Ascensión Cuesta

Alberto Zambrano

María Royo

Dr Angel Pascual
email: apascual@iib.uam.es

The tumor suppressor p53 regulates the expression of the beta-amyloid precursor protein

The expression of the β-amyloid precursor protein (APP), which plays a key role in the development of Alzheimer's disease, is regulated by a variety of cellular mediators in a cell-dependent manner. In the present study, we present evidence that p53 regulates the expression of the APP gene in neuroblastoma cells. Transient expression of ectopic p53, as well as the activation of endogenous p53 by the DNA-damaging drug camptothecin or MDM2 depletion, decrease intracellular levels of APP in murine N2aβ neuroblastoma cells. This effect was also observed in primary cultures of rat neurons as well as in SH-SY5Y cells, a human neuroblastoma cell line. Transient transfection studies using plasmids which contain progressive deletions of the 5' region of the gene, demonstrate that p53 represses APP promoter activity through a mechanism that is mediated by DNA sequences located downstream of the transcription start site (+55/+101). Accordingly, expression of a dominant negative p53 mutant significantly increases the transcriptional activity of the APP promoter. In addition, data obtained in gel mobility shift assays show that p53 does not bind to the +55/+101 APP region, although reduces binding of the transcription factor Sp1. Reduction of Sp1 binding after activation of p53 with camptothecin was also observed in chromatin immunoprecipitation assays. All together our results strongly suggest a mechanism by which p53 precludes binding of Sp1 to DNA, and therefore the stimulation of the APP promoter by this transcription factor.

INTRODUCTION

Alzheimer's disease is a degenerative disorder of the central nervous system which causes mental deterioration and progressive dementia, and is accompanied by neuropathologic lesions including the presence of senile plaques of which the β-amyloid protein (Aβ), a hydrophobic 39-43 residues amino acid peptide, is the major component [START_REF] Mattson | Evidence for excitoprotective and intraneuronal calciumregulating roles for secreted forms of the beta-amyloid precursor protein[END_REF][START_REF] Selkoe | Cell biology of the amyloid beta-protein precursor and the mechanism of Alzheimer's disease[END_REF]. The β-amyloid protein is proteolytically derived from a set of alternatively spliced β-amyloid precursor proteins (APP), which are encoded by a single gene located on human chromosome [START_REF] Villa | A response unit in the first exon of the beta-amyloid precursor protein gene containing thyroid hormone receptor and Sp1 binding sites mediates negative regulation by 3,5,3'triiodothyronine[END_REF]. APP plays a central role in Alzheimer's disease, and it has been suggested that an increase in the production of this protein might actively contribute to the development of this pathology [START_REF] Fukuchi | Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity[END_REF][START_REF] Yoshikawa | Degeneration in vitro of post-mitotic neurons overexpressing the Alzheimer amyloid protein precursor[END_REF], likely by inducing the synthesis and deposition of β-amyloid [START_REF] Yoshikawa | Degeneration in vitro of post-mitotic neurons overexpressing the Alzheimer amyloid protein precursor[END_REF][START_REF] Desvergne | Functional characterization and receptor binding studies of the malic enzyme thyroid hormone response element[END_REF], and the subsequent neurotoxicity [START_REF] Fukuchi | Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity[END_REF]. In contrast, it has been also reported that physiological APP levels appear to be involved in neurotrophic events [START_REF] Mattson | Evidence for excitoprotective and intraneuronal calciumregulating roles for secreted forms of the beta-amyloid precursor protein[END_REF], thus protecting cells against neurodegeneration. APP is ubiquitously expressed in mammalian tissues, and its expression has been proved to be regulated by a variety of cellular mediators, including growth factors [START_REF] Cosgaya | Nerve growth factor and ras regulate beta-amyloid precursor protein gene expression in PC12 cells[END_REF][START_REF] Ohyagi | Effect of growth factors and cytokines on expression of amyloid beta protein precursor mRNAs in cultured neural cells[END_REF], phorbol esters [START_REF] Trejo | A direct role for protein kinase C and the transcription factor Jun/AP-1 in the regulation of the Alzheimer's beta-amyloid precursor protein gene[END_REF][START_REF] Yoshikai | Genomic organization of the human amyloid beta-protein precursor gene[END_REF] or ligands of the superfamily of steroid/thyroid nuclear hormone receptors [START_REF] Belandia | Thyroid hormone negatively regulates the transcriptional activity of the beta-amyloid precursor protein gene[END_REF][START_REF] Konig | Retinoic acid induced differentiated neuroblastoma cells show increased expression of the beta A4 amyloid gene of Alzheimer's disease and an altered splicing pattern[END_REF].

On the other hand, it has been described that p53 levels are significantly higher in Alzheimer's brains than in aged-matched controls [START_REF] Kitamura | Changes of p53 in the brains of patients with Alzheimer's disease[END_REF], and that p53associated apoptosis may be directly involved in the development of this pathology. Thus, β-amyloid deposits are associated with local neuritic degeneration, and it has been suggested that intracellular β-amyloid would be selectively cytotoxic to human neurons through a p53-associated cell death pathway [START_REF] Zhang | Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons[END_REF]. In addition, intracellular β-amyloid may directly activate the p53 promoter and the apoptosis process induced by this factor [START_REF] Ohyagi | Intracellular Abeta42 activates p53 promoter: a pathway to neurodegeneration in Alzheimer's disease[END_REF], and a correlation between amyloid accumulation and p53 activation in neurons has been also previously described [START_REF] Laferla | Extracellular deposition of beta-amyloid upon p53-dependent neuronal cell death in transgenic mice[END_REF].

Physiological levels of APP protect neuronal cells against degeneration induced by several apoptosis-controlling factors including p53, an effect that has been proposed to involve the control of p53-mediated gene transactivation [START_REF] Xu | Wild-type but not Alzheimer-mutant amyloid precursor protein confers resistance against p53-mediated apoptosis[END_REF]. Furthermore, it has been more recently reported that AICD, a secretase-derived β-amyloid intracellular c-terminal domain, controls p53 at a transcriptional level, in vitro and in vivo [START_REF] Alves Da Costa | Presenilin-dependent gamma-secretase-mediated control of p53-associated cell death in Alzheimer's disease[END_REF][START_REF] Checler | The gamma/epsilon-secretase-derived APP intracellular domain fragments regulate p53[END_REF].

In the present study we present evidence that ectopic expression of p53 or activation of endogenous p53 by treatment with camptothecin, or MDM2 depletion, regulate APP expression, and specifically decrease the intracellular levels of APP in N2aβ cells, a subclone of the mouse N2a neuroblastoma cell line that has been previously used to analyze the effect of thyroid hormones on APP [START_REF] Belandia | Thyroid hormone negatively regulates the transcriptional activity of the beta-amyloid precursor protein gene[END_REF]. The effect of p53 on APP expression appears to be mediated at least in part at the transcriptional level, since transient transfection studies demonstrate that p53 inhibits APP promoter activity. This inhibition requires DNA sequences located within the first exon of the APP gene, between positions +55 and +101, a region that nevertheless does not bind p53. In contrast, this fragment contains sequences that bind Sp1, and p53 appears to inhibit binding of this factor. Taken together, our results suggest a novel mechanism by which the tumor suppressor p53 might decrease APP gene expression by interfering with the activation induced by the transcription factor Sp1, which binds sequences of DNA located downstream the transcriptional initiation site.

MATERIALS AND METHODS

Chemicals and antibodies

Dulbecco's modified Eagle's medium (DMEM) was purchased from Biowhittaker (Verviers. Belgium). Fetal bovine serum was from Gibco.Life technologies Ltd. (Paislay. UK). Polyclonal antibody 369A against the cytoplasmic domain of APP [START_REF] Jaffe | Estrogen regulates metabolism of Alzheimer amyloid beta precursor protein[END_REF] was a generous gift of Dr. Samuel E. Gandy (Mount Sinai School of Medicine. N.York). Polyclonal antibody against phospho-p53 was from Cell Signaling Technology, Inc (USA). Anti β-catenin antibody was from BD Biosciences (Belgium). The second biotinylated anti-rabbit antibody and the peroxidase-conjugated streptavidine used in Western blot analysis were from Amersham International plc (England). Camptothecin was from Sigma-Aldrich (USA). siRNA against mRNA encoding Mdm2 was obtained from Dharmacon Research (Lafayette, CO). Sequence analyses and primer design were performed on-line by using the nucleotide sequence databases provided by the EMBL (http://www.ebi.ac.uk/embl).

Cell culture

Murine N2aβ neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum as previously described [START_REF] Lebel | Overexpression of the beta 1 thyroid receptor induces differentiation in neuro-2a cells[END_REF][START_REF] Villa | A response unit in the first exon of the beta-amyloid precursor protein gene containing thyroid hormone receptor and Sp1 binding sites mediates negative regulation by 3,5,3'triiodothyronine[END_REF]. Human SH-SY5Y neuroblastoma cells were cultured in RPMI medium containing 10% fetal bovine serum. Primary cultures of rat cortical neurons were kindly prepared by Dr. Diaz-Guerra's group as previously described [START_REF] Gascon | Transcription of the NR1 subunit of the N-methyl-D-aspartate receptor is down-regulated by excitotoxic stimulation and cerebral ischemia[END_REF]. Briefly, the plates were treated with poly-L-lysine (100 µg/ml) and laminin (4 µg/ml) overnight at 37 °C before seeding. Cerebral cortices from 18day-old rat embryos were dissected and mechanically dissociated in culture medium (Eagle's minimum medium supplemented with 28.5 mM NaHCO3, 22.2 mM glucose, 0.1 mM glutamine, 5% fetal bovine serum, and 5% donor horse serum). The cells were seeded at a density of 0.3X10 5 cells/cm 2 in the same medium.

Reporter plasmids and expression vectors

The chloramphenicol acetyl transferase (CAT) reporter plasmid containing the -1099/+101 fragment of the human APP gene has been previously described [START_REF] Belandia | Thyroid hormone negatively regulates the transcriptional activity of the beta-amyloid precursor protein gene[END_REF][START_REF] Lahiri | The promoter activity of the gene encoding Alzheimer beta-amyloid precursor protein (APP) is regulated by two blocks of upstream sequences[END_REF]. Progressive 5' deletions to -487, -307, -15 bp were prepared by polymerase chain reaction from the original -1099/+105 bp fragment, kindly provided by Dr. Lahiri's laboratory, and subcloned into the BamH1 site of pBL-CAT8, a plasmid which lacks the AP-1 binding site present in the pUC backbone. The reporter construct +55/+101 consists of a single copy of this APP fragment inserted in front of a tk promoter driving the expression of the CAT gene [START_REF] Belandia | Thyroid hormone negatively regulates the transcriptional activity of the beta-amyloid precursor protein gene[END_REF]. Wild-type and mutant (V143A) human p53 plasmids, respectively, utilize a pCMV-Neo-Bam or a T7 polymerase-transcribed pBluescript vector [START_REF] Qi | Constitutive activation of gene expression by thyroid hormone receptor results from reversal of p53-mediated repression[END_REF].

