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Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro.

Cinti A, De Giorgi M, Chisci E, Arena C, Galimberti G, Farina L, Bugarin C, Rivolta I, Gaipa G, Smolenski RT, Cerrito MG, Lavitrano M, Giovannoni R - PLoS ONE (2015)

Bottom Line: The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells.The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells.Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy.

ABSTRACT
Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

No MeSH data available.


Related in: MedlinePlus

All the three exogenous proteins were correctly localized in pCX TRI 2A-transfected cells.WT and pCX-TRI-2A-transfected cells were co-stained with anti-hHO1 and anti-hE5NT antibodies (A) or with anti-hHO1and anti-hENTPD1antibodies (B). Transfected cells positive to hE5NT or hENTPD1 (red) were also positive to hHO1 (green). hE5NT and hENTPD1 localized on the cell surface, while hHO1 had a perinuclear and/or ER membranes cytoplasmic localization. (C-D) Plot of the signal intensity for hE5NT and hENTPD1 (red) and hHO1 (green) along the line drawn in A and B indicated that the most intense hE5NT and hENTPD1 signals are not colocalized with hHO1 signal.
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pone.0141933.g003: All the three exogenous proteins were correctly localized in pCX TRI 2A-transfected cells.WT and pCX-TRI-2A-transfected cells were co-stained with anti-hHO1 and anti-hE5NT antibodies (A) or with anti-hHO1and anti-hENTPD1antibodies (B). Transfected cells positive to hE5NT or hENTPD1 (red) were also positive to hHO1 (green). hE5NT and hENTPD1 localized on the cell surface, while hHO1 had a perinuclear and/or ER membranes cytoplasmic localization. (C-D) Plot of the signal intensity for hE5NT and hENTPD1 (red) and hHO1 (green) along the line drawn in A and B indicated that the most intense hE5NT and hENTPD1 signals are not colocalized with hHO1 signal.

Mentions: Since it has been demonstrated that different subcellular localization might influence the expression pattern of target genes coupled to the 2A peptide [38], we investigated if the human proteins encoded by the muticistronic 2A-based transgene had a correct subcellular localization by immunofluorescence and confocal analysis. Co-staining analysis for hE5NT and hHO1 or for hENTPD1 and hHO1 indicated that in pCX-TRI-2A transfected cells both the hENTPD1 and hE5NT signals had, as expected, a distribution pattern similar to that of plasma membrane proteins, whereas hHO1 signal was detected mainly in the perinuclear area suggesting, for this protein, a cytoplasmic localization related to the ER (Fig 3A and 3B). Pixel intensity analysis further confirmed the correct localization of the exogenous proteins (Fig 3C and 3D)


Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro.

Cinti A, De Giorgi M, Chisci E, Arena C, Galimberti G, Farina L, Bugarin C, Rivolta I, Gaipa G, Smolenski RT, Cerrito MG, Lavitrano M, Giovannoni R - PLoS ONE (2015)

All the three exogenous proteins were correctly localized in pCX TRI 2A-transfected cells.WT and pCX-TRI-2A-transfected cells were co-stained with anti-hHO1 and anti-hE5NT antibodies (A) or with anti-hHO1and anti-hENTPD1antibodies (B). Transfected cells positive to hE5NT or hENTPD1 (red) were also positive to hHO1 (green). hE5NT and hENTPD1 localized on the cell surface, while hHO1 had a perinuclear and/or ER membranes cytoplasmic localization. (C-D) Plot of the signal intensity for hE5NT and hENTPD1 (red) and hHO1 (green) along the line drawn in A and B indicated that the most intense hE5NT and hENTPD1 signals are not colocalized with hHO1 signal.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4626094&req=5

pone.0141933.g003: All the three exogenous proteins were correctly localized in pCX TRI 2A-transfected cells.WT and pCX-TRI-2A-transfected cells were co-stained with anti-hHO1 and anti-hE5NT antibodies (A) or with anti-hHO1and anti-hENTPD1antibodies (B). Transfected cells positive to hE5NT or hENTPD1 (red) were also positive to hHO1 (green). hE5NT and hENTPD1 localized on the cell surface, while hHO1 had a perinuclear and/or ER membranes cytoplasmic localization. (C-D) Plot of the signal intensity for hE5NT and hENTPD1 (red) and hHO1 (green) along the line drawn in A and B indicated that the most intense hE5NT and hENTPD1 signals are not colocalized with hHO1 signal.
Mentions: Since it has been demonstrated that different subcellular localization might influence the expression pattern of target genes coupled to the 2A peptide [38], we investigated if the human proteins encoded by the muticistronic 2A-based transgene had a correct subcellular localization by immunofluorescence and confocal analysis. Co-staining analysis for hE5NT and hHO1 or for hENTPD1 and hHO1 indicated that in pCX-TRI-2A transfected cells both the hENTPD1 and hE5NT signals had, as expected, a distribution pattern similar to that of plasma membrane proteins, whereas hHO1 signal was detected mainly in the perinuclear area suggesting, for this protein, a cytoplasmic localization related to the ER (Fig 3A and 3B). Pixel intensity analysis further confirmed the correct localization of the exogenous proteins (Fig 3C and 3D)

Bottom Line: The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells.The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells.Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Translational Medicine, University of Milano-Bicocca, Monza, Italy.

ABSTRACT
Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

No MeSH data available.


Related in: MedlinePlus