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RGS2 Is a Feedback Inhibitor of Melatonin Production in the Pineal Gland ⇑ Masahiro Matsuo, Steven L

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RGS2 Is a Feedback Inhibitor of Melatonin Production in the Pineal Gland ⇑ Masahiro Matsuo, Steven L View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 587 (2013) 1392–1398 journal homepage: www.FEBSLetters.org RGS2 is a feedback inhibitor of melatonin production in the pineal gland ⇑ Masahiro Matsuo, Steven L. Coon, David C. Klein The Section on Neuroendocrinology, Program in Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, United States article info abstract Article history: The 24-h rhythmic production of melatonin by the pineal gland is essential for coordinating circa- Received 22 January 2013 dian physiology. Melatonin production increases at night in response to the release of norepineph- Revised 6 March 2013 rine from sympathetic nerve processes which innervate the pineal gland. This signal is transduced Accepted 9 March 2013 through G-protein-coupled adrenergic receptors. Here, we found that the abundance of regulator Available online 21 March 2013 of G-protein signaling 2 (RGS2) increases at night, that expression is increased by norepinephrine Edited by Ivan Sadowski and that this protein has a negative feedback effect on melatonin production. These data are consis- tent with the conclusion that RGS2 functions on a daily basis to negatively modulate melatonin production. Keywords: Melatonin Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. Pineal gland Regulator of G-protein signaling RGS2 Norepinephrine 1. Introduction signaling is activated by binding of GTP; hydrolysis of bound GTP by intrinsic GTPase activity of the Ga subunit inactivates G-protein Daily rhythms characterize essentially all physiological func- signaling. This hydrolysis is accelerated by GTPase activating pro- tions in animals [1]. Disruption of normal 24-h rhythms in man teins (GAPs), and through this mechanism, GAPs accelerate G-pro- can cause sleep disorders and a wide range of metabolic and psy- tein deactivation, thereby downregulates the signaling. The chiatric diseases [2,3]. An essential element in the circadian system regulator of G-protein signaling (RGS) family of proteins act as is melatonin, which is produced by the pineal gland and acts to GAPs and accelerate the otherwise slow intrinsic GTPase activity. optimize circadian biology. The 24-h pattern in melatonin produc- Transcription of some RGS family members is dynamically regu- tion and release is controlled by the suprachiasmatic nucleus lated [5], and RGS2 proteins provide feedback regulation of G-pro- (SCN), the central circadian clock in mammals [4]. Time-of-day tein signaling [6,7]. Here we describe the expression of RGS family information from SCN is transmitted to the pineal gland by a neu- members in the rat pineal gland, and report that one negatively ral circuit which includes central and peripheral neural structures, regulates melatonin production. terminating in sympathetic fibers. At night, neural signals from the SCN elicit norepinephrine re- lease from these fibers into the pineal perivascular space; norepi- 2. Materials and methods nephrine acts through G-protein-coupled adrenergic receptors to increase cAMP. This increases melatonin production by increasing 2.1. Animals and tissue collection the activity of the penultimate enzyme in melatonin synthesis, aryl- alkylamine N-acetyltransferase (Aanat), which acetylates serotonin Pineal glands were prepared from Sprague Dawley rats (female, to produce N-acetylserotonin, the melatonin precursor. In rodents, 180–250 g; Taconic Farms Inc., Germantown, NY), that had been the increase in Aanat activity reflects in part cAMP-dependent entrained to a 14:10 light:dark (L:D) cycle for at least one week. induction of Aanat transcription, in addition to phosphorylation [4]. Six pineal glands were pooled and used for RGS family member Here we have focused on a critical element of the regulation of expression screening; the day pool was obtained at Zeitgeber time melatonin production, signaling through G-proteins. G-Protein (ZT) 7 and the night pool at ZT19. Euthanasia at ZT19 was done un- der a dim red light. Animal use and care protocols were approved by local ethical review and were in accordance with National Insti- ⇑ Corresponding author. Address: National Institutes of Health, Bldg. 49, Rm. tutes of Health guidelines. 6A82, Bethesda, MD 20892, United States. Fax: +1 301 480 3526. E-mail address: [email protected] (D.C. Klein). 