DNA transfection

For transient transfection assays cells were incubated with expression vectors for wild-type or mutated p53, or an empty vector, together with chloramphenicol acetyl transferase (CAT) reporter plasmids [START_REF] Belandia | Thyroid hormone negatively regulates the transcriptional activity of the beta-amyloid precursor protein gene[END_REF]. Cells were transfected with Lipofectamine 2000 following the instructions of the manufacturer (Invitrogen, Carlsbad, CA), and one hundred nanograms of a luciferase reference vector were simultaneously used as an internal control of the transfection efficiency. Transfected cells were then incubated for an additional 48 hours period. Assays were performed in duplicate cultures that normally showed less than 5-15% variation in CAT activity which was determined by incubation of [ 14 C]chloramphenicol with cell lysate protein. After autoradiography, the nonacetylated and acetylated [ 14 C]-chloramphenicol were quantified and the data expressed as the mean ± standard deviation (SD) of the percent of acetylated forms after each treatment. Each experiment was repeated at least 2-3 times with similar relative differences in regulated expression.

Western blot analysis

Cells transiently transfected 48 hours earlier with the p53 expression vector, or treated for six hours with 10 µM camptothecin, were washed twice with PBS and cellular proteins extracted by lysis with a buffer (150 mM NaCl, 50 mM Tris pH8, 2 mM EDTA, 1% Triton, 0.1% SDS) containing the protease inhibitors PMSF (1 mM) and leupeptin (10 µg/ml). The protein content of cells was determined by using the BCA assay (Pierce. Illinois), following the manufacturer's instructions. Equal amounts, 40 µg, of cell extracts were then electrophoresed in an 8% SDSpolyacrylamide gel and transferred to an immobilon polyvinylidine difluoride membrane. Non specific binding was blocked with 5% nonfat dried milk in TBS-T (Tris buffered saline, 0.1% Tween 20) for 2-3 h at room temperature and the cellular APP detected with a 1/1,500 dilution of the rabbit polyclonal antibody 369A, raised against the carboxi-terminal domain of APP. After 1 hour incubation at room temperature, the membrane was washed and incubated with a second biotinylated anti-rabbit antibody (1/2,000) for one additional hour, washed again and finally incubated for 1 hour with 1/2,000 peroxidase-conjugated streptavidine. All incubations took place at room temperature, and detection by enhanced chemiluminiscence (ECL, Amersham International plc. England) was carried out according to manufacturer's indications. The loading control β-catenin was detected with a 1/1500 dilution of a specific antibody obtained from BD Biosciences. Phopho-p53 was detected by using a 1/1000 dilution of the corresponding antibody and a 1/5000 dilutions of a second biotynilated anti-rabbit antibody.

The apparent molecular mass (kDa) of the detected bands was always determined by using a wide range protein standard (Mark 12 from Novex.-S. Diego. CA).

Quantitative real-time PCR assays.

Total RNA was extracted using Tri Reagent (Sigma) and mRNA levels analyzed by quantitative RT-PCR. RT was performed with 2 µg of RNA following A c c e p t e d M a n u s c r i p t specifications of SuperScriptTM First-Strand Synthesis System (Invitrogen Life Technologies). PCRs reactions were performed using a MX3005P instrument (Stratagene) and detected with Sybr Green. Data analysis was done using the comparative CT method and data were corrected with the GAPDH mRNA levels, which were not affected by any of the treatments used. The following PCR primers were used: APP, 5'-GGCCCTCGAGAATTACATCA-3', and 5'-GTTCATGCGCTCGTAGATCA-3'); GAPDH, 5'-ACAGTCCATGCCATCACTGCC-3', and 5'-GCCTGCTTCACCACCTTCTTG-3').

Small Interfering RNA (siRNA). Mdm2 (mouse double minute 2) expression was lowered using siRNA targeting mouse MDM2 mRNA. MDM2 siRNA and a control non-targeting siRNA (L-041098-00 and D-001206-13, respectively) were purchased from Dharmacon (Lafayette, CO). Cells were seeded in six well plates and the siRNAs were transfected into cells using Lipofectamine 2000. RNA was extracted after 72 hours and APP-mRNA levels were then analyzed by using quantitative RT-PCR.

Electrophoretic mobility shift assays

Synthetic oligonucleotides containing the +55 to +101 sequence of the human APP promoter, or a consensus p53 binding site [START_REF] Peterson | p53-mediated repression of DNA methyltransferase 1 expression by specific DNA binding[END_REF] were end-labeled with [ 32 P]ATP using T4-polynucleotide kinase and then incubated with either nuclear extracts obtained from p53-transfected cells or with in vitro translated p53 and/or recombinant Sp1 proteins. Nuclear extracts, obtained by the method of Andrews and Faller [START_REF] Andrews | A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells[END_REF], or p53 and Sp1, were incubated on ice for 15 min in a buffer (20 mM Tris HCl pH=7.5, 75 mM KCl, 1mM DTT, 5 µg/ml BSA, 13% glycerol) containing 3 µg poly (dI-dC), and then for 15-20 min at room temperature with approximately 70,000 cpm of the double-stranded labeled oligonucleotide.

For competition experiments, a 25 or 50 excess of unlabeled probe, the oligonucleotide containing the consensus sequence for p53 binding or an unrelated DNA were added together with the nuclear extracts to the binding reaction mixture.

For gel retardation assays with p53 and Sp1, cDNA for p53 was transcribed and translated in vitro with the TNT kit (Promega) following the manufacturer´s recommendations, and the Sp1 recombinant protein obtained from Promega (USA). In these assays an unprogrammed reticulocyte lysate was used as a control for nonspecific binding.

DNA-protein complexes were resolved on 7% nondenaturating polyacrylamide gels containing 0,5% TBE buffer, and the gels were dried and autoradiographed at -70ºC.

Chromatin immunoprecipitation (ChIP) and PCR amplification

For ChIP assays, cells were fixed with formaldehyde lysed and subsequently sonicated. Twenty 30 seconds bursts of sonication were applied with 30 seconds pauses to avoid overheating. Chromatin was precipitated with 2 µg of antibodies against Sp1 (Santa Cruz Biotechnology, Inc. USA), p53 (Cell Signaling Technology), or a control IgG, purified and subjected to PCR analysis. Primers used to detect binding to the -99 to +213 region of the APP gene were as follows:

Statistics

Unless indicated otherwise, all the data points are the mean of two independent experiments performed in duplicate. Significance of the differences was calculated with the Student's test, and is indicated in the figures with an asterisk: * (p < 0.05)

RESULTS

Effects of p53 on cell-associated APP isoforms

Cells were transiently transfected with an expression vector for p53, or with a control noncoding vector. Forty eight hours later, the cell-associated APP isoforms were detected by Western blot using the polyclonal antibody 369A. Previous to that, we first confirmed that transfection with the expression vector for p53 is actually followed by an increase in the activation of this transcription factor. As shown in figure 1A, the levels of phosphorylated-p53 (P-p53) were significantly higher in lysates obtained from p53-transfected cells. In addition, transient expression of p53 induces a reproducible decrease in the levels of intracellular APP. As previously reported [START_REF] Belandia | Thyroid hormone negatively regulates the transcriptional activity of the beta-amyloid precursor protein gene[END_REF][START_REF] Buxbaum | Processing of Alzheimer beta/A4 amyloid precursor protein: modulation by agents that regulate protein phosphorylation[END_REF][START_REF] Buxbaum | Cholinergic agonists and interleukin 1 regulate processing and secretion of the Alzheimer beta/A4 amyloid protein precursor[END_REF][START_REF] Weidemann | Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein[END_REF], the various bands observed in the western blots represent the mature and immature forms of the three major APP isoforms, APP 770 , APP 751 and APP 695 , which result by alternative splicing from the APP gene. The faster migrating bands representing immature APP and the slower migrating the mature isoforms. A densitometric quantification of the total bands is illustrated in the panel B of the figure and clearly evidenced that p53 significantly reduces the total level of APP isoforms.

Depletion of MDM2 decreases APP gene expression

The inhibitory effect of the tumor suppressor p53 on APP gene expression was also analyzed in cells depleted of MDM2, the major negative regulator of p53 in cells. MDM2 acts as an E3 ligase to ubiquitinate p53 and drive its proteasomal degradation. Thus, knock-down of MDM2 should be followed by a significant increase of cellular p53, and therefore by a reduced expression of APP in the cells. To prove this, cells were depleted of MDM2 by means of siRNA. Seventytwo hours after transfection, RNA was extracted and MDM2-and APP-mRNA levels were analyzed by using quantitative RT-PCR. Results are shown in figure 2. As expected, MDM2-mRNA levels were drastically reduced (panel A), p53 was accumulated in the cells (panel B), and APP-mRNA levels were significantly reduced (panel C) in cells exposed to 30 nM MDM2-siRNA, thus confirming the inhibitory effect of p53 on APP expression.

Activation of p53 by camptothecin reduces APP expression

To further confirm that p53 activation reduces APP expression, we also analyzed the influence of the topoisomerase-1 inhibitor camptothecin, a DNA-damaging agent that induces a strong accumulation and activation of p53 [START_REF] El-Deiry | Definition of a consensus binding site for p53[END_REF][START_REF] De La Monte | Correlates of p53-and Fas (CD95)-mediated apoptosis in Alzheimer's disease[END_REF] A c c e p t e d M a n u s c r i p t levels. Cells were incubated for 6 hours in the presence or in the absence of 10 µM camptothecin, and the levels of activated (phosphorylated)-p53, APP and βcatenin (as a loading control) were determined by Western blot. The results obtained are illustrated in figure 3. As expected, camptothecin induced a strong activation of endogenous p53 that was accompanied by a marked decrease in the levels of intracellular APP, an effect that was even more evident than that induced by p53 transfection (figure 1). Additionally, the effect of camptothecin on APP mRNA levels was also analyzed by quantitative real time PCR. APP mRNA was significantly reduced in camptothecin treated cells, thus confirming that activation of p53 indeed leads to a reduced expression of the APP gene. Moreover, to confirm whether or not these results obtained in N2a cells, a tumor cell line of murine origin, can be replicated in primary cultures and also in samples of human origin, we have analyzed the effect of camptothecin in primary cultures of rat cortical neurons, as well as in SH-SY5Y human neuroblastoma cells. As shown in figure 4, the results were in both cases very similar to those obtained in N2a cells, thus discarding a cell type specific response.

p53-mediated repression of APP promoter activity

Transient transfection assays were carried out to determine whether or not activation of p53 affects the transcriptional activity of the APP gene in neuroblastoma cells. N2aβ cells were transiently cotransfected with a chimeric plasmid containing the -1099 to +101 bp fragment of the APP gene linked to the CAT reporter gene, and an expression vector for p53, a dominant negative p53 mutant, or an empty noncoding vector. Cells were collected 48 hours later, and CAT activity was determined. As shown in figure 5, reporter activity was significantly inhibited in p53-transfected cells and increased in those cells transiently expressing the dominant negative p53 mutant, thus demonstrating that p53 indeed represses the transcriptional activity of the APP gene.

