Barbara Pavan

Alessandro Dalpiaz

Carla Biondi

Marzia Nieddu

Puttur Antonella De Luca

D Prasad

Guglielmo Paganetto

Bartolo Favaloro

Puttur D Prasad

RPE cell line as a useful in

Keywords: RARβ, HRPE cells, [ 3 H]CD367, senescence, telomere, culture passage

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

Retinoic acid (RA) and its natural derivatives (retinoids) are important vitamin A metabolites that regulate cellular growth and differentiation [START_REF] Swisshelm | Downregulation of retinoic acid receptor beta in mammary carcinoma cell lines and its upregulation in senescing normal mammary epithelial cells[END_REF]. Intracellularly, RA specifically interacts with two distinct types of proteins. In the cytoplasm, RA interacts with cytosolic retinoic acid binding proteins (CRABPs), which are primarily involved in the storage, intracellular transport, and metabolism of retinoic acid [START_REF] Si | Effect of cellular senescence and retinoic acid on the expression of cellular retinoic acid binding proteins in skin fibroblasts[END_REF]. In the nucleus, RA interacts with nuclear retinoic acid receptors, which are proteins that function as ligand-dependent transcription factors that are related to the steroid and thyroid hormone receptor family [START_REF] Gronemeyer | Principles for modulation of the nuclear receptor superfamily[END_REF]. Upon ligand binding, the receptors are activated and induce the transcription of target genes by binding to RA-responsive elements (RARE) in the promoter regions. There are two distinct classes of nuclear retinoic acid receptors: the Retinoic Acid Receptor (RAR or NR1A) and the Retinoid X Receptor (RXR or NR2B), [START_REF] Si | Effect of cellular senescence and retinoic acid on the expression of cellular retinoic acid binding proteins in skin fibroblasts[END_REF][START_REF] Greiner | Differential ligand-dependent protein-protein interactions between nuclear receptors and a neuronal-specific cofactor[END_REF]. Both RAR and RXR have three subtypes, α, β, and γ that are encoded by separate genes located at distinct chromosomal loci [START_REF] Chambon | A decade of molecular biology of retinoic acid receptors[END_REF]. The nuclear retinoic acid receptor subtypes are differentially expressed both spatially as well as temporally [START_REF] Chambon | A decade of molecular biology of retinoic acid receptors[END_REF][START_REF] Dollè | Differential expression of genes encoding alpha, beta, and gamma retinoic receptors and CRABP in the developing limbs of the mouse[END_REF][START_REF] Mangelsdorf | The nuclear receptor superfamily: the second decade[END_REF]. In humans, RARα is expressed in most tissues, RARβ mainly in neural tissues with limited expression in skin, RARγ is expressed predominantly in skin, RXRα, in liver, kidney, spleen, and skin, RXRβ in almost all tissues and RXRγ expression in mostly muscle and brain.

Among the nuclear retinoic acid receptors, the RARβ gene is of particular interest to cancer biologists because it regulates metabolic pathways associated with tumor-suppressive effects mediated by retinoids [START_REF] Geisen | High-level expression of the retinoic acid receptor beta gene in normal cells of the uterine cervix is regulated by the retinoic acid receptor alpha and is abnormally down-regulated in cervical carcinoma cells[END_REF][START_REF] Pavan | Nuclear retinoic acid receptor beta as a tool in chemoprevention trials[END_REF]. The RARβ gene contains a RARE and thus the expression of RARβ is regulated by RA. Though loss of expression of several of the nuclear retinoid receptors has been detected in various cancer cell lines, loss of RARβ is the most common in a variety of cancer, including that of head and neck, breast, lung, esophagus, pancreas, cervix and prostate, and its expression correlates inversely with cancer progression and tumor grade [START_REF] Xu | Tumor-suppressive activity of retinoic acid receptor-beta in cancer[END_REF]. Epigenetic modifications of the RARβ promoter has been linked to the loss of RARβ expression in cancer cells [START_REF] Pavan | Nuclear retinoic acid receptor beta as a tool in chemoprevention trials[END_REF]. Recent studies by Geisen et al [START_REF] Geisen | High-level expression of the retinoic acid receptor beta gene in normal cells of the uterine cervix is regulated by the retinoic acid receptor alpha and is abnormally down-regulated in cervical carcinoma cells[END_REF][START_REF] Geisen | Growth inhibition of cervical cancer cells by the human retinoic acid receptor beta gene[END_REF] have shown that RARβ may have a role to play in cellular senescence as well. They observed that both basal and RA-induced RARβ mRNA levels in normal cells tend to increase with senescence. Similar observation was made by Lee and cowokers in aging fibroblasts with decreased proliferative capacity, who observed a selective up-regulation of RARβ expression in response to RA and its derivatives in these cells [START_REF] Lee | Cellular aging and transformation suppression: a role for retinoic acid receptor beta[END_REF]. This suggests that genetic events important in regulating cellular senescence may also play a significant role in tumor suppression in humans. Indeed, RARβ expression is up-regulated with serial passage in senescing normal mammary epithelial cells while it is down-regulated in mammary cancer cell lines [START_REF] Swisshelm | Downregulation of retinoic acid receptor beta in mammary carcinoma cell lines and its upregulation in senescing normal mammary epithelial cells[END_REF]. RARβ, therefore, is of great interest as a potential target in the design of anti-tumoral drugs and to study the mechanisms of their effects [START_REF] Pavan | Nuclear retinoic acid receptor beta as a tool in chemoprevention trials[END_REF]. Availability of a standardized and sensitive method to characterize ligand binding specifically to RARβ would therefore be of great utility. However, the accuracy and reproducibility of different methods for studying binding affinities of specific compound to RARs can be compromised by several factors which can be summarized as (i) a low abundance of RARs in natural cells and tissues resulting in a low ratio of specific versus nonspecific binding sites; (ii) the chemical instability of the natural ligand, RA; and (iii) the difficulty of designing a perfect method to separate free from bound ligand [START_REF] Sablonnière | Physicochemical parameters affecting the charcoal adsorption assay for quantitative retinoid-binding measurement[END_REF].

We report here an effective method to perform RARβ-specific ligand binding studies using human retinal pigment epithelial (HRPE) cells which overcomes most of the drawbacks listed above. Since retinal pigment epithelial cells perform a number of physiological functions essential for vision, namely the uptake, processing, transport and release of vitamin A (retinol) and its visual cycle intermediates retinoids [START_REF] Janssen | Retinoic acid receptors and retinoic X receptors in the mature retina: Subtype determination and cellular distribution[END_REF], we first investigated the expression of retinoid receptors in HRPE cells. Results presented here show for the first time that HRPE cells natively and selectively express appreciable levels of only RARβ. We also provide evidence to show that RARβ expression in these cells increases with increasing passage of the cells. We have next standardized a RARβ 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.

receptor assay using nuclear extracts from late passage HRPE cells and [ 3 H]CD367, a stable synthetic ligand for the RAR subtypes [START_REF] Delescluse | Selective high affinity retinoic acid receptor a or b-g ligands[END_REF]. The efficacy of our method allowed us to distinguish between different levels of native RARβ expression in serially passaged of HRPE cells and characterize the affinity of several flavonoids to RARβ.

EXPERIMENTAL Materials

[ 3 H]CD367 (specific activity 42 Ci/mmol) and CD367 were kindly provided by GALDERMA (Sophia Antipolis, France). [ 3 H]9-cis-RA (specific activity 43.0 Ci/mmol) was obtained from Amersham Life Sciences (Milan, Italy). 9-cis-RA, genistein, quercetin, daidzein, equol, dithiothreitol (DTT), dextran, bovine serum albumin (BSA), diethyl-pyrocarbonate (DEPC) and the protease inhibitors phenylmethylsulfonyl fluoride, aprotinin and leupeptin were purchased from Sigma (Milan, Italy). Glycerol, thioglycerol, isopropyl alcohol and activated charcoal were obtained from Merck (Milan, Italy). All tissue culture media, fetal bovine serum and RT-PCR reagents were purchased from Invitrogen (Milan, Italy).

HRPE cell culture

The human retinal pigment epithelial (HRPE) cell line was routinely grown at 37 °C in an atmosphere of 5% CO 2 /95% air, in a mixture of Ham's F12 and Dulbecco's modified Eagle medium (F12/DMEM) (1:1 vol/vol) supplemented with 10% fetal bovine serum (10% FBS), 100 µg/ml streptomycin, and 10 IU/ml penicillin. The population doubling time was 24 hours. Cells were seeded every 4 to 5 days by trypsin-EDTA dispersion and grown to confluence. Early and late passage HRPE were defined here as those cultures receiving less than 5 trypsin passages and more than 35 trypsin passages, respectively.

