Neuropharmacology 39 (2000) 1628–1636 www.elsevier.com/locate/neuropharm
Retinoic acid negatively regulates neuropeptide Y expression in human neuroblastoma cells Paolo Magni *, Elena Beretta, Eugenia Scaccianoce, Marcella Motta Center for Endocrinological Oncology, Institute of Endocrinology, University of Milan, Milan, Italy Accepted 8 November 1999
Abstract Retinoids are involved in the regulation of development and differentiation in many tissues, including the nervous system, where they have been associated with some neurotransmitter systems. In the present study, we evaluated the effects of all-trans retinoic acid (RA) on the biosynthesis and secretion of neuropeptide Y (NPY), a widely expressed neuroregulatory peptide. The SH-SY5Y human neuroblastoma cell line has been used as the in vitro model system. Treatment with 10 µM RA induced a marked decrease in NPY gene expression after as little as 3–6 h of incubation and resulted in its almost complete suppression at 12–24 h and after a 6-day differentiating treatment. The NPY content in cell extracts and the NPY secreted and accumulated in the culture medium were also reduced by exposure to 10 µM RA at 12 and 24 h and at 6 days. Moreover, RA treatment for 6 days, but not for 24 h, resulted in a marked stimulation of proNPY processing to mature NPY. The presence of negative retinoic acid-response elements in the human NPY promoter (up to ⫺1078 bp) was excluded by a computer search. When SH-SY5Y cells were treated simultaneously with 20 nM TPA and 10 µM RA for 24 h, the marked stimulatory effect of TPA alone was completely suppressed. These observations suggest that the expression of NPY in SH-SY5Y human neuroblastoma cells is negatively regulated by RA at the level of gene expression, probably by mechanisms involving the interaction of activated RARs with transcription factors (such as AP-1). 2000 Elsevier Science Ltd. All rights reserved. Keywords: Retinoic acid; Neuropeptide Y gene expression; Neuropeptide Y biosynthesis; Neuroblastoma; Cell differentiation; Phorbol ester
1. Introduction Vitamin A and the retinoids, its metabolic derivatives, play a crucial role in the development and differentiation of several organs and tissues, including the nervous system. A correct embryogenesis requires precise timing of the exposure of embryonal structures to retinoic acid and a co-ordinated pattern of expression of the retinoic acid receptor (RAR) and the retinoic X receptor (RXR) isoforms (Smith et al., 1988). At the molecular level, this process results, for example, in the precise regulation of the expression of Hox homeobox genes (Marshall et al., 1996). In addition to these developmental actions, retinoids also play an important role in the function of the adult brain, which has been shown to synthesize retinoic
* Corresponding author. Tel.: +39-02-205213219; fax: +39-0229404927. E-mail address:
[email protected] (P. Magni).
acid (Dev et al., 1993), and to express retinoid receptors, as well as cellular retinoid-binding proteins, in many regions (Zetterstrom et al., 1994). Interestingly, the process of brain aging is associated in the mouse with reduced levels of RAR and RXR, and this decrease is reversed by retinoic acid treatment (Enderlin et al., 1997). Retinoids have also been shown to interact in a specific manner with some neurotransmitter systems. Retinoic acid stimulates the expression of dopamine D2 receptors (Samad et al., 1997) in rat pituitary cells, and promotes the selective survival of spinal cord cholinergic neurons, but does not affect GABAergic neurons (Waring and Sidel, 1991). Neuropeptide Y (NPY) is another widely expressed neuromodulator, which displays a pleiotropic spectrum of functions, including the regulation of cognitive functions, of food intake, and of energy balance (Wettstein et al., 1995). Interestingly, retinoic acid is also involved in the complex regulation of thermogenesis and energy expenditure, since it has been shown to modulate uncoupling protein 1 and leptin
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gene expression (Alvarez et al., 1995; Kumar and Scarpace, 1998). Several agents are known to control the expression of NPY: classical neurotransmitters (Magni et al., 1998), peptide and steroid hormones (Chan et al., 1996; Higuchi et al., 1988a), and growth factors (Allen et al., 1987; Barnea et al., 1993; Minth-Worby, 1994). However, the possible direct effects of retinoic acid on NPY expression, which might be relevant in the regulation of different brain functions, have not yet been explored. The present study was therefore designed to evaluate the actions of all-trans retinoic acid (RA) on some steps of NPY biosynthesis. The SH-SY5Y human neuroblastoma cell line, utilized in this study as an in vitro model of human neurons, has the capability to synthesize and secrete NPY in a regulated fashion (Magni et al., 1998). Moreover, these cells are responsive to treatment with RA, which induces them to differentiate to mature cholinergic neurons (Pa˚hlman et al., 1984). The effect of RA treatment on NPY biosynthesis and secretion in SH-SY5Y cells has been evaluated at different levels, such as NPY gene expression, NPY peptide synthesis and secretion, as well as processing of the precursor pro-NPY to mature NPY. In addition, some possible molecular mechanisms of RA action on NPY gene expression have been investigated.
