Sirt1 inhibits the transcription factor CREB to ... - The FASEB Journal

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Sirt1 inhibits the transcription factor CREB to regulate pituitary growth hormone synthesis Jose Monteserin-Garcia,* Omar Al-Massadi,†,‡ Luisa M. Seoane,†,‡ Clara V. Alvarez,§ Bing Shan,* Johanna Stalla,* Marcelo Paez-Pereda,* Felipe F. Casanueva,‡ Günter K. Stalla,* and Marily Theodoropoulou*,1 *Department of Endocrinology, Max Planck Institute of Psychiatry, Munich, Germany; †Endocrine Physiopathology Group, University Hospital Complex of Santiago de Compostela/Servizo Galego de Saúde (SERGAS), Santiago de Compostela, Spain; and ‡Department of Medicine and §Centro de Investigaciones Medicas e Instituto de Investigaciones Sanitarias (CIMUS-IDIS), Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain Growth hormone (GH) is a major anabolic hormone and the primary regulator of organism growth. Its transcription is triggered by GH-releasing hormone (GHRH) through the transcription factor cAMP response element-binding protein (CREB) and by caloric intake. In contrast, the deacetylase Sirt1 is activated by caloric restriction. Therefore, the present study investigates how Sirt1 affects CREB function and GH synthesis. Sirt1 pharmacological activation with resveratrol (IC50ⴝ87 ␮M) suppressed GHRH-induced GH secretion from rat anterior pituitary cells in vivo and in vitro, while vehicle controls showed no effect. Resveratrol’s effects were abolished after knocking down Sirt1 with RNA interference, but not in control scrambled siRNA-transfected rat somatotrophs, confirming the Sirt1 specificity. Sirt1 activation and overexpression suppressed forskolin-induced CREB-Ser133 phosphorylation, but no effect was seen with vehicle and empty plasmid controls. The deacetylase-dead mutant Sirt1 retained CREB-Ser133 phosphorylation by keeping protein phosphatase protein phosphatase 1 activity low. Sirt1 activation suppressed glycogen synthase kinase 3 ␤ acetylation, and a mutation on the GSK3␤-Lys205 residue mimicking a hypoacetylated form revealed increased activity. In summary, this is a novel mechanism through which Sirt1 intercepts the cAMP pathway by suppressing CREB transcriptional activation, ABSTRACT

Abbreviations: ␤Gal, ␤-galactosidase; ACTH, adrenocorticotrophic hormone; AUC, area under the curve; CRE, cAMPresponsive element; CREB, CRE-binding protein; FSH, follicle-stimulating hormone; GH, growth hormone; GHRH, growth hormone-releasing hormone; GS, glycogen synthase; GSK3␤, glycogen synthase kinase 3␤; HDAC1, histone deacetylase 1; IGF-I, insulin-like growth factor I; I-2, inhibitor 2; Luc, luciferase; mTOR, mammalian target of rapamycin; mTORC1, mammalian target of rapamycin complex 1; NAD, nicotinamide adenine dinucleotide; PDK1, 3-phosphoinositide dependent protein kinase 1; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PP1, protein phosphatase 1; PRL, prolactin; RSV, resveratrol; S6K, S6 kinase; siRNA, single interfering RNA; TSC2, tuberus sclerosis complex 2; TSH, thyroid-stimulating hormone 0892-6638/13/0027-1561 © FASEB

resulting in decreased GH synthesis.—Monteserin-Garcia, J., Al-Massadi, O., Seoane, L. M., Alvarez, C. V., Shan, B., Stalla, J., Paez-Pereda, M., Casanueva, F. F., Stalla, G. K., Theodoropoulou, M. Sirt1 inhibits the transcription factor CREB to regulate pituitary growth hormone synthesis. FASEB J. 27, 1561–1571 (2013). www.fasebj.org Key Words: resveratrol 䡠 GSK3␤ 䡠 phosphatase 䡠 acetylation The growth hormone (GH)/insulin-like growth factor I (IGF-I) axis is the most important neuroendocrine regulator of growth, metabolism, and life span. GH is synthesized and secreted by somatotroph cells of the anterior pituitary gland and acts directly or indirectly though hepatically produced IGF-I (1, 2). Chronic GH excess, as seen in patients with acromegaly-associated pituitary adenomas, is accompanied by cardiovascular complications, insulin resistance, and increased mortality (3, 4). In contrast, genetic inhibition of the GH/ IGF-I axis improves overall metabolism and increases life span (5, 6). Similarly, patients with blunted GH response due to GH receptor deficiency have no incidence of type II diabetes mellitus compared to their unaffected relatives, despite the higher occurrence of obesity (7). Pituitary GH synthesis is stimulated by the hypothalamic growth hormone-releasing hormone (GHRH) through the cAMP pathway (2). In brief, GHRH-induced cAMP levels activate protein kinase A (PKA) that phosphorylates cAMP-responsive element (CRE)-binding protein (CREB) at Ser133. CREB triggers human GH transcription directly and indirectly through the pituitary transcription factor Pit-1 (8 –11). In contrast, rat GH promoter lacks CRE and depends exclusively on Pit-1 to mediate the stimulatory cAMP signal (12, 13). 1 Correspondence: Department of Endocrinology, Max Planck Institute of Psychiatry, Kraepelinstrasse 10, Munich 80804, Germany. E-mail: [email protected] doi: 10.1096/fj.12-220129