Semi-quantitative RT-PCR

Total RNA was isolated from HRPE cells using Trizol reagent (1 ml reagent/10 6 cells) following the manufacturer's instructions. Total RNA was treated with DNase I (Invitrogen, Milan, Italy) and used in oligo(dT)-primed first strand cDNA synthesis (SuperScript II RT, Invitrogen, Milan, Italy). Primers and PCR conditions for the different RARs used are presented in Table 1. Relative amount of the mRNA of RAR was determined by semi-quantitative RT-PCR as described before [START_REF] Pavan | Circadian clocks regulate adenylyl cyclase activity rhythms in human RPE cells[END_REF]. The PCR products were resolved in 1.5% agarose gel containing ethidium bromide and the intensity of the cDNA bands relative to background was determined using Quantity One system (BioRad, Milan, Italy). The linear range of PCR was empirically determined in initial experiments where by PCR was set up with each primer set with different number of amplification cycles (15 to 40 cycles). The relationship between the intensity of the signal and the PCR cycle number was analyzed to determine the linear range for the PCR product formation. The intensities of the signals within the linear range were used for data analysis. The RARβ amplified linearly between cycles 28 to 34. When quantitative RT-PCR was performed, the housekeeping gene 18S rRNA (linear amplification between 18 to 22 cycles) was amplified simultaneously, its specific primers being added to the PCR reaction after the first 10 cycles and used for normalization of target gene values throughout experiments because its level did not vary significantly in relation to passage number [START_REF] Pavan | Circadian clocks regulate adenylyl cyclase activity rhythms in human RPE cells[END_REF]. Under the PCR conditions described above, the amount of PCR product obtained showed a linear correlation with the amount of cDNA added to the PCR mixture, i.e., ratios of RARβ mRNA to 18S mRNA remained almost constant. In each PCR reaction, -RT (to rule out amplification of genomic DNA) and DEPC-treated water instead of cDNA were used as negative controls.

Western blot analysis

Protein extracts were obtained from 80% confluent HRPE cells at early and late passages, using Passive Lysis Buffer (Promega). Crude extracts (40 μg) were analysed by SDS/PAGE on 12.5% (w/v) polyacrylamide gels. Proteins were transferred electrophoretically to nitrocellulose filter (for 120 min at 0.3 A) using an immunoblot transfer apparatus (Bio-Rad). After transfer, the filter was incubated for 180 min at room temperature in 10% (w/v) non fat milk in Tris-buffered saline (TBS; 500 mM NaCl and 20 mM Tris/HCl, pH 7.5), supplemented with 0.05% (v/v) Tween-20 to block non-specific binding. The blot was incubated overnight at 4°C with 10% non fat milk in TBS, supplemented with 0.05% (v/v) Tween-20, containing rabbit polyclonal anti-RARβ (Santa Cruz Biotechnology) at a dilution of 1:1000. After three washes with TBS containing 0.05% (v/v) Tween-20, the blot was incubated for 60 min at room temperature with peroxidase-conjugated goat anti-rabbit immunoglobulin (Calbiochem) diluted at 1:2000 in 10% non fat milk in TBS, supplemented with 0.05% (v/v) Tween-20. The blot was again washed three times with TBS containing 0.05% (v/v) Tween-20. Antibodies were visualized using a chemiluminescence detection system (Western Blotting Luminol Reagent, Santa Cruz Biotechnology).

Telomere length

Genomic DNA from HRPE cells was isolated using Wizard genomic DNA purification kit (Promega). Telomere length comparisons were done using the Telo TAGGG Telomere Length Assay kit (Roche Diagnostics, Mannheim, Germany). Genomic DNA (2 μg) was digested with RsaI and HinfI restriction enzymes, size-fractionated on a 0.8% w/v agarose gel, and transferred to nylon membranes in 20×SSC by capillary blotting. Controls were digoxigenin-(DIG) labelled DNA molecular weight markers, and high and low molecular weight un-labelled DNA markers. The blotted DNA fragments were hybridized to a DIG-labelled TTAGGG probe and incubated with DIG-specific antibody covalently coupled to alkaline phosphatase. The immobilised telomere probe was visualized using CDP-Star, a highly sensitive chemiluminescence substrate of alkaline phosphatase. Blots were documented and analysed with the GelDoc imaging system and the Quantity One software from BioRad (München, Germany). The mean length of the telomere restriction fragment was calculated according to the manufacturers instructions (Telo TAGGG telomere length assay).

Preparation of cytosolic and nuclear extracts

The preparation of nuclear and cytosolic extracts was based on the procedure described by Nervi et al. [START_REF] Nervi | Identification and characterization of nuclear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells[END_REF], with several modifications. Briefly, HRPE cells (~ 1-5 x 10 8 ) from 10-15 confluent 150 cm 2 tissue culture dishes at 35 th or higher passage were trypsinized and collected in growth medium by centrifugation at 1000 g for 5 min and cell pellet was rinsed twice with ice-cold phosphatebuffered saline (PBS) containing 2 mM EDTA. Pellet was then resuspended in 3 ml of ice-cold lysis buffer (10 mM Tris-HCl, 1.5 mM EDTA, 1 mM DTT, 10% v/v glycerol, pH 7.6) in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin and 20 μg/ml leupeptin) and placed on ice for 15 min. Cells were then homogenized with a glass Dounce homogenizer (pestle B, 60-90 strokes) and the homogenate was centrifuged at 4° C for 30 min at 100000 g. The resulting supernatant was the cytosolic extract. The pellet, containing the nuclei, was resuspended in a volume equal to 2/3 the cell pellet volume of extraction buffer (10 mM Tris-HCl, 1.5 mM EDTA, 1 mM DTT, 10 mM thioglycerol, 0.8 M KCl, 10% v/v glycerol, pH 8.00) containing the protease inhibitors. The suspension was incubated for 1 hr on ice with repeated resuspension every 10 min, rehomogenized with Dounce homogenizer (pestle B) and then centrifuged at 100000 g for 1 hour at 4° C. The resulting supernatant was referred to as the nuclear extract. Both cytosolic and nuclear extracts were used either immediately or after storage at -80°C and aliquots of both were assayed for protein concentration according to Lowry et al. [START_REF] Lowry | Protein measurement with the folin reagent[END_REF] with BSA as standard.

Dextran-charcoal adsorption binding assay

Nuclear or cytosolic extracts containing 200 μg of proteins were incubated in 200 μl of 20 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, pH 8.00 at 4°C for 4 hours. Saturation experiments were carried out using 10 different concentrations of [ 3 H]CD367 ranging from 1 to 80 nM. Displacement experiments were performed using nine different concentrations of cold drug in the presence of 10 nM [ 3 H]CD367. Binding assays with [ 3 H]9-cis-RA were done at a final concentration of 10 nM. Nonspecific binding was measured in the presence of 200-fold excess of unlabeled CD367 or 9-cis-RA. Separation of bound from free radioligand was performed essentially as previously described by Sablonnière et al [START_REF] Sablonnière | Physicochemical parameters affecting the charcoal adsorption assay for quantitative retinoid-binding measurement[END_REF]. Briefly, 100 μl of ice-cold charcoal/dextran suspension (3% water-washed activated charcoal Merk/0.3% dextran in 20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 0.1 % BSA, pH 8.00) were added to the incubation mixture. After 10 min of incubation at 4°C, the samples were centrifuged at 6000 g for 5 min and 150 μl of clear supernatant was counted for radioactivity. In all assays, total bound radioligand never exceeded 10% of the radioligand added. The percent of specific binding was ~53% and ~35% of the total bound radioactivity for

Statistical analyses

All values were expressed as means ± S.E.M. Statistical significance was determined by t-test (Prism 4.0; GraphPad Software). P values less than 0.05 were considered statistically significant.