Identification of the DNA region mediating the repression of APP promoter activity by p53

To map the DNA sequences involved in the p53-induced response, progressively deleted fragments (-1099, -487, -307, -15 and +55) of the APP promoter were linked to the upstream region of the CAT gene and transfected into N2aβ cells. As shown in panel A of figure 6,  the negative effect of p53 was maintained even in cells transfected with the shortest (+55/+101 bp) fragment, which only contains sequences located in the first exon of the gene. In addition, we also analyzed the effect of p53 in cells cotransfected with a -15 to +101 promoter fragment either wild-type or mutated in positions 81 to 84 (AACT instead of GGGC) or 86 to 90 (AACTC instead of GAGCA) which correspond to the proposed p53 responsive region. As shown in panel B of the figure the inhibitory effect of p53 was observed in cells expressing the wild-type fragment but not in cells transfected with the mutant constructs. All together these results suggest the existence of a p53 response element located downstream of the transcription start site of the gene. To further confirm that sequences located within the +55 to +101 bp fragment mediate the effect of p53 on the APP promoter activity, the cells were transiently cotransfected with the +55 to +101 bp fragment of the APP gene and A c c e p t e d M a n u s c r i p t an expression vector for p53, a p53 dominant negative mutant, or an empty noncoding vector. As shown in figure 7, CAT activity determined 48 hours later was significantly inhibited in p53-transfected cells and increased in those cells transiently expressing the dominant negative mutant, thus demonstrating that this DNA fragment indeed mediates the repression of APP promoter activity by p53. Of interest, a computer-assisted study revealed that the +55/+101 responsive fragment that, as previously reported contains a Sp1-binding site [START_REF] Villa | A response unit in the first exon of the beta-amyloid precursor protein gene containing thyroid hormone receptor and Sp1 binding sites mediates negative regulation by 3,5,3'triiodothyronine[END_REF], also contains a sequence that largely resembles a half-site of the consensus core sequence established for the p53 binding site [START_REF] Peterson | p53-mediated repression of DNA methyltransferase 1 expression by specific DNA binding[END_REF][START_REF] Albihn | Camptothecininduced apoptosis is enhanced by Myc and involves PKCdelta signaling[END_REF].

p53 interferes with binding of Sp1 to the +55 to +101 bp fragment of the APP gene

Despite the results presented in the previous figures, this fragment was unable to bind p53 as proved by gel mobility shift assays performed with a radiolabeled probe containing the +55 to +101 responsive sequence of the APP gene and nuclear extracts obtained from p53-transfected cells or in vitro translated p53. Unexpectedly, no bands compatible with p53-containing mobility complexes were detected in any of the samples analyzed. As shown in figure 8A, the retarded bands obtained in a representative assay carried out with nuclear extracts were specifically competed by an excess of unlabelled probe. However, they were not displaced by competition with an unrelated oligonucleotide or in the presence of an oligonucleotide containing the consensus p53 binding site 5'-AGCTTGAACATGTCCCAACATGGTGA-3' [START_REF] Peterson | p53-mediated repression of DNA methyltransferase 1 expression by specific DNA binding[END_REF][START_REF] Albihn | Camptothecininduced apoptosis is enhanced by Myc and involves PKCdelta signaling[END_REF]. Furthermore, as illustrated in figure 8B, p53 binds very efficiently to the oligonucleotide containing the consensus p53 binding site. However, no specific bands compatible with p53containing complexes were detected when the radiolabeled +55/+101 probe was exposed to in vitro translated p53, whereas as expected [START_REF] Villa | A response unit in the first exon of the beta-amyloid precursor protein gene containing thyroid hormone receptor and Sp1 binding sites mediates negative regulation by 3,5,3'triiodothyronine[END_REF] a specific retarded band running in a position compatible with a Sp1-containing complex was detected in assays performed with recombinant Sp1. Remarkably, although p53 does not bind to this fragment, it was very effective decreasing the binding of Sp1 to this region of the APP gene. The intensity of that band was clearly reduced in the presence of p53, thus suggesting a mechanism in which p53 decreases the accessibility of Sp1 to DNA, and consequently the constitutive stimulation of the APP promoter induced by this factor. In addition, it should be mentioned that the mobility of the Sp1-associated band was not altered in the presence of p53, thus discarding "in vitro" binding of a p53-Sp1 complex to the Sp1-binding site.

The effect of p53 on "in vivo" binding of Sp1 to the APP gene was analyzed in ChIP assays performed in cells treated with camptothecin to activate endogenous p53. As shown in figure 8C, binding of p53, which was essentially undetectable in control cells, was increased in camptothecin-treated cells. In addition, binding of Sp1 to the APP responsive region was markedly decreased after camptothecin-induced p53 activation, confirming the reduction of Sp1 binding observed in the in vitro assays. In this report we present evidence that activation of the tumor suppressor p53 decreases the intracellular APP levels in a murine neuroblastoma cell line. Transient expression of p53 results in a reliable increase in the cell content of total and phosphorylated p53 that in turn generates a decrease in the intracellular levels of APP isoforms. This effect of p53 on APP was confirmed in cells depleted of MDM2, a negative regulator of p53, and also in cells exposed to the DNA-damaging agent camptothecin, which activates the endogenous p53. Furthermore, the inhibitory effect of p53 activation on APP expression has been proved to occur not only in murine N2aβ cells, but also in neuronal primary cultures and in neuroblastoma cells of human origin, thus discarding a potential cell type specific response. Our results together with the increase of p53 levels observed in Alzheimer brain [START_REF] Kitamura | Changes of p53 in the brains of patients with Alzheimer's disease[END_REF][START_REF] De Sarno | Muscarinic receptor activation protects cells from apoptotic effects of DNA damage, oxidative stress, and mitochondrial inhibition[END_REF] suggest a role of this protein in Alzheimer's disease.

DISCUSSION

The reduction of intracellular levels that follows the p53 activation could be secondary to a p53-mediated reduction of the transcriptional activity of the APP gene, and this possibility has been actually proved in transient transfection assays performed with a CAT reporter plasmid containing the -1099/+101 fragment of the human APP gene. Transient expression of p53 significantly reduces CAT activity, while expression of a dominant negative mutant of p53 increases activity above that observed in control cells.

Additional assays carried out with pBL-CAT plasmids containing progressive deletions of the APP promoter evidenced a mechanism of p53 repression mediated by DNA sequences located downstream of the transcription initiation site, in the +55/+101 region of the APP gene. Surprisingly, this region was unable to bind p53 suggesting that, as reported by other repressed promoters lacking a p53 consensus binding site [START_REF] Yap | Modulation of the transcriptional activity of thyroid hormone receptors by the tumor suppressor p53[END_REF], the repressive effect of this protein could be mediated by a functional interaction with other specific DNAbinding factor/s that stimulate the promoter through those sequences. At this respect, we have previously reported that the basal transcription factor Sp1 can stimulate APP promoter activity by binding to sequences located in the region of the gene responsible for the effect of p53 [START_REF] Villa | A response unit in the first exon of the beta-amyloid precursor protein gene containing thyroid hormone receptor and Sp1 binding sites mediates negative regulation by 3,5,3'triiodothyronine[END_REF], and the existence of a physical interaction between p53 and Sp1 has been also demonstrated [START_REF] Esteve | Molecular mechanisms of transactivation and doxorubicin-mediated repression of survivin gene in cancer cells[END_REF][START_REF] Koutsodontis | Physical and functional interactions between members of the tumour suppressor p53 and the Sp families of transcription factors: importance for the regulation of genes involved in cell-cycle arrest and apoptosis[END_REF]. The tumor suppressor p53 appears to cooperate with Sp1 in the activation of several promoters, such as p21, hTERT or IGF-IR [START_REF] Kanaya | Adenoviral expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription[END_REF][START_REF] Lagger | The tumor suppressor p53 and histone deacetylase 1 are antagonistic regulators of the cyclin-dependent kinase inhibitor p21/WAF1/CIP1 gene[END_REF][START_REF] Ohlsson | p53 regulates insulin-like growth factor-I (IGF-I) receptor expression and IGF-I-induced tyrosine phosphorylation in an osteosarcoma cell line: interaction between p53 and Sp1[END_REF]. However, it has not been yet clarified how that interaction may contribute to control promoter activity. As previously reported Sp1-p53 complexes could bind to Sp1-recognition sites in the promoters of different genes [START_REF] Torgeman | Sp1-p53 heterocomplex mediates activation of HTLV-I long terminal repeat by 12-O-tetradecanoylphorbol-13-acetate that is antagonized by protein kinase C[END_REF]. Nevertheless, it has been also reported that interaction of both transcription factors may result in a reduced ability of Sp1 to bind to DNA [START_REF] Bargonetti | p53 represses Sp1 DNA binding and HIV-LTR directed transcription[END_REF].

Our results demonstrate that although p53 does not bind directly to the +55/+101 region of the APP gene, indeed decreases the binding of Sp1 to that region of DNA, thus suggesting a mechanism involving a functional interaction between the tumor suppressor p53 and the basal transcription factor Sp1. Moreover, immunoprecipitation of fragmented chromatin with an anti-p53 antibody reflects the presence of this factor in the protein complex associated to the responsive APP region. Therefore, p53 may affect expression of APP by All together these results open a new and interesting link between a neurodegenerative pathology and the tumor suppressor p53. Data obtained from in vivo and in vitro models have previously demonstrated that p53 plays an important role in the neuronal death induced by the β-amyloid protein in Alzheimer' disease brains. It has been described that Aβ 1-42 activates the p53 promoter, thus inducing p53-dependent apoptosis and neurotoxicity [START_REF] Zhang | Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons[END_REF][START_REF] Ohyagi | Intracellular Abeta42 activates p53 promoter: a pathway to neurodegeneration in Alzheimer's disease[END_REF][START_REF] Laferla | Extracellular deposition of beta-amyloid upon p53-dependent neuronal cell death in transgenic mice[END_REF]. On the other hand, the β-amyloid precursor APP appears to play a dual function in this pathology since it can prevent neuronal apoptosis by inhibiting p53 activation [START_REF] Xu | Wild-type but not Alzheimer-mutant amyloid precursor protein confers resistance against p53-mediated apoptosis[END_REF], but it can also contribute to neurodegeneration if overexpressed [START_REF] Fukuchi | Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity[END_REF][START_REF] Yoshikawa | Degeneration in vitro of post-mitotic neurons overexpressing the Alzheimer amyloid protein precursor[END_REF]. In the present work we demonstrate that p53 activation represses APP expression and that therefore this tumor suppressor could also play two different and opposite functions in the neurodegenerative processes that characterize the Alzheimer's disease. According to our results, p53, a transcription factor that induces neuronal apoptosis could also play a positive role in preventing Alzheimer's disease, since it can reduce APP expression and therefore the production and accumulation of Aβ 1-42 , the most neurotoxic isoform of the βamyloid protein [START_REF] Zhang | Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons[END_REF].

