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Uptake and Trafficking of DNA in Keratinocytes

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Uptake and Trafficking of DNA in Keratinocytes Gene Therapy (2004) 11, 765–774 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt RESEARCH ARTICLE Uptake and trafficking of DNA in keratinocytes: evidence for DNA-binding proteins E Basner-Tschakarjan, A Mirmohammadsadegh, A Baer and UR Hengge Department of Dermatology, Heinrich Heine-University Du¨sseldorf, Du¨sseldorf, Germany The skin is an interesting organ for human gene therapy not be identified in FACS experiments. To detect the due to accessibility, immunologic potential and synthesis potential DNA receptors on the keratinocyte surface, capabilities. In this study, we attempted to visualize and membrane proteins were extracted and subjected to measure the uptake of naked FITC-labeled plasmid by South-Western blotting using digoxigenin-labeled calf thy- FACS analysis detecting up to 15% internalization in a dose- mus and l-phage DNA. Two DNA-binding proteins, ezrin and time-dependent manner. Cycloheximide treatment in- and moesin, known as plasma membrane-actin linkers, hibited the uptake by 490%, suggesting a protein-mediated were identified by one- and two-dimensional-South-Western uptake. The inhibition of different internalization pathways blots and matrix-assisted laser desorption and ionization- demonstrated that blocking macropinocytosis (by amiloride mass spectrometry. Ezrin and moesin are functionally and N,N-dimethylamylorid) reduced DNA uptake by 485%, associated with a number of transmembrane receptors while the inhibition of clathrin-coated pits (by chlorpromazine) such as the EGF, CD44 or ICAM-1 receptor. Taken and caveolae (by nystatin/filipin III) did not limit the together, naked plasmid DNA seems to enter human uptake. Colocalization studies using confocal laser keratinocytes through different pathways, mainly by macro- microscopy revealed a time-dependent accumulation of pinocytosis. Two DNA-binding proteins were identified plasmid DNA in endosomes and lysosomes. When a green that seemed to be involved in binding/trafficking of inter- fluorescent protein (GFP) expression vector was used, nalized DNA. specific GFP-RNA became detectable by reverse transcrip- Gene Therapy (2004) 11, 765–774. doi:10.1038/sj.gt.3302221 tase-PCR, whereas measurable amounts of protein could Published online 15 January 2004 Keywords: plasmid DNA; trafficking; DNA-binding proteins; ezrin; receptor-mediated uptake; keratinocytes Introduction or horseradish peroxidase. Several pieces of evidence point to receptor-mediated DNA uptake through DNA- Epidermal keratinocytes and dendritic cells have the binding proteins in lymphocytes, monocytes, neutrophils ability to take up and express naked plasmid DNA upon and skeletal muscle cells.21–23 On human lymphocytes, intradermal in vivo injection and topical application,1–7 Gasparro et al24 have described three DNA-binding making them attractive targets for gene therapy and proteins (28, 59 and 79 kDa) following the hybridization DNA vaccination. with 32P-labeled calf thymus DNA. On rabbit skeletal Various distinct forms of endocytosis were implicated muscle, three DNA-binding proteins of 28, 60 and 95 kDa in DNA uptake.8–11 Besides phagocytosis and pinocytosis were identified using a 32P-labeled double-stranded that serve to internalize particles up to 2 mm in size,12 linearized pBluescript probe.25,26 receptor-mediated endocytosis represents a highly spe- Since gene therapy via the epidermis becomes increa- cific import mechanism by receptor–ligand interactions singly important,27 we attempted to elucidate the mecha- (eg the epithelial growth factor receptor),13,14 or by nism by which plasmids surmount the keratinocyte cell binding to more promiscuous scavenger receptors such membrane and study their intracellular trafficking, as as asialoglycoprotein or lectins.15,16 Inside the cytoplasm, well as to identify DNA-binding membrane proteins. the complex is packaged and transported as a clathrin- coated vesicle to the endosomes, where disassembly occurs.17,18 Recent data have demonstrated the uptake of Materials and methods DNA into muscle cells by caveolae (potocytosis).8,9,18,19 20 Keratinocyte culture Alternatively, McNeil et al suggested that cells Primary human keratinocytes were derived as described undergo transient membrane microdisruptions that earlier.28 Briefly, epidermis was separated by dispase occur, for example, in gastric epithelial cells or skin treatment of normal human skin followed by trypsin epidermal cells, when internalizing fluorescent dextrin treatment to obtain a single-cell suspension of keratino- cytes. Primary keratinocytes were cultured in keratino- cyte growth medium (KGM) (Clonetics, San Diego, CA, Correspondence: UR Hengge, Department of Dermatology, Heinrich-Heine- 1 University Du¨sseldorf, Moorenstrasse 5, Du¨sseldorf D-40225, Germany USA) in a humidified atmosphere at 37 C and 5% CO2. Received 11 February 2003; accepted 8 October 2003; published For confocal microscopy, keratinocytes were cultured on online 15 January 2004 four-well chamber slides (Life Technologies). DNA-binding