Regulation of Prion Gene Expression by Transcription Factors SP1 and Metal Transcription Factor-1*

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 proteinonly model of prion propagation, these diseases are associated with the conformational conversion of the host-encoded cellular prion protein (PrP C ) into an abnormal pathogenic isoform (PrP Sc ) (1). PrP C and PrP Sc both have the same primary sequence and are encoded for by a single gene, PRNP (2). PrP C 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 (3), and this protective phenotype can be inhibited when transgenes expressing PrP C are reintroduced (4).
PrP C is a cell surface glycoprotein anchored at the plasma membrane by a glycosylphosphatidylinositol anchor (5). Expression is most abundant in the central nervous system, primarily in neuronal (6) and glial cells (7). PrP C is also expressed in many non-neuronal tissues, including blood lymphocytes, gastroepithelial cells, heart, kidney, and muscle (8,9). Although this widespread expression pattern has suggested potential functional roles for PrP C in a variety of cellular mechanisms (10), the cellular functions of PrP C are still poorly understood.
Several studies have demonstrated that a major function of PrP C relates to the maintenance of intracellular copper homeostasis. PrP C contains two distinct copper-binding domains. The primary copper-binding domain is located in the N-terminal region between residues 60 -91 (11), consists of four to six octapeptide repeats of the sequence Pro-His-Gly-Gly-Gly-Trp-Gly-Gln (12)(13)(14), and binds copper ions with between femtoand nanomolar affinities (15). The secondary copper-binding domain is located between residues 91 and 111 just outside of the octapeptide repeat region (16 -18). This domain is coordinated by two histidine residues, His 96 and His 111 , and is suggested to have a much lower affinity for copper than the primary copper-binding domain (15,19). These copperbinding regions may have functional significance, because they are very highly conserved among a wide variety of mammalian species (11), and insertions of one or more octapep-tide repeat units are associated with familial forms of prion disease in humans (20,21).
Copper binding to the octapeptide repeat region induces the endocytosis of PrP C from the cell surface in a reversible manner, which suggests PrP C may act as a recycling receptor for cellular uptake or efflux of copper (22,23). Furthermore, it has been shown in PRNP null mice that copper levels in cerebellar cells are significantly reduced compared with cells from ageand sex-matched controls (13), suggesting that PrP C is an important component for maintaining brain copper homeostasis. In addition, the octapeptide region of human PrP C reduces Cu 2ϩ to Cu ϩ in vitro, which is an important step required for cellular copper uptake (24,25). 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) 3 DNA sequences (26). 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 copperdependent transcriptional regulation (27,28) and the PRNP promoter region is highly conserved among several species (29),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 (29). The active promoter region has been determined to be within a 273-bp region, Ϫ148 to ϩ125, relative to the cap start site (29,30). Metal-responsive transcription factor-1 (MTF-1) can bind to MRE sequences to regulate genes encoding metallothioneins (31)(32)(33)(34), a family of conserved metal detoxification proteins (35). In Drosophila, dMTF-1 can also regulate the expression of copper detoxification metallothioneins (36) and paradoxically control the expression of copper import and export proteins DmCtr1b (37) and DmATP7 (38), 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 (39). 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 (40). 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 (41)(42)(43). 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 (40,(43)(44)(45).
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 PrP C 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 Culture Conditions-MNK(Del) and normal human fibroblast cells (43) 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(ϩϩ) cell lines were maintained in 10% Eagle's basal medium prepared as above with the addition of 500 g/ml geneticin (Invitrogen). All of the cells were incubated at 37°C in a 5% CO 2 atmosphere.
Preparation of Expression Constructs-Full-length True-Clone TM 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 ϳ1 ϫ 10 5 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 g of pSP1 and/or 4 g of pMTF-1/well. 24 h post-transfection, the medium was changed to either basal medium or 100 M CuCl 2 , 50 M ZnCl 2 , or 50 M FeCl 3 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 TM 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.