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CREB activation is transient, and CREB-Ser133 gets dephosphorylated by the serine/threonine protein phosphatase 1 (PP1) recruited by histone deacetylase 1 (HDAC1) (14, 15). PP1 is activated when it dissociates from its inhibitor, inhibitor 2 (I-2). I-2 phosphorylation at Thr72 is a prerequisite for this to happen and is primarily carried out by glycogen synthase (GS) kinase 3␤ (GSK3␤) (16, 17). Phosphorylation at the GSK3␤-Ser9 residue downstreams to mitogenic stimuli blocks the substrate’s access to the active site and renders GSK3␤ inactive (18). Two major kinases that phosphorylate Ser9 are Akt (18) and S6 kinase (S6K; 19), which are activated by the phosphatidylinositol 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase 1 (PDK1) and the mammalian target of rapamycin complex 1 (mTORC1), respectively. PDK1 also phosphorylates S6K at a site different from mTORC1, while Akt activates mTORC1 by phosphorylating and incapacitating the mTOR inhibitor, tuberus sclerosis complex 2 (TSC2). The favorable physiological changes during genetic inhibition of the GH/IGF-I axis are shared by caloric restriction. GH and IGF-I levels are suppressed during moderately restricted caloric intake (20, 21), indicating that intracellular proteins activated by low energy availability may disrupt hormonal regulatory input to fine-tune GH synthesis. There is increasing evidence that the evolutionary conserved sirtuin family of nicotinamide adenine dinucleotide⫹ (NAD⫹)-dependent deacetylases mediates, at least in part, the beneficial effects of moderate caloric restriction (22–24). Sirt1 and its orthologs sense perturbations in the NAD⫹ to NADH ratio indicative of increased respiration (25). Therefore, they act as intracellular energy sensors activated by low energy availability to deacetylate histones and a wide range of proteins involved in pathways regulating metabolism and improving glucose homeostasis (26 –30). The pharmacological Sirt1 activation by resveratrol (3,4=,5 trihydroxystilbene; RSV) improves insulin sensitivity and general health in mice fed with a high-fat diet (31). The present study investigates how Sirt1 cross-talks with the cAMP cascade at the level of CREB to regulate GH synthesis in pituitary somatotroph cells.

MATERIALS AND METHODS Compounds and antibodies GHRH, forskolin, SB-415286, and I-2 were obtained from Sigma (St. Louis, MO, USA); resveratrol, sirtinol, and okadaic acid were from Calbiochem (Bad Soden, Germany). Resveratrol, sirtinol, and SB-415286 were dissolved in DMSO and forskolin in ethanol. Antibodies used were anti-CREB, pGSK3␤-Ser9, GSK3␤, pGS-Ser641, pPDK1-Ser241, PDK1, pAktThr308, Akt, pS6K-Thr389, S6K (all rabbit; Cell Signaling, Frankfurt am Main, Germany); pCREB-Ser133 (rabbit; EnoGene Biotech Co., New York, NY, USA); pS6K-Thr229 (rabbit; R&D Systems, Minneapolis, MN, USA); PP1␣ (rabbit; Upstate, Billerica, MA, USA); I-2 (mouse; R&D Systems); pI-2Thr72 (rabbit; MBL International, Woburn, MA, USA); Pit-1 (X-7, sc442; rabbit, Santa Cruz Biotechnology, Santa Cruz, CA, USA); Sirt1 (mouse; Upstate); pan-phosphorylated thre1562