RESULTS AND DISCUSSION

Expression of RARβ in HRPE cells

Expression of the six nuclear retinoid receptor in HRPE cells was first investigated by RT-PCR using previously validated primers [START_REF] Janssen | Retinoic acid receptors and retinoic X receptors in the mature retina: Subtype determination and cellular distribution[END_REF][START_REF] Pasquali | Retinoic acid receptors α, β, γ, and cellular retinol binding protein-I expression in breast fibrocystic disease and cancer[END_REF]. Only RARβ-specific primers produced a 400 bp amplification product of the expected size (Fig. 1). Primer pairs specific for RARα, RARγ, RXRα, RXRβ and RXRγ did not yield any amplification product (data not shown), suggesting that only RARβ is expressed in HRPE cells. As mentioned earlier, expression of nuclear retinoid receptors is not ubiquitous. For instance, hematopoietic cells have been reported to express exclusively RARα, whereas RARβ transcripts are particularly abundant in brain and areas undergoing programmed cell death [START_REF] Dollè | Differential expression of genes encoding alpha, beta, and gamma retinoic receptors and CRABP in the developing limbs of the mouse[END_REF]. This intriguing phenomenon suggested that cell propagation conditions should be considered as significant variable influencing the biological behaviour. The replicative senescence of established cell lines had been well characterized [START_REF] Matsuo | Ploidy of human embryonic fibroblasts during in vitro aging[END_REF]. Proteins which show a simple increase or a simple decrease may participate in a hypothetical mechanism of "biological clock" in the cultured cells [START_REF] Toda | TMIG-2DPAGE: a new concept of two-dimensional gel protein database for research on aging[END_REF]. At this regard, aging of RPE cells in vitro has been the subject of several studies. First of all, Wang et al. [START_REF] Wang | Differential gene expression of early and late passage retinal pigment epithelial cells[END_REF] examined changes in gene expression profiles of RPE normal cells, which were aged in vitro by repeated passage, but little information on the role in the aging process for most of the regulated genes was provided. Furthemore, it has been shown that in ARPE 19 cells, a A c c e p t e d M a n u s c r i p t spontaneously arising human RPE cell line, in normal cell culture conditions most of the retinoic acid receptors were expressed and RARβ expression was modulated by a proliferative treatment [START_REF] Tombran-Tink | Retinoic acid and dexamethasone regulate the expression of PEDF in retinal and endothelial cells[END_REF].

Since previous studies have shown changes in the expression of RARβ related to the passage number, we next evaluated the effect of passage number on RARβ expression in HRPE cells. Towards this, we measured the steady-state levels of RARβ mRNA in early and late passage HRPE cells by semi-quantitative RT-PCR. The transcription of RARβ was significantly up-regulated in late passage HRPE cells compared to early passage HRPE cells (Fig. 2). The expression of RARβ was also confirmed by immunoblot analysis using an anti-RARβ serum as described in the Materials and Methods section (Fig. 3). Crude extracts of HRPE cells obtained from 5 th and 35 th passage analyzed for RARβ expression showed an increase in the expression levels of RARβ in extracts from the 35 th passage, compared to that obtained from the 5 th passage. Thus, the increased level of the RARβ observed by western blot analysis in late passage HRPE cells was consistent with increased levels of transcript detected by semi-quantitative RT-PCR in RNA isolated from corresponding cells.

Effect of cell passage on telomere length

The shortening of telomere has been proposed as the most probable mechanism of the cellular senescence by several Authors [START_REF] Harley | The telomere hypothesis of cellular aging[END_REF][START_REF] Matsunaga | Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells[END_REF], but not all phenotypes of the senescent cells could be ascribed to the shortening of telomere [START_REF] Sherr | Cellular senescence: mitotic clock or culture shock?[END_REF]. We next investigated the relationship between RARβ expression and telomere length in HRPE cells. The effect of replicative aging of HRPE cells in multiple passaging was evaluated by telomere length assay at either early or late passages to determine telomeric DNA loss because of cell divisions in multiple passaging [START_REF] Velicescu | Aneuploidy and telomere attrition are independent determinants of crisis in SV40 transformed epithelial cells[END_REF]. Figure 4 shows a representative autoradiograph of smears of genomic DNA isolated from early and late passage HRPE cells hybridized with DIG labelled Telo TAGGG probe. The HRPE cells showed a stabilized telomere at around 3.1 kb in both low and high passage cells, indicating that the length of the telomere is not affected by passage number in HRPE cells. When telomeres reach a critical shortening in length senescence is induced resulting into permanent proliferation arrest [START_REF] Allsopp | Telomere length predicts replicative capacity of human fibroblast[END_REF]. In our work, for the first time, a typical biomarker of cellular senescence such as RARβ has been shown to be up-regulated by repeated passage in a well-established cell line. Furthermore, this up-regulation is not due to or associated with shortening of telomere. Taken together, these results suggest that the increase in RARβ mRNA and protein levels detected in late passage HRPE cells does not appear to be due to a senescence condition.

In vitro binding assay

We first measured the specific binding of retinoic acid in cytosolic and nuclear extracts isolated from HRPE cells at 35 th or higher passage by performing assays with 10 nM [ 3 H]9-cis-RA (agonist of both RAR and RXR subtypes) and saturation experiments (1-80 nM) of [ 3 H]CD367 (agonist of only RAR subtypes). No detectable binding was observed in cytosolic extracts using both the radioligands (data not shown). This could be explained assuming that HRPE cells do not contain detectable levels of cellular retinaldehyde binding protein (CRABP). Indeed, it was already reported that RPE cultures established from adult donors, maintained in DMEM with FBS on a tissue culture substrate, lose RPE specific properties such as pigmentation, expression of tyrosinase, CRABP and bestrophin [START_REF] Campochiaro | Corneal endothelial cell matrix promotes expression of differentiated features of retinal pigmented epithelial cells: implication of laminin and basic fibroblast growth factor as active components[END_REF]. Moreover, it was demonstrated that CRABP is expressed in the tissue of human adults, but not in the derived RPE cell lines [START_REF] Dunn | ARPE-19, a human retinal pigment epithelial cell line with differentiated properties[END_REF]. Similar loss of CRABP was also reported to occur in HL-60 cells, where total RA-binding was found to be associated with the nuclear extract of which were found to contain only nuclear RARα and no cytosolic binding proteins [START_REF] Nervi | Identification and characterization of nuclear retinoic acid-binding activity in human myeloblastic leukemia HL-60 cells[END_REF].

On the other hand, specific binding was observed with both [ 3 H]9-cis-RA and [ 3 H]CD367. The specific bound observed for [ 3 H]9-cis RA was 133 ± 9 fmol/mg of protein (data not shown).

Figure 5 shows the saturable binding of [ 3 H]CD367 to the late passage HRPE cell nuclear extract. Scatchard analysis (Fig. 5, inset) of the data was linear in the concentration range investigated (r = 0.982, p < 0.0001, n =10) and, analogously, computer analysis of the saturation experiments suggested a one-site, rather than a two-sites binding model. The dissociation binding constant (K D ) and B max values derived from the saturation experiments of [ 3 H]CD367 were 9.6 ± 0.6 nM and 780 ± 14 fmol/mg of protein, respectively.

The binding data obtained with radiolabeled ligands corroborate the specific expression of RARβ in HRPE cells. 9-cis RA is known to be a high affinity ligand of both RAR and RXR subtypes [START_REF] Allenby | Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids[END_REF][START_REF] Yang | Efficient inhibition of activation-induced fas ligand up-regulation and T cell apoptosis by retinoids requires occupancy of both retinoid X receptors and retinoic acid receptors[END_REF]; on the other hand RT-PCR and Western blot data reported above show that HRPE cells selectively express the RARβ subtype. Taking into account that CD367 is a relatively stable ligand and more specific to RAR subtype than [ 3 H]9-cis RA [START_REF] Sablonnière | Physicochemical parameters affecting the charcoal adsorption assay for quantitative retinoid-binding measurement[END_REF], we have therefore chosen [ 3 H]CD367 to characterize the binding properties of nuclear RA receptors. The linearity of the Scatchard plot reported in Fig. 5 suggests that [ 3 H]CD367 is binding to only one RAR subtype in HRPE cell nuclear extract which, based on our RT-PCR and western blot results, is RARβ. The CD367 affinity (K D = 9.6 nM) found in HRPE cells appears in agreement with the values obtained by previous binding studies towards RAR receptors [START_REF] Delescluse | Selective high affinity retinoic acid receptor a or b-g ligands[END_REF]. Furthermore, the B max value found in HRPE cells (~ 800 fmol/mg of protein) is in the same order of magnitude of that obtained in COS cells expressing RARβ cDNA (900 fmol/mg protein) [START_REF] Delescluse | Selective high affinity retinoic acid receptor a or b-g ligands[END_REF]. We have performed the binding studies on nuclear extracts of HRPE cells at 35 th or higher passage, when the cells show a significant upregulation of RARβ as demonstrated by RT-PCR (Fig. 2) and Western blot analysis (Fig. 3), respectively. Taken collectively, our results indicate that HRPE cell is a good model of an established cell line for characterizing RARβ ligand specificity. Since the expression of RARβ increase with increasing passage of the cells, the problem of poorly detectable RARβ in native cells can be overcome by using late passage HRPE cells instead of overexpressing RARβ by recombinant techniques.

