IGF regulation of neutral amino acid transport in the BeWo choriocarcinoma cell line (b30 clone): Evidence for MAP kinase-dependent and MAP kinase-independent mechanisms
IGF regulation of neutral amino acid transport in the BeWo choriocarcinoma cell line (b30 clone): Evidence for MAP kinase-dependent and MAP kinase-independent mechanisms
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DOI：
10.1016/j.ghir.2006.08.002
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发表时间：
2006-10-01
影响因子：
1.4
通讯作者：
Fant, M. E.
中科院分区：
文献类型：
--
作者：
Fang, J.;Mao, D.;Fant, M. E.
目的：IGF-1和IGF-1受体是胎儿生长的主要决定因素，主要在人胎盘中合胞滋养细胞膜面向母体的表面表达。IGF-1部分通过调节氨基酸在胎盘中的转运来调节胎儿生长。这些研究的目的是研究IGF-1及其信号通路在人滋养细胞培养模型中调节中性氨基酸运输中的作用。设计：利用非代谢氨基酸类似物[H-3]- α -氨基异丁酸（AIB）研究了IGF-1在培养的BeWo（b30）绒毛膜癌细胞中对中性氨基酸运输的调节。没有Na的转运被用来区分系统L和总AIB转运。同样，过量甲基aib （MeAIB）存在下的na依赖性转运允许系统A （MeAIB敏感）和ASC （MeAIB不敏感）的区分。然后使用细胞内信号通路的特异性抑制剂来确定IGFs调节每个氨基酸运输系统所利用的信号通路。通过p70s6激酶活性和MAP激酶激活的特异性标记来评估抑制的特异性。结果：IGF-1的最大刺激浓度（100 ng/ml）仅通过系统a刺激At B转运30-40%。Wortmannin (100 nM)， pi -3激酶活性抑制剂，抑制所有IGF-1刺激的转运。p70s6激酶抑制剂雷帕霉素（100 ng/ml）和蛋白激酶C （PKC）抑制剂双吲哚酰马来酰亚胺没有作用。MAP激酶激活抑制剂PD-098059 （50 μ M）抑制了20-30%的AIB基础转运，但不抑制igf -1刺激的转运。IGF-1没有增加系统A转运体，SNAT1和SNAT2的稳态mRNA水平，这表明IGF-1通过转录后机制刺激转运。结论：这些数据表明，在BeWo（b30）细胞系中，lGF-1通过pi3激酶依赖的转录后途径刺激中性氨基酸转运系统A。此外，系统A活性似乎对不受IGFs调节的MAP激酶依赖途径敏感。(c) 2006 Elsevier Ltd.版权所有。
Objective: IGF-1 and IGF-1 receptors are major determinants of fetal growth and are expressed primarily on the maternal-facing surface of the syncytiotrophoblast cell membrane in the human placenta. IGF-1 regulates fetal growth, in part, by regulating amino acid transport across the placenta. The objective of these studies was to study the role of IGF-1 and its signaling pathway in regulating neutral amino acid transport in a human trophoblast cell culture model.Design: The regulation of neutral amino acid transport by IGF-1 was studied in cultured BeWo(b30) choriocarcinoma cells using the non-metabolizing amino acid analog, [H-3]-alpha-aminoisobutyric acid (AIB). Transport in the absence of Na was used to distinguish system L from total AIB transport. Similarly, Na-dependent transport in the presence of excess methyl-AIB (MeAIB) permitted discrimination of systems A (MeAIB-sensitive) and ASC (MeAIB-insensitive). Specific inhibitors of intracellular signaling pathways were then used to determine the signaling pathway utilized by IGFs to regulate each amino acid transport system. Specificity of inhibition was assessed using specific markers of p70 S6 kinase activity and MAP kinase activation.Results: Maximal stimulating concentrations of IGF-1 (100 ng/ml) stimulated At B transport by 30-40% exclusively through system A. Wortmannin (100 nM), an inhibitor of PI-3-kinase activity, inhibited all IGF-1-stimulated transport. Rapamycin (100 ng/ml), an inhibitor of p70 S6 kinase, and bisindolylmaleimide, an inhibitor of protein kinase C (PKC), had no effect. PD-098059 (50 mu M), an inhibitor of MAP kinase activation, inhibited 20-30% of basal AIB transport but did not inhibit IGF-1-stimulated transport under the conditions studied. IGF-1 did not increase steady state mRNA levels of the system A transporters, SNAT1 and SNAT2, suggesting IGF-1 stimulates transport via post-transcriptional mechanisms.Conclusions: These data demonstrate that lGF-1 stimulates neutral amino acid transport system A by a PI3-kinase dependent, post-transcriptional pathway in the BeWo(b30) cell line. Additionally, system A activity appear to be sensitive to MAP kinase-dependent pathways not regulated by IGFs. (c) 2006 Elsevier Ltd. All rights reserved.