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onine and ␤-actin (mouse; Chemicon, Billerica, MA, USA); and pan-acetyl lysine (mouse; Cell Signaling). Animals Adult male Sprague-Dawley rats (200-250 g) were housed in the animal facility of the University of Santiago de Compostela; cannulated and serial blood samples were withdrawn after resveratrol (5 mg/kg i.p.) and GHRH (10 ␮g/kg i.v.) treatment as described previously (32). The animal studies were performed in agreement with the rules of laboratory animal care and international law on animal experimentation and approved by the ethics committee of the University of Santiago de Compostela. Cell culture Pituitary glands were removed from adult male SpragueDawley rats and processed and put in primary cell cultures as described previously (33). GH3 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in 10% FCS DMEM supplemented with 2.2 g/L NaHCO3, 10 mM HEPES, 2 nM glutamine, 2.5 mg/L amphotericin B, and 105 U/L penicillin-streptomycin. Treatments were done in serum-free DMEM, and the carriers in which the substances are dissolved were used as vehicle controls. GH determination Supernatant and plasma GH were measured as described previously (34). For plasma GH, the area under the curve (AUC) was determined by trapezoidal approximation. Resveratrol may have antiproliferative properties, so cell proliferation was determined in the same samples using the nonradioactive WST-1 assay (Roche Molecular Biochemicals, Mannheim, Germany), and in vitro GH data are presented as nanograms per milliliter per OD450 nm. cAMP radioimmunoassay cAMP was determined with a commercial RIA kit (NEN Life Science Products, Boston, MA, USA; ref. 33). Transfection, RNA interference, and plasmids GH3 cells were transfected using SuperFect (Qiagen, Hilden, Germany; ref. 35). Single interfering RNAs (siRNAs) were against Sirt1, a nonspecific scrambled control (both Santa Cruz Biotechnology) and TSC2 (5=-GCUGGAAGCUGAUGCGAAA-3=; Eurofins MWG Operon, Ebersberg, Germany) and were used at 100 nM final concentration. The GH-luc promoter-driven luciferase reporter construct has the proximal (_593) rat GH promoter upstream to the luciferase gene (pA3GHluc; kind gift of A. Gutierrez-Hartmann, University of Colorado, Denver, CO, USA). The pCREluc construct (Mercury pathway profiling system; Clontech Laboratories, Mountain View, CA, USA) has the cAMPresponsive element upstream to the TATA box of the herpes simplex virus thymidine kinase promoter and the reporter gene luciferase. The 231pit1-Luc, ⫺92pit1-luc, ⫺194pit1-luc, ⫺231mutDCREpit1-luc, and ⫺231mutPCREpit1-luc (the last two bearing mutation on the CRE binding element in the distal and proximal Pit-1 promoter, respectively) were described previously (36). Sirt1, Sirt1 H363Y, and GSK3␤ were obtained by Addgene [deposited by M. Greenberg (37) and J. Woodgett (38)]. In vitro mutagenesis on GSK3␤ changed the Lys205 to arginine (QuickChange II-Direct Mutagenesis Kit,

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Agilent Technologies, Santa Clara, CA, USA), and constructs were verified by sequencing (Sequiserve, Vaterstetten Germany).

CAAATAAAGTTTCTGTTTT-3= and 5=-TGTTAACCCGAACTGTCTTTCTTAC-3= (Eurofins MWG Operon). Protein phosphatase activity

Immunoblotting and immunoprecipitation GH3 cells were lysed in ice-cold lysis buffer (150 mM NaCl; 50 mM Hepes, pH7.4; 1 mM sodium orthovanadate; 2 mM EDTA; 2 mM phenylmethylsulfonyl fluoride; and 1% Nonidet P-40; supplemented with protease inhibitor cocktail and 10 mM nicotinamide), and protein was precleared and immunoprecipitated using Protein A/G agarose (Santa Cruz Biotechnology) and the primary antibody as indicated. Antibodies used were anti-CREB, anti-GSK3␤ (rabbit polyclonal; Cell Signaling), anti-Sirt1 (mouse monoclonal; Upstate), and antipan-acetyl lysine (mouse monoclonal; Cell Signaling). Protein immunoprecipitated with a control rabbit or mouse IgG and antibody solution without cell lysates were used as controls. Immunoblotting and immunoprecipitation were performed as described previously (35). Each immunoprecipitation experiment was repeated twice. Chromatin immunoprecipitation GH3 cells were processed with the EZ ChIP chromatin immunoprecipitation kit (Upstate), using rabbit anti-Pit-1 (Santa Cruz Biotechnology) or anti-CREB (Cell Signaling) antibody. Rabbit IgGs was used as a negative control. Primers against the rat GH promoter were described previously (39). Primers against the rat Pit-1 promoter were 5=-TGACGT-

Phosphatase activity was measured using the Ser/Thr phosphatase assay kit 1 (Upstate), following the manufacturer’s instructions, at OD630 nm. Immunohistochemistry and immunofluorescence Six human pituitary glands were obtained from autopsy cases of sudden death without any evidence for endocrine diseases, taken 8-12 h after demise. The ethics committee of the MaxPlanck-Institute approved this part of the study, and informed consent was received from the relatives. Tissue processing and immunohistochemistry were performed as described previously (40) using mouse anti-Sirt1 (Upstate) and anti-GH (kind gift of M. Bidlingmaier, Ludwig-Maximilians University, Munich, Germany), adrenocorticotrophic hormone (ACTH; Dako Diagnostika, Hamburg, Germany), thyroid-stimulating hormone (TSH), prolactin (PRL), or follicle-stimulating hormone (FSH; Immunotech, Karlsruhe, Germany) antibodies. GH3 cells were cultured in Falcon culture slides (BD Biosciences), treated as indicated, and fixed in 4% paraformaldehyde. After blocking in 5% goat serum with 0.1% (v/v) Triton X-100, slides were incubated with pCREB-Ser133 antibody (rabbit, EnoGene Biotech) and Alexa Fluor 594 goat anti-rabbit antibody (Invitrogen, Darmstadt, Germany), and slides were cover slipped with ProLong Gold antifade reagent including DAPI