2. Methods 2.1. Cell cultures and experimental design SH-SY5Y cells (kindly provided by Dr June Biedler, Memorial Sloan-Kettering Cancer Center, New York, NY, USA) were grown at 37°C in a humidified CO2 incubator in monolayer in Minimum Essential Medium containing non-essential amino acids, 1 mM sodium pyruvate, 100 µg streptomycin/ml, 100 UI penicillin/ml and 10 mg/l Phenol Red (Biochrom, Berlin, Germany). Medium was supplemented with 10% FCS (fetal calf serum; Gibco, Grand Island, NY, USA) and was replaced at 3-day intervals. Confluent cells were harvested with 0.05/0.02% trypsin/EDTA (Biochrom, Berlin, Germany) and were seeded in Petri dishes (100 mm diameter; 0.8x106 cells/dish; Becton-Dickinson, Plymouth, England, UK) for gene expression experiments or in 6-well plates for peptide studies. Experiments were performed using subconfluent cell cultures (typically, 80–90% confluence after seven days in culture). For short-term treatments (3–24 h), six days after plating the culture medium was replaced with FCS-free medium containing the experimental substances (RA, 12-O-tetradecanoylphorbol-13-acetate (TPA); Sigma Chemicals, St Louis, MO, USA) and the cell cultures were carried on for the required time. The 6-day incubation protocol was as follows: two days after plating, the culture medium was replaced with 13 ml (100 mm Petri dishes) or 3 ml
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(6-well plates) experimental medium containing the test substances. After five days, the medium was replaced with identical fresh medium without FCS (10 ml for 100 mm Petri dishes and 0.7 ml for 6-well plates) and the incubation was carried on for another 24 h. For gel-filtration chromatography studies, each experiment included cells treated for either six days or for 24 h, as well as one set of controls, which represents the same group of control for both 24-h and 6-day treated cells; all cells were collected the same day. For peptide studies, medium was collected, boiled for 10 min, centrifuged for 5 min at 1,000 g; the supernatant was stored at ⫺20°C until NPY assay. Cells were washed with cold phosphate-buffered saline (PBS), collected in PBS with a rubber policeman, boiled for 10 min, disrupted by sonication and stored at ⫺20°C until NPY and protein assay. For RNA studies, cells were washed with cold PBS, collected with a rubber policeman, snap-frozen in liquid nitrogen and stored at ⫺80°C until RNA extraction. 2.2. Total RNA extraction and Northern blot analysis Cellular pellets were placed into a guanidium thiocyanate denaturing solution and total RNA was purified from contaminants by phenol/chloroform extraction and isopropanol precipitation (Chomczinski and Sacchi, 1987). Total RNA (30 µg/lane) was fractionated by 1.2% agarose gel electrophoresis and transferred by the capillarity method onto nylon membrane (Hybond N; Amersham Italia, Milan, Italy), as previously described (Magni and Barnea, 1993). RNA was cross-linked to the membrane by baking at 80°C for 2 h. NPY mRNA was hybridized using a 32P-cDNA probe, obtained by random-prime labeling of the ECORI-ECORI insert (0.540 kb) of the pNPY3-75 plasmid (kindly donated by Dr Carolyn Worby, University of Michigan, Ann Arbor, MI, USA). Equal loading of different lanes was assessed by hybridization of the same blots with a 1.46 kb cDNA probe, random-prime labeled with 32P, specific for the housekeeping gene encoding for GAPDH (glyceraldehyde 3-phosphate dehydrogenase; American Type Culture Collection, Rockville, MD, USA). The hybridization protocol included a 4-h pre-hybridization at 42°C, in a buffer containing 50% formamide, 5×SSPE, 5×Denhardt’s solution, 0.5% sodium dodecyl sulfate (SDS) and 100 mg/ml yeast tRNA (Boehringer Mannheim Italia, Milan, Italy), followed by a 16-h hybridization (106 c.p.m./ml) at 42°C in the same buffer. Blots were then washed with 2×standard saline citrate (SSC)/0.1% SDS, 1×SSC/0.1% SDS and 0.5×SSC/0.1% SDS (1×SSC=150 mM NaCl, 15 mM sodium citrate, pH 7.0); each wash was carried on at room temperature and for 30 min. Blots were exposed to autoradiographic films (Hyperfilm; Amersham Italia, Milan, Italy) for 24–72 h at ⫺70°C. NPY mRNA data were normalized by
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GAPDH mRNA data, in order to compensate for unequal lane loading. Each Northern blot experiment was performed at least three times for each treatment. Autoradiographic bands were subjected to densitometric analysis, using a Macintosh computer and scanner, and the Image 1.60 program (National Institutes of Health, Bethesda, MD, USA).
