SERANI, ANGELO
(2017)
IDENTIFICATION AND CHARACTERIZATION OF THE
PROMOTER AND REGULATORY ELEMENTS OF THE CEREBRAL SODIUM/CALCIUM EXCHANGER ISOFORM 2 (NCX2) GENE IN RAT PHEOCHROMOCYTOMA CELLS.
[Tesi di dottorato]
| Tipologia del documento: |
Tesi di dottorato
|
| Lingua: |
English |
| Titolo: |
IDENTIFICATION AND CHARACTERIZATION OF THE
PROMOTER AND REGULATORY ELEMENTS OF THE CEREBRAL SODIUM/CALCIUM EXCHANGER ISOFORM 2 (NCX2) GENE IN RAT PHEOCHROMOCYTOMA CELLS |
| Autori: |
| Autore | Email |
|---|
| SERANI, ANGELO | angelo.serani@gmail.com |
|
| Data: |
27 Febbraio 2017 |
| Numero di pagine: |
121 |
| Istituzione: |
Università degli Studi di Napoli Federico II |
| Dipartimento: |
Neuroscienze e Scienze Riproduttive ed Odontostomatologiche |
| Dottorato: |
Neuroscienze |
| Ciclo di dottorato: |
29 |
| Coordinatore del Corso di dottorato: |
| nome | email |
|---|
| ANNUNZIATO, LUCIO | lannunzi@unina.it |
|
| Tutor: |
| nome | email |
|---|
| MOLINARO, PASQUALE | [non definito] |
|
| Data: |
27 Febbraio 2017 |
| Numero di pagine: |
121 |
| Parole chiave: |
ncx2, PROMOTER, EPIGENETIC, TRANSCRIPTION FACTORS |
| Settori scientifico-disciplinari del MIUR: |
Area 05 - Scienze biologiche > BIO/14 - Farmacologia |
[error in script]
[error in script]
| Depositato il: |
20 Apr 2017 08:15 |
| Ultima modifica: |
13 Mar 2018 11:37 |
| URI: |
http://www.fedoa.unina.it/id/eprint/11862 |
| DOI: |
10.6093/UNINA/FEDOA/11862 |
Abstract
The isoform 2 (NCX2) of sodium–calcium exchanger family is involved in the regulation of the sodium and calcium homoeostasis of neuronal and glial cells. In particular, NCX2 participates in cytosolic Ca2+ clearance after spike and synaptic plasticity, whereas under pathophysiological conditions it exerts a neuroprotective effect in stroke.
To investigate the genetic regulation of NCX2, we identified its minimal promoter in the region of 1200 bp, localized between slc8a2 and kptn genes.
This sequence induced the transcription of a reporter gene in two different neuronal cell lines, PC12 and SHSY, that express the endogenous NCX2 mRNA. By contrast, the promoter failed to induce the transcription in BHK and U87 cell lines that do not express NCX2 under control conditions. These results reinforced the similarity observed in the transcription activity of the endogenous NCX2 promoter and the region we identified and cloned. In addition, we found, by in silico analysis, a great number of putative binding sites for transcription factors in the promoter sequence. These predicted sites showed high matrix identities and were found conserved among three considered species, including rat, mouse, and human genome. Interestingly, many of these TFs are expressed in the CNS where NCX2 is localized, suggesting a possible role in the regulation
of the expression of this exchanger under physiological and pathophysiological conditions. In particular, the overexpression of the transcription factors Sp1, Sp4, and CREB displayed a stimulatory effect on NCX2 promoter activity under control conditions as measured by luciferase reporter assay, whereas SREBP1 exerted an inhibitory effect. Notably, we found that Sp1 and Sp4 share the same molecular determinant localized very close to the transcription start site of the NCX2 promoter, while CREB1 exerted its effect at the beginning of the cloned sequence. We also evaluated the possible subregion where SREBP1 exerted its inhibitory effect on the transcription activity.
Interestingly, the transfection of these TFs in U87 cell line failed to express the endogenous NCX2, thus letting us hypothesize the participation of other regulatory mechanisms, such as epigenetic, in the of NCX2 transcription. In fact, we found some epigenetic differences between PC12 and L6 cells, expressing or not expressing, respectively, the endogenous NCX2 gene.
In particular, we reported significant differences in CpG methylation in the above-mentioned cell lines on the antiporter promoter. In this regard, 5’- Azacytidine, by imparing genomic DNA methylation, induced the expression of NCX2 in U87 cells.
In conclusion, all these results showed that: (1) the basal expression of NCX2 is dependent on the methylation status of its promoter and (2) NCX2 expression can be up-regulated or down-regulated by several transcription factors found in the CNS.
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