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. PrP C 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 4Ј,6Ј-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 TM 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 g 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 (47).
siRNA Knockdown-The cells were post-seeded at ϳ1 ϫ 10 5 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 TM accession number AJ289875) was analyzed for the presence of metal response element (48) (MRE consensus sequence 5Ј-TGCRCNC-3Ј) consensus sequences using TESS: Transcription Element Search Software (49). In addition, promoters were searched for MRE-like sequences (MLS), with no more than one base mismatch from the last three MRE consensus residues (5Ј-TGCRCNC-3Ј).
Statistical Analysis-The results were expressed as the means Ϯ 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 Ͻ 0.05.

The Human PRNP Gene Promoter Contains a Number of
Putative Metal Regulatory Sequences-The human PRNP gene promoter (GenBank TM accession code AJ289875) was analyzed for the presence of MREs and MRE-like sequences found in the mammalian copper detoxification gene, metallothionein (MT) promoter (48). 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 (Ϫ148 to ϩ125) active promoter region (29) contained tandem MTF-1-and SP1-binding sites immediately prior to the suggested 5Ј-untranslated region cap start site (30). 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 (50,51). Mutations in the MNK gene, ATP7A, lead to Menkes disease in humans associated with increased intracellular copper in cultured cells from Menkes patients (52), whereas overexpression of MNK results in copper resistance and reduced intracellular copper (42). Immortalized human fibroblasts isolated from a Menkes disease patient (43), herein referred to as MNK(Del) cells, and MNK(Del) cells stably transfected and overexpressed with the MNK efflux protein or the empty mammalian expression vector (43), herein referred to as MNK(ϩϩ) and MNK(v/o) cells, respectively, represent powerful tools to manipulate intracellular copper concentrations. Analysis of total cellular copper, zinc, and iron levels demonstrates that MNK(Del) and MNK(v/o) control cell lines have significantly increased copper levels, whereas MNK(ϩϩ) cells have significantly decreased copper levels compared with normal fibroblasts ( Fig. 2A). No difference is observed for zinc and iron levels (Fig. 2, B and C, respectively). This is in agreement with previous studies that have consistently demonstrated that MNK(Del) and MNK(ϩϩ) cell lines have contrasting high and low intracellular copper levels, respectively (40,(43)(44)(45). This facilitates investigation of the hypothesis that copper levels modulate human PRNP gene expression in this study.
MNK Overexpressed Fibroblasts Have Decreased Cellular Prion Protein Levels-MNK(Del), MNK(v/o) control, and MNK(ϩϩ) cell lines were analyzed for MNK, APP, and PrP C protein expression via Western blot analysis. MNK protein was not detectable in the MNK(Del) and MNK(v/o) control cell lines, whereas MNK protein was overexpressed in the MNK(ϩϩ) cell line (Fig. 3A), as previously reported (40,(43)(44)(45). APP protein was detected in MNK(Del) and MNK(v/o) control cell lines and not detectable in the MNK(ϩϩ) cell line (Fig. 3A). This is consistent with our previous work with this model MNK cell line system where we demonstrated copperdependent regulation of the human APP gene (40).
PrP C protein was detected in MNK(Del) and MNK(v/o) control cell lines as immunoreactive bands between 38 and 17 kDa (Fig. 3A). Interestingly, PrP C protein was undetectable in MNK(ϩϩ) cells (Fig. 3A). PrP C protein expression in fibroblast lines was also examined using immunofluorescence analysis of confocal microscopy images. Although MNK(Del) and MNK(v/o) control fibroblasts all had detectable PrP C levels, PrP C was not detectable in MNK(ϩϩ) cells (Fig. 3B).

MNK Overexpressed Fibroblasts Have Decreased Cellular Prion mRNA
Levels-To determine whether decreased PrP C levels observed in MNK(ϩϩ) cells is a result of decreased mRNA transcript levels, we performed quantitative real time RT-PCR (Fig. 3C). Quantitation of human PRNP mRNA levels in MNK(Del), MNK(v/o), and MNK(ϩϩ) cells demonstrated that PRNP expression is undetectable in MNK(ϩϩ) cells compared with MNK(Del) cells (Fig. 3C). Because MNK(Del) and MNK(ϩϩ) cells have high and low copper levels, respectively, and no change in zinc and iron levels ( Fig. 2), these results are consistent with the hypothesis that copper levels regulate the expression of the human PRNP gene. Furthermore, the MNK fibroblast cell lines represent an ideal cellular system to test the hypothesis that transcription factors MTF-1 and SP1 can regulate the expression of the human PRNP gene under varying copper conditions.