Figure 1. Resveratrol suppresses GH. A) Resveratrol (RSV; 5 mg/kg i.p.) suppresses GHRH (10 ␮g/kg i.v.)-induced mean plasma GH levels in male adult rats (n⫽8/group). Data are means ⫾ se and represent AUC. veh, vehicle. B) Resveratrol (50 ␮M) treatment for 24 h suppresses GHRH (10 nM)-induced GH secretion from rat anterior pituitaries in primary cell culture. C) Resveratrol dose response (1-50 ␮M) on basal GH secretion from rat GH3 cells. For all cell culture experiments, each GH RIA value was divided to cell viability counts as determined by WST-1 at OD450 nm. Data are means ⫾ se from 3 experiments, presented as percentage of vehicle control. D) Resveratrol (50 ␮M) treatment suppressed GH promoter activity in GH3 cells transfected with the rat GH-luc plasmid (pA3GHluc). Luc/␤Gal, luciferase to ␤-galactosidase ratio. Data are means ⫾ se from 3 experiments, presented as percentage of vehicle control. *P ⬍ 0.05; **P ⬍ 0.001. E) Chromatin immunoprecipitation showing that 3 h resveratrol treatment decreases forskolin (5 ␮M for 30 min)-induced Pit-1 binding to the internal GH promoter in rat GH3 cells (␣Pit1 RSV vs. vehicle control). Rabbit IgG was used as control. F) Effect of 50 ␮M resveratrol treatment (1– 6 h) on Pit-1 in GH3 cells as determined by immunoblotting. G) Pit-1 levels in pituitaries from rats treated with GHRH (10 ␮g/kg) and resveratrol (5 mg/kg). SiRT1 INHIBITS GROWTH HORMONE SYNTHESIS

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(Invitrogen). The primary antibody was omitted as a control. Images were obtained using the FluoView FV1000 microscope (Olympus, Hamburg, Germany). Statistical analysis Results are expressed as means ⫾ se. Differences were assessed by Student’s t test and Mann-Whitney test. Values of P ⬍ 0.05 were considered significant.

RESULTS Sirt1 activation with resveratrol suppresses GH levels and synthesis in pituitary cells In adult male rats, intraperitoneal administration of the Sirt1 activator resveratrol (5 mg/kg), a treatment that improves general health in mice (31), significantly suppressed GHRH-induced GH pulse amplitude (resveratrol: 492⫾37 ng/ml vs. vehicle: 1125⫾130 ng/ ml; P⫽0.005) and total GHRH-induced GH secretion (AUC0-60 1455⫾107 vs. vehicle-treated 5861⫾2967;

P⫽0.007, Fig. 1A). Basal GH levels were slightly reduced in resveratrol-treated animals (resveratroltreated 10⫾2 ng/ml vs. vehicle-treated 14⫾10 ng/ ml). Resveratrol decreased GHRH-induced GH secretion from isolated rat anterior pituitary cells in primary cell culture and basal GH levels from the GH-secreting GH3 cells and suppressed GH promoter activity (Fig. 1B–D). GH transcription depends on Pit-1 and resveratrol reduced Pit-1 availability to the rat GH promoter by suppressing its levels in vitro and in vivo (Fig. 1E–G). Sirt1 immunoreactivity was detected in the nuclei of all anterior pituitary secretory cells where it also colocalized with GH (Fig. 2A). Sirt1 knockdown abolished resveratrol’s effect on GH and Pit-1 levels (Fig. 2B, C), revealing a Sirt1-specific effect. In contrast, it did not completely abolish resveratrol’s action on GH promoter activity, which is due to the compound’s unspecific suppressive action on basal luciferase activity independently of the inserted upstream promoter (41). Nevertheless, Sirt1 knockdown increased basal GH promoter activity (Fig. 2D), potentiated GHRH effect

