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

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 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. The Na /Ca antiporter, an integral protein belonging to the plasma membrane cation/Ca exchanger superfamily, consists of nine transmembrane segments that can mediate Ca and Na fluxes across the neuronal membrane, depending on the intracellular concentration of Ca ([Ca ]i) and Na ([Na ]i). In particular, NCX can operate either in the “forward mode” by coupling the uphill extrusion of Ca with the entrance of Na ions, or in the “reverse mode” by mediating the extrusion of Na to the entrance of Ca 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., This study was supported by grants from the Italian Ministry of Health Programma Speciale art. 12bis comma 6, D. Lgs. 229/99; COFIN 2004 (to L.A.), Regione Campania GEAR, Ricerca Finalizzata Ministero della Salute legge 502/92 “Geni di vulnerabilità e di riparazione DNA” (to L.A.) and POP and legge 41 from Regione Campania, Italian Ministry of Health Programma Speciale art. 12bis comma 6, D. Lgs. 229/99; and 12th Italian-Chinese executive program for scientific and technological cooperation for the period 2006– 2009 (to L.A.) from the Italian Foreign Ministry. L.F. and M.S. contributed equally to this work. 1 Current affiliation: University of Sannio, Benevento, Italy. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.042549. ABBREVIATIONS: NCX, Na /Ca 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 -ATPase; RT-PCR, reverse transcription-polymerase chain reaction; HPRT, hypoxanthine-guanine phosphoribosyl transferase; KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate derivative. 0026-895X/08/7303-727–737$20.00 MOLECULAR PHARMACOLOGY Vol. 73, No. 3 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 42549/3309509 Mol Pharmacol 73:727–737, 2008 Printed in U.S.A. 727 at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from 2004). During anoxic conditions, owing to the two plasma membrane ATP-dependent pumps—Na /K ATPase and Ca ATPase—being compromised, NCX assumes a relevant role in controlling the intracellular homeostasis of these two cations. It has recently been proposed that whereas in in vitro and in in vivo models of anoxia and ischemia, respectively, the pharmacological paninhibition of the NCX gene products, or the more specific antisense knockdown of NCX1 and NCX3 transcripts, can compromise neuronal survival, the activation of the exchanger yields potentially beneficial effects not only on anoxic neurons and glial cells but also in cerebral ischemia (Amoroso et al., 1997, 2000; Annunziato et al., 2004; Pignataro et al., 2004). Conversely, the Akt/PKB signaling pathway is now largely recognized as one of the most relevant pathways in regulating neuronal survival (Song et al., 2005). Indeed, it has various functions: 1) it regulates glucose metabolism; 2) it inactivates the mitochondrial death pathway (i.e., BAD and caspase 9) and FOXO pathway; 3) it activates CREB phosphorylation, which regulates the expression of genes critical for survival, such as those encoding for cytokines and brainderived neurotrophic factor; and 4) it activates the antiapoptotic factor nuclear factorB through the activation of I B kinase complex (Fukunaga and Kawano, 2003). In addition, Akt regulates Ca homeostasis through the potentiation of L-type voltage-gated calcium channels (Blair et al., 1999), sarco(endo)plasmatic Ca ATPase (Kim et al., 2003), and intracellular ligand-gated ion channels (Barac et al., 2005), thus enhancing neuronal survival. In the present study, we examined whether Akt1, an isoform that is ubiquitously and highly expressed in the brain, can, in addition to affecting the other prosurvival cascades, also exert its neuroprotective effect by modulating the expression and activity of NCX1, NCX2, and NCX3 gene products. By means