Nesfatin-1 induces the phosphorylation levels of cAMP response element-binding protein for intracellular signaling in a neural cell line.
Nesfatin-1 induces the phosphorylation levels of cAMP response element-binding protein for intracellular signaling in a neural cell line.
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NESFATIN-1诱导神经细胞系中的细胞内信号传导cAMP反应元件结合蛋白的磷酸化水平。
DOI：
10.1371/journal.pone.0050918
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Mori M
中科院分区：
文献类型：
--
作者：
Ishida E;Hashimoto K;Shimizu H;Okada S;Satoh T;Kato I;Yamada M;Mori M
Nesfatin-1是一种新型的厌食肽，通过脑室内或腹腔内给药可减少啮齿动物的食物摄入量。然而，通过Nesfatin-1进行细胞内信号传导的分子机制尚不清楚。在本研究中，我们研究了不同神经细胞系对Nesfatin-1的响应能力，并进一步阐明了Nesfatin-1的信号转导途径。为了实现这一目标，我们用含有不同类型应答元件的报告载体的不同组合转染了几种细胞系，并用Nesfatin-1进行了报告分析，Nesfatin-1的活性中间段编码30个氨基酸残基（M30）和M30衍生突变体。值得注意的是，我们发现Nesfatin-1和M30都显著增加了小鼠神经母细胞瘤细胞系NB41A3中cAMP反应元件（CRE）报告因子的活性。黑素皮素3/4受体拮抗剂SHU9119使启动子活性终止，而体内无厌食作用的突变体M30在NB41A3细胞中未诱导CRE报告子活性。Western blotting分析显示，Nesfatin-1和M30显著增加cre结合蛋白（CREB）的磷酸化水平，而不改变细胞内cAMP水平。此外，我们的研究表明，丝裂原活化蛋白激酶（MAPK）激酶抑制剂和l型钙（Ca2+）通道阻滞剂可以消除m30诱导的CREB磷酸化。此外，放射受体实验显示，125I-Nesfatin-1以饱和方式与小鼠下丘脑和NB41A3细胞的膜组分结合，Kd值分别为0.79 nM和0.17 nM。总之，我们的研究结果表明，在NB41A3细胞和小鼠下丘脑的细胞表面存在nesfatin -1特异性受体。我们的研究强调Nesfatin-1通过其受体诱导CREB磷酸化，从而激活神经元细胞内信号级联。
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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