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 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 -coated pits (by chlorpromazine) such as the EGF, CD44 or ICAM-1 receptor. Taken and (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 . 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 were implicated muscle, three DNA-binding proteins of 28, 60 and 95 kDa in DNA uptake.8–11 Besides 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 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. Finally, the pellet was under a confocal laser microscope (Carl Zeiss LSM510, dissolved in PBS containing 0.05 mM phenylmethylsul- Axiovert 100 M, Jena, Germany). Further contrast en- fonylfluoride (PMSF, Sigma). hancement was carried out with Photoshop (Adobe, Mountain View, CA, USA). Nuclei were labeled with One (1D)- and two (2D)-dimensional gel Hoechst 33258 (Sigma). electrophoresis Isolated protein extracts (20 mg) were prepared for Inhibition of various internalization pathways electrophoresis by mixing 1:1 with sample buffer Keratinocyte cultures were incubated for 1 h with several (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% inhibitors of internalization prior to the addition of FITC- bromophenol blue, 5% b-mercaptoethanol, BioRad, labeled plasmid DNA. Incubation was performed with Munich, Germany), boiled for 10 min and then chilled amiloride (3 mM, Sigma) and N,N-dimethylamiloride on ice. Proteins were separated using precast 4–20%

Gene Therapy DNA-binding proteins in keratinocytes E Basner-Tschakarjan et al 767 Tris-HCl gradient gels (BioRad, Munich, Germany) antibody (0.25 mg/ml, Transduction Laboratories, and running buffer (25 mM Tris, 192 mM glycine and Lexington, KY, USA). After several washes with 1% 0.1% SDS). BSA in PBS, a secondary anti-mouse-IgG (1:30) was added 2D gel electrophoresis was performed with commer- with the staining being completed according to the cially available isoelectric focusing strips at a nonlinear alkaline phosphatase/antialkaline phosphatase method.36 pH gradient from pH 3 to 10 using an IPGphor focusing device (Amersham Pharmacia Biotech Europe, Freiburg, Uptake inhibition by polyclonal antibodies 34 m Germany). Protein extract (200 g) were solubilized in To assess the effect of transmembrane receptors, known 250 ml solubilization buffer (7 M urea, 2 M thiourea, to be associated with ezrin and moesin, blocking 150 mM DTT, 4% (w/v) CHAPS, 0.5 (v/v) carrier antibodies were added to the keratinocyte cultures 1 h ampholyte, pH 3.5–10 (Pharmacia BioTech, Sweden)). prior to the addition of labeled plasmid DNA. Anti- Undissolved proteins were removed by centrifugation at 1 bodies (CD44 monoclonal, Biozol, Munich, Germany; 15 000 g,4 C for 15 min. After the hydration of isoelectric CD44 polyclonal, Chemicon, International, Temecula, focusing strips, the protein extracts were subjected to an CA, USA), caveolin (polyclonal, Transduction Labora- 11 h separation protocol with increasing voltage from 150 tories, Lexington, KY, USA), clathrin heavy chain m to 2500 V and 50 A/strip. Following the isoelectric (monoclonal, Transduction Laboratories, Lexington, KY, focusing, the strip was equilibrated in buffer containing USA), and ezrin (polyclonal, Santa Cruz Biotechnology, 6 M urea, 2% SDS, 50 mM Tris-HCl, 30% glycerol and CA, USA), ezrin (monoclonal, Transduction Laboratories, 0.03% bromophenol blue, pH 8.8. DTT was added to a Lexington, KY, USA), moesin (monoclonal, Transduction final concentration of 65 mM. The strip was transferred Laboratories, Lexington, KY, USA), triadin (monoclonal, onto an 8–16% gradient gel and separation was mouse, Affinity Bioreagents, Golden, CO, USA), and performed in the second dimension at 40 mA/gel at 1 villin (monoclonal, BioDesign International, USA) were 20 C in running buffer (25 mM Tris, 192 mM