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+ and Ca2+ 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.
Footnotes
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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.
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L.F. and M.S. contributed equally to this work.
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ABBREVIATIONS: NCX, Na+/Ca2+ 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 Ca2+-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.
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↵1 Current affiliation: University of Sannio, Benevento, Italy.
- Received October 9, 2007.
- Accepted December 12, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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