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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

Heme oxygenase-1 and ectonucleotidases functional assays.(A) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. *p<0.05 versus wt and mock-transfected cells. (B) ENTPD1-mediated AMP production and (C) E5NT-mediated adenosine production by wild type, mock and pCX-TRI-2A transfected-cells. Cells were incubated with 50 μM ATP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC as detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *p<0.05 versus all groups. (D) E5NT-mediated adenosine production by wild type, mock and transfected-cells. Cells were incubated with 50 μM AMP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC ad detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *p<0.05 versus all groups.
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pone.0141933.g004: Heme oxygenase-1 and ectonucleotidases functional assays.(A) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. *p<0.05 versus wt and mock-transfected cells. (B) ENTPD1-mediated AMP production and (C) E5NT-mediated adenosine production by wild type, mock and pCX-TRI-2A transfected-cells. Cells were incubated with 50 μM ATP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC as detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *p<0.05 versus all groups. (D) E5NT-mediated adenosine production by wild type, mock and transfected-cells. Cells were incubated with 50 μM AMP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC ad detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *p<0.05 versus all groups.

Mentions: The enzymatic activity of HO-1 in pCX-TRI-2A-transfected cells was evaluated by measuring the fluorescence of bilirubin during the incubation of lysates from WT, mock and pCX-TRI-2A-transfected cells with hemin. As shown in Fig 4A, the HO-1 activity in pCX-TRI-2A-transfected cells was about 2.5 fold higher than the basal activity seen in controls (1.43 ± 0.08 nmol/h/mg versus 0.54 ± 0.08 and 0.58 ± 0.1 nmol/h/mg respectively in WT and mock transfected-cells, p<0.05). Moreover, no significant differences were observed between pCX-TRI-2A and control cell lines (WT and mock-transfected cells) previously treated with CoPP. These data showed that the exogenous expression of hHO-1, encoded by pCX-TRI-2A plasmid, was comparable to the CoPP induced expression levels of endogenous HO-1.


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)

Heme oxygenase-1 and ectonucleotidases functional assays.(A) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. *p<0.05 versus wt and mock-transfected cells. (B) ENTPD1-mediated AMP production and (C) E5NT-mediated adenosine production by wild type, mock and pCX-TRI-2A transfected-cells. Cells were incubated with 50 μM ATP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC as detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *p<0.05 versus all groups. (D) E5NT-mediated adenosine production by wild type, mock and transfected-cells. Cells were incubated with 50 μM AMP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC ad detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *p<0.05 versus all groups.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0141933.g004: Heme oxygenase-1 and ectonucleotidases functional assays.(A) Heme Oxygenase 1 activity assay on NIH3T3 cells. Lysates from WT, mock- and pCX-TRI-2A-transfected cells were incubated for 2 hours with Hemin, BSA, Biliverdin Reductase A in reaction buffer as described in Materials and Methods. As positive control of the assay, wt or mock-transfected cells were pre-stimulated with 50 μM of HO1 inducer, Cobalt Protoporphyrin for 24 hours (wt CoPP, mock CoPP). Enzymatic activity is reported as nanomoles of bilirubin per hours per milligrams of protein extract. Data are expressed as mean ± SEM of 3–4 independent experiments. *p<0.05 versus wt and mock-transfected cells. (B) ENTPD1-mediated AMP production and (C) E5NT-mediated adenosine production by wild type, mock and pCX-TRI-2A transfected-cells. Cells were incubated with 50 μM ATP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC as detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *p<0.05 versus all groups. (D) E5NT-mediated adenosine production by wild type, mock and transfected-cells. Cells were incubated with 50 μM AMP for 30 min. The nucleotide content of supernatants collected at 0, 5, 15, 30 min time points was measured by reverse phase-HPLC ad detailed in Material and Methods. Data shown are mean ± S.D. (n = 3). *p<0.05 versus all groups.
Mentions: The enzymatic activity of HO-1 in pCX-TRI-2A-transfected cells was evaluated by measuring the fluorescence of bilirubin during the incubation of lysates from WT, mock and pCX-TRI-2A-transfected cells with hemin. As shown in Fig 4A, the HO-1 activity in pCX-TRI-2A-transfected cells was about 2.5 fold higher than the basal activity seen in controls (1.43 ± 0.08 nmol/h/mg versus 0.54 ± 0.08 and 0.58 ± 0.1 nmol/h/mg respectively in WT and mock transfected-cells, p<0.05). Moreover, no significant differences were observed between pCX-TRI-2A and control cell lines (WT and mock-transfected cells) previously treated with CoPP. These data showed that the exogenous expression of hHO-1, encoded by pCX-TRI-2A plasmid, was comparable to the CoPP induced expression levels of endogenous HO-1.

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