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Secretogranin II Binds to Secretogranin III and Forms Secretory Granules with Orexin, Neuropeptide Y, and POMC

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Secretogranin II Binds to Secretogranin III and Forms Secretory Granules with Orexin, Neuropeptide Y, and POMC 111 Secretogranin II binds to secretogranin III and forms secretory granules with orexin, neuropeptide Y, and POMC Kikuko Hotta*, Masahiro Hosaka1,*, Atsushi Tanabe and Toshiyuki Takeuchi1 Laboratory for Endocrinology and Metabolism, Center for Genomic Medicine, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan 1Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan (Correspondence should be addressed to K Hotta; Email: [email protected]) *(K Hotta and M Hosaka contributed equally to this work) Abstract Functional variations in the secretogranin III (SCG3) gene are structures with SCG3, orexin, NPY,or POMC. SCG3 bound associated with susceptibility to obesity. SCG3 forms to POMC; however, it did not bind to orexin, MCH, or NPY. secretory granules with orexin, melanin-concentrating By contrast, SCG2 formed aggregates with orexin, MCH, hormone (MCH), neuropeptide Y (NPY), and POMC in NPY, and POMC. SCG2 may act as a hormone carrier for the hypothalamus. In this study, we screened proteins for orexin, MCH, NPY, and POMC by binding with SCG3, SCG3-binding activity and identified secretogranin II (SCG2) which targets proteins to the secretory granules. SCG2 using a yeast two-hybrid system. Immunoprecipitation mRNA levels increased along with those of SCG3, orexin, revealed that SCG2 interacts with SCG3. In situ hybridization MCH, and NPY after a 24-h fast, suggesting that the and immunohistochemistry indicated that SCG2 was highly SCG2/SCG3 system may respond in an adaptive manner to expressed in the lateral hypothalamic area, paraventricular acute body weight changes. However, this SCG2/SCG3 nucleus, and arcuate nucleus of the hypothalamus. Double- system appears to be unresponsive to chronic body weight labeling immunohistochemical analysis demonstrated that changes, such as diet-induced obesity or obesity in ob/ob mice. SCG2 was expressed in orexin-, MCH-, NPY-, and We suggest that SCG2, as well as SCG3, may be a potential POMC-expressing neurons. SCG2 was also coexpressed regulator of food intake based on its capacity to accumulate with SCG3. Upon introduction into neuroblastoma cells, appetite-related hormones into secretory granules. SCG2 was expressed in the cytosol and formed granule-like Journal of Endocrinology (2009) 202, 111–121 Introduction Many genes expressed in the hypothalamus play important roles in the regulation of food intake and are involved in the Obesity is a major risk factor for diabetes, dyslipidemia, and monogenic and common forms of obesity. For example, the hypertension (Kopelman 2000). A combination of these fat mass and obesity-associated (FTO) gene is highly expressed metabolic disorders is defined as the metabolic syndrome in the hypothalamus and its expression is regulated by fasting (Matsuzawa et al. 1999, Wilson & Grundy 2003), and and leptin (Frayling et al. 2007, Gerken et al. 2007). Three comorbidity of these conditions significantly increases the risk groups have reported an association between SNPs in the of cardiovascular diseases. Both genetic and environmental FTO gene and obesity (Dina et al. 2007, Frayling et al. 2007, factors contribute to the development of obesity (Maes et al. Scuteri et al. 2007). Variations in the FTO gene have also been 1997, Barsh et al.2000, Rankinen et al. 2006). Genetic studies associated with obesity in Japanese subjects (Hotta et al. 2008). on mice have suggested that mutations in several genes such as SCG3 is also expressed in the hypothalamus, as well as in the leptin, proopiomelanocortin (POMC), and melanocortin-4 many endocrine cells, such as pancreatic b-cells and pituitary receptor genes are implicated in a monogenic form of inherited cells (Taupenot et al. 2003, Tanabe et al. 2007). SCG3 belongs obesity (Barsh et al.2000, Rankinen et al. 2006). However, most to a family of acidic secretory proteins known as granins, cases of obesity are caused by polygenic disorders; moreover, which are widely expressed in endocrine and neuronal cells genetic susceptibility may differ among various ethnic groups (Taupenot et al. 2003). SCG3 mRNA has been cloned from (Barsh et al.2000, Rankinen et al.2006). We have recently brain- and pituitary-specific mRNA (Ottiger et al. 1990), and reported that single-nucleotide polymorphisms (SNPs) in the is expressed in the paraventricular nucleus (PVN), lateral secretogranin III (SCG3) and myotubularin-related protein 9 hypothalamic area (LHA), and arcuate nucleus (ARC) of the (MTMR9) genes are significantly associated with obesity hypothalamus, which are important regions for appetite (Tanabe et al.2007, Ya na g i ya et al.2007). regulation (Tanabe et al. 2007). SCG3 is coexpressed with Journal of Endocrinology (2009) 202, 111–121 DOI: 10.1677/JOE-08-0531 0022–0795/09/0202–111 