Formisano Luigi, Saggese Mariangela, Secondo Agnese, Sirabella Rossana, Vito Pasquale, Valsecchi Valeria, Molinaro Pasquale, Di Renzo Gianfranco, Annunziato Lucio
Division of Pharmacology, Department of Neuroscience, School of Medicine, University of Naples "Federico II", Via Pansini 5, 80131 Naples, Italy.
Mol Pharmacol. 2008 Mar;73(3):727-37. doi: 10.1124/mol.107.042549. Epub 2007 Dec 13.
The proteins NCX1, NCX2, and NCX3 expressed on the plasma membrane of neurons play a crucial role in ionic regulation because they are the major bidirectional system promoting the efflux and influx of Na(+) and Ca(2+) ions. Here, we demonstrate that NCX1 and NCX3 proteins are novel additional targets for the survival action of the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. Indeed, the doxycycline-dependent overexpression of constitutively active Akt1 in tetracycline (Tet)-Off PC-12 positive mutants and the exposure of Tet-Off PC-12 wild type to nerve growth factor induced an up-regulation of NCX1 and NCX3 proteins. NCX1 up-regulation induced by Akt1 activation occurred at the transcriptional level because NCX1 mRNA increased, and it was counteracted by cAMP response element-binding protein 1 inhibition through small interfering RNA strategy. In contrast, Akt1-induced NCX3 up-regulation recognized a post-transcriptional mechanism occurring at the proteasome level because 1) NCX3 transcript did not increase and 2) the proteasome inhibitor N-benzyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) did not further enhance NCX3 protein levels in Akt1 active mutants as it would be expected if the ubiquitin-proteasome complex was not already blocked by Akt1 pathway. As expected, in PC-12 Tet-Off wild-type cells MG-132 enhanced NCX3 protein levels. This up-regulation produced an increased activity of NCX function. Furthermore, NCX1 and NCX3 up-regulation contributed to the survival action of Akt1 during chemical hypoxia because both the silencing of NCX1 or NCX3 and the pharmacological paninhibition of NCX isoforms reduced the prosurvival property of Akt1. Together, these results indicated that NCX1 and NCX3 represent novel additional molecular targets for the prosurvival action of PI3-K/Akt pathway.
神经元质膜上表达的蛋白质NCX1、NCX2和NCX3在离子调节中起关键作用，因为它们是促进Na(+)和Ca(2+)离子外流和内流的主要双向系统。在此，我们证明NCX1和NCX3蛋白是磷脂酰肌醇3激酶（PI3-K）/Akt信号通路存活作用的新的额外靶点。事实上，在四环素（Tet）-Off PC-12阳性突变体中强力霉素依赖性组成型活性Akt1的过表达以及将Tet-Off PC-12野生型暴露于神经生长因子会诱导NCX1和NCX3蛋白上调。Akt1激活诱导的NCX1上调发生在转录水平，因为NCX1 mRNA增加，并且通过小干扰RNA策略抑制环磷酸腺苷反应元件结合蛋白1可抵消这种上调。相反，Akt1诱导的NCX3上调识别出一种发生在蛋白酶体水平的转录后机制，原因如下：1）NCX3转录本未增加；2）蛋白酶体抑制剂N-苄氧羰基（Z）-亮氨酰-亮氨酰-亮氨酸（MG-132）并未如预期那样在Akt1活性突变体中进一步提高NCX3蛋白水平，如果泛素-蛋白酶体复合物尚未被Akt1信号通路阻断则会出现这种预期效果。正如预期的那样，在PC-12 Tet-Off野生型细胞中，MG-132提高了NCX3蛋白水平。这种上调导致NCX功能活性增加。此外，NCX1和NCX3上调有助于Akt1在化学性缺氧期间的存活作用，因为NCX1或NCX3的沉默以及NCX亚型的药理学全面抑制均降低了Akt1的促存活特性。总之，这些结果表明NCX1和NCX3代表了PI3-K/Akt信号通路促存活作用的新的额外分子靶点。