In summary, our results suggest that in addition to the previously reported function of p53 in the Aβ-induced neurotoxicity, this protein could also reduce the Aβ 1-42  generation by decreasing APP gene expression. These functions very likely may depend on the cellular levels of p53, and it is evident that new experiments should be performed to fully understand the role of this protein in Alzheimer's disease and the precise mechanisms that mediate the repression of APP by p53. A c c e p t e d M a n u s c r i p t negative mutant or the empty non coding vector. Data, expressed as % of CAT activity obtained in control cells transfected with the -1099/+101 fragment, are the mean ± SD activities obtained from two independent assays performed in duplicate.

Fig. 6 Identification of DNA sequences that mediate the negative effect of p53 on the APP gene promoter. A) N2aβ cells were transiently transfected with pBL-CAT plasmids containing progressive deletions of the APP promoter. CAT activity was determined 48 hours after these plasmids were cotransfected with an expression vector encodind the tumor suppressor p53, or the empty noncoding vector. B) Cells were transiently transfected with a pBL-CAT plasmid containing the wild type -15/+101 fragment of the APP promoter, or two mutants in positions +81 to +84 (mut 1) or +86 to +90 (mut 2). Data are expressed relative to CAT activity obtained with the largest -1099/+101 (panel A), or the wild type -15/+101 (panel) fragments, and are the mean ± SD of CAT activities obtained from three separate experiments performed in duplicate. Fig. 7 The +55 to +101 bp fragment contains DNA sequences that mediate the effect of p53 on the APP promoter. Cell were transiently cotransfected with a pBL-tkCAT plasmid containing, or not, the +55 to +101 bp fragment of the APP gene promoter and expression vectors encoding wild type p53, a dominant negative p53 mutant or the empty non coding vector. CAT activity was determined 48 hours later. Data are the mean ± SD activities obtained from two independent assays performed in duplicate, and are expressed relative to the CAT activity obtained in control cells transfected with the +55/+101 fragment.

Fig. 8 Gel retardation analysis of p53 and Sp1 binding to the +55 to +101 region of the APP promoter.

Illustrated at the top of the figure are the nucleotide sequence of the +55 to +101 bp region, and the positions of the Sp1 binding site and a putative p53 half-site. A) Gel retardation assay performed with 10 µg of nuclear extracts in the absence of competitor (C) or in the presence of increasing amounts of a consensus p53 binding site, the +55/+101 oligonucleotide, or an unrelated oligonucleotide. B) At the left, a consensus p53 binding site was incubated with or without 2 µl of in vitro translated p53. At the right, the +55/+101 oligonucleotide was incubated with 2 µl of in vitro translated protein and 0,2 µl of recombinant Sp1 alone or in combination. The same amount (6 µl) of unprogrammed retyculocyte lysate was always present in the binding reaction. C) ChIP assay of the APP promoter in cells treated with or without camptothecin (CPT). Chromatin was immunoprecipitated with specific antibodies against p53 and Sp1, and analyzed by PCR. At the top is a scheme of the amplified fragment. The corresponding inputs as well as a negative control (IgG antibody) are also included in the figure. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793

Biochemical

  Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.

  , on APP 6 Biochemical Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793

7

  Biochemical Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793

8

  Biochemical Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t interacting with Sp1 and/or other unidentified factor, which would mediate its recruitment to chromatin.

BiochemicalFig. 1

 1 Fig. 1 Western blot analysis of phosphorylated-p53 and cell-associated APP isoforms. Cell lysates obtained from N2a cells transfected with an expression vector encoding p53 (Trans), or with an empty noncoding vector (C), were analyzed for p53 activation (phosphorylation) and for cell-associated APP proteins. A) Representative results obtained in Western blots performed with the specific antibodies mentioned under Experimental Procedures. β-catenin was used as a loading control. B). Densitometric quantification of the intracellular APP bands. Data are the average ± SD from two separate experiments performed in duplicate and are expressed relative to the corresponding control values.

Fig. 2 Fig. 3 DNA

 23 Fig. 2 Depletion of MDM2 decreases APP gene expression. MDM2 mRNA (panel A), phosphorylated-p53 (panel B) and APP mRNA levels (panel C) were analyzed in control untransfected cells (C), and in cells transfected with a control siRNA (siC) or with siRNA for MDM2 (siMDM2). Data represent the mean ± SD from two independent experiments performed in duplicate. The inset in panel B shows a representative blot of phosphorylated p53 (P-p53)

Fig. 4

 4 Fig. 4 Activation of p53 inhibits APP expression in primary cultures of cortical neurons and in human neuroblastoma cells.p53 phosphorylation and cell-associated levels of APP were analyzed in protein extracts obtained from cells treated with or without 10 µM camptothecin (CPT). Representative blots obtained from primary neuronal cultures and SH-SY5Y cells are illustrated in the upper panel. The densitometric quantification of the intracellular APP bands shown in the lower panel represents the mean ± SD from two independent experiments performed in duplicate.

Fig. 5

 5 Fig. 5 Negative regulation of APP promoter activity by p53.CAT activity was determined in N2aβ cells transiently cotransfected with a pBL-CAT plasmid containing, or not, the -1099 to +101 bp fragment of the APP gene promoter and an expression vector encoding wild type p53, a p53 dominant

Biochemical

  Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p tLicenced copy. Copying is not permitted, except with prior permission and as allowed by law.

*

  Biochemical Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited Biochemical Journal Immediate Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited C Publication. Published on 03 Dec 2008 as manuscript BJ20081793 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081793 A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

© 2008 The Authors Journal compilation © 2008 Portland Press Limited

Acknowledgments

This study has been supported by grants SAF2003-01646 and SAF2006-05577 from the Spanish Comision Interministerial de Ciencia y Tecnologia. We thank Prof. Diaz-Guerra by the preparation of the primary culture of cortical neurons.


Sara Rodríguez-Acebes

Paloma De

La Cueva

Carlos Fernández-Hernando

Antonio J Ferruelo

Miguel A Lasunción

Robert B Rawson

Javier Martínez-Botas

Diego Gómez-Coronado

Paloma De La Cueva

Desmosterol can replace

Keywords: cell proliferation, sterol biosynthesis, cholest-5,22-trans-dien-3β-ol, 25-hydroxycholesterol, SREBP-targeted genes, LXR-targeted genes Abca1, ATP-binding cassette, subfamily A, member 1, Cypb, cyclophilin B, DHCR24, sterol-Δ 24 -reductase, ER, endoplasmic reticulum, Fas, fatty acid synthase, FBS, fetal bovine serum, Gapdh, glyceraldehyde-3-phosphate dehydrogenase, 25HC, 25-hydroxycholesterol

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

As a key molecule for maintaining cell membrane architecture and biophysical properties, cholesterol is essential for mammalian cell viability and function. Cell proliferation requires membrane synthesis and this, in turn, depends critically on the availability of cholesterol. We have demonstrated that cholesterol is required for the G2-to-M phase transition and completion of mitosis [1][2][3][4]. On the other hand, excess unesterified cholesterol is cytotoxic [5]. Therefore, cholesterol homeostasis is subjected to strict control, and cells have developed a complex regulatory system to maintain the cellular content of cholesterol within a narrow range [6]. Moreover, in order to preserve the biophysical properties of cell membranes, the metabolism of cholesterol is regulated in concert with that of fatty acids and acylglycerols. The sterol regulatory element-binging protein (SREBP) family of transcription factors is crucial for the control of lipid homeostasis [7, 8] and a functional SREBP pathway is required for cell survival and proliferation [8,9]. Cholesterol itself and certain cholesterol-derived oxysterols, such as 25-hydroxycholesterol (25HC), control this regulatory system.

In the present study we focussed on these two essential and tightly linked functions of cholesterol, namely, support of cell proliferation and regulation of the SREBP pathway.

The cholesterol required for cell proliferation is either obtained from plasma lipoproteins or synthesized intracellularly from acetyl-coenzyme A. 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-the target of the hypocholesterolemic statin drugs -catalyzes the conversion of HMG-CoA into mevalonate, the rate-limiting step in cholesterol biosynthesis. There is a stringent requirement for cholesterol to support optimal mammalian cell growth [3,[10][11][12]. Some sterols, such as certain cholesterol precursors and phytosterols, can support cell growth provided that a suboptimal amount of cholesterol is also available [3, 12-14], a feature that is reminiscent of the synergism between sterols first observed in lower eukaryotes [15,16]. This is interpreted as a dual role of sterols in cell growth: a bulk membrane requirement, which can be satisfied by different sterols with broad specificity, and a regulatory role that is highly specific and fulfilled by small amounts of the selected sterol, cholesterol in mammalian cells.

Rothblat et al reported that L-cell mouse fibroblasts, which are defective in cholesterol biosynthesis and accumulate desmosterol, are able to survive and proliferate indefinitely in lipoprotein-deficient serum [17]. This indicated that in L-cells cholesterol can be replaced by desmosterol. Desmosterol, the immediate unsaturated precursor, is converted to cholesterol through a reaction catalyzed by sterol-Δ 24 -reductase (DHCR24, also known as Seladin-1). Desmosterol only differs from cholesterol in the presence of a double bond in the side chain at C24. Nevertheless, it has been suggested that, aside from the structural stringency of sterols, the ability of cells to compensate through changes in the composition of membrane lipids is also important in order to adapt to growth without cholesterol [13]. Whether this adaptability is a peculiarity of certain cells, such as fibroblasts, or it is a common phenomenon is unknown.