glycine, 0.1% used at the indicated concentrations. SDS). Gels were stained with Gel Code Blue Stain Reagent (Pierce, Rockford, IL, USA) with colloidal Coomassie G-250 (Pierce) or silver stain kit (Biorad, Munich) according to the manufacturer’s instructions. Results Visualization of plasmid uptake Hybridization with digoxigenin (DIG)-labeled DNA The uptake of various FITC-, rhodamine- or Cy5-labeled Following gel electrophoresis, proteins were transferred DNA fragments (without the presence of transfection onto a PVDF membrane (Roche). HindIII-digested l- agents) of 20 bp to 7.2 kb in size was analyzed using phage DNA and sonicated calf thymus DNA (Sigma) confocal microscopy. While ODN were rapidly and were labeled with a DIG DNA-Labeling and Detection almost completely taken up into the nucleus of kerati- Kit (Roche). Briefly, the denatured DNA is incubated nocytes (Figure 1a, c and e), only a low amount of FITC- with Klenow polymerase using a deoxyribonucleoside labeled (Figure 1b) or Cy5-labeled plasmid (Figure 1e phosphate mix that contains DIG-11-dUTP. The labeled and f) (each 1 mg/ml) could be detected in the cytoplasm probe was denatured and hybridized to the blocked and around the nucleus after 6 h. When the effect of the membrane (10 mM HEPES, pH 8.0, 5% blocking reagent; lysosomotropic agent chloroquine (10 mM) was analyzed, Roche) for 2 h in 15 ml hybridization buffer (10 mM an increased accumulation of plasmid DNA in the cells HEPES, pH 8.0, 50 mM NaCl, 10 mM MgCl2, 0.1 mM and nuclei became evident (Figure 1g–j). EDTA, 1 mM DTT, 0.25% blocking reagent) at a concen- tration of 35 ng/ml.35 The detection of the DNA probe Dependence of DNA uptake on concentration, time, that bound to separated membrane proteins was performed with alkaline phosphatase-labeled anti-DIG temperature and protein synthesis antibodies using the DIG DNA-Labeling and Detection The uptake was dependent on the concentration of the Kit (Roche). added plasmid DNA (Table 1). At 1 mg/ml, 4.2% of keratinocytes (mean fluorescence intensity: 0.36) were FITC positive as revealed by flow cytometry; at 2 mg/ml: Protein identification by matrix-assisted laser 10.4% (mean fluorescence intensity: 0.53); and at 5 mg/ desorption and ionization (MALDI)-mass spectrometry ml: 12.6% (mean fluorescence intensity: 0.6), respectively, A parallel 2D SDS-PAGE was stained with Coomassie when analyzed after 4 h of incubation at 371C. The time blue and used for the excision of corresponding protein dependence of plasmid uptake was clearly evident with spots that were identified to bind DNA in South-Western a maximum of 14.9% at 24 h (Table 1). The mean analysis. The gel spots containing the respective proteins fluorescence intensity was 0.36 at 4 h, 0.38 at 8 h and were digested with trypsin and trifluoroacetic acid and 1.35 at 24 h, respectively, with no further increase commercially analyzed using mass spectrometry (Reflex- thereafter. The uptake was also dependent on the III, Bruker-Franzen, Bremen, Germany) at the Institute of temperature, since at 41C the mean fluorescence intensity Physiological Chemistry, Ruhr-University, Bochum, Ger- was 0.49 as compared with 1.14 at 371C. Next, we many. The comparison of MS spectra with known investigated whether the uptake of plasmid DNA into proteins was performed using the software ProFound keratinocytes depended on active protein synthesis as (http://prowl.rockefeller.edu/cgi-bin/ProFound). revealed by the protein synthesis inhibitor cyclohexi- mide. While the mean fluorescence intensity was 0.83 at Immunohistology 4h (5mg/ml plasmid DNA) without the addition of Paraffin-embedded sections of normal skin were used cycloheximide, it declined to 0.47 (n ¼ 3) at 4 h when cells for immunohistology using a monoclonal anti-ezrin were exposed to cycloheximide following trypsinization.