To confirm the feasibility of using HRPE cells in characterizing RARβ ligands, we have measured the affinity of several flavonoids to RARβ. We chose these compounds because of their xenoestrogenic properties and their presence in natural food sources. Retinoids are considered to be agents of chemoprevention and differentiation in several proliferative diseases, including benign and malignant breast diseases. Knowledge of how retinoids interrupt proliferative processes pharmacologically or through diet, and how they induce differentiation and programmed cell death, will probably lead to new treatment strategies, especially in the prevention of breast cancer [START_REF] Pasquali | Retinoic acid receptors α, β, γ, and cellular retinol binding protein-I expression in breast fibrocystic disease and cancer[END_REF]. Therefore, to investigate the interaction of flavonoids with RARβ, we studied the effect of these flavonoids on the binding of [ 3 H]CD367 to nuclear extracts of HRPE cells (Fig. 6). The results obtained suggest flavonoids do interact with RARβ, however, only weakly. The K i values obtained from the analysis of the curves are presented in Table 2. It can be observed that the RA affinity for RARβ was in the nanomolar range (9.1 ± 0.6 nM) whereas the binding of the flavonoids was relatively weak, with K i values ranging from 0.9 ± 0.5 µM to 30 ± 2 µM, and thus may not be able to significantly interfere with the endogenous activity of RARβ. But, what is important is that the methodology that we report here can be utilized to characterize even the relatively weak interaction between flavonoids and RARβ.

In conclusion, we report here on the specific expression of RARβ in HRPE cells, and this expression increases with increasing passage of the cells. The increased RARβ expression in aging cells is not associated with decrease in telomere length. We also show that the increased expression in high passage cells can be exploited to develop a specific assay to characterize RARβ-ligand interactions.

FIGURE LEGENDS

Figure . 1. RT-PCR of RARβ isoform expression relative to the constitutive gene 18S. Total RNA was reverse-transcribed followed by 35-cycle PCR of cDNA using the primers described in Table 1. The RNA samples used were obtained from HRPE cells at the 35 th passage. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  [ 3 H]CD367 and [ 3 H]9-cis-RA, respectively. Saturation experiments data (K D and B max values) were obtained by computer analysis of saturation curves. Linear regression of Scatchard plot was performed according to the equation [Bound]/[Free] = B max K A -K A [Bound], were K A is 1/K D and [Bound] are the specific binding values of the radiolabelled ligand obtained at the [Free] incubation concentrations. The concentrations of the unlabelled drugs needed to displace 50% of the bound radioligand (IC 50 ) values were obtained by non-linear regression analysis of the displacement curves. Inhibitory binding constants (K i values) were calculated from the IC 50 values according to the Cheng and Prusoff [19] equation K i = IC 50 /(1+[C*]/K D *, where [C*] is the concentration of the radioligand and K D its dissociation constant. All binding data were analysed using the software Graph Pad Prism (Graph Pad, San Diego, CA, USA).

Bioscience

  Reports Immediate Publication. Published on 01 Aug 2008 as manuscript BSR20080103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080103

Figure 2 .

 2 Figure 2. Semi-quantitative RT-PCR of RARβ. cDNA of RARβ and the constitutive gene 18S rRNA, derived from HRPE cells at 5 th and 35 th passage, were co-amplificated by semi-quantitative RT-PCR. (A) Intensity of RARβ transcript is higher in HRPE cells at 35 th passage. (B) RARβ mRNA expression was normalized on the basis of 18S RNA expression. RARβ mRNA normalized with 18S RNA in separated and co-amplificated RT-PCR was represented. Mean ± SEM at 5 th and 35 th passage obtained in three independent experiments is shown. * P < 0.001.

Figure 3 .

 3 Figure 3. Immunoblotting of HRPE extracts with a rabbit polyclonal anti-RARβ antibody. The RAR protein levels in HRPE cells were analysed using Chemi Doc System (Bio-Rad) and normalized to β-actin signal intensities of the corresponding lanes. Values are reported as mean ± S.D. of at least three different immunoblotting experiments. * P < 0.001.

Figure 4 .

 4 Figure 4. Telomere length inHRPE cells compared at 5 th and 35 th passage. Telomere length comparisons was done using the Telo TAGGG Telomere Length Assay kit (Roche Diagnostics, Mannheim, Germany). The signal intensities show the size distribution of terminal restriction fragments. M, marker in kbp; lane 1, DNA from HRPE cells passage 5; lane 2, DNA from HRPE cells passage 35; lane 3, control, low molecular weight DNA; lane 4, control, high molecular weight DNA.

Figure 5 .

 5 Figure 5. Saturation curve and its Scatchard plot (bound/free versus bound, inset) obtained at 4° for the equilibrium binding of [ 3 H]CD367 to HRPE nuclear extracts. The linearity of the Scatchard plot (r = 0.982, P < 0.0001) and computer analysis of the saturation plots indicate the presence of a single class of binding sites. Data shown are from a single representative experiment performed in duplicate.

Figure 6 .

 6 Figure 6. Competition experiments of Retinoic Acid (RA) and several flavonoids for specific [ 3 H]CD363 binding carried out at 4°C on HRPE nuclear extracts. Data shown are from a single representative experiment performed in duplicate.

2 .

 2 tgc tgg agg cgc taa agg tc-3' 5'-tct gtc caa gga gtc gct gcc-3tgc ctt tgg aaa tgg atg aca c-3' 5'-tga ctg acc cca ctg ttt tcc-3act gcc tga ttg aca agc g-3' 5'-aca agc tcc gtc agc accgcc ccc ctg aag atg tga agc c-3' 5'-cca tcc cca tcc ttg tcc ttt cgca gtt cag agg aca tac agc c-3' 5'-gcc tca ctc tca gct cgc tct ctca aga acg aaa gtc gga gg-3' 5'-gga cat cta agg gca tca caof annealing; § = designed by ç Ambion Inc., Texas, USA (www.ambion.com) Bioscience Reports Immediate Publication. Published on 01 Aug 2008 as manuscript BSR20080103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080103 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 Biochemical Society Table Inhibitory constants, Ki (nM), for retinoic acid and several flavonoids obtained by displacement of 10 nM [ 3 H]CD367 from RARβ of HRPE nuclear extracts. Values ± SEM are from three independent experiments performed in duplicate. These values represent the mean of three determinations. Publication. Published on 01 Aug 2008 as manuscript BSR20080103 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080103 A c c e p t e d M a n u s c r i p t

  Bioscience Reports Immediate Publication. Published on 01 Aug 2008 as manuscript BSR20080103

  THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080103

© 2008 The Authors Journal compilation © 2008 Biochemical Society

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

Acknowledgments

This research was supported by the University of Ferrara and of Chieti research grants (C. Biondi and B. Favaloro).


Annette Heinrich

Ulrike Böer

Mladen Tzvetkov

Dr Elke Oetjen
email: eoetjen@med.uni-goettingen.de

Willhart Knepel

Stimulation by lithium of the interaction between the transcription factor CREB and its coactivator TORC

Keywords: cAMP-response element binding protein (CREB), transducer of regulated CREB (TORC), Lithium, protein-protein interaction, mammalian two-hybrid assay, GST pull-down assay

come

INTRODUCTION

Lithium salts are clinically important drugs used to treat bipolar mood disorder and other mental diseases. Although some biochemical effects of lithium are known, such as inhibition of glycogen synthase kinase 3 (GSK3) 1 , inositol monophosphatase (IMPase), and adenylate cyclase (AC), the molecular mechanism that underlies the clinical effects of lithium has remained unclear [START_REF] Mota De Freitas | Is competition between Li+ and Mg2+ the underlying theme in the proposed mechanisms for the pharmacological action of lithium salts in bipolar disorder?[END_REF][START_REF] Quiroz | Molecular effects of lithium[END_REF]. Noteworthy, the mood stabilizing effect of lithium develops slowly, over weeks and months of lithium treatment [START_REF] Manji | Enhancing neuronal plasticity and cellular resilience to develop novel, improved therapeutics for difficult-to-treat depression[END_REF], suggesting that the clinical effects of lithium are secondary to neuronal adaptation and based on a lithium-induced new pattern of gene expression [START_REF] Manji | Enhancing neuronal plasticity and cellular resilience to develop novel, improved therapeutics for difficult-to-treat depression[END_REF][START_REF] Zarate | Regulation of cellular plasticity cascades in the pathophysiology and treatment of mood disorders: role of the glutamatergic system[END_REF]. The transcription factor CREB has been implicated in adaptive responses of the brain to various stimuli [START_REF] Mcclung | Neuroplasticity mediated by altered gene expression[END_REF] and has been suggested to mediate also lithium-induced

