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Lipopolysaccharide recognition protein, MD‐2, facilitates cellular uptake of E. coli ‐derived plasmid DNA in synovium
Author(s) -
Kolka Jacquelyn A.,
Vreede Andrew P.,
Roessler Blake J.
Publication year - 2005
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.743
Subject(s) - luciferase , transfection , microbiology and biotechnology , in vivo , biology , plasmid , lipopolysaccharide binding protein , cd14 , chemistry , dna , gene , biochemistry , flow cytometry
Background Several cell types are susceptible to transfection in vivo using naked plasmid DNA. The mechanisms involved in mediating in vivo transfection are incompletely known, but evidence suggests that receptor‐mediated endocytosis is important for specific types of cells. In this study we tested the hypothesis that residual Escherichia coli lipopolysaccharide (LPS) forms a non‐covalent complex with expression plasmid DNA, and host‐cell‐derived soluble LPS‐binding proteins bind to the DNA‐LPS complexes in order to facilitate receptor‐mediated endocytosis. Methods Cells from the murine synovial lining were used as an in vivo model system and in vivo luciferase imaging was used to quantify levels of transgene expression. Using a series of gene‐deleted mice, the roles of LPS recognition complex proteins, lipopolysaccharide‐binding protein (LBP), CD14 and MD‐2, in the process of in vivo transfection were determined. Results Luciferase expression assays revealed that mice lacking LBP or CD14 had increased luciferase expression ( p < 0.023 and < 0.165, respectively), while mice deleted of MD‐2 had significant reductions in luciferase expression ( p < 0.001). Gene deletion of hyaluronic acid binding protein CD44 was used as a control and had no statistically significant effect on transgene expression in vivo . In muscle tissue, where neither cell surface nor soluble MD‐2 is expressed, no MD‐2 dependence of plasmid transfection was identified, suggesting the role of MD‐2 is tissue or cell type specific. Additionally, depleting mice of macrophages showed that luciferase expression is occurring within fibroblast‐like synoviocytes. Conclusions Our data support a physical association between LPS and E. coli ‐derived plasmid DNA, and that in vivo transfection of fibroblast‐like synoviocytes is dependent on the soluble form of the LPS‐binding protein MD‐2. Copyright © 2005 John Wiley & Sons, Ltd.