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Valeria Valsecchi,
Giuseppe Pignataro,
Rossana Sirabella,
Carmela Matrone,
Francesca Boscia,
Antonella Scorziello,
Maria Josè Sisalli,
Elga Esposito,
Nicola Zambrano,
Mauro Cataldi,
Gianfranco Di Renzo,
Lucio Annunziato
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ABSTRACT: The ubiquitous sodium-calcium exchanger isoform 1 (NCX1) is a -bidirectional transporter that plays a relevant role under physiological and pathophysiological conditions including brain ischemia by regulating intraneuronal Ca(2+) and Na(+) homeostasis. Although changes in ncx1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still largely unexplored. Here, we reviewed our recent findings on several transcription factors including cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-κB), and hypoxia-inducible factor-1 (HIF-1) in the control of the ncx1 gene expression in neuronal cells.
Advances in experimental medicine and biology 01/2013; 961:137-45. · 1.09 Impact Factor
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Luigi Formisano,
Natascia Guida, Valeria Valsecchi,
Giuseppe Pignataro,
Antonio Vinciguerra,
Anna Pannaccione,
Agnese Secondo,
Francesca Boscia,
Pasquale Molinaro,
Maria Jose Sisalli,
Rossana Sirabella,
Antonella Casamassa,
Lorella M T Canzoniero,
Gianfranco Di Renzo,
Lucio Annunziato
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ABSTRACT: The Na(+)-Ca(2+) exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasmamembrane, is known to be involved in brain ischemia. Since the RE1-silencing transcription factor (REST) is a key modulator of neuronal gene expression in several neurological conditions, we studied the possible involvement of REST in regulating NCX1 gene expression and activity in stroke. We found that: (1) REST binds in a sequence specific manner and represses through H4 deacetylation, ncx1 gene in neuronal cells by recruting CoREST, but not mSin3A. (2)In neurons and in SH-SY5Y cells REST silencing by siRNA and site-direct mutagenesis of REST consensus sequence on NCX1 brain promoter determined an increase in NCX1 promoter activity. (3)By contrast, REST overexpression caused a reduction in NCX1 protein expression and activity. (4)Interestingly, in rats subjected to transient middle cerebral artery occlusion (tMCAO) and in organotypic hippocampal slices or SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD) plus reoxygenation (RX), the increase in REST was associated with a decrease in NCX1. However, this reduction was reverted by REST silencing. (5)REST knocking down, along with the deriving NCX1overexpression in the deep V and VIb cortical layers caused a marked reduction in infarct volume after tMCAO. Double silencing of REST and NCX1 completely abolished neuroprotection induced by siREST administration. Collectively, these results demonstrate that REST, by regulating NCX1 expression, may represent a potential druggable target for the treatment of brain ischemia.
Neurobiology of Disease 10/2012; · 5.40 Impact Factor
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Valeria Valsecchi,
Giuseppe Pignataro,
Annalisa Del Prete,
Rossana Sirabella,
Carmela Matrone,
Francesca Boscia,
Antonella Scorziello,
Maria Josè Sisalli,
Elga Esposito,
Nicola Zambrano,
Gianfranco Di Renzo,
Lucio Annunziato
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ABSTRACT: The sodium-calcium exchanger-1 (NCX1) represents a key mediator for maintaining [Na(+)](i) and [Ca(2+)](i) homeostasis. Although changes in NCX1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still unknown. Thus far, however, there is evidence that hypoxia-inducible factor-1 (HIF-1) is a nuclear factor required for transcriptional activation of several genes implicated in stroke. The main objective of this study was to investigate whether NCX1 gene might be a novel target of HIF-1 in the brain.
Here we report that: (1) in neuronal cells, NCX1 increased expression after oxygen and glucose deprivation or cobalt-induced HIF-1 activation was prevented by silencing HIF-1; (2) the brain NCX1 promoter cloned upstream of the firefly-luciferase gene contained 2 regions of HIF-1 target genes called hypoxia-responsive elements that are sensitive to oxygen and glucose deprivation or cobalt chloride; (3) HIF-1 specifically bound hypoxia-responsive elements on brain NCX1, as demonstrated by band-shift and chromatin immunoprecipitation assays; (4) HIF-1α silencing prevented NCX1 upregulation and neuroprotection induced by ischemic preconditioning; and (5) NCX1 silencing partially reverted the preconditioning-induced neuroprotection in rats.
