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• W2016809324 abstract "Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37·DNA complex uptake was a saturable time- and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans. Antimicrobial peptides, such as LL-37, are found both in nonvertebrates and vertebrates, where they represent important components of innate immunity. Bacterial infections at epithelial surfaces are associated with substantial induction of LL-37 expression, which allows efficient lysis of the invading microbes. Peptide-mediated lysis results in the release of bacterial nucleic acids with potential pathobiological activity in the host. Here, we demonstrate that LL-37 targets extracellular DNA plasmid to the nuclear compartment of mammalian cells, where it is expressed. DNA transfer occurred at physiological LL-37 concentrations that killed bacterial cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed in mammalian cells. Furthermore, LL-37 protected DNA from serum nuclease degradation. LL-37·DNA complex uptake was a saturable time- and temperature-dependent process and was sensitive to cholesterol-depleting agents that are known to disrupt lipid rafts and caveolae, as shown by flow cytometry. Confocal fluorescence microscopy studies showed localization of internalized DNA to compartments stained by cholera toxin B, a marker of lipid rafts, but failed to demonstrate any co-localization of internalized DNA with caveolin-positive endocytotic vesicles. Moreover, LL-37-mediated plasmid uptake and reporter gene expression were strictly dependent on cell surface proteoglycans. We conclude that the human antimicrobial peptide LL-37 binds to, protects, and efficiently targets DNA plasmid to the nuclei of mammalian cells through caveolae-independent membrane raft endocytosis and cell surface proteoglycans. Several in vitro and in vivo studies have provided compelling evidence for the importance of antimicrobial peptides in the innate host defense of multicellular organisms against microbial intruders (1Boman H.G. Scand. J. Immunol. 1998; 48: 15-25Crossref PubMed Scopus (202) Google Scholar, 2Bals R. Weiner D.J. Mosciono A.D. Meegalla R.L. Wilson J.M. Infect. Immun. 1999; 67: 6084-6089Crossref PubMed Google Scholar, 3Lehrer R.I. Ganz T. Curr. Opin. Immunol. 1999; 11: 23-27Crossref PubMed Scopus (643) Google Scholar, 4Hancock R.E. Scott M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8856-8861Crossref PubMed Scopus (813) Google Scholar, 5Zasloff M. Nature. 2002; 415: 389-395Crossref PubMed Scopus (6655) Google Scholar). Antimicrobial peptides were initially isolated from insects and subsequently also in higher organisms in which an adaptive immune system has evolved (6Boman H.G. Annu. Rev. Immunol. 1995; 13: 61-92Crossref PubMed Scopus (1497) Google Scholar, 7Gudmundsson G.H. Agerberth B. J. Immunol. Methods. 1999; 232: 45-54Crossref PubMed Scopus (152) Google Scholar). A common characteristic among antimicrobial peptides (>700 described so far) is the ability to adopt an amphipathic conformation where clusters of hydrophobic and cationic amino acids are spatially organized in discrete sections of the molecule. The relative sensitivity of bacterial cells to peptide cytotoxicity has generally been attributed to the high content of negatively charged phospholipids in the outer membrane leaflet as opposed to that of animal and plant cells, which principally consist of electroneutral lipids (8Ganz T. J. Clin. Investig. 2002; 109: 693-697Crossref PubMed Scopus (180) Google Scholar).The defensins and the cathelicidins are the two major families of antimicrobial peptides in mammals. Cathelicidins consist of a highly conserved N-terminal cathelin domain and a more diverse antimicrobial C terminus (9Zanetti M. Del Sal G. Storici P. Schneider C. Romeo D. J. Biol. Chem. 1993; 268: 522-526Abstract Full Text PDF PubMed Google Scholar, 10Zanetti M. Gennaro R. Romeo D. FEBS Lett. 1995; 374: 1-5Crossref PubMed Scopus (598) Google Scholar). LL-37, a 37-amino acid peptide with two N-terminal leucines, is the only known human cathelin-associated antimicrobial peptide. The precursor of LL-37, hCAP-18, and its mouse homolog, CRAMP, are primarily expressed in bone marrow cells but are also broadly expressed in nonmyeloid tissues, including epididymis, spermatids, and epithelial cells of a number of organs, emphasizing their role in primary host defense (11Agerberth B. Gunne H. Odeberg J. Kogner P. Boman H.G. Gudmundsson G.