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Figure 1 Visualization of plasmid uptake. Primary human keratinocytes or HaCaT keratinocytes were incubated in the presence of FITC- or rhodamine- labeled ODN (20 mer; 4 mM) or naked FITC- or Cy5-labeled plasmid DNA (1 mg/ml), respectively. While ODN accumulated around the nuclei within 30 min (a, c and d), only limited perinuclear accumulation of FITC-plasmid DNA was observed at 6 h (b, e and f). The addition of chloroquine (10 mM) was found to augment the fluorescence by inhibiting acid digestion of plasmid DNA (g–j) at 24 h. To visualize nuclei, costaining with Hoechst 33258 was performed (d, f, h and j). Original magnification  630; some images (e–h) were digitally processed to achieve higher or lower magnification.

Table 1 Dependence of concentration, time and temperature and were studied (Figure 2a–e). In order to evaluate the protein synthesis relevance of several potential internalization processes, FITC- and Cy5-labeled plasmid DNA was applied Mean together with specific inhibitors of macropinocytosis % of positive fluorescence 30 Variable keratinocytes intensity (amilorid and N,N-dimethylamilorid), potocytosis (nystatin and filipin)18 and clathrin-coated pits (chlor- promazine) (Figure 2).31 The possibility that plasmid Concentration (4 h; 371C) 1 mg/ml 4.2 0.36 DNA was recruited into macropinocytotic vesicles was 2 mg/ml 10.4 0.53 probed with amiloride and N,N-dimethylamiloride, 5 mg/ml 12.6 0.6 two potent inhibitors of macropinocytosis.30 Amiloride (and the more potent inhibitor N,N-dimethylamiloride) Time (371C; 1 mg/ml) 4 h 1.75 0.36 dramatically reduced the mean intracellular fluorescence 8 h 2.5 0.38 by 10-fold (Figure 2b and e, column 2 with amiloride 24 h 14.9 1.35 and 3 with N,N-dimethylamiloride, respectively; Temperature (24 h; Po0.05 as compared to column 1). Since keratinocytes 1 mg/ml) 41C 0.55 0.49 are known to have caveolae on their basolateral 371C 12.3 1.14 surfaces,37 nystatin and filipin III, two inhibitors of caveolae, were found not to inhibit the uptake of plasmid Protein synthesis (24 h; DNA in keratinocytes (Figure 2d and e, column 4 with 5 mg/ml) + cycloheximide 6.95 0.83 À cycloheximide 1.44 0.47 nystatin and 5 with filipin III, respectively), indicating that potocytosis does not play a significant role in uptake Quantification of several uptake characteristics was performed using and transport of plasmid DNA in keratinocytes. The flow cytometry. The dependence of concentration was evident (at addition of chlorpromazine, an inhibitor of clathrin- 1 mg/ml 4.2% of keratinocytes were FITC positive; at 2 mg/ml: 10.4%; coated pits, led to a three-fold increase in mean at 5 mg/ml: 12.6%, respectively), when analyzed after incubation for intracellular fluorescence in five independent experi- 4 h at 371C. The time dependence of plasmid uptake was shown with a maximum at 24 h. The mean fluorescence intensity was 0.36 ments (Figure 2c and e, columns 6 and 7), suggesting that at 4 h, 0.38 at 8 h and 1.35 at 24 h, respectively, with no further clathrin-coated pits were not involved in the uptake of increase thereafter. The uptake was also dependent on the naked plasmid DNA. The observed signal intensification temperature, since at 41C the mean fluorescence intensity was 0.49 was probably due to toxic effects that led to keratinocyte as compared with 1.14 at 371C. The addition of the protein synthesis differentiation as suggested by enlargement of keratino- inhibitor cycloheximide also reduced the intracellular fluorescence. cytes (increased forward scatter in FACS analysis; data Each n ¼ 4–5 experiments; keratinocytes were derived from different patients at the first or second passage, if not indicated otherwise. not shown). Intracellular trafficking of plasmid DNA Inhibition studies of several internalization When intracellular trafficking of Cy5-labeled plasmid mechanisms DNA was analyzed together with the 10-kDa FITC– Several established mechanisms of endocytosis, utilizing dextran, a commonly used fluid-phase endocytosis clathrin-coated pits, macropinocytosis and caveolae, marker,38 several colocalization signals were detected as

Gene Therapy DNA-binding proteins in keratinocytes E Basner-Tschakarjan et al 769 (4.2 kb) expressing green fluorescent protein (GFP) was added to keratinocyte cultures (without transfection agents). In these studies, a dose-dependent expression pattern was seen. At the highest dose (80 mg/ml) keratinocytes contained transcriptionally active GFP as assessed by RT-PCR performed on DNAse-treated RNA extracted from keratinocyte cultures (Figure 4a, lane 3). A clear signal was seen for naked plasmid next to the same transfections in the presence of lipofectin and polyethylenimine. The appropriate controls, where the extracted RNA was used without reverse transcriptase, revealed no signal (Figure 4e, lane 8). The integrity of RNA was confirmed by b-actin control reactions (data not shown). Corresponding FACS experiments trying to demonstrate visible amounts of GFP revealed less than 1% positive cells (data not shown).

