Simultaneous overexpression of functional human HO-1, E5NT and ENTPD1 protects murine fibroblasts against TNF-α-induced injury in vitro

Alessandro Cinti, Marco De Giorgi, Elisa Chisci, Claudia Arena, Gloria Galimberti, Laura Farina, Cristina Bugarin, Ilaria Rivolta, Giuseppe Gaipa, Ryszard Tom Smolenski, Maria Grazia Cerrito, Marialuisa Lavitrano, Roberto Giovannoni

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article numbere0141933
JournalPLoS One
Volume10
Issue number10
DOIs
Publication statusPublished - Oct 29 2015

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

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