Transcription Factors SP1, MTF-1, and Extracellular Copper Increase Cellular Prion Protein Levels-To investigate the role of the transcription factors SP1 and MTF-1 in regulating PRNP gene expression, MNK-(Del) and MNK(ϩϩ) cells were transiently transfected in basal media for 48 h with SP1 and MTF-1 (Fig. 4A).
To control for metal specificity, MNK(Del) cells were transiently transfected with SP1 and MTF-1 in 50 M zinc-or ironsupplemented medium (Fig. 4C). In MNK(Del) cells, transfec-   JANUARY 9, 2009 • VOLUME 284 • NUMBER 2 tion of SP1 and/or MTF-1 in 50 M zinc-or iron-supplemented medium resulted in an increase in PrP C levels compared with zinc-or iron-supplemented mock transfected cells (Fig. 4C, compare lane 5 with lanes 6 -8 and compare lane 9 with lanes 10 -12, respectively). However, the increase in PrP C levels from transfection of SP1 and/or MTF-1 in the presence of zinc or iron appeared to be similar to that observed under basal conditions (Fig. 4C, compare lanes 6 -8 and  lanes 10 -12 with lanes 2-4).
Therefore, transfection of SP1 and MTF-1 in the presence of extracellular zinc or iron did not result in any significant increase in PrP C levels, whereas the addition of extracellular copper resulted in increased PrP C levels from that observed under basal transfection conditions. Similar results were obtained in normal human fibroblasts transfected under the same conditions (supplemental Fig. S1).
To compare the effect of additional extracellular copper on the ability of SP1 and MTF-1 transcription factors to increase PrP C protein levels, densitometry data were normalized to cells  SP1 and MTF-1 Knockdown Decreases Human PRNP Gene Expression and Cellular Prion Protein Levels-To further support our hypothesis that human PRNP gene expression can be modulated by transcription factors SP1 and MTF-1, we performed siRNA knockdown in MNK(Del) cells and determined the relative gene expression levels by quantitative real time RT-PCR. Transfection of two independent SP1 and MTF-1 siRNA sequences resulted in a significant decrease in SP1 mRNA levels ( Fig. 6A; **, p Ͻ 0.01) and MTF-1 mRNA levels ( Fig. 6B; **, p Ͻ 0.01). Importantly, PRNP mRNA levels were significantly decreased as a result of SP1 and MTF-1 knockdown (Fig. 6C; **, p Ͻ 0.01). To confirm that knockdown of SP1 and MTF-1 reduced PrP C protein levels, siRNA knockdown cell lysates were analyzed for PrP C protein expression via Western blot analysis (Fig. 7A). Densitometric analysis of MNK(Del) cells knocked down with two independent SP1 and MTF-1 siRNAs resulted in a significant decrease in PrP C protein levels, com-  pared with negative control siRNA cells ( Fig. 7B; *, p Ͻ 0.05; **, p Ͻ 0.01). Overall, these data provide strong support for copper regulation of human PRNP gene expression being mediated by the transcription factors SP1 and MTF-1.

DISCUSSION
An important cellular function of the prion protein has been suggested to be the maintenance of copper homeostasis (11,13,22,23). A recent study has also demonstrated that copper also regulates expression of the rat prion gene via putative MRE DNA sequences (26). However, the transcription factors involved in this copper regulation remained to be determined.
MTF-1 is the most characterized metal-dependent mammalian transcription factor. MTF-1 is activated by a variety of stimuli, including copper and zinc, and binds to MREs to regulate the expression of copper detoxification metallothioneins (31)(32)(33)(34). SP1, a general transcriptional activator, can also bind to MREs to regulate the expression of metallothioneins (39). Because bioinformatics analysis of the human PRNP gene promoter revealed several putative MTF-1 MREs, as well as a number of predicted SP1-binding sites (Fig. 1), we hypothesized that transcription factors SP1 and MTF-1 play a significant role in copper regulation of the PRNP gene.
