title: Nesfatin-1 induces the phosphorylation levels of cAMP response element-binding protein for intracellular signaling in a neural cell line.
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 (Ca(2+)) channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that (125)I-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.
authors.affiliation: Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Ishida
authors.forename: Koshi
authors.initials: K
authors.lastname: Hashimoto
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Shimizu
authors.forename: Shuichi
authors.initials: S
authors.lastname: Okada
authors.forename: Tetsurou
authors.initials: T
authors.lastname: Satoh
authors.forename: Ikuo
authors.initials: I
authors.lastname: Kato
authors.forename: Masanobu
authors.initials: M
authors.lastname: Yamada
authors.forename: Masatomo
authors.initials: M
authors.lastname: Mori
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D045744:Cell Line, Tumor; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D000080866:Nucleobindins; D010766:Phosphorylation / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014162:Transfection
chemical_list: D002135:Calcium-Binding Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; C000630651:Nucb1 protein, mouse; D000080866:Nucleobindins; D000242:Cyclic AMP