The SREBP family consists of three members, designated SREBP-1a, SREBP-1c and SREBP-2, which are encoded by two genes, Srebf1 -that gives rise to SREBP-1a and SREBP-1cand Srebf2. The SREBPs are synthesized as precursor proteins that are embedded in the membrane of the endoplasmic reticulum (ER). In this location, SREBPs bind to the SREBP cleavage-activating protein (Scap), which serves as both a cholesterol sensor and an escort protein. When cells are depleted of cholesterol, Scap escorts SREBP from the ER to the Golgi apparatus via COPII-coated vesicles. In the Golgi apparatus, SREBPs are cleaved sequentially by the Site-1 and Site-2 proteases, releasing the NH 2 -terminal domain of the protein, which is the active transcription factor. This mature form of SREBPs enters the nucleus and binds to specific nucleotide sequences known as sterol regulatory elements (SRE) located in the promoters of target genes, thus activating their transcription. When the cholesterol content of the ER membrane rises, cholesterol binds to Scap and induces a conformational change that allows Scap to bind to Insig-1 or Insig-2, which are resident membrane proteins of the ER. This interaction results in the retention of Scap/SREBP complexes in the ER by preventing their incorporation into transport vesicles and subsequent SREBP cleavage in the Golgi apparatus [8,18]. Different sterols can induce a conformational change in Scap in cell membrane suspensions [19] and/or can inhibit SREBP processing in intact cells [20]. Certain cholesterol-derived oxysterols, such as 25HC, are potent inhibitors of SREBP processing. In this case, the inhibition is initiated by its binding to Insig but not to Scap [19,21], causing Insig to bind to Scap and thereby blocking the export of Scap/SREBP complexes from the ER [20,[START_REF] Sun | Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins[END_REF].

As master regulators of lipid homeostasis, SREBPs induce the transcription of many genes involved in both cholesterol and fatty acid metabolism. SREBP-1a and, especially, SREBP-1c preferentially activate genes for fatty acid biosynthesis and their incorporation into triglycerides and phospholipids, whereas SREBP-2 principally activates genes involved in cholesterol biosynthesis and uptake [START_REF] Horton | SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver[END_REF][START_REF] Horton | Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes[END_REF]. Moreover, SREBPs stimulate the expression of genes of the regulatory machinery, such as those encoding SREBP-1, SREBP-2 and Insig-1 [18, [START_REF] Horton | SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver[END_REF][START_REF] Horton | Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes[END_REF].

Recently, DHCR24 and its substrate, desmosterol, have generated great interest due to the down-regulation of DHCR24 in regions of the brain affected by Alzheimer's disease [START_REF] Greeve | The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer's disease-associated neurodegeneration and oxidative stress[END_REF]. By contrast with other cholesterol analogues, desmosterol is an abundant component of the membrane of certain mammalian cells such as spermatozoa [START_REF] Lin | Unique lipids of primate spermatozoa: desmosterol and docosahexaenoic acid[END_REF] and astrocytes [27], and high desmosterol levels have been detected in developing brain [START_REF] Fumagalli | The identification and significance of desmosterol in the developing human and animal brain[END_REF]. Mice with a targeted disruption of DHCR24 (Dhcr24 -/-) were viable but infertile, although they had no gross morphological defects [29]. This is in marked contrast to the multiple severe inborn defects appearing in human subjects with desmosterolosis, who carry a mutation in DHCR24 [30,[START_REF] Fitzpatrick | Clinical phenotype of desmosterolosis[END_REF]. It should be noted, however, that analysis of sterol content in embryos of Dhcr24 -/-mice revealed that the animals represent a low-cholesterol, high-desmosterol model rather than being cholesterol-free [29,[START_REF] Brown | Of cholesterol-free mice and men[END_REF]. Furthermore, there is no evidence that either of the two cases of desmosterolosis reported to date were cholesterol-free during development [30,[START_REF] Fitzpatrick | Clinical phenotype of desmosterolosis[END_REF]. Thus, a possible contribution of cholesterol to cell proliferation and other cellular processes in these situations can not be ruled out. J774 mouse monocyte/macrophages have been reported to be unable to convert desmosterol into cholesterol [START_REF] Tabas | The reactivity of desmosterol and other shellfish-and xanthomatosis-associated sterols in the macrophage sterol esterification reaction[END_REF]. Using this cell line, in the present study we addressed the capacity of desmosterol to replace cholesterol in sustaining cell proliferation and regulating the SREBP pathway.

MATERIALS AND METHODS

Materials

J774A.1 mouse monocyte/macrophages (ATCC TIB-67) and HL-60 human promyeloblasts (ATCC CCL-240) were obtained from the American Type Culture Collection. Fetal bovine serum (FBS), RPMI, antibiotics and L-glutamine were purchased from Gibco BRL. Lipoprotein deficient serum (LPDS) was obtained by ultracentrifugation of FBS at a density of 1.21 kg/L. DCCM-1 was purchased from Biological Industries. Lovastatin was from Merck, Sharp and Dohme. Cholesterol, mevalonate and 5-fluorodeoxyuridine were purchased from Sigma and desmosterol, cholest-5,22trans-dien-3β-ol and 25-hydroxycholesterol (25HC) were from Steraloids. Hydroxypropyl-βcyclodextrin (HPCD) and methyl-β-cyclodextrin (MCD) were obtained from Sigma.

[ 3 H-methyl]thymidine (5 Ci/mmol) and [2-14 C]acetate (53 mCi/mmol) were from Amersham Biosciences. Multiscreen-HV culture plates were purchased from Millipore. All other chemicals were of analytical grade.

Cell cultures

J774 and HL-60 cells were maintained in RPMI containing 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin and 10 µg/ml gentamicin at 37 ºC in a humidified atmosphere containing 5% CO 2 . J774 cells were adapted to grow in the cholesterol-free medium DCCM-1 supplemented with 2 mM L-glutamine and the above mentioned antibiotics. For this, the regular maintenance medium was gradually replaced by increasing the proportion of DCCM-1 every two days, until complete withdrawal of the maintenance medium was achieved on the eighth day. Thereafter, the cells were grown in DCCM-1 and passaged every three days.

Proliferation assays

DCCM-1 was replaced with fresh medium and the cells were plated in sterile 96-well Multiscreen-HV plates at 2 x 10 4 cells in a final volume of 225 µL. The medium was supplemented with lovastatin (final concentration, 0.5 µM) dissolved in dimethylsulfoxide (final concentration, 0.044%), pure sterols dissolved in ethanol (final concentration, 0.44%), and/or the corresponding vehicles, as indicated. At 71 h of incubation, the medium was supplemented with 10 mM 5fluorodeoxyuridine and 1 h later with 0.5 µCi [ 3 H-methyl]thymidine. Following a further 18 h of incubation the cells were processed for the measurement of radioactivity incorporated into DNA, as described previously [1]. Cells (2.5 x 10 5 /ml) were also incubated in 12-well plates in the indicated conditions for 72 h to determine cell number and viability by trypan blue dye exclusion using a hemocytometer.

Analysis of sterol biosynthesis and content by HPLC

To study sterol biosynthesis, 10 7 cells were incubated in 10 ml of the indicated medium supplemented with 40 µCi [2-14 C]acetate for 10 h (in RPMI containing 10% LPDS) or 72 h (in DCCM-1). Subsequently, cells were washed twice with PBS and lysed with 0.5 ml of 10% KOH. Protein concentration was determined in an aliquot as described above. The nonsaponifiable lipid fraction was isolated and analyzed by HPLC and radioactivity counting as described previously [11]. Briefly, ergosterol was added to the samples as an internal standard and the lipid fraction was extracted with chloroform/methanol (2:1, v/v). Lipids were subjected to saponification and the nonsaponifiable fraction was extracted and resuspended in hexane. Sterol separation was accomplished by reverse-phase HPLC with a Luna 5 μm C18 column (Phenomenex). The eluate was monitored simultaneously by UV absorption using a Beckman 168 variable-wavelength detector (Beckman Instruments) and online radioactivity detection using an LB-506 C-1 detector (Berthold). Peaks were identified by comparing their retention time and UV spectrum with those of pure sterol standards. Sterols were quantified by measurement of the area under the peak at 206 nm. The lowest limit of detection (LOD) for cholesterol was less than 60 ng/ml.

Analysis of Dhcr24 expression

Total RNA was extracted with TriReagent (Sigma). For Northern blot analysis, 80 μg of total RNA were subjected to electrophoresis in a 1.2% agarose gel containing 2.2 M formaldehyde, transferred to Hybond-N+ membranes (Amersham) and cross linked with UV light. The blots were then hybridized with Dhcr24 and Gapdh cDNA probes labelled with digoxygenin using the DIG High Prime DNA Labelling and Detection Starter Kit (Roche). The detection of the probes was carried out with an alkaline phosphatase-labelled antibody against digoxygenin and developed by chemiluminescence following the instructions given by the manufacturer. The cDNA probes were prepared by RT-PCR (see below) using primers no. 2 and 3 for Dhcr24 detection and primers no. 9 and 10 for Gapdh detection (Supplementary Table S1).

For RT-PCR analysis of Dhcr24, 2 µg of total RNA were reverse-transcribed with 200 U of M-MLV-RT enzyme (Promega) using random hexamers (Promega). Two µl of cDNA were then amplified by PCR with 2.5 U of TaqGold DNA Polymerase (Applied Biosystems) by using primers no. 1 and 2 (Supplementary Table S1). Gapdh was used as an internal control (primers no. 9 and 10, Supplementary Table S1). To amplify Dhcr24 cDNA fragments of increasing length, total RNA was reverse-transcribed as above, and cDNA was subjected to a set of PCRs in which a sense primer whose sequence was comprised in exon 1 of Dhcr24 mRNA (primer no. 3, Supplementary Table S1) was combined with different antisense primers whose sequences were located in exons 2 to 7 (primers no. 4-8, respectively; Supplementary Table S1). The PCR products were resolved in a 1% agarose gel and were photographed using a gel documentation system (Kodak 1D Software).

Analysis of SREBP processing

On day 0, cells were plated at 10 6 cells/well in 6-well plates. On day 1, they were washed with PBS and switched to medium containing 2.5% LPDS (J774 and L cells) or to fresh DCCM-1 (J774-D cells), both supplemented with 1% (HPCD, Sigma), 2.5 µM lovastatin and 0.1 mM mevalonate, and incubated for 1 h. Next, cells were washed twice with PBS and switched to the same medium but without HPCD in the absence or presence of 0.5 µg/mL 25HC dissolved in ethanol, 25 µg/mL cholesterol or desmosterol dissolved in ethanol or 11.6 µg/mL cholesterol or desmosterol complexed with MCD. The sterol-MCD complexes were prepared as described elsewhere [19]. After incubation for 5 h, cells were washed with PBS, harvested and processed for western blot analysis and RNA isolation.