DNA-binding proteins Since we gained evidence that plasmid DNA could be taken up by keratinocytes upon in vitro exposure, we attempted to analyze the existence of potential DNA- binding proteins on keratinocyte . Therefore, membrane proteins were prepared from epidermal lysates and compared for DNA binding with cytosolic and nuclear extracts of epidermal keratinocytes using South-Western analysis. Hybridization was performed Figure 2 Inhibition of several internalization mechanisms. FITC-labeled plasmid DNA (a, 1 mg/ml) was applied together with specific inhibitors of with DIG-labeled sonicated calf thymus DNA or macropinocytosis (amiloride; b), clathrin-coated pits (chlorpromazine; c) a DIG-labeled l-phage DNA probe digested with potocytosis (nystatin; d), respectively. Original magnification  200. HindIII. Using this technique, we were able to detect Quantification of four to five independent experiments using FACS three membrane proteins with DNA-binding ability revealed a six-fold reduction of plasmid uptake with amiloride (e, column (Figure 5a). In 1D-South-Western, these proteins were 2) and a 10-fold reduction with the more potent N,N-dimethylamiloride (e, estimated to have a molecular weight of 78, 80 column 3; Po0.05 as compared to column 1). Column 1: uninhibited uptake; column 4: nystatin; column 5: filipin III; columns 6 and 7: and 98 kDa, respectively (Figure 5a, lane 1). These chlorpromazine. proteins also bound double-stranded linearized pcDNA3 under denaturing conditions (data not shown). In the , essentially no DNA-binding proteins were soon as 8 h after incubation (Figure 3a–c). Cells with the found (Figure 5a, lane 2), whereas in nuclear and most active dextran uptake (Figure 3a) internalized membrane extracts several smaller bands o42 kDa were usually more plasmid DNA (Figure 3band c). When detected (Figure 5a, lane 1 and 3), which probably plasmid DNA was compared with calf thymus DNA, correspond to histones (Figure 5a, lane 4). Interestingly, the latter also colocalized with endosomes (data not in porcine mucosa, the same and additional bands of shown). Colocalization with FITC-labeled transferrin 200 and 130 kDa were detected to bind DNA (Figure 5a, as an indicator of receptor-mediated uptake and cla- lane 5). thrin-coated pits showed no colocalization (data not To confirm these results, 2D gel electrophoresis was shown). performed on 200 mg of keratinocyte membrane extracts. In order to identify plasmid transport into lysosomes, In 2D-South-Western blots, three DNA-binding proteins Cy5-labeled plasmid DNA was visualized along with spots were identified (Figure 5b, insert). When these lysosomes stained with LysoTracker Greent (Molecular spots were located and excised in a corresponding Probes, Eugene, OR, USA) known to accumulate in acid Coomassie-stained gel, their sequence could be analyzed cellular compartments (Figure 3d-i). At 24 h, significant using MALDI-mass spectrometry. Using this approach, colocalization was detected around the nuclei (Figure 3f), the DNA-binding proteins were identified as ezrin and with quantitatively less plasmid DNA being present in moesin, which occurred as two isoforms. Western blots lysosomes when compared with Cy5-labeled calf thymus with monoclonal anti-ezrin- and anti-moesin antibodies DNA at 24 h (Figure 3f and i). The large size of the DNA- using human epidermal extracts next to a South-Western positive vesicles and the fact that high molecular weight experiment revealed bands at the same position (data polysaccharides like dextrans are preferentially taken not shown). into macropinosomes rather than micropinosomes,38 To assess the effect of transmembrane receptors, suggested that DNA-containing vesicles were formed known to be associated with ezrin and moesin, several via macropinocytosis. blocking antibodies were added to the keratinocyte cultures 1 h prior to the addition of labeled plasmid DNA. Polyclonal antibodies against ICAM-Z and ezrin In vitro uptake of plasmid DNA leads to expression reduced intracellular fluorescence by 40% each, while In order to find out, whether internalized plasmid DNA antibodies against caveolin, clathrin, triadin and villin also permitted expression, the indicator plasmid pEGFP had no effect (data not shown).