mean±S.E.M.; n=number of replicates within an experiment or a pool of separate experiments. Significance of differences between treatment groups was evaluated by analysis of variance, followed by Tukey test.
2.3. NPY radioimmunoassay and protein assay
3.1. Effect of treatment with RA on NPY gene expression in SH-SY5Y neuroblastoma cells
NPY was quantified by radioimmunoassay using a PBS buffer containing 0.25% bovine serum albumin (BSA). The antiserum CHO (kindly supplied by Dr Ayalla Barnea, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, Dallas, TX, USA), which recognizes both mature NPY and its precursor proNPY, was previously chacterized (Barnea and Cho, 1993) and was used at a final dilution of 1:133,000; synthetic hNPY (Peninsula Laboratories, Belmont, CA) was the reference standard and [125I]pNPY (Amersham Italia, Milan, Italy) was used as the tracer. Samples/standard were first incubated with the antibody for 24 h at 4°C and then the tracer was added (about 7,000 c.p.m.) for additional 24 h. Bound and free NPY were separated by precipitation with 80% ethanol. The lowest level of sensitivity of the assay was 3.9 pg/tube. Protein was assayed by the method of Bradford (1976), using BSA as the standard. 2.4. Gel-filtration chromatography Aliquots of cell extracts or medium were lyophilized and redissolved in a buffer containing 1% formic acid and 0.1% BSA, and then fractionated by gel filtration on columns (60×1 cm) of Sephadex G50 SF (Pharmacia Biotech Italia, Milan, Italy), with running buffer of 1% formic acid and 0.1% BSA; 0.7-ml fractions were collected, lyophilized and then assayed for NPY (de Quidt and Emson, 1986). Recovery of NPY-IR after gel filtration was 79% and the column was calibrated with Dextran Blue (void volume), cytochrome C (molecular weight 12,400 Da, eluted in fraction 6), aprotinin (molecular weight 6,500 Da, eluted in fraction 13), synthetic human NPY (Peninsula Laboratories, Belmont, CA, USA) and Phenol Red (salt volume). In order to obtain well-defined chromatographic peaks, the aliquots loaded onto the column contained similar amounts of total NPY-immunoreactivity (IR). For this reason, gelchromatographic profiles related to controls and to RAtreated cells do not show quantitative variations, but give qualitative information (Fig. 5). 2.5. Analysis of the data Statistical analysis was performed by using the Systat statistical analysis package. Data are given as
3. Results
Retinoids are believed to exert their effects by acting at the genomic level. Thus, we started to evaluate the effects of RA on NPY synthesis at the level of gene expression. Non-differentiated SH-SY5Y cells express NPY mRNA transcripts of the expected size (0.8 kb), at a level detectable by Northern blot analysis (Fig. 1). Fig. 2 shows the effect of a 24-h treatment with different concentrations of RA on NPY gene expression, with 10 µM RA being the most effective. Treatment with 10 µM RA (Fig. 1) resulted in a progressive reduction of NPY mRNA levels, that was already detectable at 3 and 6 h (about ⫺40% at both time-points, as quantified by densitometric analysis of the autoradiographic bands; data not shown). By 12 and 24 h the NPY gene expression was actually almost completely abolished (⫺80%). An inhibition of NPY gene expression was observed after a 6day treatment with RA also; in this case, the decrease brought the residual NPY gene expression below the detection limit of the technique (Fig. 1). In addition the 6-day exposure to RA induced a marked differentiation of neuroblasts towards a more neuron-like morphology (Fig. 3), in accordance with the information present in the literature (Pa˚hlman et al., 1984; Preis et al., 1988). 