Figure 2. Resveratrol’s suppressive action is mediated by Sirt1. A) Immunohistochemistry on cryostat anterior pituitary sections showing nuclear Sirt1 immunoreactivity (diaminobenzidine; brown) in all endocrine cells, but not in endothelial cells. Anterior pituitary hormones (GH, ACTH, TSH, PRL, and FSH) are stained with VectorRed (red). Inset: Parallel sections omitting the antiSirt1 antibody. Nuclei are counterstained with toluidine blue. B) Transfection with 100 nM Sirt1 siRNA for 48 h abolishes the inhibitory effect of resveratrol (RSV) at 50 ␮M on GH release from GH3 cells. Here 100 nM scrambled siRNA was used as control. GH RIA values of each condition were divided to the cell viability counts as determined by WST-1 at OD450 nm. Data are means ⫾ se of 3 experiments, presented as percentage of each vehicle control. C) Sirt1 siRNA (si) abolishes resveratrol’s effect on Pit-1 protein. Inset: knockdown efficacy. D) Sirt1 siRNA on basal GH promoter activity. *P ⬍ 0.01. E) Sirt1 siRNA potentiates GHRH (10 nM)-induced GH promoter activity. F) Sirt1 siRNA increases basal GH secretion. G) Sirt1 inhibitor sirtinol (10 ␮M) abolishes suppressive effect of resveratrol (50 ␮M) on GH release. *P ⬍ 0.05 vs. vehicle control; #P ⬍ 0.001 vs. RSV. H) Sirtinol treatment increases GH promoter activity. Data obtained in GH3 cells as means ⫾ se of 3 experiments, presented as luciferase to ␤-galactosidase ratio and as percentage of vehicle control. 1564

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on GH promoter (Fig. 2E), and increased basal GH secretion (Fig. 2F), and similar results were obtained with the Sirt1 inhibitor sirtinol (Fig. 2G, H). Sirt1 deacetylates CREB and suppresses its phosphorylation through PP1 Resveratrol treatment did not affect basal CREB levels but decreased forskolin-induced phosphorylated CREB-Ser133 phosphorylation levels (Fig. 3A, B) and CREB binding to the Pit-1 promoter (Fig. 3C). Sirt1 knockdown abolished resveratrol’s suppressive effect on CREB-Ser133 phosphorylation and strongly up-regulated CRE transcriptional activity (Fig. 3D, E). Sirt1 siRNA increased Pit-1 promoter activity but had no effect on Pit-1 promoter constructs lacking CRE (Fig. 3F), showing that it acts at the CREB level. Sirt1 overexpression decreased CRE transcriptional and GH promoter activities (Fig. 4A, B) and suppressed forskolin-induced CREB-Ser133 phosphorylation in GH3 cells (Fig. 4C). However, neither resveratrol nor Sirt1

siRNA significantly affected basal and forskolin-induced cAMP levels (Fig. 4D). In contrast, Sirt1 increased PP1 activity and its inhibition with siRNA, or the deacetylasedead Sirt1 H363Y suppressed it (Fig. 4E). In fact, Sirt1 H363Y overexpression kept forskolin-induced pCREBSer133 phosphorylation levels high even after 6 h forskolin treatment, by which time they were back to basal in cells transfected with the control empty plasmid (Fig. 4F). In addition, Sirt1 H363Y increased CRE transcriptional activity (Fig. 4G). Sirt1 coimmunoprecipitated with PP1 and CREB, and resveratrol treatment reduced the CREB levels detected in acetyl lysine immunoprecipitates (Fig. 4H). Pretreatment with okadaic acid or introducing the PP inhibitor I-2 abolished resveratrol’s suppressive action on pCREB-Ser133 phosphorylation (Fig. 4I). GSK3␤ mediates Sirt1’s effects on CREB dephosphorylation Resveratrol increased I-2-Thr72 phosphorylation, and this was abolished by the specific GSK3 inhibitor SB-

Figure 3. Sirt1 activation inhibits CREB. A, B) Resveratrol (RSV; 50 ␮M) suppresses forskolin (5 ␮M)-induced pCREB-Ser133 levels in GH3 cells as determined by immunoblotting (A) and immunofluorescence (B). pCREB-Ser133 is shown in red; nuclei are stained with DAPI blue. Images were obtained with a confocal microscope using ⫻60 objectives. Each image is representative of 5 areas. C) Chromatin immunoprecipitation showing that 3 h resveratrol treatment reduces forskolin (5 ␮M, 30 min pretreatment)-induced CREB binding to Pit-1 promoter in GH3 cells. Rabbit IgG was used as control. D) Sirt1 siRNA on basal pCREB-Ser133 levels in GH3 cells treated with vehicle or resveratrol (50 ␮M) for 3 h. Due to low signal intensity, the pCREB-Ser133 image was overexposed and enhanced. All immunoblots were repeated 3 times. E) Sirt1 siRNA increases CRE transcriptional activity. F) Sirt1 siRNA (si) increases intact Pit-1 promoter activity but not constructs having mutation in the CRE (⫺231mutDPit1 and ⫺231mutPPit1) or lacking both or one of the two2 CRE (⫺92 pit1-luc and ⫺194 pit1-luc). Luc/␤Gal, luciferase to ␤-galactosidase ratio. Data are means ⫾ se of 3 experiments, expressed as percentage of each scrambled siRNA control. *P ⬍ 0.05. SiRT1 INHIBITS GROWTH HORMONE SYNTHESIS