Western blot analysis

For LDL receptor detection cells were lysed in 20 mM Tris-HCl buffer, pH 8, containing 120 mM KCl, 1 mM DTT, 1 mM Na 2 -EDTA, 2 mM EGTA, 0,1 % Triton X-100, 0,5 % Nonidet P40, 1 mM benzamidine, 10 µg/mL antipain, 1 µg/mL leupeptin, 40 µg/mL aprotinin, 100 mM NaF, 20 mM sodium molibdate, 20 mM β-glycerophosphate, 2 mM sodium ortovanadate and 1 mM PMSF and subsequently sonicated. For SREBP processing analysis, the above mentioned lysis buffer was supplemented with 1 µg/mL of the caspase-3 inhibitor Ac-DMQD-CHO (Alexis), and cells were passed through a 22-gauge needle 10 times. For HMG-CoA reductase detection the lysis buffer was 1M PBS, pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 0.01% sodium azide, 5 mM Na 2 -EDTA and 0.1 M NaCl. After passing the cells through a 22-gauge needle 10 times, proteins were precipitated with trichloroacetic acid and resuspended in Laemmli sample buffer [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF]. The protein concentration of each cell lysate was measured by using the BCA kit (Pierce), and equal amounts of protein were subjected to 10% SDS-PAGE. The proteins were transferred to a nitrocellulose filter and probed with rabbit polyclonal antibodies against LDL receptor [START_REF] Russell | cDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA[END_REF], HMG-CoA reductase [START_REF] Sato | Replacement of serine-871 of hamster 3-hydroxy-3methylglutaryl-CoA reductase prevents phosphorylation by AMP-activated kinase and blocks inhibition of sterol synthesis induced by ATP depletion[END_REF], SREBP-1 [37], SREBP-2 [START_REF] Shimano | Elevated levels of SREBP-2 and cholesterol synthesis in livers of mice homozygous for a targeted disruption of the SREBP-1 gene[END_REF], or actin as the loading control (Amersham). Bound antibodies were visualized by chemiluminescence (ECL from Amersham or Inmun-Star HRP from Bio-Rad) and exposure to X-ray film (Hyperfilm, Amersham). Optical density of the bands was quantified by using the ImageQuant TL software (Amersham).

Quantitative real-time PCR

Total RNA was extracted with TriReagent (Sigma). Two µg of total RNA were reversetranscribed with 200 U of M-MVL reverse transcriptase enzyme (Promega) using oligo(dT) primers (Promega). The cDNA was then subjected to quantitative real-time PCR using the FastStart DNA Master SYBR Green I kit and LightCycler 2.0 equipment, and data were analyzed with the manufacturer's software version 4.05 (Roche Diagnostics GmbH). Analyses were performed in duplicate using the cyclophilin B gene (Cypb) as the invariant control. Primer sequences for the different genes analyzed are shown in Supplementary Table S1.

Statistical analyses

Statistical comparisons were performed by paired t-test using SigmaStat, version 2.3 (Jandel Corporation, San Rafael, CA). Statistical significance was set at P < 0.05.

RESULTS

It has been reported that J774 cells are unable to synthesize cholesterol and instead accumulate desmosterol, suggesting a deficiency of DHCR24 activity [START_REF] Tabas | The reactivity of desmosterol and other shellfish-and xanthomatosis-associated sterols in the macrophage sterol esterification reaction[END_REF]. This was confirmed in the J774 cells used in the present study by analyzing the incorporation of [ 14 C]acetate into sterols. As shown in Figure 1A, HL-60 cells, which were used as a reference, accumulated most of the radioactivity in cholesterol. By contrast, no incorporation of 14 C into cholesterol was detected in J774 cells; instead 14 C accumulation was observed in desmosterol (Figure 1A).

To explore the defect underlying the deficiency of DHCR24 activity, we first analyzed RNA extracts from J774 cells and mouse liver as control by Northern blot. As shown in Figure 1B, no Dhcr24 mRNA was detected in J774 cells. Then, the mRNA sequence corresponding to exons 8 and 9 of Dhcr24 was subjected to RT-PCR. Again, no cDNA was detected (Figure 1C). To further increase the sensitivity of the assay, quantitative real-time RT-PCR was performed to amplify different regions of Dhcr24, but no amplification product was obtained from J774 cells in any case (results not shown). Next, we tested for possible truncated mRNAs. Total RNA was reverse-transcribed and cDNA fragments of increasing length were amplified by combining a sense primer located in exon 1 of Dhcr24 mRNA with different antisense primers located in exons 2 to 7. In contrast to liver samples, only a faint band was detectable in J774 cells. This band comprised sequences up through exon 2. No detectable amplification product was obtained for any of the longer fragments studied (Figure 1D). These results demonstrate that J774 cells are defective in the production of a normal Dhcr24 transcript.

Next, we analyzed whether desmosterol is able to substitute for cholesterol for the survival and proliferation of J774 cells. Cholesterol was the predominant sterol in cells growing in 10% FBS. After 3 days growing in 10% LPDS, desmosterol became the major sterol (Figure 2A). Despite the inability of J774 cells to synthesize cholesterol, cholesterol levels were still approximately one-fifth of total sterol content after 10 days in 10% LPDS (Figure 2A). This observation may be explained by the low concentration of cholesterol usually present in 10% LPDS (approximately 0.2 µg/ml). To test whether biosynthetic desmosterol could completely replace cell cholesterol in viable J774 cells, the regular maintenance medium containing 10% FBS was replaced with a cholesterol-free medium (DCCM-1). As shown in Figure 2A, J774 cells have been growing in this medium, with passages every three days, for as long as 13 months, the time at which the culture was interrupted. We designated these J774-D cells. They were unable to express the normal Dhcr24 transcript (Figure 1D) or to synthesize cholesterol. Instead, they accumulated biosynthetic desmosterol, as measured by incubation with [2-14 C]acetate for 72 h (Figure 2B). Consistently, cellular cholesterol was undetectable (less than 0.017 % of total sterols in J774 cells) from 10 days after the complete withdrawal of serum; desmosterol was the only quantifiable sterol (Figures 2A and2C). Replacement of the regular maintenance medium was performed on multiple occasions and in each case the cells successfully adapted to growth in cholesterol-free medium, indicating that this was not the result of selecting a specific cell mutant. On average, cellular levels of desmosterol in cells growing in DCCM-1 were similar to the sum of cholesterol plus desmosterol levels in cells growing in 10% FBS (Figure 2D). Therefore, J774 cells were able to grow indefinitely after the virtually complete replacement of cholesterol by desmosterol. These cells were used for subsequent studies at least three weeks after complete withdrawal of FBS.

To assess the effect of the addition of cholesterol or desmosterol on the proliferation of J774-D cells, incorporation of [ 3 H-methyl]thymidine was analyzed in DCCM-1 medium. The addition of either cholesterol or desmosterol up to a final concentration of 10 µg/ml slightly increased [ 3 H-methyl]thymidine incorporation in the control condition (Figure 3A). Treatment with 0.5 µM lovastatin completely abolished cell proliferation (Figure 3A). However, a dose of 2.5 µg/ml of either cholesterol or desmosterol was sufficient to completely overcome the inhibitory effect of the drug (Figure 3A). Similar results were obtained by counting the number of viable cells (Figure 3B), indicating that the [ 3 H-methyl]thymidine incorporation assay reflected actual effects on cell proliferation. These findings indicate that desmosterol can sustain J774 cell proliferation in the absence of cholesterol.

Cholest-5,22-trans-dien-3β-ol, a structural analog of desmosterol that is not converted into cholesterol [START_REF] Koroly | Synthesis of delta 5,22-cholestadien-3 beta-ol from delta 5,7,22-cholestatrien-3 beta-ol by a liver enzyme[END_REF], has also been shown to support the growth of S 2 cells, a sterol auxotrophic cell line derived from L cells and which fails to demethylate lanosterol in the course of cholesterol biosynthesis [13]. Given the ability of J774-D cells to proliferate with only desmosterol, we tested the effect of cholest-5,22-trans-dien-3β-ol on proliferation of J774-D cells. As shown in Figure 3, this sterol partially prevented the inhibitory effect of lovastatin, but it was unable to restore the proliferation rate to control values. Thus, cholest-5,22-trans-dien-3β-ol was not equivalent to its analogue desmosterol in supporting J774-D cell proliferation.

A functional SREBP pathway is required for cell survival and proliferation [9]. The SREBP family of transcription factors regulates the expression of many genes involved in lipid homeostasis in response to changes in cellular cholesterol content. Thus, we next analyzed the ability of desmosterol, cholesterol and 25HC to inhibit the SREBP pathway in J774-D cells. For this, we tested the shortterm effect of these sterols on SREBP-1 and -2 processing and on SREBP-targeted gene expression when added to sterol-deprived J774-D cells. As shown in Figure 4A, 25HC markedly decreased the ratio between the mature (M) and the precursor (P) forms of both SREBP-1 and -2, indicating an inhibition of their processing as compared with the controls. Accordingly, the mRNA levels of HMG-CoA reductase (Hmgcr), the LDL receptor (Ldlr) and Insig1 were lower in 25HC-treated cells than in controls (Figure 4B). Such effects of 25HC on Hmgcr and Ldlr mRNAs were paralleled by reductions of HMG-CoA reductase and LDL receptor protein levels (Figure 4C). Cholesterol and desmosterol were added to the cells dissolved in ethanol, as was 25HC, or complexed with MCD to increase their solubility. Cholesterol, either dissolved in ethanol or complexed with MCD, inhibited SREBP-1 and SREBP-2 processing (Figure 4A) and decreased Hmgcr, Ldlr and Insig1 mRNA levels (Figure 4B), although less potently than 25HC. Cholesterol also caused a reduction of HMG-CoA reductase and LDL receptor protein levels, although the effect on the former protein was null when cholesterol was added in ethanol (Figure 4C). Desmosterol, added in any of the two vehicles, suppressed SREBP-1 and SREBP-2 processing and the expression of the above mentioned genes and proteins at least as efficiently as cholesterol did (Figure 4).

The inhibition of SREBP processing and targeted gene expression by desmosterol was also observed in sterol-deprived parental J774 cells (Supplementary Figure S1 responses of J774-D cells to desmosterol were not a consequence of adapting the cells to growth in the cholesterol-free medium. We then questioned whether desmosterol addition would be able to regulate the SREBP pathway in L-cell mouse fibroblasts, another cell model defective in DHCR24 activity [17]. Similarly to J774-D and parental J774 cells, in sterol-deprived L cells, desmosterol was at least as efficient as cholesterol suppressing SREBP-1 and SREBP-2 processing as well as Hmgcr, Ldlr and Insig1 expression (Supplementary Figure S2). Together, these findings demonstrate that desmosterol inhibits SREBP processing and the expression of target genes, and that cholesterol-free J774 cells retain the ability to respond to sterols through the suppression of the SREBP pathway.

A noticeable finding arising from the analysis of SREBP processing in J774-D (Figure 4A), parental J774 (Supplementary Figure S1A) and L cells (Supplementary Figure S2A) is that the absolute amount of the precursor form of SREBP-1 (P-SREBP-1) was markedly increased by the addition of 25HC, cholesterol or desmosterol as compared with control cells, whereas the absolute amount of P-SREBP-2 was not changed appreciably by sterol addition. Such an accumulation of P-SREBP-1 may be a direct consequence of the sterol-induced inhibition of its cleavage. However, it may also result from an increased expression of the SREBP-1c isoform through the activation of the liver X receptor (LXR), which is known to stimulate Srebf1c transcription [40]. We then analyzed the effect of those sterols on the levels of Srebf1c mRNA and, for comparison, those of Srebf2 mRNA. As shown in Figure 5, when desmosterol was delivered complexed with MCD it significantly increased Srebf1c mRNA levels as compared with control cells, whereas no effect was produced by other additions. Neither addition influenced Srebf2 mRNA levels (Figure 5). The expression of another LXR-targeted gene, Abca1, was also analyzed. It was found that 25HC and desmosterol, when added either dissolved in ethanol or complexed with MCD, increased Abca1 mRNA levels (Figure 5). Therefore, desmosterol can stimulate the expression of LXR-targeted genes in J774-D cells.