3.2. Effect of treatment with RA on the production and secretion of NPY in SH-SY5Y neuroblastoma cells It was then evaluated whether the above-reported inhibition of NPY gene expression was reflected by a reduction in the production of the peptide. The effect of RA on the regulation of NPY production and accumulation in the culture medium was studied by treating SHSY5Y cells with 10 µM RA for a time ranging between 3 and 24 h, and for six days. The NPY-IR (NPYimmunoreactivity) present in cell extracts and in the culture medium at the end of the incubation was measured by RIA. Untreated SH-SY5Y cells synthesize and secrete NPY-IR (about 50 ng/mg prot/24 h) with a residual content in the cells of about 8–15 ng/mg protein. Exposure to 10 µM RA resulted in a significant progressive decrease of the NPY-IR in cell extracts starting at 12 h and becoming greater at 24 h and at six days (Fig. 4, panel A). The NPY-IR accumulated in the culture medium was also significantly reduced after 24-h and 6day incubations (Fig. 4, panel B).
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Fig. 1. Effect of treatment with RA on NPY gene expression (Northern blot analysis). Cells were treated for 3, 6, 12, 24 h and six days with 10 µM RA. Total RNA (30 µg/lane) was analyzed by Northern blot hybridization (C=control; RA=all-trans retinoic acid). The stripped blots were rehybridized for GAPDH mRNA for normalization. One Northern blot, representative of three separate experiments, is shown.
Fig. 2. Effect of the combined treatment with RA and TPA on NPY gene expression (Northern blot analysis). Cells were treated for 24 h with RA at different concentrations, in the presence of 20 nM TPA, or in its absence. Total RNA (30 µg/lane) was analyzed by Northern blot hybridization (C=control; RA=all-trans retinoic acid). The stripped blots were rehybridized for GAPDH mRNA for normalization. One Northern blot, representative of three separate experiments, is shown.
Fig. 3. Morphological changes of SH-SY5Y human neuroblastoma cells after differentiation with RA. Cells were treated for six days with 10 µM RA (all-trans retinoic acid) or solvent (C=control). The effect of treatment with 20 nM TPA for six days is shown for comparison.
3.3. Effect of treatment with RA on the posttranslational cleavage of the precursor proNPY to NPY in SH-SY5Y neuroblastoma cells The NPY-IR produced by SH-SY5Y cells has been characterized by size-fractionation by means of gel-fil-
tration chromatography. It was found that untreated SHSY5Y cells actually contain and secrete into the medium two NPY-IR molecular species, both recognized by the antiserum used in the radioimmunoassay (RIA): one of about 4.2 kDa, coeluting with synthetic human NPY (which has been used as the standard), and another of
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Fig. 4. Effect of treatment with RA on NPY synthesis/secretion. Cells were treated for 3, 6, 12, 24 h and six days with 10 µM RA and the NPYIR content in cell extracts and in the medium was quantified by RIA. Panel A: effect of RA treatment on NPY-IR content in cell extracts. Panel B: effect of RA treatment on NPY-IR content in the medium. One experiment representative of three or four separate experiments is shown. Data are expressed as mean±S.E.M. (percent of controls=100). C=control; RA=all-trans retinoic acid; n=6; *P⬍0.05 vs C; **P⬍0.01 vs C; ***P⬍0.001 vs C (ANOVA).
about 8.0 kDa, which most likely represents the precursor pro-NPY (Fig. 5, dotted lines). The ratio NPY/proNPY is about 30/70% in cell extracts and 50/50% in the medium. A 24-h treatment with 10 µM RA did not affect the relative ratio of proNPY to NPY in either the cell extracts or the conditioned medium. However, a 6-day treatment resulted in a relative increase of the 4.2 kDa immunoreactive species (mature NPY), which reached 50% in the cell extracts and 70% in the medium (Fig. 5).
