The Two Isoforms of the Na⁺/Ca²⁺Exchanger, NCX1 and NCX3, Constitute Novel Additional Targets for the Prosurvival Action of Akt/Protein Kinase B Pathway

Abstract

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 ϩ and Ca 2ϩ 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. with the entrance of Na ϩ ions, or in the "reverse mode" by mediating the extrusion of Na ϩ to the entrance of Ca 2ϩ ions (Blaustein and Lederer, 1999;Philipson and Nicoll, 2000;Annunziato et al., 2004). To date, three ncx genes-ncx1, ncx2, ncx3-have been identified and cloned. Whereas NCX1 is ubiquitously expressed, NCX2 and NCX3 are expressed exclusively in the brain and in the skeletal muscle (Lee et al., 1994). Specifically, NCX1, NCX2, and NCX3 are expressed in neurons, astrocytes, oligodendrocytes, and microglia (Quednau et al., 1997;Thurneysen et al., 2002;Nagano et al., ABBREVIATIONS: NCX, Na ϩ /Ca 2ϩ exchanger; PKB, protein kinase B; Tet, tetracycline; siRNA, small interfering RNA; FBS, fetal bovine serum; CREB, cAMP response element-binding; PI3-K, phosphatidylinositol 3-kinase; p-, phosphorylated; ECL, enhanced chemiluminescence; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; HA, hemagglutinin; PI, propidium iodide; FDA, fluorescein diacetate; NGF, nerve growth factor; MG-132, N-benzyloxycarbonyl (Z)-Leu-Leu-leucinal; tTA, tetracycline-controlled transactivator; EGFP, enhanced green fluorescent protein; GSK, glycogen synthase kinase; PMCA, plasma membrane Ca 2ϩ -ATPase; RT-PCR, reverse transcr...

Endoplasmic reticulum refilling and mitochondrial calcium extrusion promoted in neurons by NCX1 and NCX3 in ischemic preconditioning are determinant for neuroprotection

“…As NCX1 and NCX3 constitute novel additional targets for the prosurvival action of the Akt/Protein Kinase B pathway, 12 and as IPC induces NO production and Akt activation, 13 the involvement of the NO/Akt pathway in the regulation of NCX expression was explored in cortical neurons during IPC. Interestingly, IPC caused an increase in the phosphorylated form of Akt, which was abolished after the treatment with nNOS inhibitors L-NAME 14 and 7-Nitroindazole (7NI) 15 ( Figure 1e, Supplementary Figure S1C).…”

“…To demonstrate that NO-induced Akt activation was responsible for the increased expression of NCX1 and NCX3 during IPC, further experiments were performed in cortical neurons first treated with L-NAME (500 mM) and 7NI (500 mM) to inhibit nNOS, or with LY294002 (25 mM), wortmannin (WMN, 1 mM) and Akt-negative dominant (Akt D-) to inhibit the PI3K/Akt pathway, 12 and then exposed to IPC. As reported in Figure 2 and Supplementary Figure S1, the inhibition of nNOS prevents IPC-induced NCX1 and NCX3 expression, whereas L-NAME prevented only IPC-induced NCX3 overexpression (Figures 2a and b, Supplementary Figures S1A and B).…”

“…1 Indeed, when rats subjected to middle cerebral artery occlusion (MCAO) were intracerebroventricularly (ICV) treated with the PI3K inhibitor LY294002, 12 the expression of NCX1 and NCX3 was downregulated. Interestingly, the novel aspect of the present study was that IPC through the mediation of NO, PI3K/Akt and NCX1 and NCX3 interferes with ER refilling and mitochondrial calcium handling.…”

“…To prepare siRNA-NCX1, a 60-base oligonucleotide and another oligonucleotide with the complementary sequence were annealed and inserted into pSUPER.retro.puro as previously reported. 12 A mismatch sequence cloned in the same vector was used as an experimental control. To knock down NCX3, the nucleotide sequence corresponding to the first nucleotide of the start codon( þ 124 À 142) of rat NCX3 (GenBank accession no.…”

Dofetilide as Activator of Na/Ca Exchange: New Perspectives on an ‘Old’ Drug

“…In the brain, activation of NCX is apparently part of a protective pathway during ischemia [28,29] and pharmacological activators of NCX would be an interesting tool to further test this pathway. With dofetilide, in the correct experimental circumstances, we could have such a tool.…”