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 195-199Crossref PubMed Scopus (431) Google Scholar, 12Larrick J. Hirata M. Balint R. Lee J. Zhong J. Wright S. Infect. Immun. 1995; 63: 1291-1297Crossref PubMed Google Scholar, 13Cowland J.B. Johnsen A.H. Borregaard N. FEBS Lett. 1995; 368: 173-176Crossref PubMed Scopus (301) Google Scholar, 14Gallo R.L. Kim K.J. Bernfield M. Kozak C.A. Zanetti M. Merluzzi L. Gennaro R. J. Biol. Chem. 1997; 272: 13088-13093Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 15Bals R. Wang X. Zasloff M. Wilson J.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9541-9546Crossref PubMed Scopus (606) Google Scholar). The LL-37 concentration in adult sweat and the bronchoalveolar lavage fluid of infants is ∼1 μm (16Davidson D.J. Currie A.J. Reid G.S. Bowdish D.M. MacDonald K.L. Ma R.C. Hancock R.E. Speert D.P. J. Immunol. 2004; 172: 1146-1156Crossref PubMed Scopus (344) Google Scholar). Importantly, expression and excretion of LL-37 are induced severalfold upon infectious or inflammatory stimuli, both in keratinocytes and in epithelial cells at other sites (17Frohm M. Agerberth B. Ahangari G. Ståhle-Bäckdahl M. Lidén S. Wigzell H. Gudmundsson G.H. J. Biol. Chem. 1997; 272: 15258-15263Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 18Dorschner R.A. Pestonjamasp V. Tamakuwala S. Ohtake T. Rudisill J. Nizet V. Agerberth B. Gudmundsson G.H. Gallo R.L. J. Investig. Dermatol. 2001; 117: 91-97Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar), e.g. the LL-37 concentration in skin lesions from patients with psoriasis reaches the impressive median value of 304 μm (16Davidson D.J. Currie A.J. Reid G.S. Bowdish D.M. MacDonald K.L. Ma R.C. Hancock R.E. Speert D.P. J. Immunol. 2004; 172: 1146-1156Crossref PubMed Scopus (344) Google Scholar). A wide range of Gram-negative as well as Gram-positive bacteria is sensitive to the cytotoxic activity of LL-37 at substantially lower peptide concentrations (19Lehrer R.I. Ganz T. Curr. Opin. Hematol. 2002; 9: 18-22Crossref PubMed Scopus (277) Google Scholar). The antibacterial activity is regulated by environmental changes in osmolarity or pH, which relates to the fact that LL-37 adopts an α-helical oligomeric conformation in its active state (20Johansson J. Gudmundsson G.H. Rottenberg M.E. Berndt K.D. Agerberth B. J. Biol. Chem. 1998; 273: 3718-3724Abstract Full Text Full Text PDF PubMed Scopus (511) Google Scholar). Accordingly, LL-37 activity was reduced in the respiratory epithelia of mice suffering from cystic fibrosis, a condition associated with increased extracellular osmolarity, and virus-mediated over-expression of LL-37 restored bacterial killing to normal levels in cystic fibrosis xenografts (21Bals R. Weiner D.J. Meegalla R.L. Wilson J.M. J. Clin. Investig. 1999; 103: 1113-1117Crossref PubMed Scopus (178) Google Scholar). In addition to bacterial cell lysis, LL-37 has been reported to neutralize bacterial endotoxin and to possess chemoattractive effects on leukocytes, which may provide additional mechanisms by which LL-37 combats microbial invasion (12Larrick J. Hirata M. Balint R. Lee J. Zhong J. Wright S. Infect. Immun. 1995; 63: 1291-1297Crossref PubMed Google Scholar, 23De Yang B. Chen Q. Schmidt A.P. Anderson M. Wang J.M. Wooters J. Oppenheim J.J. Chertov O. J. Exp. Med. 2000; 192: 1069-1074Crossref PubMed Scopus (987) Google Scholar, 24Agerberth B. Charo J. Werr J. Olsson B. Idali F. Lindbom L. Kiessling R. Jörnvall H. Wigzell H. Gudmundsson G.H. Blood. 