Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins

Abstract

The skin is an interesting organ for human gene therapy due to accessibility, immunologic potential and synthesis capabilities. In this study, we attempted to visualize and measure the uptake of naked FITC-labeled plasmid by FACS analysis detecting up to 15% internalization in a doseand time-dependent manner. Cycloheximide treatment inhibited the uptake by 490%, suggesting a protein-mediated uptake. The inhibition of different internalization pathways demonstrated that blocking macropinocytosis (by amiloride and N,N-dimethylamylorid) reduced DNA uptake by 485%, while the inhibition of clathrin-coated pits (by chlorpromazine) and caveolae (by nystatin/filipin III) did not limit the uptake. Colocalization studies using confocal laser microscopy revealed a time-dependent accumulation of plasmid DNA in endosomes and lysosomes. When a green fluorescent protein (GFP) expression vector was used, specific GFP-RNA became detectable by reverse transcriptase-PCR, whereas measurable amounts of protein could not be identified in FACS experiments. To detect the potential DNA receptors on the keratinocyte surface, membrane proteins were extracted and subjected to South-Western blotting using digoxigenin-labeled calf thymus and l-phage DNA. Two DNA-binding proteins, ezrin and moesin, known as plasma membrane-actin linkers, were identified by one-and two-dimensional-South-Western blots and matrix-assisted laser desorption and ionizationmass spectrometry. Ezrin and moesin are functionally associated with a number of transmembrane receptors such as the EGF, CD44 or ICAM-1 receptor. Taken together, naked plasmid DNA seems to enter human keratinocytes through different pathways, mainly by macropinocytosis. Two DNA-binding proteins were identified that seemed to be involved in binding/trafficking of internalized DNA.

Characterization of bacterial DNA binding to human neutrophil surface

“…37 In human keratinocytes, two plasma membrane-actin linkers, ezrin and moesin, were identified as DNA-binding proteins. 38 Using southwestern analysis and gel shift and supershift assays, the authors found that these proteins were able to bind plasmid DNA, l-phage DNA, ODNs irrespective of their sequences and calf thymus DNA, but not RNA. On the other hand, Benimetskaya et al 39 demonstrated the ability of Mac-1 to bind ODNs.…”

“…[36][37][38][39][40] It has been reported that an 80 kDa protein mediates the transport of ODNs in HL-60 cells. 36 The authors reported that ODNs binding was sequence independent and could be inhibited by 60-80% by twofold excess (25 mM) of unlabeled ODNs, as well as by desoxyribonucleotides, ribonucleotides, plasmid DNA and yeast tRNA.…”

Gene therapy progress and prospects: the skin – easily accessible, but still far away

“…FITC-and Cy5-labeled plasmid DNA was applied together with specific inhibitors of macropinocytosis (amilorid and N,N-dimethylamilorid), potocytosis (nystatin and filipin) and clathrin-coated pits (chlorpromazine). 51 These experiments suggested that macropinocytosis was the predominant mode of DNA uptake by keratinocytes.…”

“…pinocytosis) seem to be involved. 51 In this study, we attempted to visualize and measure uptake of naked FITC-labeled plasmid by FACS analysis detecting up to 15% internalization in a dose-and time-dependent manner. While oligodeoxynucleotides were rapidly and almost completely taken up into the nucleus of keratinocytes, only low amounts of FITC-labeled plasmid could be detected in the cytoplasm and around the nucleus after 6 h (Figure 4).…”

“…The uptake depended on the concentration, time, temperature and on protein synthesis. 51 Several established mechanisms of endocytosis, utilizing clathrin-coated pits, macropinocytosis and caveolae, were studied. FITC-and Cy5-labeled plasmid DNA was applied together with specific inhibitors of macropinocytosis (amilorid and N,N-dimethylamilorid), potocytosis (nystatin and filipin) and clathrin-coated pits (chlorpromazine).…”