3.4. RA treatment interferes with TPA-induced expression of the NPY gene The preceding observation that RA treatment results in a marked inhibition of NPY gene expression prompted us to begin to study the molecular mechanisms underlying this phenomenon. Retinoids have been reported to exert negative effects at the genomic level by acting through different mechanisms, like the interaction of the RAR/RXR with a negative retinoic acid-response
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Fig. 5. Effect of 24-h and 6-day treatments with 10 µM RA on NPY precursor processing (cell extracts and conditioned medium). One experiment representative of 3–5 separate experiments is shown. Cell extracts (panel A) and conditioned media (panel B) were size-fractionated by gel filtration chromatography and the NPY-IR in each fraction was quantified by RIA. Dotted line: C (control); solid line: RA (all-trans retinoic acid). Please note that, for the purpose of clarity, the elution profiles of samples from cells treated for 24 h and for six days are separately superimposed to the same control elution profile (dotted line). In the upper left and in the lower right profiles is indicated the elution position of synthetic human NPY (“NPY std”), used as the reference standard, and the immunoreactive peak corresponding to the proNPY-like substance (“proNPY-like”). The table under each panel summarizes the data of gel filtration chromatography obtained in all the experiments performed and includes the representative chromatograms shown above them. The data (mean±S.E.M.) relative to the proNPY-like and to the NPY peaks are expressed as % of total NPYIR (total NPY-IR=sum of the areas under the curve relative to the two chromatographic peaks — proNPY-like and NPY —).
element (nRARE) (Lipkin et al., 1992) or the interference of these receptors with the action of transcription factors such as AP-1 (Schuele et al., 1991). By means of a CBIL-TESS computer search, we could exclude the presence of a nRARE in the 5⬘ promoter region (up to 1078 bp upstream of the transcription start site) of the human NPY gene (Minth and Dixon, 1990). However,
the NPY promoter contains a proximal AP-1 responsive element, which is required for the activation of transcription of this gene by phorbol esters (Andersson et al., 1994; Jalava and Mai, 1994; Minth et al., 1986). To verify whether the stimulatory effect of the phorbol ester TPA might be affected by the simultaneous treatment with RA, SH-SY5Y cells were treated with 10 µM RA
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or 20 nM TPA, or with the two agents in association. As shown in Fig. 6, TPA treatment for 24 h markedly stimulated the expression of the NPY gene (+400%); such an effect was totally abolished by the simultaneous exposure to 10 µM RA, which brought the NPY mRNA levels to about 50% of the controls, according to the densitometric analysis of the autoradiogram shown in Fig. 6. TPA stimulation of NPY gene expression was also blocked or reduced at lower concentrations of RA (10 nM RA; Fig. 2). It is interesting to note that, in the presence of both TPA and RA, cells displayed a morphology more similar to that obtained with TPA alone (see Fig. 3), even if the effect of RA on NPY gene expression was prevalent.
4. Discussion The findings of the present study indicate that RA exerts a potent negative control on the expression of the human NPY gene in the neuroblastoma cell line SHSY5Y. To our knowledge, this observation is reported here for the first time. Possibly as a consequence of this action, RA treatment results also in a marked reduction in NPY biosynthesis and secretion. The effect on NPY gene expression as the primary event is suggested by the known mechanism of action of retinoids, and by the time kinetics of the inhibition exerted by RA on NPY dynamics, with the reduction of gene expression preceding that of NPY cell content and NPY accumulation in the medium. The expression of RARs and RXRs has been described in several RA-responsive neuroblastoma cell lines, including the SH-SY5Y (Haussler et al., 1983). Retinoids, like glucocorticoids (Reichardt and Schutz, 1998), may down-regulate the expression of several responsive genes in different ways. Thus, different possibilities may be considered to explain the observed inhibitory action of RA on NPY gene expression. At least up to ⫺1078 bp, a putative proximal nRARE does not seem