2000; 96: 3086-3093Crossref PubMed Google Scholar).In a recent report by Islam et al. (25Islam D. Bandholtz B. Nilsson J. Wigzell H. Christensson B. Agerberth B. Gudmundsson H.G. Nat. Med. 2001; 7: 180-185Crossref PubMed Scopus (358) Google Scholar), it was shown that in bacterial infections with Shigella, expression of LL-37 and β-defensin-1 is reduced or turned off, which could partly explain the chronic inflammatory response associated with Shigella infections. Interestingly, the study further demonstrated that plasmid DNA, released from lysed bacteria by the action of LL-37, was a major mediator of antimicrobial peptide down-regulation. Treatment of bacterial lysates with DNase blocked down-regulation of LL-37, whereas pure bacterial plasmid mediated the effect.This study investigates the ability of the antimicrobial peptide LL-37 to interact with extracellular DNA and to mediate uptake and nuclear transfer of functional plasmids in mammalian cells. The involvement of cholesterol-rich plasma membrane domains (26Nichols B.J. Lippincott-Schwartz J. Trends Cell Biol. 2001; 11: 406-412Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar) and cell surface proteoglycan (PG) 1The abbreviations used are: PG, proteoglycan; CHO, Chinese hamster ovary; CTxB, cholera toxin subunit B; FCM, flow cytometry; HS, heparan sulfate; MCD, methyl-β-cyclodextrin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HIV, human immunodeficiency virus; CDTA, 1,2-cyclohexylenedinitrilotetraacetic acid. 1The abbreviations used are: PG, proteoglycan; CHO, Chinese hamster ovary; CTxB, cholera toxin subunit B; FCM, flow cytometry; HS, heparan sulfate; MCD, methyl-β-cyclodextrin; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HIV, human immunodeficiency virus; CDTA, 1,2-cyclohexylenedinitrilotetraacetic acid. in LL-37-mediated DNA internalization is also explored.EXPERIMENTAL PROCEDURESMaterials—LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES-C-amide) was synthesized by Innovagen AB, Lund, Sweden. The peptide sequence was confirmed by reverse phase high pressure liquid chromatography and mass spectrometry. pGL3 control vector (luciferase reporter gene plasmid) was provided by Dr. Oldberg (Lund University, Sweden), and the luciferase assay system was purchased from Promega. DNA was labeled with YOYO-1 fluorophore (Molecular Probes) according to the standard protocol provided by the manufacturer. Rabbit anti-caveolin-1 antibody was from Transduction Laboratories (Lexington, KY), and goat anti-rabbit IgG antibody labeled with Texas Red fluorophore and cholera toxin subunit B (CTxB)-Alexa Fluor 594 conjugate were from Molecular Probes. Heparan sulfate (HS) preparations were the same as described previously (27Belting M. Petersson P. J. Biol. Chem. 1999; 274: 19375-19382Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Rhodamine green (Molecular Probes)-labeled HS was prepared as described by Cheng et al. (28Cheng F. Petersson P. Arroyo-Yanguas Y. Westergren-Thorsson G. J. Cell. Biochem. 2001; 83: 597-606Crossref PubMed Scopus (32) Google Scholar). LipofectAMINE reagent was from Invitrogen. PD-10 and Microspin S-200 HR columns were from Amersham Biosciences AB. All fine chemicals were from Sigma.Cell Culture—Wild-type Chinese hamster ovary cells (CHO-K1), pgsA-745 (PG-deficient CHO mutant), pgsB-618 (PG-deficient CHO mutant), COS-7, and human embryonic lung fibroblasts (HFL-1) were obtained from the American Type Culture Collection (Manassas, VA). CHO cells were routinely cultured in F12K nutrient mixture supplemented with 10% (v/v) fetal calf serum, 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin (growth medium) in a humidified 5% CO2 (37 °C) incubator. HFL-1 and COS-7 cells were maintained in Eagle's minimal essential medium with the same supplements as described above.Nuclease Protection Assay—pGL3 DNA plasmid (20 μg/ml) was incubated in minimal essential medium supplemented with 50% fetal calf serum either in the absence or in the presence of LL-37 (100 μg/ml). After incubation at 37 °C for 16 h, the incubation was terminated by the addition of sodium chloride (final concentration, 1 m), followed by repeated phenol/chloroform extraction and sodium acetate precipitation to recover the DNA. The samples were finally analyzed by gel electrophoresis (0.8% agarose, supplemented with ethidium bromide) and visualized using a trans-illuminator system from Techtum Laboratory.Cell Growth Assays—To assess mammalian cell (CHO-K1, pgsA-745, and COS-7) proliferation, the same procedure was used as described previously (29Belting M. Havsmark B. Jönsson M. Persson S. Fransson L.