017 @default.
• W1993506411 countsByYear W19935064112018 @default.
• W1993506411 countsByYear W19935064112019 @default.
• W1993506411 countsByYear W19935064112020 @default.
• W1993506411 countsByYear W19935064112021 @default.
• W1993506411 countsByYear W19935064112022 @default.
• W1993506411 crossrefType "journal-article" @default.
• W1993506411 hasAuthorship W1993506411A5012082589 @default.
• W1993506411 hasAuthorship W1993506411A5019296552 @default.
• W1993506411 hasAuthorship W1993506411A5033891416 @default.
• W1993506411 hasAuthorship W1993506411A5047385288 @default.
• W1993506411 hasAuthorship W1993506411A5061389893 @default.
• W1993506411 hasBestOaLocation W19935064111 @default.

• next