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Figure 4. Sirt1 deacetylase regulates CREB in GH3 cells. A, B) Sirt1 overexpression suppresses GH promoter (A) and CRE transcriptional activity (B). Empty vector (Ø) was used as control. Luc/␤Gal, luciferase to ␤-galactosidase ratio. Data are means ⫾ se of 3 experiments. C) Immunoblot showing that Sirt1 overexpression suppresses forskolin (5 ␮M, 1– 6 h)-induced pCREBSer133 levels. D) Resveratrol (RSV) or Sirt1 siRNA (si) does not affect basal and forskolin-induced cAMP levels. Scrambled siRNA (scra) was used as control. Data are means ⫾ se of 2 experiments. E) Sirt1 overexpression increases while Sirt1 inhibition with Sirt1 siRNA or deacetylase-dead Sirt1 H363Y decreases relative PP activity, determined at OD630. Data are means ⫾ se of 2 experiments, are presented as percentage of each individual empty plasmid (Ø) or scrambled siRNA (scra) control. *P ⬍ 0.05. F) Immunoblot showing that forskolin-induced pCREB-Ser133 remains elevated in Sirt1 H363Y overexpressing GH3 cells, while it is back to basal after 6 h in the empty plasmid control transfected cells. G) Sirt1 H363Y increases CRE transcriptional activity. Data are means ⫾ se of 3 experiments. H) Endogenous Sirt1 immunoprecipitates with CREB and PP1, and 2 h resveratrol treatment suppresses the immunoprecipitated CREB levels detected with anti-acetyl lysine. Proteins immunoprecipitated with rabbit or mouse IgG were used as controls. I) Pretreatment with 10 nM okadaic acid (OkA) for 2 h or introducing I-2 abolished resveratrol’s suppressive effect on forskolin-induced pCREB-Ser133 levels. Cells were treated with resveratrol for 3 h. Immunoblots were repeated twice.

415286 (Fig. 5A), which also blocked resveratrol’s suppressive action on CREB phosphorylation (Fig. 5B). Resveratrol decreased GSK3␤-Ser9 and increased the phosphorylation of the GSK3␤ target GS at Ser641, an effect that was abolished by SB-415286 (Fig. 5C, D). In contrast, Sirt1 knockdown increased basal GSK3␤-Ser9 and decreased GS-Ser641 phosphorylation (Fig. 5E). Resveratrol did not affect PDK1 and its target AktThr308 (which is needed for Akt to phosphorylate GSK3␤; ref. 42) and S6K-Thr229 (Fig. 5F). In contrast, it suppressed S6K-Thr389 (Fig. 5F). Sirt1 associates with TSC2 and regulates mTORC1 (43). Resveratrol increased TSC2 levels and decreased mTOR-Ser2481 autophosphorylation (Fig. 5G). TSC2 knockdown and SB415286 abolished resveratrol’s suppressive action on S6K-Thr389 phosphorylation, but it had no effect on the resveratrol-induced pGS-Ser641 phosphorylation (Fig. 5H, I). In contrast, pretreatment with okadaic acid abolished resveratrol’s suppressive effect on GSK3␤Ser9 phosphorylation (Fig. 5J). 1566

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Acetylation regulates GSK3␤ activity Sirt1 H363Y increased basal GSK3␤-Ser9 (and decreased GS-Ser641) phosphorylation (Fig. 6A). Endogenous GSK3␤ coimmunoprecipitated with Sirt1 and was detected in acetyl lysine immunoprecipitates, which were decreased after resveratrol treatment (Fig. 6B, C). The PHOSIDA database (44) predicted a putative acetylation site at Lys205. Mutating this lysine to the acetylation-resistant arginine (K205R) abolished Sirt1’s suppressive effect on CRE transcriptional and GH promoter activity (Fig. 6D, E). GSK3␤ inhibition with SB-415286 potentiated basal CRE transcriptional activity and CREB-Ser133 phosphorylation (Fig. 6F, G). GSK3␤ overexpression lowered CRE transcriptional activity in Sirt1, but not in deacetylasedead Sirt1 H363Y-transfected cells (Fig. 6H). The hypoacetylated GSK3␤ K205R suppressed basal CRE transcriptional activity and CREB-Ser133 phosphorylation levels, while it induced GS-Ser641 phosphorylation (Fig. 6I, J).