-Å. Glycobiology. 1996; 6: 121-129Crossref PubMed Scopus (34) Google Scholar). In brief, cells were seeded in 96-well microplates at 3 × 103 cells/well and allowed to adhere for 4 h in growth medium, followed by a starvation period of 24 h in serum-free medium. The medium was aspirated and cells were incubated for 72 h at 37 °C in growth medium with or without LL-37 at the indicated concentrations. Cell growth was determined from the amount of crystal violet adsorbed to the cells as measured in a Multiscan 351 photometer (Labsystems) at 595 nm. For bacterial cytotoxicity assays, Escherichia coli cells (Top 10, provided by Dr. Oldberg, Lund University, Sweden) in log growth phase were incubated in minimal essential medium with or without various concentrations of LL-37 for 4 h at 37 °C. The bacteria were then pelleted and redissolved in 2-YT broth and seeded on agar plates. Following another growth period of ∼14 h, colony-forming units were determined.Flow Cytometry (FCM)—Fluorophore-labeled DNA plasmid or labeled HS were preincubated with LL-37 at different concentrations in F12K for 30 min at room temperature. The mixture was then added to prerinsed subconfluent cell layers in 24-well plates, and the incubations were allowed to proceed for various periods of time at 37 °C or, in some cases, at 4 °C. After removal of the incubation medium and rinsing with PBS, the cells were detached with trypsin followed by extensive washing with ice-cold PBS, 1% (w/v) BSA to remove unspecific extracellular fluorophore. Finally, the cells were suspended in PBS, 1% BSA and analyzed for fluorescence in a fluorescence-activated cell sorter instrument (FACSCalibur, BD Biosciences) operated by Cell-Quest software.Luciferase Gene Expression Assay—Cells were plated on 24-well plates at 1 × 105 cells/well in 0.5 ml of growth medium for 24 h before peptide-DNA incubation. To prepare LL-37·DNA complexes, pGL3-luciferase plasmid DNA (10 μg/ml) and LL-37 (5-100 μg/ml) were mixed by inversion and incubated for 30 min at room temperature. The DNA-peptide mixture was then added to subconfluent cell layers. At the end of the 4-h incubation at 37 °Cin5%CO2, the medium was aspirated and replaced with 0.5 ml of growth medium. After an additional period of incubation for ∼36 h, the cells were washed with PBS and solubilized in 200 μl of cell lysis reagent containing 25 mm Tris-HCl, pH 7.8, 2 mm CDTA, 2 mm dithiothreitol, 10% glycerol, and 1% Triton X-100 for 1 h at 4 °C. Luciferase expression was quantified in 25 μl of the cell lysate supernatant using a luciferase assay kit. Light emission was measured by integration over 15 s at 25 °C using BMG Fluostar Optima equipment (Labvision). To investigate the involvement of cholesterol-rich membrane domains in LL-37·DNA complex uptake, cells were pretreated with either nystatin (50 μg/ml), filipin (5 μg/ml), 1 mm methyl-β-cyclodextrin (MCD), or 1-5 mm MCD-cholesterol for 30 min at 37 °C. The cells were then incubated with DNA or HS and LL-37 in the presence of the respective drug at the indicated concentrations, after which FCM and luciferase experiments were conducted as described above. Under these conditions, no cytotoxicity or changes in cell morphology as a result of drug treatment were observed.Fluorescence Microscopy—Cells were seeded at low density in 4-well chamber slides and allowed to adhere overnight and were then incubated with YOYO-1-DNA and LL-37 according to the figure legends. When used, fluorophore-conjugated CTxB (10 μg/ml) was co-incubated