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- Title
Nesfatin-1 Induces the Phosphorylation Levels of cAMP Response Element-Binding Protein for Intracellular Signaling in a Neural Cell Line.
- Authors
Ishida, Emi; Hashimoto, Koshi; Shimizu, Hiroyuki; Okada, Shuichi; Satoh, Tetsurou; Kato, Ikuo; Yamada, Masanobu; Mori, Masatomo
- 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 radioreceptor 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
- Subjects
PROTEIN kinases; PHOSPHORYLATION; CHEMICAL reactions; CELL culture; CELL lines; NERVOUS system
- Publication
PLoS ONE, 2012, Vol 7, Issue 12, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0050918