NCX1 gene is a novel HIF-1 target, and HIF-1 exerts its prosurvival role through NCX1 upregulation during brain preconditioning.
Stroke 03/2011; 42(3):754-63. · 5.73 Impact Factor
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ABSTRACT: The 3 gene products of the Na(+)/Ca(2+) exchanger (NCX), viz, NCX1, NCX2, and NCX3, may play a pivotal role in the pathophysiology of brain ischemia. The aim of this study was to investigate the transductional and posttranslational mechanisms involved in the expression of these isoforms during oxygen and glucose deprivation and their role in endoplasmic reticulum Ca(2+) refilling in cortical neurons.
NCX1, NCX2, and NCX3 transcript and protein expression was evaluated in primary cortical neurons by reverse transcriptase-polymerase chain reaction and Western blot. NCX currents (I(NCX)) and cytosolic Ca(2+) concentrations ([Ca(2+)](i)) were monitored by means of patch-clamp in whole-cell configuration and Fura-2AM single-cell video imaging, respectively.
Exposure of cortical neurons to 3 hours of oxygen and glucose deprivation yielded dissimilar effects on the 3 isoforms. First, it induced an upregulation in NCX1 transcript and protein expression. This change was exerted at the transcriptional level because the inhibition of nuclear factor kappa B translocation by small interfering RNA against p65 and SN-50 prevented oxygen and glucose deprivation-induced NCX1 upregulation. Second, it elicited a downregulation of NCX3 protein expression. This change, unlike NCX1, was exerted at the posttranscriptional level because it was prevented by the proteasome inhibitor MG-132. Finally, we found that it significantly increased I(NCX) both in the forward and reverse modes of operation and promoted an increase in ER Ca(2+) accumulation. Interestingly, such accumulation was prevented by the silencing of NCX1 and the NCX inhibitor CB-DMB that triggered caspase-12 activation.
These results suggest that nuclear factor kappa B-dependent NCX1 upregulation may play a fundamental role in Ca(2+) refilling in the endoplasmic reticulum, thus helping neurons to prevent endoplasmic reticulum stress during oxygen and glucose deprivation.
Stroke 02/2009; 40(3):922-9. · 5.73 Impact Factor
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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 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.
Molecular pharmacology 04/2008; 73(3):727-37. · 4.53 Impact Factor
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Agnese Secondo,
Rosaria Ilaria Staiano,
Antonella Scorziello,
Rossana Sirabella,
Francesca Boscia,
Annagrazia Adornetto, Valeria Valsecchi,
Pasquale Molinaro,
Lorella Maria Teresa Canzoniero,
Gianfranco Di Renzo,
Lucio Annunziato
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ABSTRACT: The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10 microM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial delta psi collapses and cell death.
Cell Calcium 01/2008; 42(6):521-35. · 3.77 Impact Factor
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ABSTRACT: K(+)-dependent Na(+)/Ca(2+) exchangers (NCKX) catalyze cytosolic Ca(2+) extrusion and are particularly important for neuronal Ca(2+) signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca(2+) and Na(+) were similar, NCKX3 and -4 displayed approximately 40-fold higher affinities for K(+) ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K(+) affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca(2+) affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K(+) and Ca(2+) affinities were reduced. These results indicate that the apparently low K(+) affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K(+) liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K(+) binding pocket, because it was not possible to recreate the K(+)-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca(2+) signaling.
Journal of Biological Chemistry 03/2007; 282(7):4453-62. · 4.77 Impact Factor
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ABSTRACT: K+-dependent Na+/Ca2+ exchangers (NCKX) catalyze cytosolic Ca2+ extrusion and are particularly important for neuronal Ca2+ signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and
-2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined
and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca2+ and Na+ were similar, NCKX3 and -4 displayed ∼40-fold higher affinities for K+ ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding
residue in NCKX4, resulted in an apparent K+ affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca2+ affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both
the K+ and Ca2+ affinities were reduced. These results indicate that the apparently low K+ affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K+ liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the
structural context of the NCKX2 K+ binding pocket, because it was not possible to recreate the K+-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information
about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca2+ signaling.
Journal of Biological Chemistry 02/2007; 282(7):4453-4462. · 4.77 Impact Factor
