Nucleolin-mediated cellular trafficking of DNA nanoparticle is lipid raft and microtubule dependent and can be modulated by glucocorticoid.
DNA nanoparticles (DNPs) are nonviral gene transfer vectors with excellent in vivo potential. Previously, we reported that cell surface nucleolin directly binds DNPs, and functions as an important receptor for DNPs. However, the fate of the nucleolin-DNP complex following cellular uptake remains elusive. In this study, we examined the role of lipid rafts in the uptake of DNPs, and found that both nucleolin and DNPs are recovered from the low-density raft fractions of the sucrose gradient. Furthermore, nucleolin colocalizes with, and coimmunoprecipitates with a raft protein, flotillin. Disruption of lipid rafts by depleting membrane cholesterol significantly inhibited DNP transfection, while inhibition of other endocytic pathways had little effect. Following the uptake, the nuclear import of the DNPs required microtubules but not F-actin. By coimmunoprecipitation in conjunction with tandem mass spectrometry, we identified glucocorticoid receptor (GCR) as a nucleolin-associated protein, and confirmed this result by western blot. Cortisone or dexamethasone increased nucleolin's association with GCR, and transfection by DNPs. Finally, we detected the expression of nucleolin on the surface of airway epithelia in vivo. Taken together, our findings shed light on important determinants of DNP trafficking in cells and support the notion that nucleolin is a good target for nonviral gene delivery.
Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
Xuguang
X
Chen
Samuel
S
Shank
Pamela B
PB
Davis
Assem G
AG
Ziady
D000199:Actins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D003348:Cortisone / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods; D004705:Endocytosis / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D053758:Nanoparticles / Q000737:chemistry*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods; D014162:Transfection / Q000379:methods; D014407:Tumor Cells, Cultured; D000097796:Nucleolin
D000199:Actins; D002784:Cholesterol; D003348:Cortisone; D004247:DNA; D003907:Dexamethasone; D008565:Membrane Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D013395:Sucrose; D000097796:Nucleolin; C106354:flotillins