with YOYO-1-DNA and LL-37 for 1 h. The cells were then cleared from unspecific extracellular fluorophore by brief rinsing with 1 m NaCl in PBS followed by extensive rinsing with PBS. The cells were then fixed in 4% (w/v) paraformaldehyde in PBS for 20 min. After fixation, the slides were washed with PBS and distilled water and then air-dried.For caveolin-1 immunostaining, cells were permeabilized using PBS, 0.2% Triton X-100 (v/v) for 20 min or 1 mm saponin in PBS for 5 min and then blocked with 1% goat serum (v/v) in PBS, 1% BSA. The cells were then rinsed (three times for 5 min with PBS, 1% BSA), incubated for 3 h with 1:500 rabbit anti-caveolin-1 antibody in PBS, 1% BSA, rinsed again, and finally incubated for 2 h with 1:500 Texas Red-labeled goat anti-rabbit IgG antibody in PBS, 1% BSA. The slides were then washed with PBS and distilled water and air-dried. The fixed cells were visualized using a Nikon Eclipse E800 microscope and a Bio-Rad MRC 1024 confocal laser scanning microscopy system. The collected data were analyzed using PC-compatible Laser-Sharp software.Statistical Analyses—Each data point in the dose activity and time course experiments is the mean ± S.E. (n = 5-8) from at least two separate experiments. In some cases the error bars were smaller than the drawn symbols.RESULTSLL-37 Peptide Targets Intact DNA Plasmid to the Nuclear Compartment of Mammalian Cells—In the absence of LL-37, DNA plasmid was degraded by serum nucleases, whereas the presence of LL-37 efficiently protected plasmid DNA against serum nuclease activity (Fig. 1). The data suggest significant complex formation between polyanionic DNA and the polybasic peptide LL-37 in 50% serum. LL-37·DNA complex formation may enable efficient internalization of DNA to mammalian cells, as has been shown previously for other polybasic peptides, e.g. polylysine and HIV-Tat (30Mislick K.A. Baldeschwieler J.D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12349-12354Crossref PubMed Scopus (713) Google Scholar, 31Sandgren S. Belting M. J. Biol. Chem. 2002; 277: 38877-38883Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Therefore, the effect of LL-37 on the uptake of plasmid DNA in COS-7 cells was studied (Fig. 2A, filled squares). Under the conditions used, LL-37 increased DNA plasmid uptake up to 6-fold. The same results were obtained with CHO cells, human embryonic fibroblasts, and bladder carcinoma cells (results not shown). The effect of LL-37 was not specific for DNA, as cellular uptake of HS (a major extracellular polyanion) was also substantially increased by LL-37 (up to ∼20-fold; Fig. 2A, filled triangles). The data indicate that LL-37 binds to DNA via nonspecific electrostatic interactions that result in the protection of DNA from serum nuclease cleavage and transfer of DNA over the plasma membrane. To test the possibility that increased DNA uptake in the presence of LL-37 was simply a result of membrane rupture and cytotoxicity, cell proliferation studies, with several of the cell lines included in this study, were performed. None of the cell lines was affected at relevant LL-37 concentrations (up to 10 μm), whereas E. coli bacteria, as expected, were efficiently killed by the LL-37 preparation used in this study (Table I).Fig. 2LL-37 mediates uptake and efficient expression of exogenous plasmid DNA in mammalian cells. A, fluorophore-labeled DNA (▪) or HS (▴) at 10 μg/ml was premixed with LL-37 (0, 2, 4, and 8 μm) and then incubated with confluent COS-7 cells for 4 h at 37 °C. After removal of the incubation medium, the cells were detached with trypsin followed by extensive washing with ice-cold PBS, 1% BSA to remove unspecific extracellular fluorophore. The cells were then suspended in PBS, 1% BSA, and intracellular DNA and HS were determined by FCM. Results are presented as means ± S.E. (error bars, n = 5). a.u., arbitrary units. B, pGL3-luciferase plasmid (10 μg/ml) and the indicated concentrations of LL-37 were premixed for 30 min at room temperature. The DNA-peptide mixture was then added to subconfluent cell layers for 4 h at 37 °C. After an additional period of incubation for ∼36 h in growth medium, luciferase activity was determined as described under “Experimental Procedures.” Results are presented as means ± S.E. (error bars, n = 8). RLU, relative light units.