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 10/02/2021 12:11:42PM via free access 112 KHOTTA, M HOSAKA and others . Secretogranin II binds to secretogranin III orexin, melanin-concentrating hormone (MCH), neuro- high-fat diet (57% fat, 23% carbohydrate, and 20% protein) peptide Y (NPY), and POMC in the hypothalamus, and it with 5.08 kcal/g. Male ob/ob and C/? mice (6 weeks old: forms granule-like structures in association with these Oriental Yeast Co., Ltd) were provided with regular neuropeptides (Tanabe et al. 2007). Furthermore, SCG3 is laboratory chow for 1 week. These mice were anesthetized known to bind chromogranin A (CGA) and targets it to with pentobarbital (10 mg/100 g of body weight). Plasma the secretory granules in pituitary corticotroph cells and samples for leptin were obtained and frozen for later assays. pancreatic b-cells (Taupenot et al. 2003, Hosaka et al. 2005, A region bordered dorsally by the thalamus, rostrally by the Han et al. 2008, Takeuchi & Hosaka 2008). CGA optic chiasm, and caudally by the mamillary bodies was coaggregates several peptide hormones in its core, and the excised and immediately frozen for RNA preparation. aggregate form is directed to secretory granules by SCG3; Each experiment was performed at least three times. These thus, SCG3 has been proposed to function as a navigator of protocols were approved by the animal ethics committee the peptide hormone carrier protein CGA to the secretory of RIKEN. granules. However, in the hypothalamus, the type of peptide hormone carrier proteins, which SCG3 directs into the Screening of proteins for SGC3-binding activity using the yeast secretory granules, is unknown. two-hybrid assay In the present study, we investigated the proteins that bind to SCG3 using a yeast two-hybrid system, and we found that The coding sequence of human SCG3 was amplified using secretogranin II (SCG2) was bound to SCG3. SCG2 was reverse transcriptase-PCR (RT-PCR) from human brain coexpressed with SCG3, orexin, and NPY, and it formed RNA (Clontech) with primers containing an additional granule-like structures with them. We investigated the roles of N-terminal EcoRI restriction site before the start codon SCG2 in carrying these appetite-related neuropeptides to the and a C-terminal SalI restriction site before the stop codon. secretory granules. The PCR product was cloned between the EcoRI and SalI sites of the pGBKT7 vector (Clontech), which was used as the bait construct; the construct was transformed into Saccharomyces cerevisiae strain AH109. A pretransformed Materials and Methods human brain Matchmaker cDNA library cloned into pGADT7-rec was purchased from Clontech. The library Cell culture host strain Y187 was mated with strain AH109, which The 293 Tet-On cell line (Clontech) was cultured in DMEM contained the bait construct, and was screened for proteins (Sigma–Aldrich Corp.) containing 10% fetal bovine serum binding to SCG3. (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 100 mg/ml G418 disulfate. BE(2)-C neuroblastoma cells were Immunoprecipitation purchased from the American Type Culture Collection (Manassas, VA, USA). The cells were cultured in advanced The coding sequences of human SCG3 and SCG2 were DMEM (Invitrogen) containing 2 mM glutamine, 5% FBS, amplified using RT-PCR from human brain RNA using 100 U/ml penicillin, and 100 mg/ml streptomycin. primers with an additional N-terminal MulI restriction site before the start codon and a C-terminal EcoRV restriction site after the stop codon. The PCR products were cloned Animals between the MulI and EcoRV sites of the pBI vector Male mice (C57BL/6, 8 weeks old) were purchased from (Clontech). The coding region of human SCG2 was also CLEA Japan (Tokyo, Japan). After anesthetization with amplified with primers that included an N-terminal NotI sodium pentobarbital (100 mg/kg), the mice were perfused site before the start codon and a C-terminal SalI site after with 10% neutral-buffered formalin. The brains were the stop codon. The PCR product was cloned between dissected, fixed with tissue fixative (Genostaff, Tokyo, the NotI and SalI sites of the pBI–SCG3 plasmid. The Japan), embedded in paraffin, and sectioned. The tissue pBI–SCG3 and pBI–SCG3–SCG2 constructs were trans- sections (6 mm) were dewaxed and used for in situ hybrid- fected into a previously established HEK293 cell line ization and immunohistochemistry. containing the pTet-On vector (Clontech) using Lipofecta- Seven-week-old male C57BL/6 mice were provided mine 2000 reagent (Invitrogen). Protein expression was regular laboratory chow (CRF-1; Oriental Yeast Co., Ltd, induced by adding 1 mg/ml doxycycline hydrochloride (ICN Tokyo, Japan) for 1 week and then deprived of food (free Biomedicals, Irvine, CA, USA). After 24 h, the cells were access to water) for 24 h. Male C57BL/6 mice (6 weeks old) collected and homogenized in 1 ml lysis buffer (50 mM were provided with regular laboratory chow or a high-fat 2-(N-morpholino)-ethanesulfonic acid (MES)-NaOH, pH diet (High Fat Diet 32; CLEA Japan Inc.) for 8 weeks.
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