EXPERIMENTAL PROCEDURES Plasmids

Expression vectors for GST-CREB and GST-CREB-R300A were generated by PCR cloning. GAL4-CREB and GAL4-CREB-R300A [START_REF] Boer | Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain[END_REF] served as template. After PCR the fragments were subcloned into pGEX2T (GE Healthcare) via BamHI and EcoRI restriction sites. The expression vector for human TORC1 under control of a T7 promoter in pcDNA3 has been described before [START_REF] Boer | Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain[END_REF]. For ChIP assays, an expression vector for N-terminally FLAG-tagged human TORC1 was generated by PCR cloning using TORC1 as template followed by subcloning into pcDNA3 (Invitrogen). The plasmid 4xSomCRET81Luc has been described before [18]. The reporter gene 5xGal4E1B-Luc [START_REF] Kruger | Inhibition of CREB-and cAMP response element-mediated gene transcription by the immunosuppressive drugs cyclosporin A and FK506 in T cells[END_REF] contains the luciferase gene under control of 5 repeats of the responsive element for the yeast transcription factor GAL4. For PCR cloning of GAL4-TORC1 and GAL4-TORC1 1-44 human TORC1 served as template. The resulting DNA was subcloned into the GAL4 DNA-binding domain-coding vector pSG424 [START_REF] Sadowski | A vector for expressing GAL4(1-147) fusions in mammalian cells[END_REF]. These constructs encode full length TORC1 or the first 44 amino acids, respectively, Cterminally fused to the DNA-binding domain (aa1-147) of GAL4. The expression plasmids for VP16-bZip and VP16-bZip-R300A, encoding the basic leucine zipper domain (CREB 262- 327 ) fused C-terminally to the transactivation domain of the viral protein VP16, were generated by PCR cloning. GAL4-bZip or GAL4-bZip-R300A, respectively, served as template followed by subcloning into the VP16 domain-coding vector pHKnt-VP16 [21]. Table S1 (supplemental material) gives an overview of all primer sequences. The correct sequence of all constructs was verified by sequencing.

Cell culture and transfection

Hamster insulinoma tumor cells (HIT-T15) [21] were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 5% horse serum, 100 units/ml penicillin and 100 µg/ml streptomycin. For ChIP-assays HIT cells were transiently transfected with 4 µg of expression vector per 6-cm dish using Metafectene (Biontex) according to the manufacturer's instructions. For mammalian two-hybrid assays the DEAE-Dextran method [21] was used to transfect the cells with 2 µg of expression vector and 1 µg cytomegalovirus green fluorescent protein expression vector per 6-cm dish to control for transfection efficiency. Co-transfections were carried out with a constant amount of DNA, which was maintained by adding the empty pBluescript vector (Stratagene). Treatment of cells in the GAL4 assay was with 2 mM 8 Br-cAMP 6 h and with 20 mM LiCl 7 h prior to harvest. For the two hybrid assay, cells were treated with 1 mM 8 Br-cAMP 6 h and with 20 mM LiCl 30 h prior to harvest. Luciferase was determined 48 h after transfection as previously described [21].

Immunocytochemistry

HIT cells were grown on coverslips in 6-well plates. Treatment with 1 mM or 2 mM 8Br-cAMP and with 20 mM LiCl was 30 min and 90 min prior to fixation, respectively. Fixation and antibody incubation were performed as described [22]. panTORC antibody (Calbiochem) was used as primary antibody against TORC proteins. The secondary antibody was anti-rabbit IgG AlexaFluor ® 488 (Invitrogen). The coverslips were mounted on slides using Vectashield mounting medium with 4',6-Diamino-2-phenylindol (DAPI) (Linaris). The slides were analyzed using a Zeiss Axiovert 200 microscope (Software: OpenLab™ 3.1 for Macintosh OS9.2 by Improvision Ltd.).

RNA extraction and reverse transcription

Total RNA was extracted from HIT cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized from total RNA by reverse transcription using oligo(dT) 15 primer and M-MLV Reverse Transcriptase (Promega) according to the instructions of the manufacturer. Analysis of the mRNA level of TORC proteins in HIT cells was performed by PCR using primers for human TORC1, TORC2, TORC3, and hamster β-actin as internal standard (sequences see table S2 -supplemental material), followed by agarose gel electrophoresis and densitometric analysis of the bands.

Chromatin Immunoprecipitation (ChIP) Assay and quantitative real time PCR (qRT-PCR)

Chromatin immunoprecipitation was performed as described [23] with some modifications. Transfected HIT cells were treated with 2 mM 8Br-cAMP and with 20 mM LiCl 30 min and 90 min, respectively, prior to cross-linking with 1% (v/v) formaldehyde. Cells were harvested, resuspended in cell lysis buffer (10 mM Tris/HCl pH 8.0, 10 mM NaCl, 0.2% (v/v) Nonidet-P40, protease inhibitors), and homogenized using a 1-ml Dounce homogenizer. After centrifugation, the pellet was resuspended in nuclei lysis buffer (50 mM Tris/HCl pH 8.1, 10 mM EDTA, 1% (w/v) SDS, protease inhibitors). The DNA was sheared by sonication and the cell debris was pelleted by centrifugation. The supernatant was collected and 10% were taken as input control. Immunoclearing, immunoprecipitation, and purification of DNA were performed as described [24]. qRT-PCR was performed using the fluorescent reporter probe method. The 4 repeats of the CRE of the 4xSomCRET81Luc were chosen as DNA sample to measure efficiency of the ChIP assay. The following primers were used: 5'-GCAATAGCATCACAAATTTCACAAA-3' (forward primer) and 5'-CCGCCCCGACTGCAT-3' (reverse primer) combined with the fluorescent reporter probe 5'-CGAATTCGCCGGATCTCGAGCTC-3' (modifications: 5'-Fluorescein; 3'-TAMRA). For the qRT-PCR, the reaction mix from Eurogentec, containing the AmpliTaq ® DNA polymerase (Applied Biosystems), was used and prepared according to the manufacturer's instruction. Each sample was measured in triplicate on a 384 well plate in each 18 µl volume using the ABI Prism 7900HT Sequence Detection System and was analysed with the SDS 2.1 Software (Applied Biosystems).

Expression and purification of GST-fusion proteins from bacteria

Competent cells from E. coli strain DH5α were transformed by heat shock with expression vectors coding for GST-CREB and GST-CREB-R300A. Bacteria were grown in 1L LB medium at 37°C. The expression of the GST-fusion proteins was induced by 1 mM isopropyl β-D-1-thio-galactopyranoside (IPTG) for 4 h. The bacteria were harvested in sonication buffer (20 mM HEPES pH 7.5, 1 M NaCl, 1 mM dithiothreitol [DTT], 1 mM phenylmethansulfonylfluoride [PMSF]) and stored at -80°C. The suspension was thawed on ice and the bacteria disrupted by sonication. The cell debris was pelleted by centrifugation and the GST-fusion proteins were extracted from the supernatant by affinity chromatography using glutathione-agarose beads. The isolated proteins immobilized on agarose were stored on ice.

[ 35 S]-labeling of proteins [ 35 S]-labeled human TORC1 was generated by in vitro transcription / translation using the TNT T7 coupled reticulocyte lysate system (Promega) according to the manufacturer's instruction including T7 polymerase and [ 35 S]methionine (1000 Ci/mmol) (GE Healthcare). Human TORC1-coding sequence under control of the T7 promoter in pcDNA3 served as template.

GST pull-down assay

The GST pull-down assay using GST-CREB (wt/R300A) and [ 35 S]TORC1 was performed as described [23]. Briefly, the agarose-bound proteins were washed with reaction buffer. Buffers contained 20 mM Tris/HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% (v/v) Nonidet-P40, 1 mM DTT, and 1 mM PMSF. Considering the valency of lithium and magnesium, the salt concentration was balanced with NaCl to 100 mM to prevent effects due to changes in ionic strength of the buffer (for further details Table S3 -supplemental material). For the binding reaction, 3.5 µl of [ 35 S]TORC1 were added to equal amounts of GST-fusion protein in 250 µl reaction buffer and incubated overnight at 4°C. The samples were analyzed by SDS-PAGE. The gel was dried and the radioactively labeled proteins were visualized using a phosphor imager (Fuji Film BAS 1800 II) and analyzed densitometrically using the software AIDA 4.15 (raytest Isotopenmessgeräte).

Statistics

Statistical data analysis (two-way ANOVA with Student's t test) was performed using the software STATISTICA 7 (Statsoft). 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.