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Figure 3 Intracellular trafficking of plasmid DNA. Confocal optical sections are shown, each with two fluorescent markers, which when colocalized yield a yellow color. When Cy5-labeled plasmid DNA (b, e) was traced along with the endosomal marker FITC–10-kDa dextran (10 mg/ml) to demonstrate bulk- phase endocytosis (a), several colocalization signals were seen as soon as 8 h after incubation (c). For comparison, calf thymus DNA also colocalized to endosomes, but the amount of DNA reaching the nucleus was significantly lower (data not shown). At 24 h, Cy5-labeled plasmid DNA (e) and calf thymus DNA (h) were visualized in lysosomes stained with LysoTrackert (d, g), with significantly more calf thymus DNA being detected in lysosomes (i) than plasmid DNA (f).

and moesin were able to inhibit/outcompete one band (Figure 6b, and manuscript in preparation). Thus, ezrin and moesin seem to be relevant for binding to both plasmid DNA and ODN.

Expression of ezrin in human epidermis In cross-sections of human epidermis, the expression of ezrin was confined to the suprabasal layers of the epidermis with a cytoplasmic pattern sparing the nuclei (Figure 7a). This pattern is reminiscent of that seen upon in vivo expression of naked DNA,1,2 where differentiating Figure 4 Detection of specific RNA following naked plasmid DNA uptake suprabasal keratinocytes express the indicator gene most in vitro. The indicator plasmid pEGFP (4.2 kb) expressing GFP was added efficiently. In hair follicles, the outer root sheath with to keratinocyte cultures (without transfection agents). At the highest dose differentiating keratinocytes was also staining for ezrin (80 mg/ml) keratinocytes contained transcriptionally active GFP plasmid (Figure 7b), while papilla keratinocytes were negative. as assessed by RT-PCR on extracted DNAse I-digested RNA without (ÀRT) and with ( þ RT) the addition of RT using standard procedures (a). M, marker, lane 1: no plasmid; lane 2: no primers; lane 3 naked pEGFP-N1 (80 mg/ml), lane 4: polyethylenimine-mediated and lane 5: lipofectin- Discussion mediated transfection of pEGFP-N1. The respective experiment without RT is shown (b); lane 6: no plasmid; lane 7: plasmid control; lane 8: naked For genes to be expressed in skin and other tissues, a pEGFP-N1; lane 9: polyethylenimine-mediated transfection; lane 10: variety of different events have to occur. First, following lipofectin-mediated transfection of pEGFP-N1. Corresponding FACS intraorgan (eg intradermal) injection, naked plasmid experiments trying to demonstrate visible amounts of GFP revealed less DNA has to be transported to cells that potentially take it than 1% positive cells (data not shown). up. Most likely, physical pressure and the concentration gradient play an important role in this regard. Secondly, when plasmid DNA reaches the basal membrane, it has Involvement of ezrin and moesin in ODN binding to overcome this electron-dense adhesion structure. Following the binding of plasmid DNA to ezrin and Thirdly, transport within the epidermis has to occur moesin, we further analyzed whether ODN also bound and plasmid DNA has to reach the keratinocyte cell to keratinocyte membrane proteins and whether ezrin/ membrane. At the , uptake into the moesin played a role in this process. In gel shift and cytoplasm may occur. When inside the cell, the majority supershift assays, we found two ODN-binding mem- of the plasmid will be degraded in endosomal and brane proteins that bound ODN independent of their lysosomal compartments.39 However, a proportion of the sequence (Figure 6a). Interestingly, antibodies to ezrin DNA will be released from the acidic compartments and