0014-5793/$36.00 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.03.016 M. Matsuo et al. / FEBS Letters 587 (2013) 1392–1398 1393 2.2. Western blotting was: 95 °C for 10 min and then 45 cycles for 95 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s. Product specificity was initially con- Protein was extracted from pineal glands for quantification of firmed by agarose gel electrophoresis and sequencing, and melting daily or induced changes in RGS2 levels; and, from pineal cells to curve analysis during every qRT-PCR thereafter. For each molecule, quantify levels of virus-directed induction of RGS2. Protein extrac- 10-fold serial dilutions of each internal standard (10 fM to 1 nM) tion was done in radio immunoprecipitation assay buffer (50 mM were used to generate a standard curve. mRNA values were nor- Tris pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycho- malized to the abundance of Gapdh, and results are shown based late, 0.1% SDS). The lysate was centrifuged (15,000Âg, 30 min, 4 °C); on three independent experiments. protein concentration of the supernatant was determined by the bicinchoninic acid assay method (Pierce, Rockford, IL, USA). Proteins 2.6. Melatonin and N-acetylserotonin determination (10 lg/lane) were resolved using 12% bis-tris SDS–polyacrylamide gel electrophoresis in 3-(N-morpholino) propanesulfonic acid A published LC/MS/MS method was used [10]. (MOPS) buffer, and electroblotted onto Immobilon-P (Millipore, Billerica, MA, USA) membrane in NuPAGE transfer buffer. The mem- 2.7. cAMP assay branes were incubated for 1 h in Odyssey Blocking buffer (Li-Cor, Lincoln, NE, USA) before overnight incubation with a polyclonal cAMP in culture media was detected by the cAMP-Glo Assay chicken anti-full-length RGS2 (GW22245F, Sigma Aldrich, St. Louis, (Promega, Madison, WI, USA) following manufacturer’s MO, USA; 1:800 dilution) and monoclonal mouse anti-b-actin instructions. (A5441, Sigma Aldrich; 1:5000 dilution) in Tris-buffered saline with 0.1% Tween20. After washing (Tris-buffered saline with 0.1% 2.8. Data presentation and statistical analysis Tween20, 5 min, 3 times), membranes were incubated (Tris-buf- fered saline, 5% non-fat milk, 0.1% Tween20 and 0.02% SDS, 1 h, All data are presented as the mean of three independent exper- room temperature) with two secondary antisera: anti-mouse IgG imental results ± standard error of the mean. Statistical analyses (IRDye 800CW, #827-08364, Li-Cor) and anti-chicken IgG (IRDye were performed with Prism 5 for Windows (GraphPad software, 680LT, #926-68028, Li-Cor). Blots were visualized and band density La Jolla, CA, USA). Statistical tests used are given in the text or in of immunopositive bands was determined with an Odyssey Infrared the figure legends. Imaging System (Li-Cor); RGS2 and b-actin immunopositive bands were of the predicted size. The RGS2 signal was normalized to b-ac- 3. Results tin. The normalized RGS2 values from three biological samples were used to determine mean intensity. 3.1. Transcript levels of RGS family members in the pineal gland 2.3. Pineal cell culture Expression levels of 17 RGS family members were examined by qRT-PCR of RNA extracted from pineal glands obtained during the Pineal cells were prepared as described previously [8]; animals day and night. Data are grouped according to subfamily member- were euthanized between ZT4 and ZT7. Approximately 100,000 ship [11] (Fig. 1A). Except for the D/R12 family, expression of at cells were added to each well of a poly-D-lysine coated 96-well least one member of all subfamilies was detected. In the C/R7 sub- plate (Corning, Tewksbury, MA, USA). Pineal cells were cultured family, Rgs7 was the only highly expressed member; expression for 48 h, and then incubated in serum-free medium for 24 h prior did not exhibit a day/night difference. In the A/RZ subfamily, to and during drug treatments. Rgs20 was highly expressed, with highest levels occurring at ZT7. Three members of the B/R4 subfamily, Rgs2, Rgs4 and Rgs5 were 2.4. Virus infection highly expressed, and expression of the first two exhibited a night/day difference. The expression pattern of Rgs2 was notable A replication-defective adenovirus encoding RGS2 was provided because it exhibited a 20-fold increase at night (ZT7 vs ZT19; by Peter Chidiac (University of Western Ontario, Canada) [9]; P < 0.05, two-tailed t-test, n = 3 each), consistent with the day/ b-galactosidase-encoding adenovirus (Ad-CMV-beta-gal, Vector night difference reported based on microarray analysis [12]. BioLabs, Philadelphia, PA, USA) was used as a control. After 24 h of incubation in complete media, pineal cells were infected by addition of adenovirus (24 h). The virus was used at a multiplicity 3.2. The Rgs2 transcript and RGS2 protein exhibit similar 24-h profiles of infection (MOI) of 40 virus particles per cell, unless otherwise stated. Subsequently, cells were maintained in serum-free medium The abundance of the Rgs2 mRNA was determined at 6 time for 24 h prior to drug treatment. points throughout a 24-h period (Fig. 1B). The peak in Rgs2 expres- sion occurred at ZT19. Expression was no more than 20% of the peak 2.5. mRNA quantification by qPCR value at other times.
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