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Figure 5. Resveratrol activates GSK3␤. A) Immunoblot showing that resveratrol (RSV) induces pI-2-Thr72 levels in GH3 cells, and this is abolished by 14 ␮M GSK3 inhibitor SB-415286. B) Forskolin-induced pCREB-Ser133 levels in GH3 cells after 3 h treatment with 50 ␮M resveratrol (RSV) in the presence of 14 ␮M SB-415286. C) Immunoblot showing that resveratrol (50 ␮M, 1– 6 h) suppresses GSK3␤-Ser-9. D) Three hours resveratrol treatment induces pGS-Ser-641 levels, and this is abolished by 14 ␮M SB-415286. E) Sirt1 siRNA (si) increases GSK3b-Ser9 and decreases pGS-Ser641 phosphorylation as shown by Western blot. Scrambled siRNA (scra) was used as control. F) Effect of 50 ␮M resveratrol on pAkt-Thr308, pPDK1-Ser241, pS6K-Thr389, and pS6K-Thr229. Each membrane was stripped and blotted for total Akt, PDK1, S6K, and ␤-actin, respectively. G) Effect of 50 ␮M resveratrol on TSC2 and pmTOR-Ser2481; each membrane was stripped and blotted for ␤-actin and total mTOR- ␤-actin, respectively. H) TSC2 siRNA abolishes effect of resveratrol (50 ␮M, 3 h) on pS6K-Thr389 but not on pGS-Ser641. I) Resveratrol’s suppressive effect on pS6K-Thr389 is abolished by 14 ␮M SB-415286. J) Pretreatment with 10 nM okadaic acid (OkA) for 2 h abolishes resveratrol’s suppressive effect on GSK3␤-Ser9.

DISCUSSION The present study defines a mechanism through which the Sirt1 deacetylase suppresses GH synthesis in pituitary somatotroph cells by inhibiting the cAMP hormonal cascade at CREB level through GSK3␤ and PP1 (Fig. 7). Sirt1 was shown to regulate TSH secretion from pituitary thyrotrophs (45). GH synthesis is tightly regulated by metabolic signals (46), so our finding of Sirt1 immunoreactivity also in the anterior pituitary somatotrophs suggests that it may act as an intrapituitary energy sensor to regulate hormone synthesis. We used the polyphenol resveratrol for the pharmacological activation of Sirt1 in vivo and in vitro. Although resveratrol was shown to have Sirt1-independent actions (47, 48), recent in vivo studies established that it acts through Sirt1 (49). In our study, Sirt1 knockdown abolished the inhibitory effect of resveratrol on GH secretion and upstream signaling targets. Interestingly, our RNAi experiments revealed a tonic effect of Sirt1 on basal GH synthesis. In addition, the Sirt1-null mouse has normal circulating GH levels despite the small pituitary size (50). Altogether, these observations indicate increased GH synthesis in the absence of Sirt1. Resveratrol was recently shown to block cAMP phosphodiesterases and increase cAMP in muscle and adipose tissue (51). In contrast, in pituitary cells, no SiRT1 INHIBITS GROWTH HORMONE SYNTHESIS

significant changes in cAMP levels were detected after resveratrol treatment or Sirt1 siRNA. Instead, Sirt1 suppressed CREB phosphorylation and transcriptional activity, and this depended on its deacetylase activity. Sirt1 deacetylates CREB directly and not through CBP (52). CREB acetylation is tightly linked to its phosphorylation and activation, as it controls the HDAC1-induced recruitment of PP1 (14, 15). HDAC1 recruits PP1 to CREB without affecting phosphatase activity (53). In contrast, our data show that Sirt1 activates PP1, revealing an additional mechanism through which CREB acetylation may control its phosphorylation. PP1 activation depended on intact Sirt1 deacetylase, indicating the presence of another deacetylation event upstream to CREB. As PP1 is not acetylated and is therefore not a direct substrate for Sirt1, we sought the signaling cascade through which Sirt1 may affect its activity. Among the factors regulating PP1, a central role for GSK3␤ on Sirt1 action was highlighted. Experiments with the deacetylase-dead Sirt1 revealed changes in basal GSK3␤ activation. However, no connection between GSK3␤ and acetylation has been previously described. The HDAC1 deacetylase was shown to regulate Sirt1 transcriptionally (54, 55), but this is not the case with Sirt1, as evidenced by the unchanged basal GSK3␤ 1567