The fact that desmosterol increased Srebf1c expression while inhibiting SREBP-1 processing led us to ask what would be the net effect on the expression of genes required for fatty acid biosynthesis, which are characteristic SREBP-1c targets [START_REF] Horton | SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver[END_REF]. Thus, we addressed the effect of desmosterol addition on the expression of fatty acid synthase (Fas) and stearoyl-CoA desaturase-1 (Scd1) in J774-D cells. As shown in Figure 6, desmosterol, 25HC and cholesterol decreased Fas mRNA levels. By contrast, the different sterols did not decrease Scd1 mRNA levels, but, instead, desmosterol and cholesterol significantly enhanced it when added complexed with MCD (Figure 6).

Finally, we tested the ability of endogenously produced desmosterol to regulate the expression of SREBP and LXR gene targets. For this, J774-D and HL-60 cells, as a control, were treated with 0.25 µM lovastatin for 24 h, which was then removed to promote a burst of de novo sterol biosynthesis [START_REF] Wong | Endogenous 24(S),25epoxycholesterol fine-tunes acute control of cellular cholesterol homeostasis[END_REF]. The expression of Hmgcr, Insig1 and Abca1 was studied up to 24 h after the removal of the drug. The results are shown in Figure 7. In J774-D cells, lovastatin increased the mRNA levels of Hmgcr and Insig1, and both returned to control levels by 8 h after lovastatin removal. As regards Abca1, lovastatin decreased its mRNA levels, which increased up to control levels after lovastatin removal. Qualitatively similar results were obtained in HL-60 cells for HMGCR, INSIG1 and ABCA1, respectively (Figure 7). These findings suggest that endogenously produced desmosterol is able to regulate the expression of SREBP and LXR gene targets in a similar manner to that of endogenously produced cholesterol.

DISCUSSION

J774 cells are unable to synthesize cholesterol and instead accumulate desmosterol. Present results show that the lack of cholesterol biosynthesis in J774 cells results from their inability to produce a normal Dhcr24 transcript. This leads to the complete absence of enzyme, and is consistent with the virtually total replacement of cholesterol by desmosterol in J774-D cells when growing in a cholesterol-free medium. Using this cell model we addressed the capacity of desmosterol to substitute for cholesterol in two essential and tightly linked functions in mammalian cells, namely, the maintenance of cell growth and the control of regulatory responses involved in the maintenance of lipid homeostasis.

The ability of J774-D cells to grow indefinitely in a cholesterol-free medium demonstrates that desmosterol is sufficient to support cell survival and proliferation. Therefore, desmosterol can perform the dual role attributed to cholesterol in mammalian cell growth, namely, the formation of membranes, an ability shared by different sterols, and the highly specific regulatory function, which is fulfilled by small amounts of the species' selected sterol, cholesterol in mammalian cells [3,4,12,13]. This finding extends previous results obtained in L-cell mouse fibroblasts [13, 17] and indicates that the ability of desmosterol to substitute for cholesterol is not a peculiarity of fibroblasts. The fact that the addition of either desmosterol or cholesterol overcomes the inhibitory effect of lovastatin on proliferation of J774-D cells further supports the suggestion that both sterols can be used interchangeably for cell growth in J774 cells.

As far as we know, desmosterol is the only sterol proven to be able to completely substitute for cholesterol in mammalian cells for an indefinite period of time. Although our observations in J774 cells and previous findings with L cells are consistent with the obvious proliferation that takes place in the Dhcr24 -/-mice and in human subjects with desmosterolosis, the Dhcr24 -/-mice actually represents a low-cholesterol, high-desmosterol model [29,[START_REF] Brown | Of cholesterol-free mice and men[END_REF], and there is no evidence that subjects with desmosterolosis were cholesterol-free during development [30,[START_REF] Fitzpatrick | Clinical phenotype of desmosterolosis[END_REF]. It is conceivable that desmosterol could replace cholesterol for cell proliferation in vivo, but the severe abnormalities associated with desmosterolosis indicate that cholesterol has a unique role in embryogenesis. Another process in which cholesterol and desmosterol appear not to be equivalent is in the lipid raft/caveolae function, since exchanging the bulk of cell cholesterol for desmosterol increases caveolin-1 phosphorylation [42] and impairs insulin signaling [43].

The ability of desmosterol to substitute for cholesterol in maintaining membrane integrity may not be surprising, since it differs from cholesterol only in the presence of a double bond at C24, and it otherwise satisfies the structural features required to support cell growth [10,11]. Interestingly, cholest-5,22-trans-dien-3β-ol, which only differs from desmosterol by the position of the double bond (C22 vs. C24) in the side chain, was not able to effectively support J774-D cell proliferation, thus demonstrating the specificity of this effect. The partial prevention of the inhibitory effect of lovastatin by cholest-5,22-trans-dien-3β-ol suggests that this compound has some capacity to cooperate with desmosterol in a process that is similar to the synergism between sterols described previously [3, 12-14]. The discrepancy between the effects of desmosterol and cholest-5,22-trans-dien-3β-ol on cell proliferation may reflect a distinct requirement for accompanying membrane lipids imposed by the distance of the side chain double bond to the steroid nucleus. Our findings contrast with those obtained by Rujanavech and Silbert in S 2 cells, an L cell-derived sterol auxotrophic cell line, which grow normally on either desmosterol or cholest-5,22-trans-dien-3β-ol [13]. The reason why J774-D cells discriminate between these two sterols whereas S 2 cells do not is unknown, but it may be due to a differing ability to undergo accompanying compensatory changes in membrane lipids.

In response to changes in cholesterol levels, SREBP-2 controls the cholesterol biosynthesis pathway and lipoprotein uptake, whereas SREBP-1, and especially the SREBP-1c isoform, regulates the biosynthesis of free fatty acids and acylglycerols, also required for membrane biosynthesis. It was predictable that J774-D cells would be able to control lipid homeostasis as to preserve cell viability and proliferation. We were, therefore, interested in studying the ability of desmosterol to substitute for cholesterol in the regulation of the SREBP pathway. We found that desmosterol is able to inhibit SREBP-1 and SREBP-2 processing, as observed both in J774 and L cells. Accordingly, in these cells the expression of Hmgcr and Ldlr, two preferential SREBP-2 target genes [START_REF] Horton | SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver[END_REF], was suppressed by desmosterol. Similarly, desmosterol reduced the expression of Insig1, whose expression can be stimulated by SREBP-1c [18]. Addition of desmosterol to J774-D cells also decreased Fas expression, which is regulated principally by SREBP-1c [START_REF] Horton | SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver[END_REF]. Consistently with these studies, the treatment of J774-D cells with lovastatin produced an up-regulation of SREBP-targeted genes, whereas after lovastatin removal the expression of these genes returned to control levels. This suggests that, like cholesterol, endogenously produced desmosterol can regulate the SREBP pathway. However, contributions to these effects by other endogenously produced sterols, notably 24(S),25epoxycholesterol, cannot be excluded [START_REF] Wong | Endogenous 24(S),25epoxycholesterol fine-tunes acute control of cellular cholesterol homeostasis[END_REF]. Such effects of desmosterol are in keeping with the finding that desmosterol, like cholesterol, can induce a conformational change in Scap when assayed in cell membrane suspensions [19]. This conformational change causes Scap to bind to Insig, thereby retaining the Scap/SREBP complex in the ER and precluding SREBP processing [8,18]. Consistent with this, desmosterol treatment of CHO cells has recently been shown to reduce SREBP-2 processing [20, 44], although the possibility existed that its conversion into cholesterol mediated such an effect.

Our findings unambiguously show that desmosterol inhibits SREBP processing in intact cells that cannot convert it to cholesterol at least as efficiently as does cholesterol added with the same vehicle.

Current results demonstrate for the first time that cells virtually free of cholesterol retain the capacity to respond to sterols such as desmosterol and cholesterol through the inhibition of the SREBP pathway. They also respond to 25HC, whose potent inhibitory effect is exerted by binding to Insig [20,[START_REF] Sun | Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Insig renders sorting signal in Scap inaccessible to COPII proteins[END_REF], but not to Scap [19,21]. Therefore, the replacement of cholesterol with desmosterol in cell membranes is compatible with the functioning of the SREBP pathway, sterols being able to suppress SREBP processing by binding to either one of the two sterol sensors, Scap or Insig.

Increased expression of both Abca1 and Srebf1c, two well known LXR targets, following addition of desmosterol to J774-D cells suggests activation of this nuclear receptor. Consistently, we also found that Abca1 is up-regulated in response to increased production of endogenous desmosterol after lovastatin pretreatment. However, the involvement of other sterols, such as 24(S),25epoxycholesterol, in the effect of lovastatin removal cannot be excluded [START_REF] Wong | Endogenous 24(S),25epoxycholesterol fine-tunes acute control of cellular cholesterol homeostasis[END_REF]. It has recently been demonstrated that desmosterol is an LXR ligand [START_REF] Yang | Sterol intermediates from cholesterol biosynthetic pathway as liver X receptor ligands[END_REF]. Moreover, all these observations are in agreement with the up-regulation of hepatic Srebf1c, Cyp7a1, Abcg5 and Abcg8, all of which are LXR targets, in the Dhcr24 -/-mouse [START_REF] Heverin | Studies on the cholesterol-free mouse: strong activation of LXR-regulated hepatic genes when replacing cholesterol with desmosterol[END_REF]. Surprisingly, hepatic Abca1 expression was not enhanced in this animal model, which may reflect a differential regulation of this gene by LXR in hepatocytes and monocyte/macrophages like J774 cells. Desmosterol-induced stimulation of Abca1 expression suggests that desmosterol itself might be released from cells through the ABCA1 transporter, thus contributing to the reported high efficiency of the efflux of biosynthetic desmosterol [START_REF] Aviram | High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages[END_REF]47].