proteins in keratinocytes E Basner-Tschakarjan et al 766 Plasmid labeling (0.5 mM, Sigma) inhibiting macropinocytosis,30 nystatin Vectors (pCMV-IL-2-b-Gal, 7.2 kb or pEGFP-N1, 4.2 kb; (10 mM, Sigma) and filipin III (23 mM, Sigma), inhibiting Clontech) were labeled using commercially available caveolae (potocytosis)18 and with chlorpromazine labeling kits (Cy5 or FITC Label It; Mirus, Madison, (10 mg/ml, Sigma) that interferes and FITC dextran WI, USA) according to the manufacturer’s instructions. clathrin-coated pits,31 respectively. FITC transferrin (Mo- The FITC label binds covalently to guanine residues of lecular Probes, Eugene, OR, USA) were used to visualize the plasmid; labeled plasmid is separated from free receptor-mediated uptake and clathrin-coated pits. FITC via a G50 microspin column.29 The labeling efficiency was confirmed by titration of the plasmid PCR from 1 mg to 50 ng on a 1% agarose gel and visualization Keratinocyte cultures incubated with pEGFP-N1 plasmid under UV light and fluorescence microscopy, and found (up to 80 mg/ml) were washed three times with PBS prior not to alter their uptake and intracellular trafficking.8,20 to the isolation of total RNA using the Trizolt method FITC-labeled oligodeoxynucleotides (ODNs) were pur- (Life Technologies, Karlsruhe, Germany) as described.32 chased from MWG Biotech, Ebersberg, Germany. ODN1: To remove contaminating DNA, RNA samples were GTC GGT AAA ACT TGA GCC CT; ODN2: AGG AAC incubated with 10 U RNAse-free DNAse I (Roche)31 per CCC TAG TGA TGG AG; ODN3: AGA TAC GAG CGC 10 mg RNA in 0.1 ml for 1 h at 371C followed by Trizolt TAC CTA GG; and ODN4: AAA AAA AAA AGG TGG (Invitrogen). PCR was performed using a RNA-PCR kit GTG GG. (Superscriptt One Step PCR-system, Roche) on 10–50 ng of total DNAse-treated RNA with primers (forward: Flow cytometry measurement of plasmid uptake ACA AGT TCA GCG TGT CCG GC; reverse: TTG TGG Proliferating first or second passage keratinocytes were CTG TTG TAG TTG TAC TCC AG) at the following incubated at 50% confluency for various intervals with conditions (cDNA synthesis: 501C, 30 min; one single FITC-labeled DNA. Following trypsinization (0.5% tryp- denaturing at 941C, 2 min; PCR: 40 cycles of 941C, 30 s, sin, 0.53 mM EDTA, Life Technologies) and washing with 601C, 30 s and 721C, 1 min). For the Àreverse transcrip- 10% fetal calf serum in phosphate-buffered saline (PBS), tase (RT) controls, DNA-PCR was performed using 1 U the cells were resuspended in FACS buffer (1 Â PBS, 1% of the Hot Start Polymeraset (Qiagen, Hilden, Germany) bovine serum albumin (BSA), 0.02% NaN3, Sigma) and per reaction according to the manufacturer’s recommen- analyzed by flow cytometry (Coulter Epicss, XL3-color dations and subsequently 35 cycles as described above. FACS). Living cells as indicated by propidium iodide To ensure high quality of RNA, b-actin controls were run exclusion were gated and the fraction of fluorescent in parallel. keratinocytes was determined. Besides the influence of concentration (1 mg and 50 mg/ml), time (0–48 h) and Membrane protein extractions temperature (incubation at 4 and 371C), the effect of Membrane protein fractions were obtained following a active protein synthesis was assessed. An aliquot of modified procedure according to Trigatti et al.33 Human keratinocytes was treated with trypsin (Sigma) to remove epidermis was frozen in homogenization buffer (1 mM surface proteins, resuspended in KGM containing 10 mg/ HEPES, pH 7.4, 1 mM EDTA, 0.25 M sucrose). After ml cycloheximide (Sigma) and compared with the homogenization (Potter S, Braun, Melsungen, Germany) untreated control following incubation for 4 and 24 h, on ice in the presence of a mix of proteinase inhibitors respectively. (Roche), a further freeze–thaw cycle was performed. Finally, the homogenate was pelleted at 10 000 g, 15 min, Confocal laser microscopy 41C and resuspended in homogenization buffer. Follow- Primary human keratinocytes were treated with FITC- or ing a second centrifugation, the pellet was resuspended Cy5-labeled plasmid DNA for up to 24 h. After incuba- in homogenization buffer and aspirated with a 27-guage tion, the cells were washed with PBS, fixed with ice-cold needle prior to separation on a sucrose gradient (0.87, methanol and subsequently washed with PBS, followed 1.1 M, 1.5 M sucrose in 10 mM HEPES, pH 7.4, 1 mM by an incubation with a polyclonal anti-ezrin antibody EDTA). Following ultracentrifugation (L-60 ultracentri- (1:100) in PBS (Santa Cruz Biotechnology, USA). After fuge, Beckmann) at 23 000 g,41C for 90 min, the plasma three washes in PBS, a 45 min incubation with Alexas- membrane fraction located to the upper interphase labeled secondary antibody (diluted 1:10 in PBS) between the 0.25 M sucrose of the homogenization buffer (Molecular Probes, Eugene, OR, USA) was performed and the 0.87 M sucrose of the gradient. This interphase at room temperature in the darkness. The cultured was removed and resuspended in the five-fold volume of keratinocytes were analyzed on the glass slip, embedded ice-cold PBS prior to another ultracentrifugation at in FluoroPreps (BioMe´rieux, France) and evaluated 100 000 g,41C for 60 min.
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