RESULTS

Nuclear translocation of TORC proteins

Nuclear translocation of TORC has been shown to be a key regulator for CREB transcriptional activity [10,11,14,[START_REF] Wu | Transducer of regulated CREB and late phase long-term synaptic potentiation[END_REF][START_REF] Zhou | Requirement of TORC1 for late-phase long-term potentiation in the hippocampus[END_REF]. By immunocytochemistry the effect of lithium and cAMP on the cellular localization of endogenous TORC proteins in HIT cells was investigated. HIT cells, like neurons, are electrically excitable [27] and share with neurons the expression of proteins like the scaffold protein JNK-interacting protein (JIP)-1b / islet-brain (IB)1 [START_REF] Hashimoto | alpha-Synuclein protects against oxidative stress via inactivation of the c-Jun N-terminal kinase stress-signaling pathway in neuronal cells[END_REF], or the transcription factors Pax6 [START_REF] Cerf | Transcription factors regulating beta-cell function[END_REF]30] and BETA2 / NeuroD [START_REF] Cerf | Transcription factors regulating beta-cell function[END_REF]31]. CREBdirected transcription has been well characterized in HIT cells [START_REF] Boer | Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain[END_REF]18,21,27,32,33] including the biology of TORC [14]. Endogenous TORC was mainly retained in the cytoplasm under resting conditions (Figure 1A, upper panel). Treatment of HIT cells with 8Br-cAMP induced in a concentration-dependent manner the translocation of TORC into the nucleus (Figure 1A, middle panel; Figure 1B). These results are consistent with other reports in which the nuclear translocation of TORC in different cell lines and also primary hippocampal neurons was inducible by treatment with forskolin, a potent activator of adenylate cyclase [10,14,[START_REF] Zhou | Requirement of TORC1 for late-phase long-term potentiation in the hippocampus[END_REF][START_REF] Kovacs | TORC1 is a calcium-and cAMP-sensitive coincidence detector involved in hippocampal long-term synaptic plasticity[END_REF]. Furthermore, the nuclear translocation of TORC was induced in HIT cells by high potassiuminduced membrane depolarization and blocked by the calcineuin inhibitor cyclosporine A (data not shown), consistent with a previous report [14]. In HIT cells, LiCl did not affect the translocation of TORC (Figure 1A, lower panel), neither alone nor in combination with 8Br-cAMP (Figure 1B). The panTORC antibody used in the immunocytochemistry experiments can detect all three isoforms of TORC as it is directed against the first 42 amino acids which are highly conserved among the TORCs [11]. To identify the isoforms expressed in HIT cells, total RNA was extracted and examined regarding the expression level of the different isoforms. This analysis revealed an 8-fold higher expression of TORC1 over TORC2 and the absence of TORC3 (Figure S1, Supplemental Material). Noteworthy, TORC1 is the isoform particularly copious also in brain tissues like hippocampus, cerebral cortex, and cerebellum [11,[START_REF] Zhou | Requirement of TORC1 for late-phase long-term potentiation in the hippocampus[END_REF][START_REF] Kovacs | TORC1 is a calcium-and cAMP-sensitive coincidence detector involved in hippocampal long-term synaptic plasticity[END_REF]. Therefore, we focussed on TORC1 in the further investigations.

Transactivation by TORC1

To investigate the effect of lithium on TORC1 transcriptional activity, the GAL4 system was employed. In a luciferase reporter gene assay, HIT cells were transfected with an expression vector coding for TORC1 fused to the DNA-binding domain of the yeast transcription factor GAL4. The promoter of the reporter gene contained 5 repeats of the GAL4-binding site (Figure 2A). Thereby TORC1 is tethered to the promoter and its own transcriptional activity can be examined independently of its binding to CREB. The GAL4 DNA-binding domain alone exhibited virtually no activity and did not show any response to the stimuli (data not shown). In contrast, GAL4-TORC1 conferred strong transcriptional activity (Figure 2), indicating the presence of a transactivation domain in TORC1 as suggested before [11,[START_REF] Iourgenko | Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells[END_REF]. Treatment with 8Br-cAMP increased the activity of TORC1 3-fold (Figure 2B), LiCl affected the transcriptional activity of TORC1 neither alone nor in combination with 8Br-cAMP (Figure 2B). In view of these results, an influence of lithium salts on either the translocation of TORC1 into the nucleus or TORC1 transactivation potential seems to be unlikely.

Effect of lithium on the recruitment of TORC1 to the promoter

To examine whether lithium influences the recruitment of TORC1 to the promoter, we employed a chromatin immunoprecipitation (ChIP) assay. HIT cells were transfected with an expression plasmid containing a promoter controlled by four repeats of the rat somatostatin CRE. It contains the consensus sequence 5'-TGACGTCA-3' constituting a high-affinity binding site for endogenous CREB [18]. An expression vector coding for TORC1 tagged with A c c e p t e d M a n u s c r i p t a FLAG epitope was co-transfected (Figure 3A). After treatment, DNA and proteins were cross-linked and precipitated with an antibody against the FLAG epitope. The amount of precipitated DNA was determined by quantitative real-time PCR. In control experiments, pBluescript as unspecific DNA was co-transfected with FLAG-TORC1 and did not result in a specific signal in real time PCR (data not shown). Treatment of HIT cells with LiCl alone or with 8Br-cAMP alone did not induce a detectable increase of promoter occupancy (Figure 3B). An increase has been shown before for TORC2 upon forskolin stimulation [START_REF] Ravnskjaer | Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression[END_REF][START_REF] Koo | The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism[END_REF]. This system might be not sensitive enough to the effect elicited by 8Br-cAMP alone. In fact, stimulation with forskolin is in general about 10-fold more potent compared to 8Br-cAMP [18,32]. However, the combination of cAMP and lithium enhanced the promoter occupancy of TORC1 2-fold (P < 0.025, n = 5) (Figure 3B).

Effect of lithium on the interaction between CREB and TORC1: mammalian twohybrid assay

A mammalian two-hybrid assay was employed to investigate the effect of lithium and cAMP directly on the interaction between CREB and TORC1. Only the interaction domains of the proteins of interest were used. HIT cells were transiently transfected with the luciferase reporter gene under control of five repeats of the GAL4-binding site and an expression vector coding for the first 44 amino acids of TORC1 as GAL4-fusion construct. Co-transfected was either the CREB bZip wild type (bZip-wt), or the mutant CREB bZip R300A (bZip-R300A), each fused to the potent viral protein VP16, or VP16 alone for control (Figure 4A). Compared to the VP16 control, VP16-bZip-wt showed a 2-fold higher basal activity (Figure 4B) indicating the interaction between bZip-wt and TORC1 1-44. LiCl strongly increased interaction between the wild type bZip and TORC1 1-44 (Figure 4B). 8Br-cAMP alone had no effect on luciferase activity, whereas treatment with LiCl plus 8Br-cAMP increased the interaction between TORC1 1-44 and bZip-wt to the same extent as LiCl alone (Figure 4B). The bZip R300A mutant (VP16-bZip-R300A) did not show significant differences from the VP16 control (Figure 4B), indicating lack of interaction between bZip-R300A and TORC1 1-44 . This confirms previous reports in which this mutation disrupted complex formation [14] and the enhancement by lithium of cAMP-induced CREB target gene transcription [START_REF] Boer | Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain[END_REF]. These data show an enhancement by lithium of the protein-protein interaction between bZip and TORC1 1-44 that is independent of cAMP. The two-hybrid assay could not be applied to investigate the interaction of the full-length proteins due to very high basal activity of GAL4-TORC1 (data not shown).

Effect of lithium on the interaction between CREB and TORC1: in vitro GST pull-down assay

Recombinant GST-CREB-wt and GST-CREB-R300A mutant were expressed in bacteria and TORC1 was labeled by in vitro transcription and translation using [ 35 S]methionine. Specificity of the interaction was examined by use of GST alone which exhibited no remarkable interaction with [ 35 S]TORC1 (6.6% of GST-CREB-wt, P < 0.002) (Figure 5A). In the presence of 20 mM LiCl, the amount of [ 35 S]TORC1 recovered from GST-CREB-wt was clearly increased (P < 0.035) (Figure 5). In contrast, no specific binding of [ 35 S]TORC1 to the mutant GST-CREB-R300A was detectable, and there was no effect of lithium (Figure 5A). A concentration-response curve of LiCl revealed significantly increased binding of [ 35 S]TORC1 to GST-CREB-wt from concentrations of 5 mM LiCl (Figure 5B). These results indicate that lithium enhances directly the interaction between CREB and TORC1 also in this cell-free system. Lithium is known to inhibit GSK3 by competition with magnesium at the non-Mg:ATP magnesium binding site [START_REF] Ryves | Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites[END_REF]36]. Therefore, the effect of increasing concentrations of magnesium on the interaction between CREB and TORC1 was investigated in the GST pull-down assay. This analysis revealed that magnesium strongly inhibited the interaction in a concentration-dependent manner (Figure 6A): Already 0.5 mM MgCl 2 reduced the interaction by 40% (P < 0.03), and in the presence of 10 mM and 20 mM MgCl 2 only 25% and 14%, respectively, of the interaction between CREB and TORC1 remained (Figure 6A). This strong inhibition by high magnesium concentrations was attenuated by 5 mM LiCl (Figure 6B). LiCl thus enhanced the interaction about 3-fold in the presence of 10 mM or 20 mM MgCl 2 (Figure 6C). These data indicate an inhibitory effect of magnesium on the interaction between CREB and TORC1, which is reversed by lithium. 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.