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Figure 5 DNA-binding proteins of the keratinocyte membrane. 1D-South-Western analysis (a) of DIG-labeled sonicated calf thymus DNA binding to membrane proteins (lane 1), cytosolic (lane 2) and nuclear (lane 3) extracts of epidermal keratinocytes. Several membrane proteins with DNA-binding ability were seen at 78, 80 and 98 kDa, respectively, as well as below 42 kDa. While cytosolic extracts contained no detectable DNA-binding proteins under the conditions used, nuclear extracts showed several protein bands colocalizing with histones H1 (lane: 4, Sigma). In porcine mucosa epithelial membranes, the same and additional bands of 130 and 200 kDa were found to bind DNA (lane: 5). Lane 6: mucosa nuclear protein extract; lane 7: mucosa by mass spectrometry cytosolic protein extract; M, markers. In 2D-South-Western blots (b, inset), three DNA-binding proteins spots were identified in a corresponding 2D gel as ezrin and moesin, which occurred as two isoforms (b, silver-stained 2D gel).

Figure 6 Involvement of ezrin and moesin in ODN binding. In gel shift assays two 20 mer ODN-binding proteins were detected irrespective of the ODN sequence (a). ODN1-4 were as described in Materials and methods (a, lanes 1–4). In supershift assays using ODN1 (lane 5), the lower ODN- binding protein could be blocked (or shifted into the upper band) by antibodies to ezrin and moesin (b, lane 6 and 7), while an unrelated antibody to cytoskeletal villin did not affect the binding (b, lane 8). Specificity of binding was confirmed by using an excess of unlabeled ODN. Other polyanions such as RNA and heparin were found not to inhibit DNA binding (data not shown). arrive at the nuclear membrane,40 where a fraction of it may be shuffled into the nucleus for transcription to occur. The active uptake of naked plasmid DNA by mammalian cells has not been described, but it is known that Gram-positive bacteria are transformed by the transport of single-stranded DNA through a protein pore in the bacterial membrane. Moreover, Gram- positive bacteria such as Hemophilus have specific membrane receptors that enable double-stranded DNA to enter small surface vesicles. Generally, it seems unlikely that there is a special endocytosis process for Figure 7 Expression of ezrin in the epidermis and hair follicles. Ezrin was DNA in mammalian cells. More likely, plasmid DNA confined to the suprabasal layers of the epidermis with a cytoplasmic may use an existing pathway of another molecule such pattern sparing the nuclei (a). In hair follicles, the outer root sheath with as potocytosis or phagocytosis.8,11,20 Whereas many differentiating keratinocytes was also stained (b), while papilla keratino- cytes (*) were negative. of the internalization processes are only incompletely

Gene Therapy DNA-binding proteins in keratinocytes E Basner-Tschakarjan et al 772 understood,39 Wolff et al8 were the first to demonstrate but also found additional, as yet unidentified, proteins. that naked plasmid DNA is taken up by skeletal muscle For comparison, a number of ODN-binding membrane via potocytosis.41 Their investigations including gold- proteins has been identified on kidney epithelial cells, labeling electron microscopic studies led to the identifi- and keratinocyte cell lines (HaCaT and A431 cells) that cation of non-nuclear double-stranded DNA-binding have a molecular weight of 35 and 61–63 kDa.49,51 Since proteins in striated muscle, among them a 95 kDa protein the proteins were also detected to a lesser extent in identified as triadin.25,42 More specifically, the uptake in nuclear extracts, the authors hypothesized that these muscle cells depended on their differentiation state with proteins might be involved in the uptake and nuclear small myotubes exhibiting the highest uptake. Consistent transport of DNA.49 To our knowledge, ezrin and moesin with our results in keratinocytes, the uptake was also have not been described to bind DNA, neither has their temperature dependent with higher uptake (and expres- involvement in intracellular trafficking of DNA been sion) occurring at 37 than at 41C.41 Our results suggest reported. that for keratinocytes, DNA uptake via caveolae does not seem to be responsible, since neither nystatin or filipin III nor antibodies to caveolin-1, the major component