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

PLoS One. 2015 Oct 29;10(10):e0141933. doi: 10.1371/journal.pone.0141933. eCollection 2015.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5'-Nucleotidase / genetics*
  • Animals
  • Antigens, CD / genetics*
  • Antigens, CD / metabolism
  • Apoptosis / drug effects
  • Apoptosis / genetics
  • Apyrase / genetics*
  • Apyrase / metabolism
  • Cells, Cultured
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Enzyme Activation
  • Fibroblasts / drug effects*
  • Fibroblasts / metabolism*
  • Gene Expression*
  • Gene Order
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • Heme Oxygenase-1 / genetics*
  • Heme Oxygenase-1 / metabolism
  • Humans
  • Mice
  • NIH 3T3 Cells
  • Protein Transport
  • Transgenes
  • Tumor Necrosis Factor-alpha / pharmacology*


  • Antigens, CD
  • DNA-Binding Proteins
  • TNIP1 protein, human
  • Tumor Necrosis Factor-alpha
  • Heme Oxygenase-1
  • 5'-Nucleotidase
  • Apyrase
  • CD39 antigen

Grant support

This work was supported by grants from the Ministero della Ricerca e dell’Università [FIRB-RBAP06LAHL to ML] and the University of Milano-Bicocca [F.A.R. 2009 and 2010 to ML and RG]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.