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IEffects of LL-37 on the growth of E. coli bacteria and mammalian cells[LL-37]GrowthE. coliCHO-K1PgsA-745COS-7μm% of control0100 ± 32100 ± 5100 ± 2100 ± 160.593 ± 1694 ± 399 ± 4115 ± 101.539 ± 18NDND122 ± 630.1 ± 0.195 ± 2100 ± 4126 ± 65NDNDND122 ± 910ND85 ± 1297 ± 6122 ± 7 Open table in a new tab In the next series of experiments, a luciferase reporter gene expression assay was employed to gain more insight into the intracellular fate of internalized LL-37·DNA complexes. As shown in Fig. 2B, LL-37 peptide substantially increased the expression of luciferase reporter plasmid in CHO cells (up to ∼1600-fold). The data indicate that LL-37 mediates significant nuclear transfer of intact DNA plasmid in cultured mammalian cells with no concomitant cytotoxic effects. This notion was supported by confocal fluorescence microscopy studies that demonstrated rapid nuclear accumulation of internalized DNA in the presence of LL-37 (Fig. 3B). At 4 h of incubation, fluorescence was restricted to well defined vesicular structures at the cell membrane and in the cytoplasm (Fig. 3B, left panel). At 24 h, internalized DNA displayed a more diffuse staining pattern in the cytosol, consistent with release from endocytotic vesicles, and could also be observed to be largely redistributed to the nuclear compartment (Fig. 3B, right panel). In the absence of peptide, a much weaker DNA signal was observed in relatively few and small vesicular structures (results not shown). LL-37-mediated DNA uptake was time-dependent (Fig. 3A), saturable, and temperature-dependent (no DNA uptake at 4 °C, results not shown), which is consistent with an endocytotic uptake mechanism.Fig. 3LL-37 targets exogenous plasmid DNA to the nuclear compartment. A, CHO-K1 cells grown to confluence were incubated with YOYO-1-DNA (10 μg/ml) and LL-37 (8 μm) for 0, 2, 4, and 8 h. The cells were then analyzed for intracellular DNA by FCM as described in Fig. 2A. Results are presented as means ± S.E. (error bars, n = 6). a.u., arbitrary units. B, CHO-K1 cells were sparsely seeded on chamber slides and allowed to adhere for a period of 24 h followed by incubation with YOYO-1-DNA (10 μg/ml, green) and LL-37 (8 μm) for 4 h (left panel) and 24 h (right panel). The cells were then processed for confocal fluorescence microscopy as described under “Experimental Procedures.” Bars indicate 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)LL-37-mediated Gene Transfer Occurs via Membrane Rafts and Cell Surface PGs—We next sought to determine the molecular mechanism of LL-37·DNA complex uptake. For this purpose, cells were treated with either MCD or nystatin, both of which represent well established cholesterol-depleting agents employed for studying the involvement of rafts/caveolae in cellular transport (32Kilsdonk E.P. Yancey P. Stoudt G.W. Bangerter F.W. Johnson W.J. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 17250-17256Abstract Full Text Full Text PDF PubMed Scopus (701) Google Scholar, 33Ono A. Freed E.O. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13925-139230Crossref PubMed Scopus (559) Google Scholar, 34Pelkmans L. Kartenbeck J. Helenius A. Nat. Cell Biol. 2001; 3: 473-483Crossref PubMed Scopus (1041) Google Scholar). Cholesterol- and sphingolipid-enriched membrane domains may exist either as small phase-separated “rafts” or, when associated with caveolin, may form flask-shaped invaginations called caveolae, which are involved in special forms of nonclathrin-dependent endocytosis (26Nichols B.J. Lippincott-Schwartz J. Trends Cell Biol. 2001; 11: 406-412Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). As shown in Fig. 4A, cholesterol depletion resulted in an ∼65% reduction of