of Ezrin and moesin as membrane–cytoskeleton linkers caveolae, affected the DNA uptake and trafficking.9 and signal transducers. Ezrin and moesin belong to a Clathrin-coated pits, the other common mechanism of highly homologous family of proteins that connect the receptor-mediated endocytosis, were also excluded as plasma membrane with the actin cytoskeleton at specific the DNA-uptake route because: (1) inhibitors of clathrin- locations.52,53 In addition, ezrin and moesin are involved coated pit formation did not reduce, but rather increase, in signal transduction54 and in growth control.55 intracellular plasmid DNA content; and (2) immuno- Although ezrin (and moesin) have no transmembrane staining for caveolae showed no colocalization with portion, they are functionally associated with a variety of DNA (data not shown). The fact that inhibitors of transmembrane receptors such as ICAM-1 and -2, CD44, endocytosis through coated pits actually lead to the CD43 and the EGF receptor.56,57 accumulation of intracellular DNA may reflect the Ezrin, moesin and radixin are about 80% homologous pleiotropic effects of the inhibitors, or the activation of in the entire amino-acid sequence.52,58 They contain an a- alternative uptake systems when one route is helix followed by a polyprolin stretch in ezrin and blocked.43,44 Instead, bulk-phase endocytosis (macropi- radixin before the C-terminus. Whereas the C-terminus nocytosis) seems to be the primary mechanism of contains binding sites for F-actin,52 the N-terminus is plasmid DNA uptake in keratinocytes. This is substan- used to interact with transmembrane receptors such as tiated by the inhibition through amiloride and CD44 through a 357 amino-acid-binding protein, named N,N-dimethylamiloride and the FITC–dextran colocali- EBP50 (ezrin-, radixin-, moesin-binding phosphopro- zation studies.38 tein),59 which occurs in the apical microvilli of polarized Very recently, a novel internalization pathway has epithelia.60 EBP50 was found to bind to the N-terminus been described for hyaluronan in a rat keratinocyte cell of ezrin, radixin and moesin. Binding sites at the N- line that uses nonclathrin-coated endosomes in a terminus (EBP50) that are expected to interact with the partially receptor-mediated way.45 In contrast to our intracytoplasmic portions of transmembrane receptors results on the uptake of plasmid DNA, amiloride did not have not yet been characterized. The N-terminus of ezrin inhibit the internalization of hyaluronan.45 contains a multivalent binding site shown to interact Our experiments also show – for the first time – that a with EBP50, Rho-GDI and to the cytoplasmic part of significant, albeit low amount of the internalized naked CD44. Besides the binding to actin, a homotypic binding plasmid DNA is being expressed in vitro as evidenced by of the C-terminus to the N-terminus has been described GFP studies. Together with the ability to take up and for moesin and ezrin masking the binding site for express plasmid DNA upon in vivo injection into the actin.61,62 dermis,1,2 these mechanistic data demonstrate a novel It has been found that ezrin serves as a target for characteristic of keratinocytes. The exact mechanism by several protein kinases such as for the EGF-receptor which keratinocytes internalize and transport plasmid tyrosine kinase.13,63,64 For example, in A431-cells, 2 min DNA remains unknown, but a protein-mediated uptake following stimulation with EGF, phosphorylation and transport seems to be involved. Besides calcium- of tyrosine residues occurs leading to the resolution of mediated transfection,46 lysosomotropic agents such as homotypic head-to-tail-binding enabling the binding chloroquine were found to improve the transfection of of the C-terminus to F-actin. This molecular signaling is mammalian cells by interfering with several steps accompanied by phenotypical cell changes forming involved in transport and release of plasmid DNA from membrane ruffles and microvilli and increased endocy- lysosomes.47 tosis.65 