DISCUSSION

CREB activity has been known for years to be induced by serine-119 phosphorylation and CBP/p300 cofactor recruitment [START_REF] Mayr | Transcriptional regulation by the phosphorylationdependent factor CREB[END_REF]37]. In contrast to CBP/p300, the more recently recognized other essential coactivator, TORC, binds to the CREB bZip domain [11,[START_REF] Iourgenko | Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells[END_REF]. This interaction is per se independent of CREB serine-119 phosphorylation and extends the functional role of CREB bZip domain to include not only DNA binding and dimerization but also coactivator recruitment. The regulation of TORC by cAMP-activated and other signaling pathways is thus as important for CREB activity as the regulation of CREB serine-119 phosphorylation [10,11,14,38]. This crucial role of the TORC family of coactivators was further emphasized by the recent identification of TORC as the target of a clinically important drug, when the mood stabilizer lithium was shown to stimulate CREB target gene expression through TORC [START_REF] Boer | Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain[END_REF]. The present study now defines what step in TORC function is influenced by lithium, and demonstrates that lithium does not affect TORC nuclear translocation and transcriptional transactivation, but increases as a cation the binding of TORC to the CREB bZip domain. Cyclic AMP-and Ca 2+ -activated signaling pathways control the nucleo-cytoplasmic shuttling of TORC [14, 39]. Under resting conditions, TORC1 and TORC2 are sequestered in the cytoplasm bound by 14-3-3 proteins [14]. This binding occurs in response to TORC phosphorylation by SIK. Elevated levels of cAMP inhibit SIK kinase activity via the protein kinase A-mediated phosphorylation of SIK at serine-587 (SIK2). In turn, the phosphorylation of TORC decreases and TORC accumulates in the nucleus [14, 39]. Our data confirm a previous report that TORC accumulates in the nucleus upon increased cAMP levels in HIT cells [14]. But no effect of lithium was detected on the translocation of TORC into the nucleus. Likewise, no effect of lithium on transactivation by TORC was found in the present study. The TORC proteins contain at their C-terminus a strong transactivation domain [11,[START_REF] Iourgenko | Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells[END_REF]. This can be studied, when TORC is fused to the heterologous GAL4 DNA-binding domain and tested for induction of a promoter with GAL4-binding sites, as has been shown previously [START_REF] Iourgenko | Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells[END_REF]. The transactivation potential of TORC may not entirely be constitutive, since cAMP stimulated GAL4-TORC1 transcriptional activity (this study). A possible explanation is offered by NONO (p54 nrb ) as cAMP has been shown to stimulate the binding of TORC to NONO, which then acts as a bridge between the CREB/TORC complex and RNA polymerase II [40]. Several lines of evidence support the view that, once TORC has reached the nucleus, lithium enhances the binding of TORC to CREB. As revealed by the ChIP assay, the stimulation by lithium of cAMP-induced CREB target gene expression [START_REF] Boer | Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain[END_REF] is accompanied by an increase in CRE promoter occupancy by TORC. Since lithium does not alter the nuclear translocation of TORC (see above), the increase in promoter occupancy suggests that lithium stimulates TORC-CREB binding. This was then directly demonstrated by the results of the mammalian two-hybrid assay and the GST pull-down assay. Noteworthy, lithium stimulates TORC-CREB binding independently of cAMP and protein kinase A activity, since in the mammalian twohybrid assay lithium induced a robust increase in the binding of the TORC N-terminus to the CREB bZip in the absence of cAMP and cAMP had no effect. This conclusion is further supported by the results of the GST pull-down. Lithium thus depends on cAMP to stimulate CRE/CREB-directed transcription [START_REF] Boer | Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain[END_REF], because cAMP provides CREB serine-119 phosphorylation / CBP recruitment as well as TORC nuclear translocation. Consistent with the previous finding that the stimulation by lithium of the CRE/CREB-directed transcription is not mediated via protein kinase A, GSK3 or IMPase [START_REF] Boer | Enhancement by lithium of cAMP-induced CRE/CREB-directed gene transcription conferred by TORC on the CREB basic leucine zipper domain[END_REF], the effect of lithium in the cell-free GST pull-down assay indicates that lithium increases TORC-CREB binding independently not only of protein kinase A but also of other enzymes and cellular proteins. Therefore, when taken together, the results of the present study strongly suggest that, once TORC has entered the nucleus, lithium as a cation directly enhances the binding of the TORC N-terminus to the CREB bZip resulting in an increase in CREB target gene expression (Figure 7). Both the CREB bZip and the TORC N-terminus are highly structured, forming a continuous α-helix and coiled-coil structure, respectively [START_REF] Iourgenko | Identification of a family of cAMP response element-binding protein coactivators by genome-scale functional analysis in mammalian cells[END_REF][START_REF] Schumacher | The structure of a CREB bZIP.somatostatin CRE complex reveals the basis for selective dimerization and divalent cation-enhanced DNA binding[END_REF]. Based on glutaraldehyde crosslinking experiments, it has been suggested that TORC binds as a tetramer to the CREB dimer [11]. The binding of a cation like lithium to such a composite structure, we speculate, may induce a conformational change that increases complex stability. The best-studied example of lithium acting as a cation is the competition of lithium with magnesium for one of the two magnesium binding sites of GSK3, the non-Mg:ATP magnesium binding site, leading to inhibition of GSK3 kinase activity [START_REF] Ryves | Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites[END_REF]36,[START_REF] Klein | A molecular mechanism for the effect of lithium on development[END_REF]. Li + and Mg 2+ have similar ionic radii [START_REF] Mota De Freitas | Is competition between Li+ and Mg2+ the underlying theme in the proposed mechanisms for the pharmacological action of lithium salts in bipolar disorder?[END_REF], and it has been suggested that this enables lithium to physically invade the magnesium binding site within GSK3, where due to its lower charge density it disrupts catalytic function [36]. A similar model could hold true also for TORC-CREB binding, since magnesium was found in the present study to inhibit the binding of TORC to CREB over a similar range of concentrations as it stimulates GSK3 activity [36] and lithium antagonized the effect of magnesium, thus increasing TORC-CREB binding by release from magnesium inhibition. Interestingly, the crystal structure of the CREB bZip revealed the presence of a hexahydrated magnesium ion in the cavity between the bifurcating basic regions [START_REF] Schumacher | The structure of a CREB bZIP.somatostatin CRE complex reveals the basis for selective dimerization and divalent cation-enhanced DNA binding[END_REF]. The role of this magnesium binding to the CREB bZip in the stimulation by lithium of TORC-CREB binding remains to be investigated. Clinical studies have demonstrated neuronal atrophy, loss of glial cells/neurons, and impairments of neuroplasticity and resilience in the brain of patients that suffer from bipolar mood disorder and suggest that chronic treatment with lithium may attain mood stabilization via neuroprotective actions and induction of neuronal adaptation [START_REF] Liang | Lithium inhibits Smad3/4 transactivation via increased CREB activity induced by enhanced PKA and AKT signaling[END_REF][START_REF] Manji | Impairments of neuroplasticity and cellular resilience in severe mood disorders: implications for the development of novel therapeutics[END_REF][45][START_REF] Chuang | Neuroprotective and neurotrophic actions of the mood stabilizer lithium: can it be used to treat neurodegenerative diseases?[END_REF]. Since CREB has major roles in mediating adaptive responses and neuroprotection in the brain [47], the discovery of the lithium enhancement of TORC-CREB binding thus suggests its therapeutic relevance. In cortical neuronal, for example, the addition of glutamate with glycine increases the free intracellular magnesium ion concentration of approximately 300 µM to levels that are almost 10-fold higher [START_REF] Brocard | Glutamate-induced increases in intracellular free Mg2+ in cultured cortical neurons[END_REF] and contribute to glutamate-induced excitotoxicity [49]. In view of the results of the present study, it is not unreasonable to suggest that such fluxes in the concentration of magnesium ion could disrupt TORC-CREB interaction and that lithium could reverse this effect and increase the binding of TORC to CREB, thus increasing CREB activity and the expression of CREB target genes like BDNF and Bcl-2 [START_REF] Chuang | Neuroprotective effects of lithium in cultured cells and animal models of diseases[END_REF][START_REF] Chuang | Neuroprotective and neurotrophic actions of the mood stabilizer lithium: can it be used to treat neurodegenerative diseases?[END_REF] that promote cell survival. Furthermore, TORC has been shown to be required for long-term synaptic plasticity in the hippocampus [START_REF] Zhou | Requirement of TORC1 for late-phase long-term potentiation in the hippocampus[END_REF][START_REF] Kovacs | TORC1 is a calcium-and cAMP-sensitive coincidence detector involved in hippocampal long-term synaptic plasticity[END_REF]. Thus the stimulation by lithium of the binding of TORC to CREB, as demonstrated by the present study, represents a novel mechanism of lithium action that may contribute to the clinical mood stabilizing effect of lithium salts. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Bioscience