to be present in the NPY gene promoter, and therefore a protein-DNA functional binding should be excluded. Consequently, it seems reasonable that a direct protein– protein interaction of negative nature might be operative in this case. It has been shown for other genes that RARAR complexes may interact with the c-Jun monomer component of the AP-1 protein dimer in a DNA-independent manner, and prevent it from activating gene transcription via binding to the AP-1 DNA response element (Schuele et al., 1991). This might be the case for the human NPY gene, which, in its promoter region, possesses an AP-1-like sequence (Minth et al., 1986), able to bind the Fos-Jun protein complex after treatment with TPA (Jalava and Mai, 1994). Our finding that the stimulatory effect of TPA on NPY gene expression (which works through AP-1) is abolished by the presence of RA clearly supports this hypothesis. It remains to be established which RAR/RXR isoforms are involved in this process, and whether they work in a monomeric or in a dimeric form. According to Schuele (Schuele et al., 1991), RARs, but not RXRs, are involved in the inhibition of AP-1 activity on the expression of the human collagenase gene. It is presently unknown whether this action of RA results from a direct modulation of NPY gene transcription or from an effect on mRNA stability, or both these events. Moreover, some direct effects of RA on NPY biosynthetic steps located downstream of gene expression (i.e., translation and secretion) might not be excluded. Particularly significant appears the stimulation of proNPY processing after RAinduced cell differentiation for six days. This seems to be related more to the differentiation of SH-SY5Y cells to neurons, than to a direct action of RA on the NPY biosynthetic process, since these effects take place within a much shorter time interval (6–24 h). It is possible that a 6-day exposure to RA results in the induction of processing enzymes, like the prohormone convertases 1 and 2, which catalyze the cleavage of peptide prohormones at dibasic sites (Steiner et al., 1992), like the
Fig. 6. Effect of the combined treatment with RA and TPA on NPY gene expression (Northern blot analysis). Cells were treated for 24 h with 10 µM RA, 20 nM TPA alone or in association. Total RNA (30 µg/lane) was analyzed by Northern blot hybridization (C=control; RA=all-trans retinoic acid). The stripped blots were rehybridized for GAPDH mRNA for normalization. One Northern blot, representative of three separate experiments, is shown.
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Lys38-Arg39 present in the proNPY sequence. In this respect, it has recently been reported that, in the human neuroblastoma cell line SK-N-BE, retinoids increase the expression of metallopeptidase N-arginine dibasic convertase, another enzyme involved in peptide processing (Draoui et al., 1997). RA seems to represent an overall downregulator of the NPY system, since, in addition to the inhibitory effect on the expression of NPY here reported, RA is also able to reduce the expression of the NPY-Y1 receptor (Y1-R) (Mannon and Kaiser, 1997). Taken together, these data suggest that common molecular mechanisms might be utilized by RA to regulate in a coordinated manner the expression of the NPY gene and of the Y1-R. The physiological and pathophysiological relevance of the inhibitory effect of NPY expression by RA remains to be clarified. Differentiation therapy with retinoids has been proposed for various malignant tumors, including neuroblastomas (Finklestein et al., 1992). RA treatment not only induces cell differentiation, but also reduces the proliferation of various neuroblastoma-derived cell lines, including the SHSY5Y (Breitman et al., 1980), with reversion of the malignant phenotype. Neuroblastomas are normally very aggressive, but, in some cases, the tumors show a spontaneous evolution towards more benign forms (stage IVS), which is accompanied by an increased expression of the NPY gene. Similarly, the induction of in vitro differentiation of SH-SY5Y cells by the phorbol ester TPA towards an adrenergic phenotype is associated with an increase of NPY synthesis (Pa˚hlman et al., 1984). On the contrary, RA-stimulated differentiation of these cells, as shown in the present study, is associated with a marked decrease of NPY expression. These observations underline the complexity of the interplay between differentiating agents, differentiation markers and proliferation factors (Melino et al., 1997). The present data might also be relevant in the context of the control of energy metabolism and food intake, whose regulation derives from a complex network of signals, which, among others, include NPY and retinoids (Kumar and Scarpace, 1998). Future researches will be addressed to unravel the fine molecular mechanisms of the RA action on NPY gene expression, as well as the significance of the RA-NPY relationship in the control of energy metabolism and of brain aging. Acknowledgements This work was supported by MURST and by AIRC. E.S. is the recipient of a research fellowship by FIRC. The authors are grateful to Prof. Luciano Martini for his precious advice and continuous support and to Dr. Roberto Maggi for much red ink on the original draft. Thanks are due to Ms. Paola Assi, Ms. Giovanna Micciche` and Ms. Ornella Mornati for their skillful technical collaboration.
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