In addition, recent data showed that the associa- Recent evidence in mice and pigs showed that other tion of ERM proteins with transmembrane receptors tissues may also take up plasmid DNA upon in vivo such as CD44 is regulated through Rho-dependent injection of a supraphysiological dose.23,48 According to pathways.62,66–69 our and others’ results, ODN seem to be internalized Ezrin and moesin are expressed in human lympho- efficiently into keratinocytes leading to rapid accumula- cytes, monocytes and neutrophils. Totaling for up to tion in the nucleus.49,50 Moreover, we analyzed whether 0.5% of total cellular protein in lymphocytes and ODN also bound to keratinocyte membrane proteins and endothelial cells. From a functional standpoint, ezrin, whether ezrin/moesin played a role in this process. In radixin and moesin seem to be redundant, since gel shift and supershift assays, we found two ODN- a moesin knockout mouse did not reveal any morpho- binding membrane proteins, of which antibodies to ezrin logical or functional alterations on the cellular and and moesin were able to inhibit/outcompete one band tissue level.70

Gene Therapy DNA-binding proteins in keratinocytes E Basner-Tschakarjan et al 773 In summary, ezrin and moesin were identified as 13 Marchese C et al. Receptor-mediated endocytosis of keratinocyte DNA-binding membrane–cytoskeleton linker proteins growth factor. J Cell Sci 1998; 111: 3517–3527. that are involved in uptake and trafficking of DNA 14 Strous GJ, Govers R. The ubiquitin–proteasome system and although – up to now – we were not able to identify endocytosis. J Cell Sci 1999; 112: 1417–1423. transmembrane DNA-binding receptors. However, ezrin 15 Fajac I, Briand P, Monsigny M, Midoux P. Sugar-mediated and moesin are functionally associated with a number of uptake of glycosylated polylysines and gene transfer into membrane receptors such as CD44, EGF and ICAM-1–3. normal and cystic fibrosis airway epithelial cells. Hum Gene Following ligand binding to some of the associated Ther 1999; 10: 395–406. receptors, phosphorylation of ezrin occurs at the C- 16 Smith RM, Wu GY. Hepatocyte-directed gene delivery by terminus by a special protein kinase, leading to the receptor-mediated endocytosis. Semin Liver Dis 1999; 19: 83–92. resolution of head-to-tail enabling the binding of actin. 17 Pishvaee B, Payne GS. Clathrin coats – threads laid bare. Cell 1998; 95: 443–446. Within 2 min significant morphological changes occur 18 Schnitzer JE, Oh P, Pinney E, Allard J. Filipin-sensitive caveolae- such as the formation of microvilli, membrane ruffles in endothelium: reduced , and increased endocytosis. The exact mechanism of the scavenger endocytosis, and capillary permeability of select transmembrane transport and the intracellular traffick- macromolecules. J Cell Biol 1994; 127: 1217–1232. ing of plasmid DNA still remain illusive. Currently, we 19 Budker V et al. Hypothesis: naked plasmid DNA is taken up by try to identify the DNA-binding domains within the cells in vivo by a receptor-mediated process. J Gene Med 2000; 2: ezrin molecule and the interactions of DNA with 76–88. transmembrane receptors to further elucidate the biolo- 20 McNeil PL, Vogel SS, Miyake K, Terasaki M. Patching plasma gical function of this newly recognized property of membrane disruptions with cytoplasmic membrane. J Cell Sci proteins known as cytoskeleton–actin linkers. 2000; 113: 1891–1902. 21 Bennett RM, Gabor GT, Merritt MM. DNA binding to human leukocytes. Evidence for a receptor-mediated association, Acknowledgements internalization, and degradation of DNA. J Clin Invest 1985; 76: 2182–2190. We are grateful to Piotr Babioch, Institute of Anatomy, 22 Hefeneider SH et al. Identification of a cell-surface DNA receptor for assistance with the confocal laser microscopy, to and its association with systemic lupus erythematodes. J Invest Hagen Apel for image processing and Nicole-C Bartosch Dermatol 1990; 94: 79S–84S. for editorial help. 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