  Reports Immediate Publication. Published on 22 Aug 2008 as manuscript BSR20080116 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080116

  Bioscience Reports Immediate Publication. Published on 22 Aug 2008 as manuscript BSR20080116 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080116

Bioscience

  Reports Immediate Publication. Published on 22 Aug 2008 as manuscript BSR20080116 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080116

Bioscience

  Reports Immediate Publication. Published on 22 Aug 2008 as manuscript BSR20080116 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080116A 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.

Figure 1

 1 Figure 1 Enhancement by cAMP but not LiCl of the nuclear accumulation of TORC in HIT cells as revealed by immunocytochemistry. (A) Co, controls without treatment; cAMP, treated with 2 mM 8Br-cAMP for 30 min; LiCl, treated with 20 mM LiCl for 90 min. (B) 300 cells per group were analyzed with respect to the localization of TORC, expressed as percentage of cells with nuclear TORC. Values are means ± SEM. * P < 0.05, ** P < 0.025, two-way ANOVA and Student's t-test.

Figure 2

 2 Figure 2 Lack of effect of LiCl on transactivation by TORC1. (A) Scheme of the GAL4fusion constructs and the luciferase reporter gene used. GAL4 represents the DNA-binding domain of the yeast transcription factor GAL4 (aa1-147). GAL4-TORC1, full length TORC1 (aa1-651) fused to GAL4. (B) GAL4-TORC1 and the luciferase reporter gene (5xGal4E1B-Luc) were transiently co-transfected into HIT cells. Cells were treated as indicated with 20 mM LiCl 7 h prior to harvest and with 2 mM 8Br-cAMP 6 h prior to harvest. Luciferase activity is expressed relative to the mean value of the control (no treatment). Values are means ± SEM of three independent experiments each performed in duplicate. *** P < 0.001, two-way ANOVA and Student's t-test.

Figure 3

 3 Figure 3 Lithium increased the recruitment of TORC1 to the promoter in the presence of cAMP as revealed by ChIP assay in HIT cells. (A) Scheme of the FLAG-tagged TORC1 construct and the reporter plasmid used. FLAG-TORC1 represents TORC1 (aa1-651) containing a FLAG epitope (DYKDDDDK) between aa1 and aa2. The reporter plasmid contains 4 repeats of the CRE as high affinity binding site for endogenous CREB. TK, thymidine kinase promoter. (B) Expression vectors were transiently co-transfected into HIT cells. Treatment with 20 mM LiCl was for 90 min and treatment with 2 mM 8Br-cAMP was for 30 min. After treatment the proteins and DNA were cross-linked and DNA was precipitated using anti-FLAG antibody. The amount of precipitated DNA was determined by quantitative real-time PCR and is expressed relative to the mean value of the control (no treatment). Values are means ± SEM of five independent experiments. * P < 0.05, ** P < 0.025, two-way ANOVA and Student's t-test.

Figure 4

 4 Figure 4Enhancement by lithium of the interaction between CREB-bZip and TORC1 1- 44 as revealed by the mammalian two-hybrid assay. (A) Scheme of expression vectors and the luciferase reporter gene used. VP16 comprises the transactivation domain (aa411-490) of the viral protein VP16. VP16-bZip-wt, CREB-bZip encompassing aa269-327 C-terminally fused to VP16. VP16-bZip-R300A, CREB-bZip mutant Arg300Ala fused to VP16. HIT cells were transiently transfected with the luciferase reporter gene 5xGal4E1B-Luc and GAL4-TORC1 1-44 representing the CREB-interaction domain of TORC1 (aa1-44) fused to the DNAbinding domain of GAL4 (aa1-147). Co-transfected was either VP16 as control, VP16-bZipwt, or VP16-bZip-R300A. (B) HIT cells were transfected as indicated and treated with 20 mM LiCl 30 h prior to harvest, and with 1 mM 8Br-cAMP 6 h prior to harvest. Luciferase acitivity is expressed relative to the control VP16. Values are means ± SEM of three independent experiments each performed in duplicate. *** P < 0.001, two-way ANOVA and Student's t-test.

Figure 5

 5 Figure 5 Enhancement by lithium of the protein-protein interaction between CREB and TORC1 in the in vitro GST pull-down assays. (A) Recombinant GST-CREB wild type or Arg300Ala mutant (GST-CREB-wt or GST-CREB-R300A, respectively) were employed in pull down assays with [ 35 S]-labeled TORC1. To control for specificity of the interaction, GST alone was also used. The samples were analyzed by SDS-PAGE and densitometry of the bands representing [ 35 S]TORC1. A typical gel is shown at the bottom. Values are expressed relative to the control in each experiment and are means ± SEM of three independent experiments. * P < 0.05, ** P < 0.025, *** P < 0.001, two-way ANOVA and Student's t-test. (B) Concentration-response curve. GST-CREB-wt and [ 35 S]TORC1 were incubated with increasing concentrations of LiCl. A typical gel is shown at the bottom. Values are means ± SEM of seven independent experiments. * P < 0.05, ** P < 0.025, two-way ANOVA and Student's t-test.

Figure 6

 6 Figure 6 Effect of magnesium on the interaction between CREB and TORC1 in the in vitro GST pull-down assays. (A) Recombinant GST-CREB and [ 35 S]-labeled TORC1 were incubated with increasing concentrations of MgCl 2 . To control for specificity, GST alone was used. The samples were analyzed by SDS-PAGE and the amount of [ 35 S]TORC1 was determined by densitometry. A typical gel is shown at the bottom. Values are expressed relative to the GST-CREB control (no MgCl 2 ) in each experiment and are means ± SEM of three independent experiments. * P < 0.05, ** P < 0.025, *** P < 0.001, two-way ANOVA and Student's t-test. (B) GST-CREB and [ 35 S]TORC1 were incubated with increasing concentrations of MgCl 2 in the presence or absence of a constant concentration of 5 mM LiCl. Values are expressed relative to the control of each group (no MgCl 2 ) and are means ± SEM of four independent experiments. * P < 0.05, two-way ANOVA and Student's t-test. (C) To demonstrate the effect of lithium on top of magnesium, the values from Figure 6B without lithium at each MgCl 2 concentration were set as 100% and the values with 5 mM LiCl were expressed relative to these.

Figure 7 A 1 Bioscience 2 Bioscience

 712 Figure 7 A new mechanism of action of lithium. Taken together, the data of the present study suggest that, once TORC has entered the nucleus, lithium stimulates the binding of TORC1 to the CREB bZip, thereby increasing cAMP-induced CREB target gene transcription. See discussion for details.

Figure 3 Bioscience

 3 Figure 3

Figure 7 Bioscience

 7 Figure 6

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

© 2008 The Authors Journal compilation © 2008 Biochemical Society

Bioscience Reports Immediate Publication. Published on 22 Aug 2008 as manuscript BSR20080116 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BSR20080116 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 Biochemical Society

ACKNOWLEDGEMENTS

The work was funded partly by grants from the Heidenreich-von Siebold-Programm of the University of Göttingen (to E.O.) and from the Deutsche Forschungsgemeinschaft, SFB 403/A3 (to W.K.). A.H. receives a Georg-Christoph-Lichtenberg thesis grant from the Government of Lower Saxony and is enrolled in the MSc/PhD Program Neurosciences at the University of Goettingen. We are grateful to Dr. Ralph Krätzner for methodological discussion provided for GST pulldown assays. The excellent technical assistance of Doris Krause is also gratefully acknowledged.

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.