To investigate the role of copper and the transcription factors SP1 and MTF-1 in modulating human PRNP gene regulation, we utilized a system where intracellular copper levels are genetically manipulated through altered expression of the MNK copper efflux protein. Cells lacking a functional MNK protein show high intracellular copper levels because of reduced copper efflux (43). Restoration of MNK function by stable transfection in a MNK deletion background restores copper efflux ability, resulting in dramatically decreased intracellular copper levels and no change in other transition metals such as zinc and iron (Fig. 2).
Therefore, MNK(Del) and MNK(ϩϩ) cells have contrasting high and low intracellular copper levels, respectively (40,(43)(44)(45). Here we report for the first time evidence that copper regulation of the human PRNP gene is mediated by the transcription factors SP1 and MTF-1. This demonstrates a previously uncharacterized aspect of regulation of the human PRNP gene and further supports a role for the cellular prion protein in copper homeostasis.
Investigation of PrP C protein levels in MNK(Del) and MNK(ϩϩ) cell lines demonstrated that PrP C is not detectable by both Western blot and immunofluorescence analysis with PrP specific antibodies in the low copper MNK(ϩϩ) cell line (Fig. 3). These data are consistent with the hypothesis that copper levels regulate the expression of the human PRNP gene and also support the findings that elevated copper regulates PRNP gene expression (26,57).
To evaluate the role of transcription factors SP1 and MTF-1, in copper regulation of PRNP gene expression, we transfected SP1 and MTF-1 into MNK(Del) and MNK(ϩϩ) cell lines. In low copper MNK(ϩϩ) cells transfection of SP1 and/or MTF-1 did not have any effect on restoring cellular prion protein levels under basal or copper-supplemented conditions (Fig. 3, A and  B). This may be due to a lack of available copper in the pool required for activating SP1 and MTF-1 or suppression of PRNP gene by an unknown mechanism that overrides the activities of SP1 and MTF-1. Moreover, it is possible that other co-factors required for transcriptional activation of the PRNP gene are either absent or down-regulated. This is supported by proteomic antibody array analysis, which identified a significant protein expression differential between high copper MNK(Del) and low copper MNK(ϩϩ) cells (44).
Under basal and copper-, zinc-, and iron-supplemented medium conditions, transfection of SP1 and/or MTF-1 resulted in elevated PrP C protein levels in MNK(Del) cells (Fig. 4, A-C). PrP C protein levels were increased by ϳ100% under basal, zinc, and iron conditions and ϳ200% under copper-supplemented conditions in MNK(Del) cells (Fig. 4D). Furthermore, transfection of SP1 and MTF-1 under copper-supplemented conditions, either alone or co-transfected, resulted in an additional ϳ30 -50% significant increase in cellular PrP C levels compared with transfections performed under basal conditions (Fig. 4E). Together, these results strongly suggest that SP1 and MTF-1 expression increases cellular prion protein levels and that copper is required as a specific co-factor.
To confirm that increased cellular prion protein, as a result of expression of SP1 and/or MTF-1 transcription factors, is due to transcriptional activation of human PRNP gene expression, we performed quantitative real time RT-PCR analysis (Fig. 5). Quantitation of human PRNP mRNA levels in MNK(Del) cells, under basal or copper-supplemented conditions, demonstrated that SP1 and/or MTF-1 significantly increased PRNP gene expression by ϳ200% (Fig. 5). Additionally, siRNA knockdown of SP1 and MTF-1 transcription factors significantly reduced PRNP gene expression levels (Fig. 6), resulting in a concomitant reduction in cellular prion protein levels (Fig. 7). Although we were unable to achieve greater than 50% knockdown of transcription factors SP1 and MTF-1 because of cell toxicity when the amount of siRNA transfected was increased above 30 nM, this is consistent with SP1 and MTF-1 being vital for cellular development with SP1 and MTF-1 null mice being embryonic lethal (58,59). Together, these data provide strong support for the mechanism that increased cellular PrP C levels are the result of transcriptional activation of the human PRNP gene by SP1 and MTF-1.