Addition of 25HC also raised Abca1 expression, although, in contrast to desmosterol, it failed to affect Srebf1c expression. The latter result is consistent with the notion that 25HC is a weak activator of LXR [START_REF] Janowski | An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha[END_REF], but other mechanisms may be involved. Besides being stimulated by LXR, Srebf1c transcription is also up-regulated by SREBPs, including SREBP-1c [START_REF] Amemiya-Kudo | Promoter analysis of the mouse sterol regulatory element-binding protein-1c gene[END_REF][START_REF] Cagen | Insulin activates the rat sterol-regulatory-element-binding protein 1c (SREBP-1c) promoter through the combinatorial actions of SREBP, LXR, Sp-1 and NF-Y cis-acting elements[END_REF]. Therefore, the lack of effect of 25HC on Srebf1c expression may be the consequence of the counterbalance between two opposite mechanisms: the activation of the LXR pathway and the suppression of the SREBP pathway. Relative to 25HC and cholesterol, excess desmosterol appears to be more effective activating the LXR pathway, thus markedly increasing the expression of both Abca1 and Srebf1c.

Apart from influencing the expression of genes involved in cholesterol homeostasis, desmosterol altered the expression of genes for fatty acid biosynthesis, such as Fas and Scd1, as well as their upstream regulator Srebf1c. Similarly to cholesterol and 25HC, desmosterol had a differential effect on Fas and Scd1 expression. While lowering Fas mRNA levels, in correspondence to the sterolinduced inhibition of SREBP-1 processing, Scd1 mRNA levels were increased (desmosterol and cholesterol) or unchanged (25HC). A possible involvement of an LXR-mediated up regulation of Scd1 can not be ruled out [51]. Similarly to our findings, it has been observed previously that feeding mice a cholesterol-rich diet can produce different responses of hepatic expression of Fas and Scd1 [40]. Considered together, these results suggest a differential regulation of both genes by sterols, in such a way that the expression of Fas is more responsive than that of Scd1 to the sterol-induced inhibition of SREBP-1 cleavage.

Current results show that desmosterol can replace cholesterol in sustaining J774 cell proliferation. These cells have a functional SREBP pathway, which, by regulating lipid homeostasis, presumably contributes to cell viability and growth. Desmosterol, like cholesterol and 25HC, is able to suppress SREBP processing and targeted gene expression. On the other hand, in comparison with cholesterol and 25HC, desmosterol is especially effective activating LXR-targeted genes. Such effects of desmosterol on SREBP and LXR pathways may be relevant for lipid homeostasis in cells and tissues with high desmosterol content, such as spermatozoa [START_REF] Lin | Unique lipids of primate spermatozoa: desmosterol and docosahexaenoic acid[END_REF], astrocytes [27] and the developing brain [START_REF] Fumagalli | The identification and significance of desmosterol in the developing human and animal brain[END_REF].

This study was supported by grants from Fondo de Investigación Sanitaria (00/0229), Ministerio de Ciencia y Tecnología (BMC2002-01262) and Ministerio de Educación y Ciencia (SAF2005-07038). S.R.A. was a recipient of a fellowship "Beca de Formación en Investigación" from the Instituto de Salud Carlos III. J.M.B. is an investigator of FIBio-HRC, supported by the Comunidad Autónoma de Madrid. The "CIBER de Fisiopatología de la Obesidad y Nutrición" is an initiative of the Instituto de Salud Carlos III. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. S1) and antisense (no. 4-8 in Supplementary Table S1) primers (filled and empty arrowheads, respectively) used for the amplifications and the resulting cDNA fragments (horizontal bars). stand., standard; bp, base pairs. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  ), indicating that such Biochemical Journal Immediate Publication. Published on 05 Mar 2009 as manuscript BJ20081909 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081909 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Figure 1 .

 1 Figure 1. Sterol biosynthesis and Dhcr24 expression in J774 cells. (A) [ 14 C]Acetate incorporation into sterols in J774 and HL-60 cells. Cells were incubated in RPMI containing 10% LPDS supplemented with 40 μCi of [ 14 C]acetate for 10 h and the cell extracts containing the nonsaponifiable lipid fraction were analyzed by reverse-phase HPLC and in-line radioactivity counting. The arrows indicate the elution times of desmosterol (des), and cholesterol (chol). nd, not detected. (B) Northern blot analysis of RNA of J774 cells and mouse liver. Total RNA was subjected to electrophoresis and blot hybridization with digoxygenin-labelled cDNA probes for mouse Dhcr24 and mouse Gapdh. (C) RT-PCR analysis of RNA of J774 cells and mouse liver for detection of Dhcr24 and Gapdh. (D) RT-PCR analysis of RNA of J774 cells and mouse liver for detection of Dhcr24 cDNA fragments of increasing length and comprising regions expanding from exon 1 up to exon 7, as indicated. J774-D cells are J774 cells grown in DCCM-1 (see text). The upper panel indicates the position of the sense (no. 3 in Supplementary TableS1) and antisense (no. 4-8 in Supplementary TableS1) primers (filled and empty arrowheads, respectively) used for the amplifications and the resulting cDNA fragments (horizontal bars). stand., standard; bp, base pairs.

Figure 2 .

 2 Figure 2. Sterol content and biosynthesis in J774 cells adapted to grow in a cholesterol-free medium. (A) Cholesterol and desmosterol content of cells at different times of growth after the complete replacement of RPMI containing 10% FBS with either RPMI containing 10% LPDS or DCCM-1. (B and C) HPLC profiles of radioactivity (B) and absorbance at 206 nm (C) of sterols from cells at day 21 of growth in DCCM-1. DCCM-1 was supplemented with 40 μCi of [ 14 C]acetate for 3 days and the cell extracts containing the nonsaponifiable lipid fraction were analyzed by reverse-phase HPLC and in-line radioactivity counting. The arrows indicate the elution times of desmosterol (des), cholesterol (chol) and ergosterol (erg). d, days; m, months; is, internal standard; nd, not detected. (D) Average cholesterol and desmosterol levels in cells maintained in RPMI containing 10% FBS or at days 21 to 32 of growth in DCCM-1. Bars show the mean and whiskers indicate the SEM of data from seven separate cell cultures.

Figure 3 .

 3 Figure3. Effect of cholesterol, desmosterol and cholest-5,22-trans-dien-3β-ol on the proliferation of lovastatin-treated J774-D cells. (A) [ 3 H-methyl]thymidine incorporation into DNA of cells incubated in DCCM-1 in the absence or presence of 0.5 μM lovastatin alone or in combination with the indicated concentrations of cholesterol, desmosterol or cholest-5,22-trans-dien-3β-ol. Data correspond to the mean ± SEM of triplicates. (B) Cells were incubated in the presence or absence of 0.5 μM lovastatin alone or in combination with 2.5 μg/ml cholesterol, desmosterol or cholest-5,22trans-dien-3β-ol. At 72 h of incubation, the cells were harvested for counting of viable cells. Data correspond to the mean ± SEM of duplicates. Data in A and B correspond to a representative experiment out of five. chol, cholesterol, des, desmosterol; C-5,22, cholest-5,22-trans-dien-3β-ol.

Figure 4 .

 4 Figure 4. Effect of 25HC, cholesterol and desmosterol on the SREBP pathway in sterol deprived J774-D cells. Cells were incubated in DCCM-1 supplemented with 1% HPCD, 2.5 µM lovastatin and 100 µM mevalonate for 1 h. Then, cells were switched to the same medium but without HPCD and containing or not (control, C) 0.5 µg/mL 25HC dissolved in ethanol, 11.6 µg/mL cholesterol (CholMCD) or desmosterol (DesMCD) complexed with MCD or 25 µg/mL cholesterol (CholEt) or desmosterol (DesEt) dissolved in ethanol, and incubated for additional 5 h. (A) Western blot analysis of SREBP-1 and SREBP-2 processing. Fifty µg of protein were subjected to SDS-PAGE electrophoresis. The precursor and mature form of SREBPs are denoted P and M, respectively. The results correspond to a representative experiment out of three. Lower panels represent the ratio between the respective optical densities of M and P bands; the ratio obtained for the control condition has been arbitrarily defined as 1. Bars show the mean and whiskers indicate the SEM of data from three experiments. *, P<0.05; and **, P<0.01 vs. the control condition by paired t-test. (B) Hmgcr, Ldlr and Insig1 mRNA levels. The results are expressed as the relative amount of mRNA compared to the level in control condition without sterol. Bars show the mean and whiskers indicate the SEM of data from three experiments. *, P<0.05; and **, P<0.01 vs. the control condition by paired t-test. (C) Western blot analysis of HMG-CoA reductase and LDLR protein levels. Fifty µg of protein were

Figure 5 .

 5 Figure 5. Effect of 25HC, cholesterol and desmosterol on Srebf1c, Srebf2 and Abca1 mRNA levels in sterol deprived J774-D cells. Cells were treated as described in Figure 4. The results are expressed as the relative amount of mRNA compared to the level in control condition without sterol (C). Bars show the mean and whiskers indicate the SEM of data from three experiments. *, P<0.05; **, P<0.01; and ***, P<0.001 vs. control condition by paired t-test.

Figure 6 .

 6 Figure 6. Effect of 25HC, cholesterol and desmosterol on Fas and Scd1 mRNA levels in sterol deprived J774-D cells. Cells were treated as described in Figure 4. The results are expressed as the relative amount of mRNA compared to the level in control condition without sterol (C). Bars show the mean and whiskers indicate the SEM of data from three experiments. *, P<0.05; and **, P<0.01 vs. control condition by paired t-test.

Figure 7 .

 7 Figure 7. Effect of endogenously produced desmosterol on the expression of SREBP and LXR gene targets. J774-D and HL-60 cells (5x10 5 cells/ml) were preincubated in the absence (control, C) or presence of 0.25 µM lovastatin for 24 h. Media with lovastatin (lov) were then removed and the cells were washed with PBS and incubated in fresh media without lovastatin for additional 8 or 24 h. The levels of Hmgcr, Insig1 and Abca1 mRNA were measured. The results are expressed as the relative amount of mRNA compared to the level in control condition. Bars show the mean and whiskers indicate the SEM of data from four experiments. *, P<0.05; **, P<0.01 and ***, P<0.001 vs. control; and #, P<0.05; ##, P<0.01 and ###, P<0.001 vs. lovastatin pretreatment by paired t-test.

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  [START_REF] Greeve | The human DIMINUTO/DWARF1 homolog seladin-1 confers resistance to Alzheimer's disease-associated neurodegeneration and oxidative stress[END_REF] µM lovastatin for 24 h. Media with lovastatin (lov) were then removed and the cells were washed with PBS and incubated in fresh media without lovastatin for additional 8 or 24 h. The levels of Hmgcr, Insig1 and Abca1 mRNA were measured. The results are expressed as the relative amount of mRNA compared to the level in control condition.Bars show the mean and whiskers indicate the SEM of data from four experiments. *, P<0.05; **, P<0.01 and ***, P<0.001 vs. control; and #, P<0.05; ##, P<0.01 and ###, P<0.001 vs. lovastatin pretreatment by paired t-test.

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

We are grateful to Angela Murúa and Lorena Crespo for excellent technical support and to Dr. Iain Patten for editorial advice on a version of the manuscript.

FUNDING