of the Tet-Off strategy, we demonstrate that in positive PC-12 Akt1 mutants, a selective increase of NCX1 and NCX3 isoform expression and activity occurs. The Akt1induced NCX1 overexpression occurred at the transcriptional level, and it was mediated by CREB activation; by contrast, NCX3 up-regulation seemed to be dependent on Akt inhibition at the level of the proteasome-ubiquitin complex. Furthermore, the selective inhibition of NCX1 and NCX3 by siRNA strategy, and its pharmacological paninhibition, markedly reverted the NCX1 and NCX3 prosurvival action exerted by Akt1. Materials and Methods Reagents. Media and sera for cell culture were purchased from Invitrogen (Milan, Italy); antibiotics for cell culture were from Sigma-Aldrich (St. Louis, MO). Tet system-approved fetal bovine serum (FBS) was from BD Biosciences (DBA srl, Milan, Italy). Hygromycin and Geneticin (G418) were from Invitrogen (Inalco, Milan, Italy). Rabbit-polyclonal AKT and p-AKT antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and phosphokinase antibodies were from New England Biolabs (Ipswich, MA). Polyclonal NCX1 antibody was from Swant (Bellinzona, Switzerland), and polyclonal NCX2 and NCX3 antibodies were from Sigma Diagnostics (San Antonio, TX) and Drs. K. D. Philipson and D. A. Nicoll. Western blotting, ECL reagents, and radiochemicals were from GE Healthcare (Milan, Italy). LY294002 was purchased from Calbiochem (San Diego, CA). pTRE-2Hyg expression vector was from Invitrogen. The positive mutant hemagglutinin (HA) antigen-tagged HA-Aktm 4-129 (Akt D ) and inactive mutant HA-Aktk179M (Akt D ) plasmids were donated by P. Formisano (University of Naples “Federico II”, Naples, Italy), as described previously (Eves et al., 1998; Trencia et al., 2003). Oligomycin, 2-deoxy-glucose, propidium iodide (PI), fluorescein diacetate (FDA), and all other reagents were from Sigma (Milan, Italy). NGF was purchased from Millipore (Billerica, MA). MG-132 was from Sigma. siRNA-CREB and siRNA-CONTROL were purchased from Dharmacon RNA Technologies (Thema Ricerca, Italy). Cloning, Cell Culture, and Development of Double-Stable Cell Lines. HA-Aktm 4-129 (Akt D ) was obtained by fusing the c-Akt and retroviral Gag protein with 21 additional amino acids derived from the translation of 63 nucleotides of the c-Akt 5 , placed in phase between Gag and Akt. The myristoylation site in the Gag sequence targets Akt to the plasma membrane, and it results in high basal kinase activity. By contrast, HA-Aktk179M (Akt D ) was mutated at the ATP binding site, and it results in an inactive kinase. Akt D and Akt D were subcloned into the Bam-EcoRI sites of the pEGFP-N1 vector (Clontech, Mountain View, CA), and then they were cloned into the Nhe1-Not1 sites of pTRE-2Hyg vector. PC-12 Tet-Off cells were obtained from Clontech. This system is based on the regulatory elements of the tetracycline-resistance operon of Escherichia coli, characterized by a tetracycline-controlled transactivator (tTA) and a tTA-dependent promoter. The latter is virtually silent in the presence of tetracycline and doxycycline, but it becomes active in their absence, as indicated in the Tet-Off and Tet-On gene expression systems and cell lines user’s manual (Clontech). These cells were grown on plastic dishes in Dulbecco’s modified Eagle’s medium composed of 10% horse serum, 5% FBS, 100 UI/ml penicillin, and 100 g/ml streptomycin. The double-stable Tet-Off PC-12 cell lines, expressing Akt D and Akt D , were obtained by transfecting pTRE-2Hyg-Akt1-EGFP vectors into PC-12 Tet-Off cells. Upon reaching 80% confluence, PC-12 Tet-Off cells were transfected with standard protocol by using 10 g of the two pTRE-2Hyg-Akt1-EGFP vectors and 30 l of Lipofectamine 2000 (Invitrogen). To select stably transfected cells, selection was carried out by isolating hygromycin-resistant clones incu0 12 1 24 48 p -A kt / A kt (O . D . p ro te in ex p re ss io n )