Overall, the regulation of PRNP gene expression described in the current studies suggests that SP1 and MTF-1 function as copper transcriptional activators to regulate PRNP expression. We therefore propose that copper regulation of human PRNP gene expression occurs by either copper-activated SP1 or MTF-1 binding to putative MREs located in the human gene promoter. This may occur independently or as a co-activator complex (Fig. 8).
This model is strongly supported by two recent studies; first, MTF-1 and SP1 form a co-activator complex to regulate metallothionein gene expression (60), and second, SP1 is a coppersensing transcription factor responsible for maintaining cellular copper homeostasis by regulating expression of the copper uptake gene hCTR1 (61). Furthermore, the active promoter region of human PRNP, Ϫ148 to ϩ125 relative to the ϩ1 transcriptional start site, contains tandem MTF-1 MRE-and SP1-binding sites in the human PRNP promoter (Fig.  1). Although it is well established that MTF-1 binds to putative MRE sequences to activate gene expression (31)(32)(33)(34), SP1 can also bind to weakly activated putative MREs in close proximity to the SP1-binding regions, possibly in competition with MTF-1, to regulate gene expression (39). Based upon sequence homology, this putative MTF-1 MRE, located Ϫ90 to Ϫ83 in the human PRNP promoter, is predicted to be weakly activated by MTF-1 (48).
Therefore, it is plausible that SP1 and MTF-1 compete for the weakly activated MTF-1 MRE located Ϫ90 to Ϫ83 in the human PRNP promoter to modulate copper regulation of human PRNP (Fig. 8). Further studies will be aimed at determining the role of SP1 and MTF-1 in activating this promoter region under a variety of copper conditions in both neuronal and non-neuronal cell lines.
These results may also have a significant impact on studies investigating the regulation of the APP. APP and the cellular prion protein are hypothesized to be involved in neuronal copper homeostasis (62). APP and PrP posses conserved histidine copper-binding domains, and interactions with metal ions such as copper have important roles in the biology of both proteins. A␤-copper interactions have been shown to mediate amyloid plaque formation (63,64). PrP has been show to coordinate copper; it has been reported that this interaction modulations PrP aggregation and conversion from PrP C to PrP Sc (65). APP gene expression is also regulated in a copper-dependent manner (40,57), with the promoter region of human APP containing a number of putative MRE-binding sites that may be regulated by SP1 or MTF-1 (40). Two key proteins, ␤-amyloid cleaving enzyme (BACE) and Tau, implicated in the pathology of Alzheimer disease are also regulated by SP1 (66,67). BACE is a key enzyme responsible for cleavage of APP into ␤-amyloid (68), whereas Tau is a microtubule associated protein that forms the major component of neurofibrillary tangles in Alzheimer disease (69,70). Because SP1 can act as a copper-sensing transcription factor (61), SP1 may be a global neuronal regulator of proteins involved in mammalian copper homeostasis.
In summary, our results strongly suggest that at least in human fibroblasts, SP1 and MTF-1 are co-factors in copper-regulated A, under normal conditions, the active promoter region Ϫ148 to ϩ125 of PRNP is activated by SP1 binding to GC box sites. B-D, under copper-replete conditions, copper-activated expression of PRNP may occur independently or as a co-activator complex. B, copper-activated expression of PRNP may occur via MTF-1 binding to putative metal response element (MRE) and MLS located in the active promoter region. C, copper-activated expression of PRNP may occur via SP1/MTF-1 co-activator complexes binding to putative metal response element (MRE) and MLS located in the active promoter region. D, copper-activated expression of PRNP may occur via SP1 acting as a copper sensing transcription factor and binding to putative metal response element (MRE) and MLS located in the active promoter region, possibly in competition with MTF-1 for weakly activated MRE sequences. JANUARY 9, 2009 • VOLUME 284 • NUMBER 2 PRNP gene regulation. The data also further support a role for PrP C in copper efflux/detoxification. The elucidation of the copper regulation mechanisms of PRNP in human neuronal cells lines and prion susceptible cell lines may provide new targets in developing therapeutic strategies in the treatment of prion diseases. These strategies would be designed to reduce the expression of the PRNP gene and the ensuing conversion of the cellular prion protein into the abnormal pathogenic PrP Sc .