LL-37-mediated internalization of both DNA and HS. Next, the effect of cholesterol depletion on the expression of internalized reporter gene plasmid was studied (Fig. 4B). Both MCD and nystatin dramatically reduced reporter gene activity after incubations in the presence of LL-37 (∼90 and 99% inhibition with MCD and nystatin, respectively). Notably, both treatments enhanced reporter gene expression following incubation of naked DNA plasmid (an ∼4.5- and 11-fold increase with MCD and nystatin, respectively). Overall, the data indicate that disruption of raft/caveolar structures by cholesterol depletion inhibits LL-37·DNA complex uptake and, accordingly, expression of internalized DNA plasmid. Similar results were obtained with filipin (results not shown), another known inhibitor of raft/caveolar endocytosis. The fact that drug treatment enhanced the expression of naked DNA plasmid makes it highly unlikely that drug effects were simply a result of cellular toxicity. We next used the method described by Roepstorff et al. (35Roepstorff K. Thomsen P. Sandvig K. van Deurs B. J. Biol. Chem. 2002; 277: 18954-18960Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) to investigate the possible enhancing effect of cholesterol loading on LL-37-mediated DNA uptake. With the concentrations of MCD-cholesterol used in this study (1-5 mm), no further enhancement was observed (results not shown).Fig. 4LL-37-mediated gene transfer involves membrane rafts. A, subconfluent CHO-K1 cells were either untreated (Control) or pretreated with MCD (1 mm) or nystatin (50 μg/ml) for 30 min at 37 °C followed by incubation with LL-37 (8 μm) and YOYO-1-DNA (10 μg/ml) or rhodamine green-HS (10 μg/ml), respectively, for 4 h. Cholesterol-depleting agents were present during the whole course of the experiments. The cells were then analyzed for intracellular DNA or HS by FCM. B, subconfluent CHO-K1 cells were pretreated with nystatin (50 μg/ml) or MCD (1 mm) for 30 min, as indicated by the table below panel B, followed by another incubation period of 4 h with pGL3-luciferase plasmid (8 μg/ml) and LL-37 (5 μm), as indicated, in the continued presence of the respective cholesterol-depleting drugs. The media were then replaced with growth medium, and the cells were incubated in the absence of cholesterol-depleting drugs for 36 h and then finally analyzed for luciferase activity. Results are presented as means ± S.E. (error bars, n = 8). RLU, relative light units. C, sparsely seeded CHO-K1 cells were incubated with YOYO-1-DNA (10 μg/ml, green) and LL-37 (8 μm) for 4 h. The cells were stained for caveolin-1 (red) and processed for confocal fluorescence microscopy as described under “Experimental Procedures.” D, sparsely seeded CHO-K1 cells were incubated with YOYO-1-DNA (10 μg/ml, green) and LL-37 (8 μm) for 1 h in the presence of CTxB (10 μg/ml, red) and then processed for confocal fluorescence microscopy as described under “Experimental Procedures.” Bars indicate 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Confocal fluorescence studies were then performed to examine possible co-localization of internalized DNA with caveolin, a well established marker of caveolae (26Nichols B.J. Lippincott-Schwartz J. Trends Cell Biol. 2001; 11: 406-412Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). As shown in Fig. 4C, there was no co-localization between DNA-positive vesicles (green) and caveolae or caveosomes (red) under the conditions used. This finding was consistent irrespective of the method of cell permeabilization" @default.
• W2016809324 created "2016-06-24" @default.
• W2016809324 creator A5000511870 @default.
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• W2016809324 date "2004-04-01" @default.
• W2016809324 modified "2023-10-16" @default.
• W2016809324 title "The Human Antimicrobial Peptide LL-37 Transfers Extracellular DNA Plasmid to the Nuclear Compartment of Mammalian Cells via Lipid Rafts and Proteoglycan-dependent Endocytosis" @default.
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