Bianchi M G, Gazzola G C, Tognazzi L, Bussolati O
Unit of General and Clinical Pathology, Department of Experimental Medicine, University of Parma, Via Volturno, 39, 43100 Parma, Italy.
Neuroscience. 2008 Feb 19;151(4):1042-52. doi: 10.1016/j.neuroscience.2007.11.055. Epub 2007 Dec 15.
The transport of excitatory amino acids (EAA) in CNS is performed by a family of high affinity, sodium dependent carriers. One of these transporters, excitatory amino acid carrier 1 (EAAC1), is known to be regulated by several mechanisms that modify carrier abundance on the plasma membrane. Much less is known on EAAC1 regulation at the level of gene expression. Here we report that, in C6 rat glioma cells, a line recently described to contain neural stem-like cells, EAAC1 is markedly induced by all trans-retinoic acid (ATRA), a well known differentiating agent. Consistently, ATRA stimulates EAA transport, with the maximal effect observed at concentrations>or=1 microM. After 4 days of treatment with 10 microM ATRA, the transport Vmax is fivefold enhanced, Slc1a1 mRNA is increased by 400% compared with control, EAAC1 carrier is sixfold overexpressed and the C6 culture is greatly enriched of cells with bipolar morphology strongly positive for EAAC1 immunoreactivity. Compared with untreated cells, ATRA-treated C6 cells express less Slc1a3 mRNA, for the transporter GLAST, but significantly higher levels of Slc1a2 mRNA, for the transporter GLT-1, although no expression of either protein is detected with Western blot in both untreated and ATRA-treated cells. Consistently, the inhibition pattern of aspartate transport and its stimulation by phorbol esters are indicative of a transport process due to EAAC1 operation. Under the conditions adopted, ATRA treatment causes the induction of proteolipid protein, an oligodendrocytic marker. These results indicate that, in C6 cells, ATRA stimulates the expression of EAAC1, possibly as a step toward oligodendrocytic differentiation, and constitute the first demonstration of the induction of this transporter by a differentiating agent.
中枢神经系统中兴奋性氨基酸（EAA）的转运由一类高亲和力、依赖钠的载体完成。其中一种转运体，即兴奋性氨基酸载体1（EAAC1），已知受多种机制调节，这些机制可改变其在质膜上的载体丰度。而关于EAAC1在基因表达水平的调控，人们了解得较少。在此我们报告，在C6大鼠胶质瘤细胞（最近报道该细胞系含有神经干细胞样细胞）中，全反式维甲酸（ATRA，一种著名的分化剂）可显著诱导EAAC1。一致的是，ATRA刺激EAA转运，在浓度≥1μM时观察到最大效应。用10μM ATRA处理4天后，转运Vmax提高了5倍，Slc1a1 mRNA比对照增加了400%，EAAC1载体过表达6倍，并且C6培养物中具有双极形态且EAAC1免疫反应性强阳性的细胞大量富集。与未处理的细胞相比，经ATRA处理的C6细胞中转运体GLAST的Slc1a3 mRNA表达较少，但转运体GLT-1的Slc1a2 mRNA水平显著更高，尽管在未处理和经ATRA处理的细胞中通过蛋白质印迹均未检测到这两种蛋白的表达。一致的是，天冬氨酸转运的抑制模式及其受佛波酯的刺激表明这是一个由EAAC1运作引起的转运过程。在所采用的条件下，ATRA处理可诱导少突胶质细胞标志物蛋白脂质蛋白的表达。这些结果表明，在C6细胞中，ATRA刺激EAAC1的表达，这可能是向少突胶质细胞分化的一个步骤，并且首次证明了分化剂可诱导这种转运体。