Figure 6. GSK3␤ acetylation regulates its activity. A) Immunoblots for GSK3␤-Ser9 and pGS-Ser641 in GH3 cells overexpressing Sirt1 or the deacetylase-dead Sirt1 H363Y. Empty vector was used as control. Protein was harvested 24 h after transfection. Each transfection experiment was repeated twice. B) Endogenous GSK3␤ immunoprecipitated with Sirt1 and acetyllysine. C) Resveratrol (RSV) treatment decreased GSK3␤ immunoprecipitates detected with anti-acetyl lysine. Rabbit IgG was used as negative control. Representative of 2 immunoprecipitations is shown. D, E) Deacetylase-dead GSK3␤ K205R abolishes the suppressive effect of Sirt1 overexpression on CRE transcriptional activity (D) and) GH promoter activity (E). Luc/␤Gal, luciferase to ␤-galactosidase ratio. Data are means ⫾ se of 3 experiments, expressed as percentage of empty plasmid (Ø) control. *P ⬍ 0.05. F) Treatment with 14 ␮M GSK3 inhibitor SB-415286 increased CRE transcriptional activity. Data are means ⫾ se of 3 experiments, expressed as percentage of vehicle control. *P ⬍ 0.0001. G) SB-415286 increases CREB-Ser133 (red) phosphorylation, as demonstrated by immunofluorescence. Nuclei are stained with DAPI blue. Images were obtained with a confocal microscope using ⫻60 objectives. Each image is representative of 5 areas. H) Effect of Sirt1 or Sirt1 H636Y on the suppressive action of GSK3␤ on CRE transcriptional activity. *P ⬍ 0.01. I) Hypoacetylated GSK3␤ K205R mutant suppresses CRE transcriptional activity. Data are means ⫾ se of 3 experiments. *P ⬍ 0.0001. G) GSK3␤ K205R suppresses pCREB-Ser133 and increases pGS-Ser641, indicative of GSK3␤ activation. Same membrane was used and reblotted after sequential strippings. Immunoblots were repeated twice.

levels during Sirt1 manipulation. Nevertheless, Sirt1 suppressed GSK3␤ inhibitory Ser9 phosphorylation and enhanced GS phosphorylation, both being markers of increased GSK3␤ activity. The PI3K pathway primarily regulates GSK3␤-Ser9 phosphorylation. Resveratrol was reported to be a class I PI3K inhibitor independently of Sirt1, while in other studies both resveratrol and Sirt1 were shown to activate PI3K signaling (56 – 60). In pituitary somatotrophs, resveratrol inhibited mTOR/ S6K-Thr389, but not PDK1 and its targets, revealing a specific mode of action. The mTOR inhibitor TSC2 was previously shown to be an acetylated protein that interacts with Sirt1 (43), so this could be the deacetylation target of Sirt1 upstream to mTOR and GSK3␤. However, TSC2 inhibition with RNA interference revealed that TSC2/mTORC1/S6K does not mediate resveratrol’s effect on GSK3␤. In contrast, our data 1568

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show that GSK3␤ mediates resveratrol’s effect on the mTOR inhibition, possibly through its well-described action on TSC2 (61). The possibility that GSK3␤ itself is a direct target of the Sirt1 deacetylase was examined, and indeed we show that it can be modified by acetylation. We focused on the Lys205 that resides in the priming pocket regulating the GK3␤ kinase activity. Hypoacetylated GSK3␤ K205R mutants displayed higher activity in terms of GS phosphorylation and CREB inhibition. Furthermore, they revealed that Lys205 deacetylation is an important step in Sirt1’s action on CREB. GSK3␤-induced PP1 dephosphorylates Ser9 in an autoregulatory loop that intensifies phosphatase activity and CREB dephosphorylation, reinforcing the inhibitory role for GSK3␤ on CREB (62– 64). Therefore, our data suggest that Sirt1induced GSK3␤ deacetylation may increase its activity,

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mechanism through which caloric restriction may mediate its beneficial effects on metabolism and life span. In fact, caloric restriction was found to affect life span and aging-related pathologies in part by reducing GH and IGF-I levels (70). However, caloric restriction had no effect on mice with targeted disruption of the GH receptor/GH-binding protein (71), indicating that it needs an intact GH response in order to deliver its beneficial effects. Therefore, the inhibition of the GH/IGF-I axis during caloric restriction may be an important endocrine change that facilitates organism adaptation. Our study provides with a novel putative mechanism through which the energy sensor Sirt1 regulates the GH/IGF-I axis upstream at the level of pituitary GH synthesis by activating GSK3␤ and PP1 and inhibiting CREB.

Figure 7. Signaling events downstream to Sirt1 activation deducted from the findings of the present work. Sirt1 binds and deacetylates CREB and GSK3␤. GSK3␤ deacetylation leads to GSK3␤ activation. The activated GSK3␤ phosphorylates the PP1 inhibitor I-2, releasing it from the PP complex. Active PP1 dephosphorylates GSK3␤ through a positive autoregulatory feedback loop and associates with CREB and dephosphorylates it. Dephosphorylated and deacetylated CREB has little DNA binding and transcriptional activity, and subsequently there is less Pit-1 transcription, which compromises GH synthesis. Solid arrows indicate activation; blunted arrows indicate inhibition. Purple line connects Sirt1 with the proteins with which it physically associates.

The authors are grateful to A. Gutierrez-Hartman (University of Colorado Health Sciences Center, Denver, CO, USA) for the pA3GHluc reporter plasmid and M. Bidlingmaier (Ludwig-Maximilians University, Munich, Germany) for the mouse monoclonal GH antibody. The study was supported in part by a Pfizer Young Investigator research fellowship to M.T. The funding source had no role in the conceptualization, interpretation, and writing of this study. The authors declare no conflicts of interest.

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