Nesfatin-1 Induces the Phosphorylation Levels of cAMP Response Element-Binding Protein for Intracellular Signaling in a Neural Cell Line |
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| Authors: | Emi Ishida Koshi Hashimoto Hiroyuki Shimizu Shuichi Okada Tetsurou Satoh Ikuo Kato Masanobu Yamada Masatomo Mori |
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| Institution: | 1. Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.; 2. Department of Health and Nutrition, Faculty of Health and Welfare, Takasaki University of Health and Welfare, Gunma, Japan.; 3. Department of Bioorganic Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa, Japan.; University of Houston, United States of America, |
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| Abstract: | Nesfatin-1 is a novel anorexic peptide that reduces the food intake of rodents when administered either intraventricularly or intraperitoneally. However, the molecular mechanism of intracellular signaling via Nesfatin-1 is yet to be resolved. In the current study, we investigated the ability of different neuronal cell lines to respond to Nesfatin-1 and further elucidated the signal transduction pathway of Nesfatin-1. To achieve this, we transfected several cell lines with various combinations of reporter vectors containing different kinds of response elements and performed reporter assays with Nesfatin-1, its active midsegment encoding 30 amino acid residues (M30) and M30-derived mutants. Notably, we found that both Nesfatin-1 as well as M30, significantly increased cAMP response element (CRE) reporter activity in a mouse neuroblastoma cell line, NB41A3. An antagonist of Melanocortin 3/4 receptor, SHU9119, aborted the promoter activity, and a mutant M30, which exerts no anorexic effect in vivo did not induce the CRE reporter activity in NB41A3 cells. Western blotting analyses revealed that Nesfatin-1 and M30 significantly increased the phosphorylation levels of CRE-binding protein (CREB), without altering the intracellular cAMP levels. Further, our study showed that a mitogen-activated protein kinase (MAPK) kinase inhibitor and an L-type Calcium (Ca2+) channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that 125I-Nesfatin-1 binds in a saturable fashion to the membrane fractions of the mouse hypothalamus and NB41A3 cells, with Kd values of 0.79 nM and 0.17 nM, respectively. Collectively, our findings indicate the presence of a Nesfatin-1-specific receptor on the cell surface of NB41A3 cells and mouse hypothalamus. Our study highlights that Nesfatin-1, via its receptor, induces the phosphorylation of CREB, thus activating the intracellular signaling cascade in neurons. |
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